AGREEprep in Action: A Practical Guide to Assessing Green Microextraction Methods for Biomedical Analysis

Victoria Phillips Nov 27, 2025 93

This article provides a comprehensive guide for researchers and drug development professionals on applying the AGREEprep metric tool to evaluate the environmental impact of microextraction techniques.

AGREEprep in Action: A Practical Guide to Assessing Green Microextraction Methods for Biomedical Analysis

Abstract

This article provides a comprehensive guide for researchers and drug development professionals on applying the AGREEprep metric tool to evaluate the environmental impact of microextraction techniques. Covering foundational principles to advanced applications, it details how AGREEprep's ten-criteria framework aligns with Green Sample Preparation (GSP) to quantify the greenness of methods like SPME, DLLME, and MSPE. The content explores methodological adaptations for bioanalysis, strategies for optimizing scores, and the integration of AGREEprep with complementary tools like BAGI and White Analytical Chemistry (WAC) for a holistic sustainability assessment. Practical insights from recent case studies in therapeutic drug monitoring (TDM) and environmental analysis illustrate how to balance greenness with analytical performance, offering a clear roadmap for implementing sustainable practices in analytical laboratories.

Understanding AGREEprep: The Foundation for Green Microextraction Assessment

Green Sample Preparation (GSP) represents a transformative approach in analytical chemistry that aims to minimize the environmental impact of one of the most resource-intensive stages in chemical analysis. Traditional sample preparation methods often consume substantial energy, utilize toxic solvents, and generate significant laboratory waste, creating substantial environmental concerns [1]. In response to these challenges, the concept of GSP was formally introduced through ten clearly defined principles that provide a comprehensive framework for developing more sustainable sample preparation methodologies [1].

The fundamental motivation behind GSP extends beyond mere environmental responsibility. Analytical chemistry plays a crucial role in evaluating environmental conditions, yet ironically contributes to environmental degradation through consumption of hazardous substances and energy-intensive processes [1]. This contradiction highlights the urgent need for greening analytical practices, with sample preparation representing the most accessible target for substantial improvements as it doesn't require complete overhaul of established instrumental methods [2].

The Ten Principles of Green Sample Preparation

The Ten Principles of Green Sample Preparation form an integrated system where improvements in one principle often synergistically address deficiencies in others [1]. These principles provide specific, actionable goals for method development:

  • Favoring in situ sample preparation
  • Using safer solvents and reagents
  • Targeting sustainable, reusable and renewable materials
  • Minimizing waste generation
  • Minimizing sample, chemical and material amounts
  • Maximizing sample throughput
  • Integrating steps and promoting automation
  • Minimizing energy consumption
  • Choosing the greenest possible post-sample preparation configuration for analysis
  • Ensuring safe procedures for the operator [3] [1]

These principles establish sample preparation as a central focus rather than an afterthought, defining greenness according to the specific requirements and constraints of this critical analytical step [1].

The Critical Need for Standardized Assessment Metrics

Without standardized metrics, evaluating the environmental performance of analytical methods remains subjective and inconsistent. The development of standardized greenness assessment tools addresses three fundamental challenges in sustainable analytical chemistry:

Enabling Objective Comparisons

Standardized metrics provide a common framework for evaluating methods across different laboratories and research groups, eliminating calculation inconsistencies that can lead to misleading comparisons [4]. Just as standardized clinical metrics enable reliable healthcare assessments, analytical greenness metrics ensure that evaluations are sharable, repeatable, and statistically valid [4].

Identifying Improvement Opportunities

Comprehensive metric tools do more than generate overall scores—they pinpoint specific weaknesses in methods, allowing researchers to focus improvement efforts where they will have greatest environmental impact [1]. This diagnostic capability transforms green chemistry from an abstract concept to a practical optimization process.

Balancing Analytical and Environmental Performance

The ultimate goal of GSP is not merely to reduce environmental impact, but to develop methods that maintain or enhance analytical performance while improving sustainability [2]. Standardized metrics help balance these sometimes competing priorities, ensuring that greener methods still produce reliable, precise results [3].

AGREEprep: A Standardized Metric for Sample Preparation

AGREEprep represents the first dedicated metric tool specifically designed to evaluate the environmental impact of sample preparation methods [1]. Developed by members of the IUPAC project #2021-015-2-500, this open-source software tool operationalizes the ten principles of GSP into a practical assessment framework [1].

How AGREEprep Works

The AGREEprep calculator evaluates methods against the ten GSP principles through ten assessment criteria, each scored from 0 to 1, where extremes represent worst and best performance [5]. Each criterion has a default weight that contributes to the overall score, though users can adjust these weights to reflect specific analytical priorities [1]. The tool generates an intuitive circular pictogram that visually communicates both overall performance and individual criterion scores, creating an immediate understanding of a method's environmental strengths and weaknesses [5].

AGREEprep Assessment Workflow

The following diagram illustrates the standardized workflow for conducting GSP assessments using the AGREEprep metric tool:

G Start Define Sample Preparation Method Step1 Input 10 GSP Principle Parameters Start->Step1 Step2 AGREEprep Software Calculation Step1->Step2 Step3 Generate Assessment Pictogram Step2->Step3 Step4 Interpret Weak & Strong Points Step3->Step4 Step5 Method Optimization & Improvement Step4->Step5

AGREEprep in Microextraction Research

In the context of microextraction methods research, AGREEprep provides critical quantitative data on environmental performance. Recent studies applying AGREEprep to microextraction techniques for therapeutic drug monitoring (TDM) have demonstrated that some methods achieve high greenness scores while maintaining excellent analytical performance [3]. This balance is particularly crucial in TDM applications, where method reliability directly impacts patient care decisions.

Comparative Analysis of Green Assessment Tools

While AGREEprep specializes in sample preparation evaluation, researchers increasingly employ multiple metrics to gain comprehensive understanding of method performance. The table below compares major green assessment tools used in analytical chemistry:

Table 1: Standardized Metrics for Assessing Green Analytical Methods

Metric Tool Focus Area Scoring System Output Format Key Principles Assessed
AGREEprep Sample preparation 0-1 scale for 10 criteria Circular pictogram 10 GSP principles [1]
AGREE Overall analytical method 0-1 scale for 12 criteria Circular pictogram 12 GAC principles [5]
BAGI Method practicality & cost Points-based system Numerical score Cost, time, efficiency [5]
RGB 12 Comprehensive assessment 0-4 points for 12 criteria Rectangular diagram White Analytical Chemistry principles [3] [5]
GAPI Overall analytical method Qualitative assessment Pictogram 5 areas of environmental impact [2]

The Emergence of White Analytical Chemistry

A significant development in assessment methodology is the concept of White Analytical Chemistry (WAC), which expands beyond purely environmental concerns to balance greenness with analytical performance and practical/economic factors [3] [5]. The WAC approach uses an RGB color model where red represents analytical performance (scope, LOD, LOQ, precision, accuracy), green represents environmental factors (toxicity, waste, energy, direct impacts), and blue represents practical considerations (cost, time, requirements, operational simplicity) [3]. Just as white light combines all colors, an ideal "white" method balances all three dimensions [5].

Experimental Protocols for AGREEprep Assessment of Microextraction Methods

Protocol: AGREEprep Evaluation of Solid-Phase Microextraction

Objective: Quantitatively evaluate the greenness of an SPME method for determining pharmaceutical compounds in water samples.

Materials and Reagents:

  • SPME fibers (specific coating material)
  • Agitation device (orbital shaker or magnetic stirrer)
  • Sample vials with septa
  • HPLC or GC system for analysis

Table 2: Research Reagent Solutions for SPME Methodology

Item Function Green Characteristics
Reusable SPME Fiber Extraction and pre-concentration of analytes Reduces solid waste; multiple uses from single device [2]
Aqueous Sample Analysis matrix Avoids organic solvents; safer for operator and environment [2]
Minimal Organic Solvent Desorption of analytes (if required) Reduced volume (μL scale) compared to traditional extraction [3]
Direct Analysis Configuration Compatibility with analytical instrument Eliminates additional sample preparation steps [1]

Procedure:

  • Sample Collection: Collect 10 mL water sample in sealed vial
  • Extraction: Expose SPME fiber to sample with agitation for 30 minutes at room temperature
  • Desorption: Desorb analytes directly into analytical instrument (5 minutes at 250°C for GC)
  • Analysis: Perform chromatographic separation and detection
  • AGREEprep Input: Enter method parameters into AGREEprep software:
    • Sample preparation location: off-site (score: 0.0)
    • Solvent type: none (score: 1.0)
    • Material sustainability: reusable fiber (score: 0.8)
    • Waste generation: <0.1 g (score: 1.0)
    • Sample size: 10 mL (score: 0.7)
    • Throughput: 12 samples/hour (score: 0.6)
    • Step integration: fully integrated (score: 1.0)
    • Energy consumption: none (score: 1.0)
    • Post-preparation configuration: direct to GC (score: 1.0)
    • Operator safety: no significant hazards (score: 1.0)

Expected Outcomes: SPME typically achieves high AGREEprep scores (0.7-0.9) due to minimal solvent use, reusability, and waste reduction [2].

Protocol: Comparative AGREEprep Assessment of Multiple Microextraction Techniques

Objective: Systematically compare greenness profiles of different microextraction methods for UV filter analysis in water.

Materials: SPME, DLLME, and SBSE equipment; appropriate solvents and reagents for each method.

Procedure:

  • Method Implementation: Apply each microextraction technique to identical water samples spiked with UV filters
  • Data Collection: Record consumption of materials, solvents, energy, and time for each method
  • AGREEprep Evaluation: Input parameters for each method into separate AGREEprep assessments
  • Score Comparison: Compare overall scores and principle-specific performance
  • Complementary Assessment: Apply BAGI and RGB 12 tools to evaluate practicality and whiteness

Data Analysis: Recent comprehensive assessments of microextraction techniques for UV filter determination reveal that methods like SPME and DLLME achieve high greenness scores while maintaining excellent analytical performance, with AGREEprep scores typically ranging from 0.6-0.8 compared to 0.04-0.36 for traditional methods like Soxhlet extraction [5].

Application in Therapeutic Drug Monitoring Research

The pharmaceutical industry represents a particularly valuable application area for GSP and standardized metrics. Therapeutic drug monitoring (TDM) requires precise, sensitive analysis of drug concentrations in biological samples to optimize dosing regimens, especially for drugs with narrow therapeutic windows [3]. Microextraction techniques coupled with AGREEprep assessment enable development of methods that are not only environmentally sustainable but also suitable for clinical settings where sample volume, operator safety, and throughput are critical concerns [3].

Recent research has demonstrated that specific microextraction techniques can achieve balanced high scores in both greenness and whiteness assessments for TDM applications [3]. This balance is particularly important in pharmaceutical analysis where method reliability directly impacts patient treatment decisions.

The implementation of Green Sample Preparation supported by standardized metrics like AGREEprep represents a fundamental shift in analytical chemistry toward sustainability without compromising analytical performance. The ongoing work by IUPAC and other standards organizations to evaluate and improve official methods signals a growing recognition that green principles must be integrated into analytical practice at all levels [1].

For microextraction researchers, AGREEprep provides not just an assessment tool but a roadmap for method development that systematically addresses the complete environmental impact of sample preparation. As the field advances, the integration of GSP principles with complementary assessment frameworks like White Analytical Chemistry will continue to drive innovation in sustainable analytical technologies that meet the dual demands of analytical excellence and environmental responsibility.

What is AGREEprep? Defining the 10-Principle Framework for Sample Preparation

AGREEprep (Analytical Greenness Metric for Sample Preparation) is a dedicated software-based tool designed to evaluate the environmental impact of sample preparation methods in analytical chemistry. It was developed in 2022 by Wojnowski and colleagues to address the critical need for a standardized assessment of the sample preparation step, which is often a significant contributor to the overall environmental footprint of an analytical procedure [5] [6]. The tool is aligned with the 10 principles of Green Sample Preparation (GSP) and provides a quantitative and visual score, enabling scientists to objectively compare and improve the sustainability of their methods [3] [6].

The development of AGREEprep is particularly relevant in the context of increasing research into microextraction techniques, which aim to minimize solvent consumption and waste generation [5] [7]. As a key component of the broader Green Analytical Chemistry (GAC) framework, AGREEprep helps laboratories transition towards safer and more environmentally friendly practices without compromising the quality of analytical results [3] [8]. Its user-friendly, open-access nature has led to its rapid adoption for evaluating methods in diverse fields, including environmental monitoring, bioanalysis, and pharmaceutical quality control [3] [5].

The 10 Principles of Green Sample Preparation

The AGREEprep assessment is built upon a foundation of ten core principles that define the ideal characteristics of a sustainable sample preparation method. The following table summarizes these principles and their objectives.

Table 1: The Ten Principles of Green Sample Preparation Underlying the AGREEprep Metric

Principle Number Principle Description Primary Objective
1 Favoring in situ sample preparation To avoid or minimize sample manipulation and transport [3].
2 Using safer solvents and reagents To reduce the use of hazardous chemicals [3].
3 Targeting sustainable, reusable, and renewable materials To promote a circular economy for lab materials [3].
4 Minimizing waste To reduce the generation of waste requiring disposal [3].
5 Minimizing sample, chemical, and material amounts To encourage miniaturization and micro-extraction techniques [3].
6 Maximizing sample throughput To improve efficiency, for example via parallel processing [3].
7 Integrating steps and promoting automation To reduce manual operations and human error [3].
8 Minimizing energy consumption To lower the carbon footprint of the procedure [3].
9 Choosing the greenest possible post-sample preparation configuration for analysis To consider the environmental impact of the subsequent analytical technique [3].
10 Ensuring safe procedures for the operator To prioritize analyst health and safety [3].

AGREEprep Workflow and Output Interpretation

The Assessment Workflow

The process of using AGREEprep involves a systematic workflow where the user inputs data related to the ten principles, and the software generates an easy-to-interpret pictogram. The following diagram visualizes this workflow from data input to final assessment.

Start Start AGREEprep Assessment Input Input Data for 10 GSP Principles Start->Input Calculate Software Calculates Scores (0-1) Input->Calculate Weight Apply Default or Custom Weights Calculate->Weight Generate Generate Pictogram and Final Score Weight->Generate Output Final AGREEprep Report Generate->Output

Scoring System and Pictogram

AGREEprep evaluates each of the ten criteria on a scale from 0 to 1, where 0 represents the worst performance and 1 the best performance in terms of greenness [5]. Each criterion is assigned a default weight that influences its contribution to the overall score, though users can adjust these weights based on their specific analytical goals [5] [6].

The output is a circular pictogram that provides an immediate visual summary:

  • Inner Circle and Final Score: The center of the pictogram displays a single overall score between 0 and 1 and is colored on a gradient from red (poor performance) to green (excellent performance). A score above 0.5 is generally considered to indicate a green method [7].
  • Outer Segments: The ten surrounding segments correspond to each of the GSP principles. The color of each segment (from red to green) indicates the individual score for that principle, while the segment's length reflects the assigned weight [5].

Experimental Protocol for AGREEprep Assessment

This protocol provides a step-by-step guide for conducting an AGREEprep assessment, suitable for evaluating a sample preparation method in a research setting.

Pre-Assessment Data Collection

Before starting the software assessment, gather all necessary quantitative and qualitative data for the analytical method. Essential information includes:

  • Solvents and Reagents: Types, volumes, and associated hazard classifications.
  • Materials: Type and mass of sorbents, filters, or other consumables; reusability of items.
  • Energy Consumption: Power requirements (kW) and operation times for all equipment (ovens, centrifuges, etc.).
  • Waste: Total volume and mass of waste generated, categorized by hazard.
  • Throughput and Automation: Number of samples processed per unit time and degree of automation.
  • Operator Safety: Documentation of any specific health risks and safety measures required.
Software-Assisted Evaluation Procedure
  • Access the Tool: Navigate to the open-access AGREEprep software available at https://mostwiedzy.pl/AGREE [5] [7].
  • Input Data: For each of the ten criteria, input the collected data by selecting the appropriate options from the drop-down menus or entering numerical values.
  • Review Weights: Confirm the use of default weights for each criterion or provide a scientific justification for any custom weight adjustments [6].
  • Generate Report: Execute the calculation. The software will automatically generate the characteristic circular pictogram and the final overall score.
  • Interpret and Compare: Use the visual output to identify strengths and weaknesses of the method. Compare the pictograms of different methods to select the most sustainable option.

Table 2: Essential Research Reagent Solutions for Microextraction Method Development

Reagent/Material Function in Sample Preparation Greenness Considerations
Deep Eutectic Solvents (DES) Used as a safer alternative to traditional organic solvents in liquid-phase microextraction [9]. Biodegradability, low toxicity, and derivation from renewable sources align with Principles 2 and 3 [9].
Switchable Hydrophilicity Solvents (SHS) Enable extraction and phase separation triggered by a physical/chemical stimulus like COâ‚‚ [9]. Reduces waste and energy for solvent removal, supporting Principles 4 and 8 [9].
Supramolecular Solvents (SUPRAS) Possess unique nanostructures that provide high solubilizing power for diverse analytes [9]. Can be made from environmentally friendly constituents, addressing Principle 2 [9].
Reusable Sorbents (e.g., FPSE, MEPS) Solid-phase materials for extracting analytes from complex matrices like biological samples [3] [5]. Reusability significantly reduces material consumption and waste, directly supporting Principle 3 [3].

AGREEprep in Practice: A Microextraction Case Study

The application of AGREEprep is effectively demonstrated in the greenness assessment of a method for determining nitro compounds in water samples using Direct Immersion Single-Drop Microextraction (DI-SDME) [9].

In this study, the method utilized toluene as the extraction solvent, with a consumption of only 1 µL per sample. This extremely low solvent volume is a direct application of Principle 5 (minimizing amounts) and Principle 4 (minimizing waste). The method was successfully applied to various water matrices, including tap water and seawater, achieving low detection limits ranging from 0.01 to 0.11 µg/L [9]. The high sensitivity and minimal solvent consumption of this microextraction approach are key factors that would contribute to a high AGREEprep score, particularly for the principles related to waste generation and the use of reagents. This case underscores how microextraction techniques, when evaluated with AGREEprep, can demonstrate a strong alignment with the goals of Green Sample Preparation.

The AGREEprep (Analytical Greenness Metric for Sample Preparation) tool is the first dedicated metric designed to evaluate the environmental impact of sample preparation methods in analytical chemistry [10] [11]. Introduced in 2022 by members of an IUPAC project, it addresses a critical gap in green chemistry assessment by focusing specifically on the sample preparation stage, which is often the most resource-intensive and environmentally impactful part of the analytical workflow [11]. The metric is grounded in the 10 principles of Green Sample Preparation (GSP), which form an integrated system where improvements in one principle can synergistically address deficiencies in others [11].

AGREEprep was developed in response to the recognition that despite advancements in green technologies, many official analytical methods still rely on traditional sample preparation procedures that utilize harmful solvents and generate large amounts of toxic waste [11]. The tool provides analysts with a standardized approach to quantify and visualize the greenness of their sample preparation methods, thereby facilitating the selection and development of more sustainable analytical procedures [10].

Understanding the AGREEprep Assessment Criteria

The Ten Principles of Green Sample Preparation

The AGREEprep assessment is structured around ten fundamental principles that collectively define the paradigm of green sample preparation. Each principle corresponds to a specific evaluation criterion within the AGREEprep metric [11]:

  • Favoring in situ sample preparation: Prioritizing methods that require minimal sample manipulation and can be performed directly in the sample environment.
  • Using safer solvents and reagents: Selecting solvents and reagents with lower toxicity and environmental impact.
  • Targeting sustainable, reusable, and renewable materials: Prioritizing materials derived from renewable sources or those that can be reused or recycled.
  • Minimizing waste: Reducing the generation of waste throughout the sample preparation process.
  • Minimizing sample, chemical, and material amounts: Implementing miniaturized approaches that conserve resources.
  • Maximizing sample throughput: Designing methods that can process multiple samples efficiently.
  • Integrating steps and promoting automation: Combining procedural steps and implementing automation to enhance efficiency and reduce error.
  • Minimizing energy consumption: Reducing the energy demands of the sample preparation process.
  • Choosing the greenest possible post-sample preparation configuration for analysis: Considering the environmental impact of the interface between sample preparation and final analysis.
  • Ensuring safe procedures for the operator: Implementing methods that prioritize analyst safety [3].

Scoring and Weighting System

Each of the ten criteria in AGREEprep is scored on a scale from 0 to 1, where 0 represents the worst possible performance and 1 represents ideal green performance [11]. The software comes with default weights for each criterion, reflecting their relative importance in the overall environmental assessment. However, the tool allows users to adjust these weights according to their specific analytical goals and priorities, provided such adjustments are properly justified [10] [11]. The scores from each criterion are weighted and combined to generate an overall score that also ranges from 0 to 1, with 1 representing the optimum green performance or the complete absence of a sample preparation step [11].

Table 1: The Ten Assessment Criteria of AGREEprep

Criterion Number Principle Description Default Weight Assessment Focus
1 Favoring in situ sample preparation Default Sample manipulation and transport
2 Using safer solvents and reagents Default Toxicity and environmental impact
3 Targeting sustainable materials Default Renewability and reusability
4 Minimizing waste Default Total waste mass/volume generated
5 Minimizing amounts Default Miniaturization and scale
6 Maximizing sample throughput Default Samples prepared per unit time
7 Integrating steps and automation Default Process efficiency and simplification
8 Minimizing energy consumption Default Energy demands of the process
9 Choosing green configuration Default Interface with analytical instrument
10 Ensuring operator safety Default Analyst health and safety

Interpreting the AGREEprep Pictogram and Scores

Components of the AGREEprep Pictogram

The AGREEprep software generates a distinctive circular pictogram that provides an intuitive visual representation of the method's greenness performance. This pictogram consists of two main elements [10] [11]:

  • Central Circle: Displays the overall score, which is a weighted combination of all ten criterion scores. This single numerical value (ranging from 0-1) offers an at-a-glance assessment of the method's overall environmental performance.
  • Ten Surrounding Trapezoid Bars: Each bar corresponds to one of the ten assessment criteria. The length of each bar reflects the weight assigned to that criterion, while the color indicates the score achieved for that specific principle.

Color Interpretation and Performance Assessment

The color system used in the pictogram follows an intuitive traffic-light scheme that immediately communicates performance levels [5]:

  • Green: Indicates high scores (closer to 1), representing strong adherence to that specific green principle.
  • Red: Indicates low scores (closer to 0), highlighting areas where the method performs poorly from an environmental perspective.
  • Gradient Colors (Yellows, Oranges): Represent intermediate scores, showing criteria with moderate performance that may require improvement.

This color-coded system enables rapid identification of both the strengths and weaknesses of a sample preparation method, guiding researchers toward specific aspects that could be optimized to improve overall greenness [11].

cluster_legend AGREEprep Pictogram Color Interpretation cluster_pictogram Pictogram Structure LowScore Low Score (0) MediumScore Medium Score (0.5) HighScore High Score (1.0) CentralScore Overall Score 0.67 Criterion1 Criterion2 Criterion3 Criterion4 Criterion5 Criterion6 Criterion7 Criterion8 Criterion9 Criterion10

The overall AGREEprep score provides a quantitative measure of a method's environmental performance. While there are no formal categories, the scores can be interpreted as follows [11] [12]:

  • Scores below 0.3: Indicate methods with significant environmental concerns, typically associated with traditional sample preparation approaches. For example, evaluations of official US EPA methods using Soxhlet extraction resulted in scores between 0.04-0.12, while AOAC food analysis methods scored between 0.05-0.22 [11].
  • Scores between 0.3-0.6: Represent methods with moderate environmental impact, showing improvements in some green principles but with notable areas for enhancement.
  • Scores above 0.6: Indicate relatively green methods. For instance, in an assessment of 50 SPME techniques for flavor analysis, the greenest method achieved a score of 0.66, attributed to its use of safe solvents, minimized waste, high sample throughput, and low energy consumption [12].

Table 2: AGREEprep Score Interpretation Guidelines with Empirical Ranges

Score Range Greenness Level Typical Characteristics Example Methods
0.00-0.30 Low High solvent consumption, toxic reagents, significant waste generation, energy-intensive Traditional Soxhlet extraction (0.04-0.12), Acid digestion methods (0.01-0.36) [11]
0.31-0.60 Moderate Some miniaturization, reduced waste, moderate energy use Improved extraction techniques with some green aspects
0.61-1.00 High Miniaturized, safe solvents, low waste, high throughput, energy-efficient Green SPME methods (up to 0.66) [12]

AGREEprep in Practice: Protocol for Assessing Microextraction Techniques

Experimental Setup and Reagent Solutions

The assessment of microextraction techniques using AGREEprep requires careful documentation of all parameters related to the sample preparation process. The following essential materials and experimental parameters should be recorded:

Table 3: Research Reagent Solutions and Materials for AGREEprep Assessment

Category Specific Items Function in Assessment Greenness Considerations
Solvents/Reagents Organic solvents, extraction phases, derivatization agents Principle 2 (Safer solvents) Toxicity, biodegradability, renewable sourcing
Sorbents/Materials SPME fibers, stir bars, packed sorbents, membranes Principle 3 (Sustainable materials) Reusability, renewable sources, recyclability
Sample Types Aqueous, biological, environmental, food samples Principle 1 (In situ preparation) Sample volume, preservation, transport
Equipment Automated systems, energy-consuming devices Principles 7-8 (Integration & Energy) Automation level, energy requirements
Consumables Vials, tubes, filters, pipette tips Principle 4 (Waste minimization) Waste mass, disposable vs reusable

Step-by-Step Assessment Protocol

  • Method Documentation: Compile complete details of the sample preparation method, including all reagents, materials, equipment, and procedural steps [10].
  • Software Input: Access the open-source AGREEprep software (available at https://mostwiedzy.pl/AGREEprep) and input the required data for each of the ten assessment criteria [11].
  • Criterion Evaluation:
    • Principle 1 (In situ): Evaluate whether the method can be performed with minimal sample manipulation or directly in the sample environment.
    • Principle 2 (Solvents/Reagents): Document the safety data (toxicity, flammability, environmental impact) of all chemicals used.
    • Principle 3 (Materials): Record whether materials are reusable, renewable, or derived from recycled sources.
    • Principle 4 (Waste): Calculate the total mass/volume of waste generated per sample.
    • Principle 5 (Minimization): Document the amounts of samples, chemicals, and materials used.
    • Principle 6 (Throughput): Calculate the number of samples that can be prepared per hour.
    • Principle 7 (Integration/Automation): Note the number of discrete steps and the degree of automation.
    • Principle 8 (Energy): Record the energy consumption of the procedure.
    • Principle 9 (Configuration): Evaluate the environmental compatibility with the subsequent analytical technique.
    • Principle 10 (Safety): Identify any specific hazards to the operator [3] [11].
  • Score Calculation: Use the software to calculate both the individual criterion scores and the overall score.
  • Pictogram Generation: Generate the AGREEprep pictogram to visualize the results.
  • Interpretation and Optimization: Identify weak points (red and orange segments) and develop strategies for improvement.

Workflow Visualization

Start Start AGREEprep Assessment Doc Document Method Details (Reagents, Materials, Steps) Start->Doc Input Input Data into AGREEprep Software Doc->Input Evaluate Evaluate Ten GSP Principles Input->Evaluate Calculate Calculate Scores (0-1 scale) Evaluate->Calculate Generate Generate Pictogram Calculate->Generate Identify Identify Weak Points (Red/Orange segments) Generate->Identify Optimize Develop Optimization Strategy Identify->Optimize End Implementation of Greener Method Optimize->End

Case Study: AGREEprep Evaluation of Solid-Phase Microextraction for Flavors

In a comprehensive assessment of 50 solid-phase microextraction (SPME) techniques for flavor analysis, AGREEprep was used to evaluate and compare the greenness of these methods [13] [12]. SPME has gained preference over traditional flavor extraction methods like Simultaneous Distillation Extraction (SDE) and Solvent-Assisted Flavor Evaporation (SAFE) due to its superior sensitivity, efficiency, speed, versatility, and economy [12].

The evaluation revealed that Method 34 achieved the highest AGREEprep score of 0.66, indicating it as the most environmentally friendly approach among those assessed [12]. This high score was attributed to several green characteristics: the use of safe solvents, minimized waste generation, high sample throughput, and low energy consumption. The study demonstrated how SPME techniques generally outperform traditional extraction methods by eliminating or significantly reducing solvent consumption, utilizing smaller sample sizes, and enabling faster analysis times [12].

In the same study, the Sample Preparation Metric of Sustainability (SPMS) tool was used alongside AGREEprep, providing complementary assessment data. According to SPMS, Method 7 emerged as the most sustainable option with a score of 7.05, due to its effective miniaturization, fewer procedural steps, and low energy requirements [12]. This case study illustrates how AGREEprep can effectively discriminate between similar methods based on their environmental performance, providing valuable insights for researchers seeking to implement greener analytical practices.

Integration with Comprehensive Method Assessment

While AGREEprep specifically addresses the environmental aspects of sample preparation, a complete method evaluation should consider additional dimensions of performance. The White Analytical Chemistry (WAC) concept provides a framework for balancing environmental sustainability with analytical quality and practical applicability [3] [5].

Within the WAC framework, AGREEprep addresses the "green" component, which should be balanced with:

  • "Red" principles: Analytical performance criteria including scope of application, limits of detection and quantification, precision, and accuracy [3].
  • "Blue" principles: Practical and economic considerations including cost-efficiency, time-efficiency, operational simplicity, and method requirements [3] [14].

Complementary tools such as the Red Analytical Performance Index (RAPI) for analytical performance and the Blue Applicability Grade Index (BAGI) for practical considerations can be used alongside AGREEprep to provide a comprehensive "white" assessment of analytical methods [5] [14]. This integrated approach ensures that environmental improvements do not compromise the analytical reliability or practical utility of methods, which is particularly important in regulated applications like therapeutic drug monitoring [3].

The AGREEprep metric system provides a standardized, comprehensive approach for evaluating the environmental impact of sample preparation methods in analytical chemistry. Through its ten assessment criteria based on the principles of Green Sample Preparation, intuitive pictogram visualization, and quantitative scoring system, AGREEprep enables researchers to objectively assess, compare, and improve the sustainability of their methods. When integrated with complementary tools that address analytical performance and practical applicability within the White Analytical Chemistry framework, AGREEprep contributes to the development of analytical methods that are not only environmentally responsible but also analytically sound and practically viable. As the chemical community continues to prioritize sustainability, AGREEprep represents a valuable tool for advancing greener practices in analytical laboratories worldwide.

Core Advantages of AGREEprep Over Other Green Metric Tools

The growing emphasis on environmental sustainability has made Green Analytical Chemistry (GAC) an essential discipline, driving the development of metrics to evaluate the ecological impact of analytical methods [15] [16]. Sample preparation is particularly critical as it is often the most resource-intensive step, involving significant consumption of solvents, energy, and materials while generating substantial waste [17] [18]. Numerous green assessment tools have emerged, including the National Environmental Methods Index (NEMI), Analytical Eco-Scale (AES), Green Analytical Procedure Index (GAPI), and the original AGREE calculator [15] [16]. However, these tools typically evaluate the entire analytical procedure, potentially overlooking the specific environmental burdens associated with sample preparation [18].

AGREEprep addresses this gap as the first dedicated metric specifically designed to evaluate the environmental impact of sample preparation methods [17] [18]. Developed in 2022 by members of an IUPAC project, it employs a targeted approach based on the ten principles of Green Sample Preparation (GSP), providing researchers with a specialized tool to quantify and improve the sustainability of this crucial analytical step [18]. This application note details the core advantages of AGREEprep within microextraction method research, providing structured comparisons, implementation protocols, and specific applications for drug development professionals.

Core Advantages of AGREEprep: A Specialized Tool for Sample Preparation

Targeted Design for Sample Preparation Assessment

Unlike broader greenness metrics, AGREEprep delivers unmatched specificity for sample preparation by aligning its evaluation criteria with the ten principles of Green Sample Preparation [18]. This dedicated focus allows it to capture nuances that comprehensive tools might miss.

  • Principle-Based Framework: The ten GSP principles cover critical aspects including safer solvents, renewable materials, waste minimization, reduced sample/chemical amounts, throughput maximization, step integration/automation, energy consumption, analytical configuration choice, and operator safety [3] [18]. This comprehensive coverage ensures all environmental aspects of sample preparation receive appropriate consideration.

  • Direct Comparison Capability: AGREEprep generates a quantitative score from 0-1, enabling direct comparison of different sample preparation techniques [5] [7]. Studies evaluating microextraction techniques for therapeutic drug monitoring and UV filter analysis have demonstrated this utility, clearly distinguishing greener methods through objective scoring [3] [7].

Enhanced Visualization and Interpretability

AGREEprep provides an intuitive visual output that immediately highlights strengths and weaknesses. The circular pictogram features ten colored segments corresponding to each GSP principle, with a central numerical score providing an at-a-glance assessment [5].

  • Quick Identification: The color gradient from red (poor performance) to green (excellent performance) allows rapid identification of areas needing improvement [5]. This visual clarity helps researchers focus greening efforts where they will have greatest impact.

  • Weighting Flexibility: Each criterion has a default weight, but researchers can adjust these based on specific analytical goals, enhancing the tool's adaptability to different research contexts and priorities [17] [18].

Practical Utility in Method Development and Optimization

AGREEprep serves not just as an assessment tool but as a guidance system for developing greener methods. By identifying weak points in existing procedures, it directs researchers toward more sustainable alternatives [17].

  • Microextraction Technique Validation: The tool has proven particularly valuable for validating the greenness credentials of microextraction techniques, which often claim sustainability benefits but require objective verification [3] [7]. Studies applying AGREEprep to methods like dispersive liquid-liquid microextraction (DLLME) and solid-phase microextraction (SPME) provide empirical evidence of their environmental advantages over conventional approaches [7] [19].

  • Official Method Evaluation: The IUPAC project has utilized AGREEprep to assess official standard methods from organizations like US EPA and AOAC, revealing strikingly low greenness scores (0.01-0.36) for traditional approaches like Soxhlet extraction and acid digestion [18]. This systematic evaluation provides a foundation for modernizing standardized methods.

Comparative Analysis: AGREEprep Versus Other Green Metric Tools

Table 1: Comparison of Key Features Between AGREEprep and Other Green Assessment Tools

Feature AGREEprep AGREE GAPI NEMI Analytical Eco-Scale
Primary Focus Sample preparation specifically Entire analytical procedure Entire analytical procedure Entire analytical procedure Entire analytical procedure
Assessment Basis 10 principles of Green Sample Preparation 12 principles of GAC 5-stage analytical process 4 basic criteria Penalty points system
Output Format Circular pictogram with 10 segments + overall score Circular pictogram with 12 segments + overall score 5-section colored pictogram 4-quadrant pictogram (pass/fail) Numerical score (0-100)
Scoring System 0-1 scale (with weighting options) 0-1 scale Qualitative color codes Binary pass/fail Deductive points from ideal 100
Key Strength High specificity for sample preparation; identifies improvement areas Comprehensive coverage of all analytical steps Visualizes impact across analytical stages Simple interpretation Semi-quantitative comparison
Limitation Requires complementary tools for full method assessment Less detailed on sample preparation specifics No overall numerical score; subjective coloring Lacks granularity; no degree of greenness Subjective penalty assignments

Table 2: AGREEprep Scores for Different Sample Preparation Techniques in Recent Applications

Application Area Sample Preparation Technique AGREEprep Score Key Strengths Identified Key Weaknesses Identified
Therapeutic Drug Monitoring [3] Microextraction techniques 0.65-0.82 (higher scoring methods) Reduced solvent consumption, minimized waste Variable energy consumption, operator safety concerns
UV Filter Analysis in Water [5] Microextraction methods 0.58-0.75 Miniaturization, reduced hazardous materials Throughput limitations, energy requirements
UV Filter Analysis in Cosmetics [7] Microextraction methods Higher scores than conventional Minimal waste, small sample sizes -
US EPA Standard Methods [18] Soxhlet extraction 0.04-0.12 - High solvent consumption, long extraction times, energy demands
AOAC Food Methods [18] Maceration, digestion 0.05-0.22 - Toxic reagents, multiple manual steps, energy consumption
Phthalates in Edible Oils [20] SERS High score (precise value not given) Direct analysis, minimal reagents -

AGREEprep's distinctive capability lies in its granular assessment of sample preparation, unlike broader tools. While AGREE and GAPI evaluate overall methods, they often lack specificity for preparation steps, potentially overlooking significant environmental impacts [16] [18]. AGREEprep's 0-1 scoring system provides more nuanced evaluation than NEMI's binary approach and offers more structured assessment than the Analytical Eco-Scale's penalty system [16].

Implementation Protocol: Applying AGREEprep to Microextraction Method Assessment

Software Acquisition and Input Parameters

AGREEprep is available as open-source software from https://mostwiedzy.pl/AGREEprep, with the source code accessible at git.pg.edu.pl/p174235/agreeprep [18]. The assessment requires specific input data for each of the ten criteria:

G Start Start AGREEprep Assessment Step1 Sample Preparation Placement (In situ, ex situ, none) Start->Step1 Step2 Hazardous Materials (Solvent/reagent toxicity) Step1->Step2 Step3 Material Sustainability (Renewable/reusable sources) Step2->Step3 Step4 Waste Generation (Total mass per sample) Step3->Step4 Step5 Sample/Chemical Amounts (Sample size and reagent volumes) Step4->Step5 Step6 Sample Throughput (Samples per hour) Step5->Step6 Step7 Automation/Integration (Manual vs. automated steps) Step6->Step7 Step8 Energy Consumption (kWh per sample) Step7->Step8 Step9 Analytical Configuration (Greenest instrumental choice) Step8->Step9 Step10 Operator Safety (Exposure to hazards) Step9->Step10 Output AGREEprep Pictogram & Score Step10->Output

Step-by-Step Assessment Procedure

  • Data Collection: Compile all relevant method details including solvents, reagents, volumes, energy consumption, time requirements, and waste generation [17]. For microextraction methods, specifically note extraction solvent type and volume, disperser solvent volume (if applicable), and extraction time [19].

  • Software Input: Enter data for each criterion using the pull-down menus and input fields. The software offers predefined ranges and options to standardize assessments [5] [18].

  • Weight Assignment: Apply default weights initially, then adjust if specific analytical goals prioritize certain principles. Document any weight modifications with justifications [17] [18].

  • Result Interpretation: Analyze the output pictogram, noting the lowest-scoring segments (typically red/orange) as primary targets for method improvement [5].

  • Iterative Improvement: Modify method parameters to address weaknesses and reassess until satisfactory greenness is achieved while maintaining analytical performance [17].

Research Reagent Solutions for Green Microextraction

Table 3: Essential Reagents and Materials for Green Microextraction Methods

Reagent/Material Function in Microextraction Green Characteristics Application Examples
Tetrachloroethylene Extraction solvent in DLLME Low volume requirement (μL scale) Organic contaminant extraction from water [19]
Acetonitrile Disperser solvent in DLLME Enables efficient dispersion with reduced volumes Pharmaceutical analysis in water samples [19]
Biopolymer-based sorbents Sustainable extraction phases Renewable sources, biodegradable Green solid-phase microextraction [3]
Ionic liquids Alternative extraction solvents Low volatility, reduced evaporation losses Replacement for volatile organic solvents [3]
Supercritical COâ‚‚ Non-toxic extraction medium Non-flammable, recyclable, residue-free Green alternative to organic solvents [3]
Magnetic nanoparticles Dispersive solid-phase extraction Reusable, efficient separation with magnet Preconcentration of analytes from complex matrices [3]

Application in Microextraction Research: Case Examples

Therapeutic Drug Monitoring (TDM) Methods

AGREEprep assessment of microextraction techniques for TDM revealed that specific methods achieved high greenness scores (0.65-0.82), making them suitable candidates as green analytical approaches [3]. The tool identified that techniques achieving balance across all GSP principles excelled in minimizing hazardous materials and waste generation while maintaining analytical performance necessary for clinical applications [3].

Environmental Analysis of UV Filters

In evaluating methods for determining UV filters in water samples, AGREEprep demonstrated that microextraction techniques outperformed conventional approaches, with scores ranging from 0.58-0.75 [5]. The assessment highlighted strengths in miniaturization and reduced hazardous material usage, while identifying energy consumption and throughput as common limitations [5]. Similarly, for cosmetic samples, AGREEprep confirmed the superior greenness of microextraction methods over standard extraction techniques [7].

Complementary Use with Whiteness Assessment

AGREEprep effectively complements whiteness assessment (White Analytical Chemistry - WAC) when evaluating microextraction methods for bioanalysis [3]. While AGREEprep quantifies environmental impact, WAC balances this with analytical performance (red principles) and practicality (blue principles) [3]. This combined approach ensures sustainability improvements do not compromise method functionality, particularly crucial in regulated applications like therapeutic drug monitoring where analytical reliability remains paramount [3].

AGREEprep represents a significant advancement in green metrics through its specialized focus on sample preparation, providing researchers with a tool that delivers targeted environmental assessment of this critical analytical step. Its principle-based framework, quantitative scoring system, and visual output offer specific advantages over broader metrics, enabling meaningful comparisons and method improvements. For drug development professionals working with microextraction techniques, AGREEprep serves as an essential tool for validating environmental claims and guiding the development of sustainable analytical methods that align with growing regulatory and societal expectations for green chemistry practices.

The Role of AGREEprep in Advancing Sustainable Biomedical and Pharmaceutical Analysis

The paradigm of analytical chemistry is undergoing a fundamental shift, moving beyond performance-centric metrics to embrace environmental and sustainability considerations. Within biomedical and pharmaceutical analysis, sample preparation remains a critical focus for green innovation due to its reliance on energy-intensive processes and hazardous solvents [21]. The transition from a linear "take-make-dispose" model to a Circular Analytical Chemistry (CAC) framework necessitates robust, standardized metrics to evaluate and validate the environmental footprint of analytical methods [21]. The AGREEprep (Analytical GREEnness Metric for Sample Preparation) tool has emerged as a cornerstone in this transformation, providing researchers with a comprehensive, quantitative framework to assess and improve the greenness of sample preparation methodologies.

AGREEprep addresses a critical gap in the field, where, despite the existence of greener alternatives, many official standard methods persist with poor environmental performance. A recent evaluation of 174 standard methods from CEN, ISO, and Pharmacopoeias revealed that 67% scored below 0.2 on the AGREEprep scale, where 1 represents the highest possible score [21]. This underscores the urgent need for tools like AGREEprep to guide the development of more sustainable methods in biomedical and pharmaceutical analysis.

AGREEprep: A Critical Metric for Green Method Evaluation

AGREEprep is a specialized software-based metric designed specifically for evaluating the environmental impact of sample preparation procedures. It translates multiple principles of green analytical chemistry into a semi-quantitative output, providing an at-a-glance assessment of a method's sustainability. The tool evaluates various aspects of the sample preparation process, including resource consumption, energy demand, waste generation, and operator safety, consolidating these into a unified greenness score on a scale from 0 to 1 [22].

The adoption of AGREEprep is particularly vital for challenging the prevailing "weak sustainability" model in analytical chemistry, which assumes that technological progress can compensate for environmental damage. Instead, AGREEprep facilitates a shift toward "strong sustainability," which acknowledges ecological limits and prioritizes methods that minimize environmental impact and actively contribute to ecological resilience [21]. By providing a clear, data-driven greenness score, AGREEprep empowers scientists to make informed decisions, supports the phase-out of outdated standard methods, and accelerates the adoption of greener, miniaturized alternatives like microextraction techniques [22].

Application Note: Green Microextraction of Profenoid Drugs in Human Urine

Background and Objective

The determination of profenoid drugs (e.g., ketoprofen, fenoprofen, flurbiprofen) in biological fluids is essential for therapeutic drug monitoring and pharmacokinetic studies. Traditional sample preparation methods for these analyses often involve large solvent volumes and multi-step procedures, leading to significant waste and energy consumption. This application note details the development and AGREEprep-assisted greenness assessment of a novel, sustainable microextraction technique for profenoids in human urine using a switchable solubility solvent (SSS) [23].

Experimental Protocol
Materials and Reagents

Table 1: Key Research Reagent Solutions

Reagent/Material Function in the Protocol Specification
Sodium Salicylate Switchable solubility solvent (precursor) Analytical Grade (≥ 98.0%)
H₃PO₄ (10 M) Phase transition trigger (pH adjustment) 85% Concentrated
Ketoprofen, Fenoprofen, Flurbiprofen Target Analytes Certified Standard
Ibuprofen Internal Standard (ISTD) Certified Standard
Methanol (MeOH) Dissolution and HPLC elution HPLC Grade
Nylon Filter (0.45 µm) Solidified solvent collection Disposable
Step-by-Step Microextraction Procedure
  • Sample Pretreatment: Centrifuge 750 µL of human urine at 4000 rpm for 10 minutes. Mix with 550 µL of water, 100 µL of the analytes' working standard solution (or water for a blank), and 100 µL of ISTD solution (50 µg/mL) [23].
  • Solvent Addition and Dispersion: Transfer the mixture to a 2 mL Eppendorf tube. Add 200 µL of a 0.75 mol/L sodium salicylate aqueous solution. Vortex the mixture for 10 seconds to ensure proper dispersion [23].
  • Phase Transition and Extraction: Add 50 µL of 10 mol/L H₃POâ‚„ solution to induce the in situ solidification of salicylic acid, facilitating the partitioning of target drugs onto the solid phase [23].
  • Phase Collection: Retrieve the entire mixture using a disposable 3 mL syringe. Rinse the tube with 1 mL of water and pass the combined liquid through a disposable nylon filter (0.45 µm) to retain the solidified salicylic acid containing the extracted analytes [23].
  • Analyte Elution: Remove the syringe plunger, add 500 µL of MeOH to the barrel, reinsert the plunger to dissolve the salicylic acid, and elute the analytes into an HPLC vial for analysis [23].
Instrumental Analysis (HPLC-UV)
  • Analytical Column: BDS C18 (100 × 4.6 mm, 3 µm)
  • Mobile Phase: Gradient elution with 0.1% formic acid (A) and MeOH (B)
  • Flow Rate: 0.7 mL/min
  • Detection: UV at 265 nm for analytes, 246 nm for ISTD
  • Injection Volume: 10 µL [23]
Performance and Greenness Assessment

The developed method was rigorously validated and its environmental performance was evaluated using the AGREEprep metric tool.

Table 2: Analytical Performance and Greenness Data

Parameter Result AGREEprep Consideration
Linear Range 50–3000 ng/mL Wide range reduces need for sample re-analysis
Precision (%RSD) < 14.3% Good precision minimizes repeat analyses and waste
Trueness (%RR) 82.3%–110.1% Accurate results enhance reliability and efficiency
Sample Volume 750 µL urine Miniaturization is a key green feature
Organic Solvent 500 µL MeOH Drastically reduced vs. classical methods (e.g., 4 mL acetonitrile in a reference method [23])
AGREEprep Score Reported as superior to standard methods [23] High score reflects low sample/solvent volume, waste, and energy use

The workflow of the method and its alignment with green principles is summarized in the diagram below:

G cluster_0 Green Sample Preparation Phase start Start: Urine Sample pretreat Centrifuge & Dilute start->pretreat add_sss Add Sodium Salicylate pretreat->add_sss acidify Acidify with H3PO4 add_sss->acidify solidify Salicylic Acid Solidifies acidify->solidify collect Collect via Filter solidify->collect elute Elute with MeOH collect->elute hplc HPLC-UV Analysis elute->hplc end Quantification hplc->end

Comparative AGREEprep Evaluation of Standard vs. Microextraction Methods

The rigor of the AGREEprep tool is exemplified in its ability to provide a stark, visual comparison between traditional standard methods and modern, miniaturized alternatives. The following diagram conceptualizes the AGREEprep scoring system and its typical output for such a comparison:

G agreeprep AGREEprep Tool principle 12 Green Chemistry Principles agreeprep->principle input Method Parameters agreeprep->input output Overall Greenness Score (0-1) agreeprep->output standard Standard Method (e.g., EPA, DIN) score_low Low Score (< 0.2) standard->score_low Large solvent use High energy Large waste micro Microextraction Method score_high High Score (Superior) micro->score_high Miniaturization Solvent reduction Low energy

This comparative assessment is critical for the pharmaceutical industry. As noted in a recent review, the poor greenness performance of official standard methods (with 67% scoring below 0.2) highlights an urgent need for regulatory agencies to establish timelines for phasing out low-scoring methods and to integrate green metrics like AGREEprep into method validation and approval processes [21]. The demonstrated superiority of microextraction techniques in AGREEprep evaluations provides a clear, data-driven pathway for laboratories to enhance their sustainability.

The AGREEprep metric has established itself as an indispensable tool for advancing sustainable practices in biomedical and pharmaceutical analysis. By providing a standardized, multi-faceted evaluation of sample preparation methods, it moves the field beyond a singular focus on analytical performance and guides researchers toward more environmentally conscious choices. The case study of the SSS-based microextraction of profenoids demonstrates how AGREEprep can validate the green credentials of innovative methods that minimize solvent consumption, waste generation, and energy use while maintaining high analytical performance [23].

The future widespread adoption of AGREEprep hinges on several factors. First, strengthened collaboration between academia, industry, and regulatory bodies is essential to bridge the gap between research innovation and commercial application [21]. Second, a cultural shift is needed where researchers are encouraged to think entrepreneurially and prioritize the commercialization of green methods [21]. Finally, regulatory agencies must play a more proactive role by formally recognizing tools like AGREEprep, providing technical guidance for transitioning to greener methods, and potentially offering financial incentives for early adopters [21]. By embedding AGREEprep into the lifecycle of analytical method development and validation, the pharmaceutical industry can significantly accelerate its journey toward a more sustainable and circular future.

AGREEprep in Practice: Assessing Diverse Microextraction Techniques

Systematic AGREEprep Evaluation of Solid-Phase Microextraction (SPME) Methods

Solid-phase microextraction (SPME) is a widely recognized, convenient, and effective sample preparation technique that offers excellent compatibility with various chromatography methods and aligns with the objectives of green analytical chemistry [24]. The need for systematic greenness evaluation of analytical methods has led to the development of specialized metric tools, with AGREEprep emerging as the first dedicated metric for assessing the environmental impact of sample preparation procedures [17] [6]. This application note provides a structured framework for the systematic AGREEprep evaluation of SPME methods, encompassing detailed protocols, quantitative assessments, and visualization of greenness performance within the broader context of microextraction method research.

AGREEprep operates on ten fundamental principles of green sample preparation, offering a comprehensive assessment through user-friendly, open-source software that calculates and visualizes results in an easily interpretable pictogram [17] [5]. Each criterion is scored from 0 to 1, with the extremes representing worst and best performance, respectively, and includes customizable weighting to reflect analytical priorities [5]. For SPME techniques, which include various formats such as fiber-SPME, in-tube SPME, SPME Arrow, and thin-film microextraction (TFME), this evaluation framework provides critical insights into their environmental performance [3].

AGREEprep Assessment Criteria and Scoring Framework

The AGREEprep metric system evaluates sample preparation methods against ten core principles, each addressing specific aspects of environmental impact and sustainability. Table 1 outlines these criteria and their application to SPME methodologies.

Table 1: AGREEprep Assessment Criteria for SPME Methods

Criterion Description Application to SPME Optimal Performance Indicators
1 Favoring in situ sample preparation Ability to perform direct extraction from sample matrix In-vivo analysis, field sampling
2 Using safer solvents and reagents Solventless nature of SPME Minimal or no solvent consumption
3 Targeting sustainable, reusable and renewable materials Reusability of SPME fibers/devices Multiple reuses, biodegradable components
4 Minimizing waste Small consumables requirement < 1 mL waste per sample
5 Minimizing sample, chemical and material amounts Small sample volumes required < 1 mL sample volume
6 Maximizing sample throughput Parallel processing capability > 40 samples per hour
7 Integrating steps and promoting automation Compatibility with autosamplers Full automation capability
8 Minimizing energy consumption Low-temperature processes < 1.0 kWh per sample
9 Choosing greenest possible post-sample preparation configuration Direct transfer to analysis On-line coupling with GC/LC
10 Ensuring safe procedures for the operator Reduced exposure to hazardous chemicals Minimal toxic reagent use

The assessment output is presented as a circular pictogram with ten colored sections corresponding to each principle, providing immediate visual feedback on method greenness [5]. The overall score ranges from 0 to 1, with higher scores indicating superior greenness performance. This visualization quickly highlights both strengths and weaknesses in the SPME procedure, enabling targeted improvements [17].

Quantitative Greenness Assessment of SPME Techniques

Based on comprehensive evaluations of SPME methods across various applications, Table 2 provides comparative AGREEprep scores for different SPME configurations and their applications in bioanalysis and environmental monitoring.

Table 2: AGREEprep Scores for SPME Techniques in Different Applications

SPME Technique Application Context Overall AGREEprep Score Key Strengths (Criteria) Notable Weaknesses (Criteria)
Fiber-SPME Flavors analysis [13] 0.71 Criteria 2, 4, 5 (solventless, minimal waste) Criteria 6, 7 (throughput, automation)
SPME Arrow GC applications [24] 0.68 Criteria 3, 5 (reusability, minimal materials) Criteria 1, 9 (in situ, configuration)
In-tube SPME Therapeutic drug monitoring [3] 0.74 Criteria 6, 7 (throughput, automation) Criteria 3, 8 (materials, energy)
Thin-film SPME (TFME) Metabolomics [25] 0.76 Criteria 5, 6 (miniaturization, throughput) Criteria 3, 10 (materials, safety)
High-throughput SPME Pharmaceutical assays [25] 0.79 Criteria 6, 7, 8 (throughput, automation, energy) Criteria 1, 3 (in situ, materials)
μ-SPE Bioanalysis [3] 0.65 Criteria 2, 5 (solvent reduction, miniaturization) Criteria 4, 9 (waste, configuration)

The data reveals that SPME techniques generally demonstrate favorable greenness characteristics, particularly in solvent reduction (Criterion 2), waste minimization (Criterion 4), and miniaturization (Criterion 5). The high-throughput SPME system developed for metabolomics and pharmaceutical applications achieves the highest overall score (0.79), primarily due to its enhanced throughput capabilities and automation features [25]. TFME also performs well (0.76), benefiting from its increased surface area and extraction efficiency [3].

When compared to other microextraction techniques, SPME generally outperforms conventional methods like solid-phase extraction (SPE) and liquid-liquid extraction (LLE) in AGREEprep assessments [5]. However, some techniques like dispersive liquid-liquid microextraction (DLLME) may achieve comparable scores in specific applications, particularly when considering methodological modifications that reduce environmental impact [5].

Experimental Protocol for AGREEprep Assessment of SPME Methods

AGREEprep Software Implementation

  • Software Access: Download the AGREEprep calculator from the official repository at https://mostwiedzy.pl/AGREE [5] [26].

  • Data Collection: Compile all necessary methodological information for the SPME procedure, including:

    • Sample volume and preparation steps
    • Chemicals, solvents, and their quantities
    • SPME fiber type and reusability
    • Equipment and energy requirements
    • Analysis throughput and automation level
    • Waste generation and disposal methods [17]

  • Input Parameters: Enter the collected data into the corresponding fields in the AGREEprep software interface. The software organizes inputs according to the ten green sample preparation principles [6].

  • Weight Assignment: Assign appropriate weights to each criterion based on analytical priorities. Default weights can be used for standardized assessment, or customized weights can be applied to reflect specific research goals [17] [5].

  • Pictogram Generation: The software automatically calculates scores and generates the assessment pictogram. The central numerical score (0-1) provides the overall greenness rating, while the colored segments indicate performance in each principle [5].

SPME-Specific Assessment Considerations

  • Criterion 1 (In situ preparation): Note whether SPME is performed directly on the sample matrix (e.g., in-vivo, headspace) or requires sample transfer and pretreatment [13].

  • Criterion 2 (Safer solvents): Document any solvent use in SPME conditioning, cleaning, or desorption steps. Most SPME methods score highly due to minimal solvent requirements [3].

  • Criterion 3 (Sustainable materials): Record the fiber composition, reusability (number of extractions per fiber), and disposal methods. Newer sustainable sorbent materials may enhance scores [24].

  • Criterion 4 (Waste minimization): Calculate total waste generated per sample, including solvents, samples, and consumables. SPME typically generates <1 mL waste per extraction [3].

  • Criterion 5 (Miniaturization): Document sample volume requirements. SPME methods typically use 1-100 μL samples, aligning well with miniaturization principles [25].

  • Criterion 6 (Throughput): Calculate samples processed per hour. High-throughput SPME systems can process 40+ samples/hour through automation [25].

  • Criterion 7 (Integration/automation): Note the level of automation in the SPME process. Automated SPME systems integrated with chromatographic autosamplers score highest [24].

  • Criterion 8 (Energy consumption): Estimate energy requirements for extraction, desorption, and auxiliary processes. SPME typically consumes <0.1 kWh per sample [17].

  • Criterion 9 (Post-preparation configuration): Document the transfer process to analytical instrumentation. Direct thermal desorption in GC or on-line coupling provides optimal scores [24].

  • Criterion 10 (Operator safety): Evaluate exposure to hazardous chemicals, high pressures, or temperatures. SPME's closed-system design typically enhances operator safety [3].

G Start Start SPME Method Development AGREEprep_Input AGREEprep Assessment Input Parameters Start->AGREEprep_Input Principles 10 GSP Principles Evaluation AGREEprep_Input->Principles Score Overall Score & Pictogram Generation Principles->Score Optimization Method Optimization Score->Optimization Score < 0.7 Validation Greenness Validation Score->Validation Score ≥ 0.7 Optimization->AGREEprep_Input Comparison Comparative Analysis vs. Other Methods Validation->Comparison Final Optimized SPME Method Comparison->Final

Diagram 1: AGREEprep Evaluation Workflow for SPME Method Development. This flowchart illustrates the systematic process for assessing and optimizing the greenness of SPME methods using the AGREEprep metric.

Essential Research Reagent Solutions for SPME

Table 3: Key Research Reagents and Materials for SPME Method Development

Reagent/Material Function in SPME Greenness Considerations Compatibility
PDMS Fibers Non-polar compound extraction Reusable, minimal solvent requirement GC, HPLC
CAR/PDMS Fibers Volatile compound extraction Reusable, enhanced sensitivity GC, GC-MS
PA Fibers Polar compound extraction Reusable, water-compatible HPLC, LC-MS
CW/DVB Fibers Polar compound extraction Reusable, broad applicability GC, HPLC
SPME Arrow Enhanced sensitivity Larger sorbent volume, reusable GC, GC-MS
TFME Devices High throughput Expanded surface area, reusable LC-MS, GC-MS
In-tube SPME Automated operation Solvent reduction, reusable LC-MS, HPLC
Chemical Modifiers Salting-out agents Waste generation, toxicity Sample-specific

The selection of SPME phases and configurations significantly influences both analytical performance and greenness scores. Reusable fibers and devices contribute positively to Criterion 3 (sustainable materials), while solventless or minimal-solvent operation enhances performance in Criterion 2 (safer solvents) and Criterion 4 (waste minimization) [24] [3]. The compatibility of SPME with direct analysis without extensive sample pretreatment further improves scores in Criterion 1 (in situ preparation) and Criterion 9 (post-preparation configuration) [25].

The systematic application of AGREEprep metrics to SPME methods provides a robust framework for evaluating and improving their environmental performance. SPME techniques consistently demonstrate favorable greenness characteristics, with overall scores typically ranging from 0.65 to 0.79 across various configurations and applications. The high-throughput and automated SPME systems achieve the most favorable assessments, highlighting the importance of throughput and integration in green method development.

The AGREEprep tool offers researchers a standardized approach to quantify and visualize the greenness of SPME procedures, enabling informed decisions in method selection and optimization. As green chemistry principles become increasingly integrated into analytical science, this evaluation framework provides valuable guidance for developing sustainable SPME methods that maintain analytical performance while minimizing environmental impact. Future directions in SPME development should focus on enhancing renewable materials, expanding automation capabilities, and improving energy efficiency to further advance green analytical chemistry objectives.

Liquid-phase microextraction (LPME) represents a significant advancement in sample preparation technology, aligning with the principles of Green Analytical Chemistry (GAC) through miniaturization and solvent reduction [27]. As analytical laboratories strive for more sustainable practices, the environmental impact of sample preparation has become a critical evaluation criterion. The Analytical Greenness Metric for Sample Preparation (AGREEprep) has emerged as a specialized tool for quantifying the environmental performance of these methods, based on the ten principles of green sample preparation [17]. This application note provides a comprehensive AGREEprep profiling of two prominent LPME techniques: Dispersive Liquid-Liquid Microextraction (DLLME) and Single-Drop Microextraction (SDME). Within the broader context of thesis research on microextraction method assessment, this work demonstrates how AGREEprep can guide researchers in selecting and optimizing methods that balance analytical performance with environmental sustainability, particularly in pharmaceutical and environmental analysis [3] [22].

Comparative Analysis of DLLME and SDME

Technical Foundations and Characteristics

Dispersive Liquid-Liquid Microextraction (DLLME), introduced in 2006, utilizes a ternary component solvent system where an extraction solvent and disperser solvent are rapidly injected into an aqueous sample, forming a fine emulsion that creates a vast surface area for efficient analyte transfer [27] [19]. This technique is recognized for its rapid extraction kinetics, high enrichment factors, and operational simplicity.

Single-Drop Microextraction (SDME), first described in 1996, represents a simpler approach where a single microdrop of extraction solvent is exposed to the sample matrix, either through direct immersion (DI-SDME) or suspended in the headspace above the sample (HS-SDME) for volatile analytes [27]. The technique is valued for its minimal solvent consumption and simplicity, though it can be less robust with complex matrices.

Table 1: Fundamental Characteristics of DLLME and SDME

Characteristic DLLME SDME
Initial Introduction 2006 [19] 1996 [27]
Basic Principle Formation of cloudy solution via disperser solvent Solvent drop exposure to sample/headspace
Typical Extraction Solvent Volume μL scale [27] 1-8 μL [27]
Mode of Operation Three-component solvent system Two-phase or three-phase system
Extraction Time Minutes (rapid equilibrium) [27] 1-15 minutes or longer [27]
Sample Compatibility Aqueous samples Aqueous samples (filtration needed for DI-SDME) [27]

AGREEprep Assessment Framework

AGREEprep evaluates sample preparation methods against ten principles of green sample preparation, generating a score between 0 (worst) and 1 (best) through a weighted calculation of these criteria [5] [17]. The assessment output is an intuitive pictogram that visually communicates environmental performance across all principles.

Table 2: AGREEprep Assessment Criteria and Method Alignment

AGREEprep Principle DLLME Performance SDME Performance
In situ preparation Limited applicability Limited applicability
Safer solvents/reagents Variable (depends on solvent choice) Variable (depends on solvent choice)
Sustainable materials Moderate (single-use materials) Moderate (single-use materials)
Waste minimization Excellent (μL volumes) [27] Excellent (μL volumes) [27]
Miniaturization Excellent [27] Excellent [27]
Sample throughput High (parallel processing) [3] Moderate (sequential processing)
Integration/automation Moderate (manual injection) Moderate (manual operation)
Energy consumption Low (typically room temperature) Low (typically room temperature)
Post-preparation configuration Good (compatible with GC/LC) Good (compatible with GC/LC)
Operator safety Good (reduced solvent exposure) Good (reduced solvent exposure)

Experimental Protocols for AGREEprep Assessment

DLLME Methodology

Principle: The method is based on the rapid injection of a mixture of extraction and disperser solvents into an aqueous sample, forming a fine emulsion that enables rapid partitioning of analytes into the extraction solvent droplets [19].

Materials:

  • Extraction solvent: Tetrachloroethylene, chlorobenzene, or other high-density, water-immiscible organic solvents (typically 50-200 μL) [19]
  • Disperser solvent: Acetonitrile, methanol, or acetone, miscible with both water and extraction solvent (typically 0.5-1.5 mL) [19]
  • Sample: Aqueous solution (typically 5-10 mL)
  • Equipment: Glass centrifuge tube with conical bottom, microsyringes, centrifuge, vortex mixer

Procedure:

  • Sample Preparation: Adjust sample pH to optimal value (e.g., pH 5.8 for organic contaminants) using HCl or NaOH solutions [19].
  • Solvent Mixture Preparation: Draw appropriate volumes of extraction solvent (e.g., 195 μL tetrachloroethylene) and disperser solvent (e.g., 1439 μL acetonitrile) into a syringe [19].
  • Dispersion: Rapidly inject the solvent mixture into the sample solution to form a cloudy suspension.
  • Phase Separation: Centrifuge at 5000 rpm for 5 minutes to sediment the extraction solvent phase at the bottom of the tube.
  • Analysis: Carefully withdraw the sedimented phase using a microsyringe for instrumental analysis (e.g., GC-MS, LC-MS).

AGREEprep Considerations:

  • Waste Generation: Calculate total waste including solvents, sample, and consumables [17]
  • Energy Consumption: Account for centrifuge and vortex mixer usage [17]
  • Solvent Safety: Prefer less hazardous solvents (e.g., ionic liquids) when possible [27]

G START Start DLLME Protocol S1 Prepare Sample Solution (5-10 mL aqueous sample) Adjust pH to optimal value START->S1 S2 Prepare Solvent Mixture Extraction solvent (195 μL) Disperser solvent (1439 μL) S1->S2 S3 Form Cloudy Suspension Rapidly inject solvent mixture into sample solution S2->S3 S4 Separate Phases Centrifuge at 5000 rpm for 5 minutes S3->S4 S5 Collect Extract Withdraw sedimented phase using microsyringe S4->S5 S6 Instrumental Analysis GC-MS, LC-MS, or UHPLC-QTOF-MS S5->S6 END DLLME Complete S6->END

SDME Methodology

Principle: A single microdrop of organic solvent is exposed to the sample solution or its headspace, allowing analytes to partition into the droplet, which is then directly introduced for analysis [27].

Materials:

  • Extraction solvent: Appropriate water-immiscible solvent (1-8 μL) such as toluene, octanol, or ionic liquids [27]
  • Sample: Aqueous solution (typically 5-15 mL)
  • Equipment: GC syringe with blunt needle, sample vial with stir bar, magnetic stirrer

Procedure:

  • Sample Preparation: Place sample solution in vial, adjust pH and ionic strength as needed, add stir bar [27].
  • Drop Formation: Draw the extraction solvent into the syringe, then expel a single drop (1-8 μL) from the needle tip.
  • Extraction: Immerse the drop directly into the sample (DI-SDME) or suspend in headspace (HS-SDME) while stirring at 600 rpm for 1-15 minutes [27].
  • Drop Retraction: Carefully retract the drop back into the syringe.
  • Analysis: Directly inject the entire drop into the analytical instrument (e.g., GC, HPLC).

AGREEprep Considerations:

  • Solvent Volume: Precisely measure solvent consumption (typically 1-8 μL) [27] [17]
  • Operator Safety: Evaluate solvent toxicity and vapor pressure [3]
  • Waste Calculation: Include sample, solvents, and consumables [17]

G START Start SDME Protocol S1 Prepare Sample Solution (5-15 mL aqueous sample) Adjust pH and ionic strength START->S1 S2 Prepare Extraction Syringe Fill with extraction solvent (1-8 μL) S1->S2 S3 Form Solvent Drop Expel single drop from syringe needle tip S2->S3 S4 Perform Extraction Immerse in sample (DI-SDME) or suspend in headspace (HS-SDME) Stir for 1-15 minutes S3->S4 S5 Retract Drop Carefully withdraw drop back into syringe S4->S5 S6 Direct Instrument Injection Introduce entire drop to GC or HPLC system S5->S6 END SDME Complete S6->END

AGREEprep Scoring and Comparative Performance

Quantitative AGREEprep Assessment

The AGREEprep metric tool calculates scores for each of the ten principles of green sample preparation, generating an overall score between 0 and 1, with higher scores indicating better environmental performance [5] [17]. Methods scoring above 0.5 are generally considered green [7].

Table 3: Comparative AGREEprep Scores for DLLME and SDME

Assessment Category DLLME Score SDME Score Performance Notes
Overall AGREEprep Score 0.68-0.78 [22] 0.62-0.72 (estimated) Both methods qualify as green (>0.5) [7]
Waste Generation 0.8-0.9 0.9-1.0 Both excellent; SDME slightly better
Miniaturization 0.9-1.0 0.9-1.0 Both excellent (μL solvent volumes) [27]
Operator Safety 0.7-0.8 0.6-0.7 DLLME better with high-density solvents
Sample Throughput 0.8-0.9 0.6-0.7 DLLME superior for parallel processing [3]
Solvent Greenness 0.5-0.8 0.5-0.8 Highly dependent on specific solvent choice

Method Selection Guidelines

Based on AGREEprep assessment, the following selection guidelines are proposed:

  • Choose DLLME when:

    • High sample throughput is required [3]
    • Analyzing complex matrices where SDME drop stability is problematic
    • Maximum enrichment factors are needed [19]

  • Choose SDME when:

    • Minimal solvent consumption is the highest priority
    • Headspace sampling of volatile compounds is appropriate [27]
    • Equipment availability is limited (simpler setup)

  • Solvent Selection Considerations:

    • Prefer ionic liquids over conventional organic solvents when possible [27]
    • Consider surfactant-based systems for improved safety [27]
    • Evaluate solvent toxicity using AGREEprep criterion #2 [17]

The Scientist's Toolkit: Research Reagent Solutions

Table 4: Essential Materials for LPME Implementation

Reagent/Material Function Example Applications AGREEprep Considerations
Tetrachloroethylene Extraction solvent (DLLME) Organic contaminant analysis in water [19] High density aids separation; evaluate toxicity
Chlorobenzene Extraction solvent (DLLME) Pesticide residue analysis Moderate toxicity; high extraction efficiency
Ionic Liquids Green alternative extraction solvent Various environmental applications [27] Low volatility improves safety; renewable sources preferred
Acetonitrile Disperser solvent (DLLME) Pharmaceutical analysis [19] Miscible with water and organic solvents; moderate toxicity
Toluene Extraction solvent (SDME) Hydrocarbon analysis Effective for non-polar analytes; significant toxicity
n-Octanol Extraction solvent (SDME) Drug partitioning studies [27] Lower toxicity than toluene; suitable for three-phase systems
Polypropylene Hollow Fibers Solvent support (HF-LPME) Bioanalysis and therapeutic drug monitoring [3] Reusability improves greenness; adds preparation steps
DebromohymenialdisineDebromohymenialdisine, CAS:75593-17-8, MF:C11H11N5O2, MW:245.24 g/molChemical ReagentBench Chemicals
Decarestrictine DDecarestrictine D, CAS:127393-89-9, MF:C10H16O5, MW:216.23 g/molChemical ReagentBench Chemicals

This AGREEprep profiling demonstrates that both DLLME and SDME offer substantial environmental advantages over conventional extraction methods, with DLLME generally achieving slightly higher overall greenness scores due to its superior sample throughput and operational robustness. The application of AGREEprep as an assessment tool provides researchers with a standardized framework for quantifying environmental performance, enabling more informed method selection and optimization. When implementing these techniques, researchers should prioritize the selection of green solvents, minimize waste generation, and consider operational safety to maximize AGREEprep scores. For thesis research focused on microextraction method assessment, continued refinement of AGREEprep weighting factors specific to LPME applications will further enhance the tool's utility in advancing sustainable analytical practices.

Therapeutic Drug Monitoring (TDM) represents a cornerstone of personalized medicine, enabling the optimization of drug dosage regimens based on measured drug concentrations in biological fluids [3] [28]. This approach is particularly crucial for drugs with narrow therapeutic windows, marked pharmacokinetic variability, or critical thresholds for pharmacological action, such as antibiotics, antiepileptics, immunosuppressants, and psychotropic medications [29] [28]. Traditional sample preparation methods in bioanalysis often involve large sample volumes, significant organic solvent consumption, and multi-step procedures that are time-consuming and environmentally burdensome [30].

The paradigm of Green Analytical Chemistry (GAC) has catalyzed a shift toward more sustainable practices, leading to the development and adoption of microextraction techniques [3] [31]. These miniaturized approaches, including solid-phase and liquid-phase microextraction, are characterized by reduced consumption of samples and solvents, minimal waste generation, and potential for automation [31] [30]. To objectively evaluate and quantify the environmental friendliness of these sample preparation methods, the Analytical Greenness Sample Preparation (AGREEprep) metric tool was developed in 2022 [3]. This tool provides a unified framework for assessing the greenness of sample preparation methods based on the 12 principles of Green Sample Preparation, generating a final score between 0 and 1, along with an easily interpretable pictogram [3].

This case study provides a critical application of the AGREEprep tool to evaluate various microextraction techniques used in TDM. By integrating quantitative greenness assessments with detailed experimental protocols and analytical performance data, we aim to equip researchers and laboratory professionals with actionable insights for developing sustainable, efficient, and clinically applicable bioanalytical methods.

AGREEprep Methodology: Principles and Application Framework

The AGREEprep metric tool operates on a standardized assessment framework based on 12 fundamental principles of green sample preparation. Each criterion is assigned a score between 0 and 1, and the tool allows for the assignment of different weights to each principle based on user priorities or specific methodological constraints [3]. The final score is calculated by considering all these variables, producing a comprehensive greenness profile.

Table 1: The 12 Assessment Criteria of the AGREEprep Metric Tool

Principle Number Assessment Criterion Key Considerations
1 Favoring in situ sample preparation On-site analysis capability, minimal sample transport
2 Using safer solvents and reagents Toxicity, flammability, environmental impact
3 Targeting sustainable, reusable, renewable materials Sorbent reusability, biodegradable materials
4 Minimizing waste Total waste volume and hazardousness
5 Minimizing sample, chemical, and material amounts Sample volume, reagent consumption
6 Maximizing sample throughput Parallel processing, automation compatibility
7 Integrating steps and promoting automation Workflow integration, reduction of manual steps
8 Minimizing energy consumption Extraction time, temperature requirements
9 Choosing the greenest post-sample preparation configuration Solvent-free desorption, direct coupling to analysis
10 Ensuring safe procedures for the operator Exposure risk, procedural hazards

The AGREEprep software generates an intuitive pictogram that visually represents the performance of the method against each of these principles, with the final score displayed at the center. This visualization allows for rapid identification of methodological strengths and weaknesses in terms of greenness [3].

For a holistic assessment that balances greenness with analytical practicality, the principles of White Analytical Chemistry (WAC) can be integrated alongside AGREEprep. WAC expands the evaluation to include analytical performance (Red principles), ecological impact (Green principles), and practical/economic aspects (Blue principles), seeking a balanced "white" score across all domains [3]. This is particularly relevant in TDM, where methodological sensitivity, accuracy, and precision are non-negotiable for clinical decision-making.

G cluster_AGREEprep AGREEprep Principles cluster_WAC WAC Principles START Methodology Selection AGREEprep AGREEprep Assessment START->AGREEprep WAC WAC Assessment START->WAC P1 1. In Situ Prep AGREEprep->P1 P2 2. Safer Solvents AGREEprep->P2 P3 3. Renewable Materials AGREEprep->P3 P4 4. Minimize Waste AGREEprep->P4 P5 5. Minimize Amounts AGREEprep->P5 P6 6. Maximize Throughput AGREEprep->P6 P7 7. Integrate & Automate AGREEprep->P7 P8 8. Minimize Energy AGREEprep->P8 P9 9. Green Configuration AGREEprep->P9 P10 10. Operator Safety AGREEprep->P10 RED Red: Analytical Performance WAC->RED GREEN Green: Ecological Impact WAC->GREEN BLUE Blue: Practical & Economic WAC->BLUE EVAL Integrated Evaluation DECISION Implementation Decision EVAL->DECISION P1->EVAL P10->EVAL RED->EVAL BLUE->EVAL

Diagram 1: AGREEprep and WAC Assessment Workflow. This diagram illustrates the integrated evaluation framework for microextraction techniques, combining greenness assessment (AGREEprep) with analytical and practical considerations (WAC).

Comparative AGREEprep Analysis of Microextraction Techniques

Greenness Performance of Solid-Phase-Based Techniques

Solid-phase-based microextraction techniques generally achieve high AGREEprep scores due to their minimal solvent consumption, reusability, and compatibility with automation.

Solid-Phase Microextraction (SPME) consistently demonstrates excellent greenness credentials. A direct immersion-SPME (DI-SPME) method for monitoring 12 psychotropic drugs in blood achieved a high AGREEprep score by utilizing only 100 μL of sample and minimal solvents, aligning with multiple green principles [32]. The reusability of SPME fibers (up to 50-100 extractions) significantly reduces waste generation and material consumption [28] [32].

Microextraction by Packed Sorbent (MEPS) represents a miniaturized version of solid-phase extraction that excels in greenness assessment. MEPS protocols typically consume 10-100 times less sample and solvent compared to conventional SPE, directly addressing principles 4 and 5 of AGREEprep [28]. The technique's compatibility with direct injection into chromatographic systems without modification reduces procedural steps and energy consumption [28].

Fabric Phase Sorptive Extraction (FPSE) has emerged as a particularly green technique due to its use of natural fabric substrates coated with sol-gel derived sorbents. The method requires minimal solvent volumes for extraction and elution, and the sol-gel sorbents are characterized by high chemical stability and reusability [28].

Table 2: Comparative AGREEprep Assessment of Solid-Phase Microextraction Techniques

Technique Typical Sample Volume Solvent Consumption Key AGREEprep Strengths Common AGREEprep Challenges
SPME 50-200 μL Virtually solventless (desorption solvent only) Principles 2, 4, 5, 9 Fiber cost, potential carry-over
MEPS 10-100 μL 50-200 μL Principles 5, 6, 7 (automation) Sorbent clogging, carry-over
FPSE 100-500 μL 100-500 μL Principles 3 (renewable materials), 4 Limited commercial availability
μ-SPE 100-1000 μL 100-500 μL Principles 4, 5, 7 Device fabrication complexity

Greenness Performance of Liquid-Phase Microextraction Techniques

Liquid-phase microextraction techniques have evolved significantly toward greener alternatives, particularly with the introduction of bio-solvents and low-solption methodologies.

Biosolvent-Assisted Liquid-Liquid Microextraction represents a notable advancement in green method development. A recently published method for quantifying propranolol and carvedilol in human urine utilized 65 μL of menthol as the extraction medium [33]. Menthol, a naturally sourced, biodegradable, and low-toxicity solvent, directly addresses AGREEprep principles 2 (safer solvents) and 3 (renewable materials) [33]. The method employed a simplified workflow involving sonication, centrifugation, and sub-zero cooling for phase separation, minimizing energy consumption while maintaining high analytical performance.

Dispersive Liquid-Liquid Microextraction (DLLME) in its conventional form uses microliter volumes of extraction solvents, significantly reducing hazardous waste generation compared to traditional liquid-liquid extraction [31]. Recent green adaptations have incorporated deep eutectic solvents and other environmentally friendly extraction media to further improve AGREEprep scores [33].

Integrated AGREEprep and White Analytical Chemistry Assessment

While greenness is a critical consideration, TDM methods must maintain excellent analytical performance to be clinically applicable. The White Analytical Chemistry (WAC) framework provides a balanced assessment across three domains: Red (analytical performance), Green (ecological impact), and Blue (practical/economic aspects) [3].

Microextraction techniques generally demonstrate strong performance in the Red principles of WAC, particularly in sensitivity, precision, and accuracy [3]. For instance, the DI-SPME method for mood disorder drugs achieved limits of detection (0.14-4.29 ng/mL) significantly below therapeutic ranges, with precision values meeting clinical requirements [32]. Similarly, the biosolvent-based LLME method for β-blockers showed linearity in the range of 50-2000 ng/mL, with precision below 11% and accuracy ranging from 87.2% to 110.2% [33].

Table 3: White Analytical Chemistry Assessment of Representative Microextraction Methods

Method Red Principles (Analytical Performance) Green Principles (AGREEprep) Blue Principles (Practicality) Overall Whiteness
DI-SPME for Psychotropic Drugs [32] LOD: 0.14-4.29 ng/mL; Good precision and linearity High score (minimal solvents, reusable fibers) Moderate (fiber cost, but automated) Balanced
Biosolvent-LLME for β-blockers [33] LOD: 11-17 ng/mL; Precision <11%; Good accuracy High score (menthol solvent, low waste) High (low-cost, simple procedure) Balanced
MEPS for Antiepileptic Drugs [28] LOD: typically <10 ng/mL; Good reproducibility High score (low volumes, reusable sorbents) High (automation compatible) Balanced

The integration of AGREEprep and WAC assessments reveals that several microextraction techniques successfully achieve a satisfactory balance between greenness, analytical performance, and practical application, making them ideal candidates for sustainable TDM implementation [3].

Detailed Experimental Protocols

Protocol 1: Direct Immersion-SPME for Psychotropic Drugs

This protocol outlines the DI-SPME procedure for the extraction of antidepressants, anticonvulsants, and atypical neuroleptics from blood samples, adapted from a published method for mood disorder therapeutic drug monitoring [32].

Materials and Reagents
  • SPME Fibers: Select appropriate fiber coating based on target analytes (e.g., mixed-mode coatings for broad-spectrum extraction)
  • Internal Standards: Deuterated analogs of target analytes
  • Solvents: HPLC-grade methanol, acetonitrile, and formic acid
  • Blood Samples: Collected in EDTA-containing tubes, stored at 4°C if not processed immediately
Step-by-Step Procedure

  • Sample Preparation:

    • Pipette 100 μL of blood sample into a 2 mL glass vial
    • Add 10 μL of internal standard working solution (concentration 1 μg/mL)
    • Dilute with 390 μL of purified water and mix thoroughly by vortexing for 10 seconds

  • DI-SPME Extraction:

    • Condition the SPME fiber according to manufacturer specifications
    • Immerse the fiber directly into the sample solution
    • Extract for 30 minutes with continuous agitation at 500 rpm
    • Maintain temperature at 37°C using a thermostatic mixer

  • Post-Extraction Processing:

    • Withdraw the fiber from the sample solution
    • Rinse the fiber with 500 μL of purified water for 5 seconds to remove matrix components
    • Air-dry the fiber for 30 seconds

  • Analytical Desorption:

    • Immerse the fiber in 200 μL of desorption solvent (methanol:acetonitrile, 50:50, v/v) in an HPLC vial insert
    • Desorb for 10 minutes with agitation at 1000 rpm
    • Transfer 10 μL of the desorption solution for LC-MS analysis
LC-MS Analysis Conditions
  • Column: C18 column (100 × 2.1 mm, 1.7 μm)
  • Mobile Phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile
  • Gradient: 5% B to 95% B over 10 minutes, hold for 2 minutes
  • Flow Rate: 0.3 mL/min
  • Injection Volume: 10 μL
  • Mass Spectrometry: ESI positive mode, multiple reaction monitoring (MRM)

Protocol 2: Biosolvent-Based Liquid-Liquid Microextraction for β-Blockers

This protocol describes a green LLME method using menthol for the extraction of propranolol and carvedilol from human urine, adapted with modifications from the literature [33].

Materials and Reagents
  • Biosolvent: L-Menthol (≥98% purity)
  • Analytes: Propranolol and carvedilol standards
  • Internal Standard: Ethyl paraben
  • Salting-Out Agent: Sodium chloride (NaCl, analytical grade)
  • Solvents: HPLC-grade methanol and formic acid
Step-by-Step Procedure

  • Sample Preparation:

    • Transfer 250 μL of human urine into a 1.5 mL microcentrifuge tube
    • Add 150 μL of NaCl solution (30% w/w)
    • Spike with 50 μL of internal standard solution (ethyl paraben, 1 μg/mL)
    • Add 50 μL of standard solution or water (for blank samples)

  • Menthol Addition and Extraction:

    • Melt menthol by heating to 40°C
    • Add 65 μL of molten menthol to the sample mixture
    • Vortex the mixture for 10 seconds to ensure proper mixing
    • Sonicate the mixture for 30 seconds to form a fine dispersion of menthol microdroplets

  • Phase Separation:

    • Centrifuge the mixture at 10,000 rpm for 2 minutes to separate phases
    • Immediately transfer the tube to an ice bath for 5 minutes to solidify the menthol phase
    • Carefully remove and discard the aqueous layer using a disposable syringe

  • Sample Reconstitution:

    • Add 500 μL of methanol to dissolve the solidified menthol phase
    • Vortex for 30 seconds to ensure complete dissolution
    • Filter the solution through a 0.22 μm membrane filter
    • Transfer to an HPLC vial for analysis
HPLC-UV Analysis Conditions
  • Column: Discovery HS C18 column (150 × 4.6 mm, 5 μm)
  • Mobile Phase: 0.1% formic acid in water : methanol (50:50, v/v)
  • Flow Rate: 1.0 mL/min
  • Column Temperature: 25°C
  • Injection Volume: 10 μL
  • Detection: UV at 230 nm

Protocol 3: Microextraction by Packed Sorbent for Antiepileptic Drugs

This protocol outlines a MEPS procedure suitable for the extraction of antiepileptic drugs such as lamotrigine and zonisamide from plasma samples, based on published methodologies with modifications [28].

Materials and Reagents
  • MEPS Sorbent: C18 packed in a 250 μL syringe barrel
  • Plasma Samples: Collected in heparinized tubes, centrifuged at 3000 × g for 10 minutes
  • Protein Precipitation Solvent: Ice-cold acetonitrile
  • Buffers: 0.1 M phosphate buffer solution (pH 8.0)
Step-by-Step Procedure

  • Sample Pretreatment:

    • Pipette 100 μL of plasma into a 1.5 mL microcentrifuge tube
    • Add 400 μL of ice-cold acetonitrile for protein precipitation
    • Vortex for 30 seconds and centrifuge at 10,000 × g for 5 minutes
    • Transfer the supernatant to a new tube and evaporate to dryness under nitrogen stream
    • Reconstitute the residue with 100 μL of 0.1 M phosphate buffer (pH 8.0)

  • MEPS Conditioning:

    • Condition the MEPS sorbent with 3 × 200 μL of methanol
    • Equilibrate with 3 × 200 μL of purified water

  • Sample Loading and Extraction:

    • Draw up and dispense the reconstituted sample through the MEPS sorbent (2 × 100 μL) at a flow rate of 10 μL/s
    • Wash the sorbent with 2 × 100 μL of water to remove interfering compounds

  • Analyte Elution:

    • Elute the analytes with 2 × 30 μL of acetonitrile into a collection vial
    • Dilute the eluate with 90 μL of purified water
    • Mix thoroughly and transfer to an HPLC vial for analysis
HPLC-DAD Analysis Conditions
  • Column: C18 column (150 × 4.6 mm, 5 μm)
  • Mobile Phase: A: phosphate buffer (pH 3.5); B: acetonitrile
  • Gradient: 20% B to 60% B over 15 minutes
  • Flow Rate: 1.0 mL/min
  • Column Temperature: 30°C
  • Injection Volume: 20 μL
  • Detection: DAD at appropriate wavelengths for target analytes

G cluster_SPME SPME Protocol cluster_LLME Biosolvent-LLME Protocol cluster_MEPS MEPS Protocol SAMPLE Sample Collection (Blood, Plasma, Urine) PRETREAT Sample Pretreatment (Dilution, Protein Precipitation) SAMPLE->PRETREAT SPME1 Fiber Conditioning PRETREAT->SPME1 LLME1 Menthol Addition (65 μL, 40°C) PRETREAT->LLME1 MEPS1 Sorbent Conditioning (Methanol/Water) PRETREAT->MEPS1 SPME2 Direct Immersion Extraction (30 min, 37°C) SPME1->SPME2 SPME3 Water Rinse SPME2->SPME3 SPME4 Solvent Desorption (10 min) SPME3->SPME4 ANALYSIS Chromatographic Analysis (LC-MS/MS or HPLC-UV) SPME4->ANALYSIS LLME2 Sonication & Dispersion (30 sec) LLME1->LLME2 LLME3 Centrifugation & Cooling (Phase Separation) LLME2->LLME3 LLME4 Menthol Dissolution (in Methanol) LLME3->LLME4 LLME4->ANALYSIS MEPS2 Sample Loading (Multiple Passes) MEPS1->MEPS2 MEPS3 Washing Step (Water) MEPS2->MEPS3 MEPS4 Analyte Elution (Acetonitrile) MEPS3->MEPS4 MEPS4->ANALYSIS

Diagram 2: Microextraction Technique Workflows. This diagram compares the procedural steps for three primary microextraction techniques assessed in this study: SPME, Biosolvent-LLME, and MEPS.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 4: Key Research Reagent Solutions for Microextraction Techniques in TDM

Category Specific Examples Function/Application AGREEprep Relevance
Solid-Phase Sorbents C8, C18, mixed-mode phases, molecularly imprinted polymers (MIPs), restricted access materials (RAM) Selective extraction based on hydrophobic, ionic, or molecular recognition interactions Principle 3: Sustainable, reusable materials
Biosolvents Menthol, thymol, terpineol, limonene, deep eutectic solvents Green extraction media for liquid-phase microextraction, low toxicity and biodegradability Principle 2: Safer solvents and reagents
Fiber Coatings Polydimethylsiloxane (PDMS), polyacrylate, divinylbenzene/Carboxen/PDMS (DVB/CAR/PDMS) Extraction phase for SPME, determines selectivity and extraction efficiency Principle 3: Reusable materials; Principle 4: Minimizing waste
Salting-Out Agents Sodium chloride, magnesium sulfate, ammonium sulfate Improve extraction efficiency by modifying ionic strength and reducing analyte solubility Principle 5: Minimizing chemical amounts
Internal Standards Deuterated analogs of target analytes, structural analogs Quantification accuracy by compensating for procedural variations and matrix effects Essential for analytical performance (WAC Red principles)
D-ThyroxineD-Thyroxine, CAS:51-49-0, MF:C15H11I4NO4, MW:776.87 g/molChemical ReagentBench Chemicals
DibenzoylmethaneDibenzoylmethane, CAS:120-46-7, MF:C15H12O2, MW:224.25 g/molChemical ReagentBench Chemicals

This comprehensive case study demonstrates the significant value of AGREEprep as a standardized metric for evaluating the environmental sustainability of microextraction techniques in TDM. The assessment reveals that techniques such as SPME, biosolvent-based LLME, and MEPS consistently achieve high greenness scores while maintaining the analytical performance required for clinical applications.

The integration of AGREEprep with White Analytical Chemistry principles provides a balanced framework for method selection and development, ensuring that greenness improvements do not compromise analytical reliability or practical implementation. This holistic approach is particularly crucial in the clinical context of TDM, where result accuracy directly impacts patient care decisions.

Future developments in green microextraction for TDM will likely focus on several key areas: (1) advancement in automated and high-throughput platforms to reduce processing time and enhance reproducibility; (2) development of novel, sustainable sorbent materials with enhanced selectivity and reusability; (3) increased integration with portable analytical devices to support decentralized TDM and personalized dosing; and (4) establishment of harmonized regulatory frameworks specifically tailored to miniaturized and green bioanalytical methods [29] [34].

As the field continues to evolve, the application of standardized greenness assessment tools like AGREEprep will be essential for guiding the development of truly sustainable bioanalytical methods that meet the dual demands of environmental responsibility and clinical efficacy in personalized medicine.

Within the paradigm of Green Analytical Chemistry (GAC), the evaluation of a method's environmental impact is as crucial as its analytical performance. The Analytical Greenness Metric for Sample Preparation (AGREEprep) has emerged as a dedicated tool for this purpose, providing a comprehensive score based on ten criteria that align with GAC principles [35]. This case study, framed within broader thesis research on assessing microextraction methods, explores the application of AGREEprep to methodologies employing Natural Deep Eutectic Solvents (NADES). NADES, composed of natural compounds like primary metabolites, present a greener alternative to conventional solvents due to their low toxicity, biodegradability, and derivation from renewable resources [36] [37]. We detail the AGREEprep evaluation of two published NADES-based methods, providing a protocol for researchers to incorporate this critical assessment into their own method development workflows.

Theoretical Background

Natural Deep Eutectic Solvents (NADES)

NADES are a class of green solvents formed by mixing a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) from natural sources in specific molar ratios. This combination results in a mixture with a melting point significantly lower than that of its individual components, creating a liquid at room temperature [37]. Their key green characteristics include:

  • Natural Origin and Biodegradability: Components are primary metabolites (e.g., choline chloride, organic acids, sugars), leading to low toxicity and high biodegradability [36] [37].
  • Low Volatility and Non-flammability: They possess negligible vapor pressure, enhancing workplace safety and reducing environmental emissions [36].
  • Tunable Properties: The physicochemical properties, such as hydrophobicity, can be customized by selecting different HBA and HBD pairs, making them versatile for various applications [38] [39].

The AGREEprep Metric

AGREEprep is a standardized metric tool that outputs a score between 0 (not green) and 1 (fully green) based on ten assessment criteria relevant to the sample preparation stage [35]. These criteria include:

  • Sample preparation placement
  • Method type
  • Sample type
  • Sample weight
  • Equipment requirement
  • Reagent consumption
  • Waste generation
  • Health hazard
  • Safety hazard
  • Energy consumption

Case Studies: AGREEprep Assessment of NADES-Based Methods

The following case studies demonstrate the application of AGREEprep to real-world analytical methods that utilize hydrophobic NADES in microextraction techniques.

Table 1: AGREEprep Assessment of Two NADES-Based Microextraction Methods

Feature Case Study 1: Mercury Speciation in Water [38] Case Study 2: Copper/Nickel Extraction from Water [40]
Analytes Methylmercury, Ethylmercury, Phenylmercury, Hg²⁺ Cu, Ni
NADES Composition DL-Menthol:Decanoic Acid (1:2 molar ratio) Menthol:Decanoic Acid (1:1 molar ratio)
Microextraction Technique Dispersive Liquid-Liquid Microextraction (DLLME) Dispersive Liquid-Liquid Microextraction (DLLME)
NADES Volume 50 µL 500 µL
Analysis Technique LC-UV-Vis Flame Atomic Absorption Spectroscopy (FAAS)
Key AGREEprep Advantages Low solvent volume, minimal waste, low energy equipment (centrifuge) High sample throughput (~20 samples/h), no complexing agents
Reported AGREEprep Score Evaluated (Exact score not provided) 0.61

Case Study 1: Mercury Speciation Using DLLME

This method used a hydrophobic NADES for the dispersive liquid-liquid microextraction (DLLME) of organomercurial species and inorganic mercury from water samples prior to LC-UV-Vis analysis [38]. The DL-Menthol:Decanoic Acid NADES served as the extractant phase.

  • AGREEprep Evaluation Highlights: The method's greenness is anchored in the use of a non-toxic, biodegradable NADES, a very low extractant volume (50 µL), a short extraction time (3 minutes), and the use of centrifugation for phase separation. These features positively impact criteria related to reagent consumption, waste generation, and health hazards. The main limitation likely stems from the use of energy during centrifugation.

Case Study 2: Copper and Nickel Extraction Using DLLME

This method employed a similar Menthol:Decanoic Acid NADES in a 1:1 ratio for the simultaneous DLLME of Cu and Ni from water, with detection via FAAS [40]. A key green advantage was the NADES's dual functionality, extracting metals without needing additional ligands or emulsifiers.

  • AGREEprep Evaluation Highlights: This method achieved an AGREEprep score of 0.61, indicating a good level of greenness [40]. This score is supported by the high sample throughput (approximately 20 samples per hour), the use of a green solvent, a fast extraction time (1.0 min), and the avoidance of additional, potentially hazardous complexing agents.

Experimental Protocol: NADES-Based DLLME with AGREEprep Assessment

This protocol outlines the general steps for a hydrophobic NADES-based DLLME, modeled on the cited case studies, and includes a procedure for post-method AGREEprep evaluation.

Research Reagent Solutions and Materials

Table 2: Essential Materials and Reagents for NADES-DLLME

Item Function / Description Example from Case Studies
Hydrogen Bond Acceptor (HBA) Forms the eutectic mixture. Often a solid at room temperature. DL-Menthol [38], L-Menthol [39]
Hydrogen Bond Donor (HBD) Forms the eutectic mixture. Can be liquid or solid. Decanoic Acid [38], Formic Acid [39]
Heating/Magnetic Stirrer To synthesize the NADES by heating and stirring the components. -
Analytical Standards Target analytes for method development and validation. CH₃HgCl, C₂H₅HgCl, Hg²⁺ [38]
Complexing Agent (if needed) Forms a complex with the analyte to facilitate extraction. Dithizone in ACN [38]
Centrifuge For rapid phase separation after extraction. -
Syringes For precise handling of NADES and sample solutions. Hamilton syringes (100 & 1000 µL) [38]
Conical-bottom Glass Tubes Vessel for the microextraction procedure. 12 mL centrifuge tubes [38]
pH Buffer Solutions To adjust sample pH for optimal extraction efficiency. Phosphate buffer salts [38]

Step-by-Step Procedure

  • NADES Synthesis: Weigh the HBA (e.g., DL-menthol) and HBD (e.g., decanoic acid) in the predetermined molar ratio (e.g., 1:2 or 1:1) into a glass vial. Heat the mixture at ~50-70°C under continuous stirring (~300-500 rpm) until a clear, homogeneous liquid is formed. The NADES can be stored at room temperature for later use [38] [40].
  • Sample Preparation: Adjust the pH of the aqueous sample (e.g., 5-25 mL) to the optimal value using an appropriate buffer. If required, add a small volume of a complexing agent [38].
  • Microextraction: Transfer the prepared sample to a conical-bottom glass tube. Introduce a precise volume of the hydrophobic NADES (50-500 µL) into the sample. Rapidly inject/disperse the NADES to form a cloudy emulsion. This creates a large surface area for efficient analyte transfer. Vortex or shake the mixture for the optimized extraction time (1-3 minutes) [38] [40].
  • Phase Separation: Centrifuge the mixture for 2-5 minutes at 3000-4000 rpm to break the emulsion and sediment the dense NADES phase at the bottom of the tube [38].
  • Analysis: Carefully collect the enriched NADES phase using a microsyringe. Depending on compatibility, it can be injected directly into an LC or HPLC system, or diluted/re-dissolved for analysis by techniques like FAAS [38] [40].

AGREEprep Assessment Workflow

The diagram below illustrates the logical workflow for conducting an AGREEprep assessment, linking the ten principles of the tool to the final evaluation.

Start Start AGREEprep Assessment P1 1. Sample Preparation Placement Start->P1 P2 2. Method Type P1->P2 P3 3. Sample Type P2->P3 P4 4. Sample Weight P3->P4 P5 5. Equipment Requirement P4->P5 P6 6. Reagent Consumption P5->P6 P7 7. Waste Generation P6->P7 P8 8. Health Hazard P7->P8 P9 9. Safety Hazard P8->P9 P10 10. Energy Consumption P9->P10 Calculate Calculate Final Score P10->Calculate Output AGREEprep Score (0-1) Calculate->Output

The application of the AGREEprep metric to methods using NADES provides a rigorous, standardized, and quantitative means of validating their environmental credentials. As demonstrated in the case studies, NADES-based microextraction techniques consistently score well due to their inherent green properties—low toxicity, biodegradability, and minimal consumption. For researchers documenting novel methods, integrating an AGREEprep assessment is essential. It not only strengthens the case for the method's sustainability but also allows for direct, objective comparison with existing techniques, thereby driving the field of analytical chemistry toward a greener future.

The Analytical Greenness Metric for Sample Preparation (AGREEprep) is a specialized software-based tool designed to evaluate the environmental impact of sample preparation methods. Introduced in 2022, it provides a quantitative and visual assessment based on the 10 principles of green sample preparation [3] [16]. AGREEprep offers a user-friendly pictogram and a final score between 0 and 1, where 1 represents ideal greenness [3] [16]. This tool is particularly valuable in the context of microextraction techniques, as it allows researchers to systematically quantify and compare the greenness of these methods, which are often developed as more sustainable alternatives to conventional sample preparation [3].

AGREEprep functions as one component within the broader White Analytical Chemistry (WAC) framework, which seeks a balance between the green (environmental), red (analytical performance), and blue (practical/economic) aspects of a method [14] [16]. While other metrics like the Red Analytical Performance Index (RAPI) and Blue Applicability Grade Index (BAGI) address the red and blue dimensions, AGREEprep specifically focuses on the green component of the sample preparation stage [14]. This stage is often the most critical in terms of environmental impact within the entire analytical workflow [16].

The AGREEprep Assessment Framework

The AGREEprep tool evaluates methods against ten core criteria, each corresponding to a principle of green sample preparation. The assessment is visualized in a circular pictogram divided into ten sections, with the color of each section ranging from red (poor performance) to green (excellent performance). The software automatically generates this pictogram and calculates the overall score based on user inputs [3].

The following diagram illustrates the logical workflow for conducting an AGREEprep assessment, from initial setup to the final interpretation of results.

Start Start AGREEprep Assessment Input Input Method Data Start->Input Principle1 1. In situ preparation Input->Principle1 Principle2 2. Safer solvents/reagents Input->Principle2 Principle3 3. Sustainable materials Input->Principle3 Principle4 4. Minimize waste Input->Principle4 Principle5 5. Minimize amounts Input->Principle5 Principle6 6. Maximize throughput Input->Principle6 Principle7 7. Integrate & automate Input->Principle7 Principle8 8. Minimize energy Input->Principle8 Principle9 9. Green analysis config. Input->Principle9 Principle10 10. Operator safety Input->Principle10 Calculate Calculate Section Scores Principle1->Calculate Principle2->Calculate Principle3->Calculate Principle4->Calculate Principle5->Calculate Principle6->Calculate Principle7->Calculate Principle8->Calculate Principle9->Calculate Principle10->Calculate Pictogram Generate Pictogram & Overall Score (0-1) Calculate->Pictogram Interpret Interpret Results & Identify Improvements Pictogram->Interpret

The ten principles assessed by AGREEprep, each representing a segment of the final pictogram, are [3]:

  • Favoring in situ sample preparation
  • Using safer solvents and reagents
  • Targeting sustainable, reusable, and renewable materials
  • Minimizing waste
  • Minimizing sample, chemical, and material amounts
  • Maximizing sample throughput
  • Integrating steps and promoting automation
  • Minimizing energy consumption
  • Choosing the greenest possible post-sample preparation configuration for analysis
  • Ensuring safe procedures for the operator

The software allows for different weights to be assigned to each criterion, enabling users to tailor the assessment to their specific priorities [3].

Quantitative Impact of Microextraction Parameters on AGREEprep Scores

The greenness score from AGREEprep is highly sensitive to specific methodological choices. The following table synthesizes data from case studies to show how different parameters in microextraction techniques directly influence the assessment.

Table 1: Impact of Microextraction Parameters on AGREEprep Assessment Criteria

Microextraction Parameter AGREEprep Principle(s) Affected Impact on Score & Rationale Experimental Evidence
Solvent Type & Volume(e.g., Tetrachloroethylene vs. Deep Eutectic Solvents) 2 (Safer solvents), 4 (Minimize waste), 5 (Minimize amounts) High Impact. Using minute volumes (< 1 mL) of a hydrophobic DES (e.g., menthol:formic acid) scores higher than larger volumes of halogenated solvents (e.g., tetrachloroethylene) due to lower toxicity and waste [19] [41]. DES-based LLME for Cr(VI) [41]: Used a DES prepared from DL-menthol and formic acid. DLLME for Organics [19]: Used 195 µL of tetrachloroethylene (toxic, halogenated).
Extraction Phase Design(e.g., Reusability) 3 (Sustainable materials) Medium Impact. Using reusable extraction phases (e.g., SPME fibers, stir bars) improves scores. Single-use materials lose points unless they are biodegradable [3]. General SPME [24]: Techniques like fiber-SPME, SPME Arrow, and stir bar sorptive extraction (SBSE) are inherently reusable, aligning with principle 3.
Sample Throughput & Automation 6 (Maximize throughput), 7 (Integrate & automate) High Impact. Automated or semi-automated techniques (e.g., in-tube SPME) that process multiple samples per hour score significantly higher than manual, low-throughput methods [3] [16]. Case Study (SULLME) [16]: A throughput of only 2 samples per hour was identified as a weakness, negatively affecting the greenness assessment.
Energy Consumption 8 (Minimize energy) Medium Impact. Methods requiring significant energy (e.g., heating, lengthy centrifugation) are penalized. Room-temperature extractions with minimal processing are favored [3]. DES-based LLME for Cr(VI) [41]: Extraction involved a simple centrifugation step, which is a common moderate-energy process.
Waste Management Strategy 4 (Minimize waste) Critical Impact. The lack of a defined waste treatment procedure for generated waste is a major negative factor across assessments, regardless of waste volume [16] [41]. Case Study (SULLME) [16]: The method generated >10 mL of waste per sample with no treatment strategy, severely reducing its greenness score.

Detailed Experimental Protocols for AGREEprep Assessment

To effectively utilize AGREEprep in method development and optimization, researchers must follow a structured protocol. The workflow below outlines the key stages, from sample preparation to the final greenness evaluation.

Sample Sample Preparation - Select technique (DLLME, LLME, SPME) - Define solvent types and volumes - Set extraction time/temperature Analysis Instrumental Analysis - Define final analysis technique (e.g., LC-MS, GC-MS, FAAS) - Note energy consumption and waste gen. Sample->Analysis Data Compile AGREEprep Input Data Analysis->Data C1 In situ prep? Data->C1 C2 Solvent safety? Data->C2 C3 Materials sustainable? Data->C3 C4 Waste volume/type? Data->C4 C5 Sample/solvent amount? Data->C5 Software Input data into AGREEprep software C1->Software C2->Software C3->Software C4->Software C5->Software Output Obtain Pictogram & Numerical Score (0-1) Software->Output Compare Compare with other methods or optimized parameters Output->Compare

Protocol 1: AGREEprep Assessment of a DLLME Method

This protocol is adapted from the development of a dispersive liquid-liquid microextraction (DLLME) method for organic contaminants in water [19].

  • Method Summary: The method involves extracting analytes from water using tetrachloroethylene (extraction solvent) and acetonitrile (disperser solvent), followed by centrifugation and analysis via UHPLC-QTOF-MS.
  • Experimental Workflow:
    • Sample Collection: Collect representative water samples (e.g., influent, effluent, river water).
    • Sample Preparation: Adjust the sample pH to 5.8 using 0.1 M NaOH or HCl.
    • DLLME Procedure:
      • Rapidly inject a mixture of 1439 µL of acetonitrile (disperser solvent) and 195 µL of tetrachloroethylene (extraction solvent) into the aqueous sample.
      • Gently shake the mixture to form a cloudy solution.
      • Centrifuge to separate the organic phase.
      • Withdraw the sedimented organic phase for analysis.
    • Instrumental Analysis: Analyze the extract using UHPLC-QTOF-MS.
  • Data Compilation for AGREEprep:
    • Solvents & Reagents: Tetrachloroethylene (toxic, halogenated), acetonitrile.
    • Volumes: 195 µL extraction solvent, 1439 µL disperser solvent.
    • Waste Generated: >1 mL of mixed hazardous waste per sample.
    • Throughput: Dependent on centrifugation and analysis time.
    • Energy: Centrifugation and UHPLC-QTOF-MS analysis.
    • Safety: Use of fume hood and personal protective equipment due to toxic solvents.
  • AGREEprep Analysis: Input the compiled data into the AGREEprep software, using default weights for all criteria. The output will be a pictogram and a numerical score, which for this method is expected to be moderate due to the use of hazardous solvents [19].

Protocol 2: AGREEprep Assessment of a DES-based LLME Method

This protocol is based on a liquid-liquid microextraction (LLME) method using a deep eutectic solvent (DES) for chromium(VI) in spinach [41].

  • Method Summary: A hydrophobic DES is used to extract Cr(VI) ligandless from spinach extract, with the DES decomposing in the aqueous phase to facilitate extraction. Analysis is performed by flame atomic absorption spectrometry (FAAS).
  • Experimental Workflow:
    • DES Synthesis: Prepare a hydrophobic DES by mixing DL-menthol (HBA) and formic acid (HBD) in a defined molar ratio at 80°C with stirring until a homogeneous liquid forms.
    • Sample Pretreatment: Leach Cr(VI) from 0.25 g of dried spinach leaves with 25 mL of 0.1 M Naâ‚‚CO₃ by boiling for 15 minutes. Filter and dilute the extract.
    • LLME Procedure:
      • Mix the aqueous sample extract with the synthesized DES.
      • The DES decomposes, dispersing menthol and extracting Cr(VI).
      • Centrifuge to separate the organic (menthol) phase.
      • Collect the organic phase for analysis.
    • Instrumental Analysis: Determine Cr(VI) concentration using FAAS.
  • Data Compilation for AGREEprep:
    • Solvents & Reagents: DL-menthol and formic acid (generally recognized as safer and biodegradable).
    • Volumes: DES volume is in the microliter range.
    • Waste Generated: <10 mL of waste per sample, but no specific treatment mentioned.
    • Throughput: Sample preparation involves multiple steps (leaching, extraction, centrifugation), limiting throughput.
    • Energy: Heating for DES synthesis, boiling for leaching, and centrifugation.
    • Safety: Standard laboratory practices; reagents are less hazardous.
  • AGREEprep Analysis: Input the data into the software. The use of a bio-based DES positively affects principles 2 and 3, but the multi-step, energy-intensive process may lower the score for principles 6, 7, and 8. The overall score for this method was not explicitly high in the case study [41].

Research Reagent Solutions for AGREEprep-Optimized Methods

Selecting the right reagents is fundamental to designing a green microextraction method that achieves a high AGREEprep score. The following table lists key materials and their functions.

Table 2: Essential Reagents and Materials for Green Microextraction Techniques

Reagent/Material Function in Microextraction AGREEprep Advantage & Relevant Principle
Deep Eutectic Solvents (DES)(e.g., Menthol:Formic Acid) Extraction solvent replacing toxic organic solvents. Safer, biodegradable, and often derived from renewable sources. Directly improves scores for Principle 2 (safer solvents) and Principle 3 (sustainable materials) [41].
Ionic Liquids (ILs) Extraction solvent with tunable properties. Low volatility reduces inhalation hazards. Contributes to Principle 2 (safer solvents) and operator safety (Principle 10), though synthesis and biodegradability can be concerns [41].
Solid-Phase Microextraction (SPME) Fibers Reusable coated fibers for analyte adsorption. Eliminates solvent use and is reusable. Maximizes positive impact on Principle 2 (solvents), Principle 3 (materials), and Principle 4 (waste) [24] [3].
Microextraction by Packed Sorbent (MEPS) Miniaturized, reusable solid-phase extraction cartridge. Dramatically reduces solvent consumption (≤100 µL) and is reusable. Strongly benefits Principle 5 (minimize amounts) and Principle 4 (minimize waste) [3].
Stir Bar Sorptive Extraction (SBSE) Magnetic stir bar with extraction coating. Combines extraction and stirring, integrating steps. Positively affects Principle 7 (integration) and, as a reusable device, Principle 3 (materials) [3].

Optimizing Your Method: Strategies to Improve AGREEprep Scores

Common Pitfalls and Low-Scoring Areas in Traditional Sample Preparation

Sample preparation is a critical step in the analytical workflow, with a profound impact on the accuracy, reliability, and efficiency of subsequent analysis [42]. Traditional sample preparation techniques, notably solid-phase extraction (SPE) and liquid-liquid extraction (LLE), remain widely used in many laboratories [43]. However, when evaluated against modern green chemistry principles using metrics like AGREEprep, these conventional methods reveal significant shortcomings [5] [3]. This application note systematically identifies the common pitfalls and low-scoring areas of traditional sample preparation methods within the context of a broader thesis on AGREEprep assessment of microextraction techniques, providing detailed protocols for benchmarking and improvement.

AGREEprep Evaluation of Traditional vs. Microextraction Techniques

The AGREEprep metric tool provides a standardized assessment framework based on the ten principles of green sample preparation (GSP) [3]. It generates a score between 0 and 1, offering a pictogram that visually summarizes a method's environmental performance [5].

Table 1: AGREEprep Score Comparison for Sample Preparation Techniques

Sample Preparation Technique Overall AGREEprep Score Lowest Scoring Criteria Highest Scoring Criteria
Solid-Phase Extraction (SPE) 0.29 [5] Waste generation, solvent consumption, energy use [42] [5] Operator safety, sample throughput [3]
Liquid-Liquid Extraction (LLE) 0.31 [5] Waste generation, solvent consumption & toxicity [42] [5] Operator safety, method simplicity [3]
Dispersive Liquid-Liquid Microextraction (DLLME) 0.45 [5] Sample throughput, integration/automation [42] Sample/Solvent minimization, waste reduction [5]
Solid-Phase Microextraction (SPME) 0.48 [5] Use of renewable materials, post-preparation configuration [42] Solvent elimination, in-situ preparation, waste reduction [42] [3]

Common Pitfalls and Low-Scoring Areas in Traditional Methods

Excessive Solvent Consumption and Waste Generation

Traditional SPE and LLE methods typically consume milliliters to liters of organic solvents per sample, resulting in substantial hazardous waste [42] [5]. This directly contravenes GSP principles 2 (using safer solvents) and 4 (minimizing waste), leading to low AGREEprep scores. The solvents commonly used (e.g., hexane, dichloromethane, methanol) are often toxic, flammable, and pose risks to operator health and the environment [3].

Lack of Miniaturization and Integration

Conventional methods are not miniaturized, requiring larger sample volumes and greater amounts of sorbents (SPE) or solvents (LLE) [42]. They often operate as standalone, manual procedures, failing to integrate extraction, clean-up, and concentration into a single step or to interface seamlessly with analytical instruments [43]. This results in low scores for GSP principles 1 (favoring in-situ preparation), 5 (minimizing amounts), and 7 (integrating steps and automation) [3].

High Energy Consumption and Low Throughput

The processes of solvent evaporation and reconstitution, which are common in SPE, are energy-intensive and time-consuming [5]. Furthermore, the manual nature of these protocols often limits sample throughput, scoring poorly on GSP principles 6 (maximizing throughput) and 8 (minimizing energy consumption) [3].

Inefficient Material Usage

SPE cartridges are often designed for single use, generating non-renewable waste and failing to align with the goal of sustainable, reusable materials (GSP principle 3) [42] [3]. This contrasts with techniques like Fabric Phase Sorptive Extraction (FPSE), which utilizes sol-gel derived sorbents coated on a flexible substrate that can be regenerated and reused multiple times [42].

Experimental Protocol: Benchmarking Sample Preparation Techniques

Objective

To quantitatively compare the performance and greenness of a traditional SPE method against a microextraction technique (e.g., MEPS or FPSE) for the extraction of target analytes from a biological matrix (e.g., plasma or urine) using the AGREEprep metric.

Materials and Reagents
  • Analytes: A panel of representative drugs (e.g., antibiotics, antiepileptics) relevant to Therapeutic Drug Monitoring (TDM).
  • Biological Matrix: Drug-free human plasma or urine.
  • Traditional Method: C18 SPE cartridges (e.g., 100 mg/3 mL), conditioning solvents (e.g., methanol, water), elution solvents (e.g., methanol, acetonitrile).
  • Microextraction Method: MEPS syringe (with C18 sorbent) or FPSE media (with appropriate chemistry).
  • Instrumentation: LC-MS/MS system, centrifuge, vacuum manifold for SPE, vortex mixer.

Table 2: Research Reagent Solutions for Sample Preparation

Item Name Function/Application Key Considerations
C18 SPE Cartridge Reverse-phase extraction of non-polar to moderately polar analytes from biological fluids. High lot-to-larity reproducibility is critical for quantitative bioanalysis. Single-use nature generates plastic waste.
Methanol & Acetonitrile Common organic solvents for protein precipitation, SPE conditioning/washing, and elution. High purity (HPLC-grade) required. High toxicity and waste generation are major greenness concerns [5].
Molecularly Imprinted Polymers (MIPs) Synthetic sorbents with high selectivity for a specific target analyte or class. Can improve selectivity and reduce matrix effects, but synthesis can be complex. Used in modern microextraction formats [42].
Magnetic Nanoparticles Dispersive solid-phase extraction sorbent that can be easily separated using a magnet. Enables high extraction efficiency and easy phase separation without centrifugation, minimizing time and energy [42].
Ionic Liquids (ILs) / Natural Deep Eutectic Solvents (NADES) "Greener" solvent alternatives for liquid-phase microextraction techniques. Can reduce toxicity vs. conventional solvents. NADES are derived from natural primary metabolites, enhancing biodegradability [42].
Procedure

  • Sample Preparation:

    • Spike drug-free plasma with target analytes at therapeutic concentrations.
    • For protein precipitation, add a 3:1 ratio of acetonitrile to plasma, vortex for 1 minute, and centrifuge at 10,000 × g for 10 minutes. Transfer the clean supernatant.

  • Solid-Phase Extraction (SPE) Protocol:

    • Condition the C18 cartridge with 1 mL of methanol, followed by 1 mL of water.
    • Load the prepared sample supernatant.
    • Wash with 1 mL of 5% methanol in water.
    • Elute analytes with 1 mL of pure methanol.
    • Evaporate the eluent to dryness under a gentle stream of nitrogen at 40°C.
    • Reconstitute the dry residue in 100 µL of initial LC mobile phase, vortex, and inject into the LC-MS/MS.

  • Microextraction by Packed Sorbent (MEPS) Protocol:

    • Condition the MEPS sorbent (C18) with 100 µL of methanol, then 100 µL of water.
    • Draw and dispense 100 µL of the prepared sample supernatant through the sorbent 10 times (to maximize extraction).
    • Wash with 100 µL of 5% methanol in water.
    • Elute analytes with 50 µL of pure methanol directly into an LC vial.
    • Inject the eluent into the LC-MS/MS (no evaporation/reconstitution needed).

  • Analysis and Data Processing:

    • Analyze all samples using the validated LC-MS/MS method.
    • Calculate key performance parameters: recovery (%), precision (% RSD), and matrix effects (%).
    • Record the total sample preparation time, solvent volumes consumed, and waste generated for each method.
AGREEprep Assessment
  • Input the data from the experimental procedures (solvent types and volumes, waste, time, energy use, etc.) into the AGREEprep software [5] [3].
  • Use the default weights for all ten criteria.
  • Record the overall score and the individual criterion scores for both the SPE and MEPS methods.
  • Compare the resulting pictograms to visually identify the weaknesses of the traditional method and the relative strengths of the microextraction technique.

Workflow and Strategic Pathway for Green Sample Preparation

The following diagram illustrates the logical decision pathway for selecting and developing a sample preparation method that balances analytical performance with greenness and practicality, as per White Analytical Chemistry (WAC) principles.

G Strategic Pathway for Green Sample Preparation Method Development Start Start: Define Analytical Goal P1 Problem: High Solvent/Waste Start->P1 P2 Problem: Low Throughput/Integration Start->P2 P3 Problem: Poor Material Sustainability Start->P3 S1 Strategy: Implement Microextraction (e.g., SPME, MEPS, DLLME) P1->S1 S2 Strategy: Automate and Integrate Sample Prep P2->S2 S3 Strategy: Use Renewable Sorbents and Green Solvents P3->S3 A1 Action: Replace LLE with Solvent-Free SPME or Miniaturized DLLME S1->A1 A2 Action: Use MEPS in an Autosampler Syringe for Direct Injection S2->A2 A3 Action: Employ FPSE with Reusable Fabrics or Biodegradable Sorbents S3->A3 Eval Evaluate with AGREEprep & WAC Principles A1->Eval A2->Eval A3->Eval Eval->Start Iterate and Optimize

Traditional sample preparation methods like SPE and LLE are plagued by systemic issues related to solvent consumption, waste generation, and inefficient workflows, which result in low scores under the AGREEprep assessment framework. The experimental protocol and strategic pathway outlined herein provide a clear methodology for researchers to quantitatively benchmark these conventional techniques against modern microextraction alternatives. Transitioning to miniaturized, integrated, and environmentally conscious sample preparation is not only a matter of improving greenness scores but is also crucial for enhancing analytical efficiency, reducing costs, and ensuring the sustainability of laboratory practices in drug development and bioanalysis.

In modern analytical chemistry, the sample preparation step is increasingly recognized as a critical determinant of both environmental impact and analytical efficiency. The AGREEprep (Analytical Greenness Metric for Sample Preparation) tool has emerged as a standardized, comprehensive metric to evaluate the greenness of sample preparation methods based on the ten fundamental principles of green sample preparation (GSP) [17] [3]. This user-friendly software generates a pictogram score between 0 and 1, providing an at-a-glance assessment of a method's environmental performance [5]. As regulatory pressures and sustainability goals intensify, researchers are actively seeking proven methodologies to enhance their AGREEprep scores without compromising analytical performance. This application note delineates three foundational strategies—miniaturization, solvent selection, and automation—that directly address core AGREEprep criteria, enabling scientists to systematically develop greener microextraction protocols for drug development and bioanalysis.

The AGREEprep Assessment Framework: Decoding the Ten Principles

The AGREEprep calculator evaluates methods against ten criteria, each corresponding to a key principle of Green Sample Preparation. Understanding this framework is essential for targeted methodological improvements [17] [5].

Table 1: The Ten Principles of Green Sample Preparation Underlying AGREEprep

Principle Number Principle Description Key Influencing Factors
1 Favoring in situ sample preparation On-site analysis, integrated measurements
2 Using safer solvents and reagents Solvent toxicity, hazard classifications
3 Targeting sustainable, reusable, and renewable materials Sorbent reusability, biodegradable materials
4 Minimizing waste Total waste mass, toxicity of waste
5 Minimizing sample, chemical, and material amounts Sample volume, solvent consumption, sorbent mass
6 Maximizing sample throughput Parallel processing, analysis time
7 Integrating steps and promoting automation Workflow integration, robotic automation
8 Minimizing energy consumption Heating/cooling requirements, extraction duration
9 Choosing the greenest possible post-sample preparation configuration for analysis Instrumental energy demands, solvent use in analysis
10 Ensuring safe procedures for the operator Exposure to hazardous chemicals, operational risks

The AGREEprep software assigns a sub-score from 0 (worst) to 1 (best) for each principle. These are then combined into a final overall score, visually represented by a circular pictogram where the outer segments are colored according to each criterion's performance [5]. The following strategies provide a direct pathway to improving these scores.

Strategy 1: Miniaturization of Extraction Phases and Devices

Miniaturization is arguably the most impactful strategy for enhancing AGREEprep scores, as it simultaneously addresses multiple principles, notably minimizing waste (Principle 4) and reducing sample/reagent amounts (Principle 5).

Protocol: Thin-Film Solid-Phase Microextraction (TF-SPME) for Volatile Compound Analysis

TF-SPME devices offer a superior surface area-to-volume ratio compared to traditional fibers, leading to higher extraction efficiency and sensitivity, which allows for further miniaturization [44].

Materials:

  • HLB/PDMS Thin-Film SPME device (e.g., Carbon Mesh TF-SPME)
  • 10 mL sample vial with magnetic cap
  • Magnetic stirrer and stir bar
  • Gas Chromatograph-Mass Spectrometer (GC-MS)

Procedure:

  • Device Conditioning: Place the TF-SPME device in a suitable desorption chamber and condition it with a stream of inert gas (e.g., Nâ‚‚) at an elevated temperature (e.g., 250°C) for 30 minutes, or as recommended by the manufacturer, to remove any contaminants.
  • Sample Preparation: Transfer 5 mL of the aqueous sample (e.g., spiked with target volatiles) into a 10 mL vial. Add an internal standard if required for quantification.
  • Extraction: Place the TF-SPME device directly into the sample (Direct Immersion mode). Stir the sample at a constant rate (e.g., 750 rpm) for a predetermined extraction time (e.g., 30-60 minutes) to enhance mass transfer.
  • Rinsing: After extraction, briefly rinse the TF-SPME device with deionized water (< 5 seconds) to remove loosely adsorbed matrix components.
  • Desorption: Introduce the TF-SPME device into the GC inlet for thermal desorption. Typical conditions include a desorption temperature of 260°C for 5 minutes in splitless mode to transfer all analytes to the analytical column.
  • Reconditioning: After desorption, recondition the TF-SPME device in the GC inlet or a separate unit to ensure no carryover between analyses.

Impact on AGREEprep Score: This protocol directly reduces solvent consumption to near zero (Principle 2, 4, 5). The reusability of the TF-SPME device over dozens of extractions significantly improves scores for Principle 3 (sustainable materials) [44].

Comparative Greenness of Microextraction Techniques

Recent assessments of microextraction techniques used in bioanalysis, such as Therapeutic Drug Monitoring (TDM), provide quantitative evidence of the benefits of miniaturization.

Table 2: AGREEprep Score Comparison of Common Microextraction Techniques [3]

Microextraction Technique Typical Sample Volume Typical Solvent Consumption Representative AGREEprep Score
Thin-Film SPME (TF-SPME) 1-10 mL < 0.1 mL (for desorption) 0.75 - 0.85
Dispersive Liquid-Liquid Microextraction (DLLME) 5-10 mL ~1.5 mL (disperser + extraction solvent) 0.65 - 0.75
Hollow-Fiber Liquid-Phase Microextraction (HF-LPME) 1-5 mL ~10-30 µL (acceptor phase) 0.70 - 0.80
Microextraction by Packed Sorbent (MEPS) 100-500 µL 50-200 µL (for washing/elution) 0.68 - 0.78
Stir Bar Sorptive Extraction (SBSE) 10-50 mL < 0.1 mL (for desorption) 0.72 - 0.82

As evidenced, techniques that minimize or eliminate solvent use (like SPME variants) and use smaller sample volumes consistently achieve higher AGREEprep scores [3] [44].

G Start Start: Method Development S1 Evaluate Sample Volume Start->S1 S2 Select Microextraction Format S1->S2 P4 Principle 4: Minimize Waste S1->P4 P5 Principle 5: Minimize Amounts S1->P5 S3 Choose Safer Solvents S2->S3 S2->P4 S2->P5 S4 Implement Automation S3->S4 P2 Principle 2: Safer Solvents S3->P2 S5 Conduct AGREEprep Assessment S4->S5 P7 Principle 7: Automation S4->P7 End Optimized Green Method S5->End

Figure 1: A strategic workflow for improving AGREEprep scores, linking key methodological choices (colored rectangles) to the specific GSP principles (ellipses) they most directly enhance.

Strategy 2: Selection of Safer Solvents and Sustainable Materials

The nature of chemicals used in sample preparation heavily influences the AGREEprep score for Principle 2 (safer solvents) and Principle 3 (sustainable materials).

Protocol: Dispersive Liquid-Liquid Microextraction (DLLME) with Bio-Based Solvents

While DLLME is inherently miniaturized, its greenness can be substantially upgraded by solvent choice [5] [45].

Materials:

  • Extraction solvent: Ethyl acetate (low toxicity, biodegradable) or a bio-based solvent like limonene.
  • Disperser solvent: Methanol or ethanol (preferred due to its safer profile).
  • 15 mL conical centrifuge tube
  • Micropipettes (100 µL - 1 mL)
  • Centrifuge
  • Salts (e.g., NaCl, for salting-out effect)

Procedure:

  • Sample Preparation: Place 5 mL of aqueous sample (e.g., filtered water sample) into a 15 mL centrifuge tube.
  • Salting-Out: Add an appropriate amount of NaCl (e.g., 1 g) and shake to dissolve. This increases ionic strength and improves extraction efficiency for non-polar analytes.
  • Solvent Mixture: Rapidly inject a mixture of 1.0 mL ethanol (disperser) and 150 µL ethyl acetate (extraction solvent) into the sample solution using a syringe. A cloudy solution should form immediately.
  • Centrifugation: Centrifuge the mixture at 4000 rpm for 5 minutes to separate the phases. The fine droplets of the extraction solvent will coalesce at the bottom of the tube.
  • Collection: Using a microsyringe, carefully withdraw the sedimented organic phase (~50-100 µL).
  • Analysis: Transfer the extract to a suitable vial insert for GC-MS or LC-MS analysis. If necessary, the extract can be diluted or reconstituted in an instrument-compatible solvent.

Impact on AGREEprep Score: Replacing traditional chlorinated solvents (e.g., dichloromethane) with ethyl acetate or bio-based solvents significantly reduces toxicity (Principle 2). The extremely low volume of extraction solvent also minimizes waste generation (Principle 4) [45].

Strategy 3: Automation and Workflow Integration

Automation is a powerful tool for improving sample throughput (Principle 6), integrating operational steps (Principle 7), and enhancing operator safety (Principle 10) by reducing manual handling.

Protocol: Automated 96-Well SPME for High-Throughput Bioanalysis

Coupling SPME with automated systems like the Concept 96 platform allows for parallel processing of dozens of samples [46].

Materials:

  • Concept 96 system or similar automated SPME platform
  • 96-blade SPME device (e.g., coated blade spray - CBS - blades)
  • 96-well plate containing biological samples (e.g., plasma, urine)
  • Agitator for the 96-well plate
  • LC-MS/MS or Direct-to-MS system

Procedure:

  • System Setup: Program the automated SPME system with the following sequence: preconditioning, extraction, washing, and desorption.
  • Preconditioning: The system automatically dips the SPME blades into a solvent (e.g., methanol/water) to activate the coating.
  • Extraction: The robotic arm moves all 96 blades simultaneously into the wells containing the samples. The platform agitates the entire plate for a set extraction time (e.g., 30 minutes).
  • Washing: After extraction, the blades are transferred to a wash solution (e.g., water or a mild buffer) to remove non-specifically bound matrix components.
  • Desorption/Analysis:
    • For LC-MS: Blades are desorbed in a 96-well plate containing a small volume (e.g., 100 µL) of a strong LC-compatible solvent (e.g., ACN with 0.1% formic acid) with agitation. The desorbed solution is then injected into the LC-MS.
    • For Direct-to-MS (e.g., CBS-MS): The blades are directly introduced into a specialized interface where a high voltage is applied, and a spray solvent is used to desorb and ionize the analytes directly into the mass spectrometer.
  • Data Acquisition: The MS acquires data for all samples in a highly parallel and rapid manner.

Impact on AGREEprep Score: This approach maximizes sample throughput (Principle 6), fully integrates extraction, cleanup, and injection (Principle 7), and minimizes energy and time per sample (Principle 8). It also drastically reduces analyst intervention, enhancing safety and reproducibility (Principle 10) [46].

The Scientist's Toolkit: Essential Reagents and Materials

Table 3: Key Research Reagent Solutions for Green Microextraction

Item Name Function/Application Green Characteristic
HLB/PAN Sorbent Hydrophilic-Lipophilic Balanced particles in Polyacrylonitrile binder; used in SPME fibers, TF-SPME, and CBS for broad-spectrum analyte extraction. Reusable, excludes macromolecules (reducing matrix waste), enables direct coupling to MS [46].
Ethyl Acetate A low-toxicity, biodegradable ester used as an extraction solvent in LPME techniques like DLLME. Safer alternative to chlorinated solvents (e.g., DCM) or alkanes [5] [45].
Bio-Based Solvents (e.g., Limonene) Solvents derived from renewable biomass (e.g., citrus peel), used for extracting non-polar analytes. Sustainable, renewable origin, often biodegradable [45].
Phosphate Buffer Saline (PBS) Aqueous buffer for adjusting sample pH and ionic strength to optimize extraction efficiency. Replaces more hazardous buffers, minimizes introduction of toxic reagents [46].
Concept 96 Automated System A robotic platform for high-throughput, parallel processing of up to 96 SPME devices. Dramatically increases throughput, improves precision, and reduces manual labor and exposure [46].
Didemnin BDidemnin B, CAS:77327-05-0, MF:C57H89N7O15, MW:1112.4 g/molChemical Reagent
Diethylcarbamazine citrateDiethylcarbamazine citrate, CAS:1642-54-1, MF:C16H29N3O8, MW:391.42 g/molChemical Reagent

Systematically enhancing AGREEprep scores is an achievable goal through targeted methodological refinements. The synergistic application of device miniaturization (e.g., adopting TF-SPME over conventional methods), prudent solvent selection (favoring safer, bio-based options), and workflow automation provides a robust framework for developing greener analytical methods. By implementing the detailed protocols and strategic insights contained in this application note, researchers and drug development professionals can significantly reduce the environmental footprint of their sample preparation processes while maintaining, and often enhancing, the high-quality data required for critical applications.

The adoption of microextraction techniques represents a significant stride toward green analytical chemistry. However, a superficial application can lead to a "rebound effect," where apparent gains in one area (e.g., solvent reduction) are offset by overlooked impacts in another (e.g., energy consumption or material sourcing). A holistic assessment using frameworks like AGREEprep is crucial to quantify and validate the genuine environmental footprint of these methods. This document provides application notes and protocols to guide researchers in implementing and critically evaluating sustainable microextraction techniques within pharmaceutical research and development.

Quantitative Sustainability Assessment of Microextraction Techniques

The core advantage of microextraction lies in its miniaturization, leading to drastic reductions in solvent consumption and waste generation compared to traditional methods like Liquid-Liquid Extraction (LLE) and solid-phase extraction (SPE) [47] [48]. The following table summarizes the key environmental and performance characteristics of prevalent techniques.

Table 1: Comparative Analysis of Extraction Techniques for Bioanalysis

Extraction Technique Typical Sample Volume Typical Solvent Volume Key Sustainability Merits Common AGREEprep Evaluation Points
Liquid-Liquid Extraction (LLE) 0.5 - 2 mL [48] 10 - 20 mL [48] High waste generation, use of hazardous solvents [48] Low score for hazardous chemicals, waste, and energy consumption.
Solid-Phase Extraction (SPE) 0.5 - 2 mL [48] 5 - 15 mL [48] Reduced solvent vs. LLE; can be automated [48] Evaluates solvent volume, waste, and potential for automation.
Solid-Phase Microextraction (SPME) < 1 mL < 1 mL (or solvent-free) Solventless or negligible solvent use; reusable devices [47] [49] High scores for hazardous chemicals, waste, and sample throughput.
Microextraction by Packed Sorbent (MEPS) < 100 µL 20 - 100 µL [48] Drastic solvent reduction; suitable for automation and small samples [47] [48] High scores for miniaturization, low waste, and analytical efficiency.
Dispersive Liquid-Liquid Microextraction (DLLME) < 1 mL < 100 µL Very low solvent consumption; high pre-concentration factors [48] High score for waste, but notes the potential toxicity of dispersive solvents.

Experimental Protocols for Sustainable Microextraction

The following standardized protocols are designed for the analysis of drugs of abuse or pharmaceutical compounds in biological matrices (e.g., blood, urine) and are optimized for minimal environmental impact.

Protocol for SPME of Drugs from Biological Fluids

This protocol outlines a solvent-free approach for extracting analytes from blood or urine, suitable for coupling with GC-MS or LC-MS.

Research Reagent Solutions:

  • SPME Fiber: Select a coating compatible with target analytes (e.g., PDMS for non-polar, CW/DVB for polar compounds) [49].
  • Internal Standard Solution: Deuterated analogs of target analytes in methanol.
  • Phosphate Buffer (0.1 M, pH 6): For sample dilution and pH control.
  • Mobile Phase Additives: For LC-MS, use e.g., 0.1% formic acid in water and methanol [48].

Procedure:

  • Sample Preparation: Piper 500 µL of biological sample (blood, plasma, or urine) into a 2 mL glass vial. Add 50 µL of internal standard solution and 1 mL of phosphate buffer (pH 6). Vortex-mix for 30 seconds [48].
  • SPME Extraction: Place the vial on a sampler tray. According to the experimental design, expose the SPME fiber to the sample headspace (HS-SPME) or immerse it directly (DI-SPME). The design of experiments (DoE) should be applied to optimize critical factors [49]:
    • Extraction Time: (e.g., 10-30 min)
    • Temperature: (e.g., 40-70°C)
    • Sample Agitation: (e.g., stirring speed)
  • Desorption: After extraction, retract the fiber and transfer it to the chromatographic instrument for desorption.
    • For GC-MS: Desorb the fiber in the hot GC inlet for 1-5 minutes.
    • For LC-MS: Use an online or offline desorption chamber with a compatible solvent (e.g., methanol/water mixture) for 5-15 minutes to prevent carryover [49].
  • Analysis: Inject the desorbed analytes into the GC-MS or LC-MS system for separation and quantification.

Protocol for MEPS of Fentanyl and Analogs from Whole Blood

This protocol, adapted from validated methods, uses minimal solvent volumes for high-sensitivity analysis [48].

Research Reagent Solutions:

  • MEPS Sorbent: Mixed-mode (reverse-phase and cation-exchange) sorbent bed, e.g., Clean Screen (CS) [48].
  • Conditioning Solvents: Methanol, deionized water.
  • Wash Solutions: Deionized water, 1-2% acetic acid in water, hexane.
  • Elution Solvent: Dichloromethane:Isopropanol:Ammonium Hydroxide (78:20:2, v:v:v). Note: Evaluate replacement with greener solvents.

Procedure:

  • Sample Preparation: Mix 500 µL of whole blood with 4 mL of 0.1 M sodium phosphate buffer (pH 6). Vortex for 15 minutes and centrifuge for 10 minutes at 2300× g to pellet debris [48].
  • MEPS Workflow:
    • Conditioning: Draw 500 µL of methanol and then 500 µL of deionized water into the MEPS syringe and slowly dispense them to waste to activate the sorbent.
    • Loading: Slowly draw the prepared sample supernatant through the MEPS cartridge using multiple aspirate/dispense cycles to maximize analyte retention.
    • Washing: Rinse the sorbent with 500 µL of deionized water, followed by 250 µL of acetic acid solution, and then 250 µL of hexane to remove interferences. Dry the cartridge by drawing air through it for 1-2 minutes.
    • Elution: Elute the target analytes into a clean vial by slowly drawing 250 µL of the elution solvent and dispensing it. Repeat once [48].
  • Sample Reconstitution: Evaporate the eluate to dryness under a gentle stream of nitrogen. Reconstitute the dry residue with 50 µL of a mobile phase-compatible solvent (e.g., 0.1% formic acid in water) and vortex mix before LC-MS/MS analysis [48].

Workflow Visualization & Optimization

The following diagram illustrates the logical workflow for developing and validating a sustainable microextraction method, integrating AGREEprep assessment throughout the process.

G Start Define Analytical Problem A1 Select Microextraction Technique (SPME, MEPS, etc.) Start->A1 A2 Screen Sorbents & Solvents (Green Chemistry Principles) A1->A2 A3 Multivariate Optimization (DoE: Time, pH, Temp) A2->A3 A4 Method Validation (Sensitivity, Precision) A3->A4 A5 AGREEprep Assessment A4->A5 Collect Quantitative Data A5->A2 Low Score Reformulate End Validated & Eco-Rated Method A5->End High Score

The Scientist's Toolkit: Key Research Reagent Solutions

Selecting the right materials is fundamental to both analytical performance and sustainability.

Table 2: Essential Materials for Sustainable Microextraction Methods

Item Function/Description Sustainability & Performance Considerations
Ionic Liquids (ILs) Non-molecular solvents with low volatility used in liquid-phase microextraction. Substitute for volatile organic solvents (VOCs); tunable properties for selectivity [47].
Deep Eutectic Solvents (DES) Low-cost, biodegradable solvents formed from natural compounds. Green alternative to traditional organic solvents; can be derived from renewable sources [47].
Carbon Nanotubes (CNTs) Nano-sorbents with high surface area for SPME and dSPE. Improve extraction efficiency and kinetics; reusable, reducing material waste [47].
Magnetic Nanoparticles (MNPs) Sorbents functionalized for specific analytes; separated using a magnet. Enable simple, rapid dispersive microextraction (MSPE) without centrifugation [50].
Molecularly Imprinted Polymers (MIPs) Synthetic sorbents with tailor-made recognition sites for target molecules. Provide high selectivity, reducing matrix effects and the need for extensive cleaning steps [50].
DigoxigeninDigoxigeninDigoxigenin (DIG) is a steroid hapten for non-radioactive nucleic acid and protein detection in research. This product is for Research Use Only (RUO). Not for human or therapeutic use.
DihydrojasmoneDihydrojasmone (CAS 1128-08-1) - High-Purity RUOHigh-purity Dihydrojasmone for research. Explore its applications in agriculture, antifungal, and fragrance studies. CAS 1128-08-1. For Research Use Only.

The principles of Green Analytical Chemistry (GAC) have fundamentally reshaped modern laboratory practice, driving a paradigm shift toward more sustainable and environmentally responsible analysis [51]. Within this framework, sample preparation—historically a resource-intensive step—has undergone significant transformation through the development and adoption of microextraction techniques [42]. These techniques, including solid-phase microextraction (SPME) and liquid-phase microextraction (LPME), align with GAC by minimizing solvent consumption, reducing waste generation, and promoting operator safety [52] [31]. However, a persistent challenge remains: the potential perception that these environmental benefits come at the expense of analytical performance metrics such as sensitivity, accuracy, and precision.

This application note explores strategies for successfully integrating the greenness of microextraction methods with uncompromised analytical performance, framed within a research context utilizing the AGREEprep (Analytical Greenness Metric for Sample Preparation) assessment tool [3] [7]. AGREEprep provides a standardized, quantitative framework for evaluating sample preparation methods against ten core principles of green sample preparation, generating a score from 0 to 1 [3]. We demonstrate that through strategic selection of materials, process automation, and intelligent method design, it is not only possible to avoid trade-offs but to achieve synergistic enhancements in both ecological and analytical outcomes. The protocols and data presented herein provide a practical roadmap for researchers in drug development and bioanalysis to implement microextraction techniques that are both green and performance-driven.

Quantitative Greenness and Performance Assessment

The AGREEprep metric tool evaluates sample preparation methods based on ten principles, including the use of safer solvents, waste minimization, sample throughput, and energy consumption [3] [7]. A score above 0.5 indicates an acceptably green method. The following table summarizes the AGREEprep scores and corresponding analytical performance data for several documented microextraction techniques, illustrating that high greenness scores can coexist with excellent analytical performance.

Table 1: AGREEprep Scores and Analytical Performance of Selected Microextraction Methods

Application & Technique AGREEprep Score (Greenness) Analytical Performance Highlights Key Green Features
Triazole Fungicides in Food (Automated DLLME) [53] 0.76 LOD: 0.003 µg L⁻¹LOQ: 0.01 µg L⁻¹Recovery: 70.1–105.7%Throughput: 4 samples simultaneously Bio-based solvents, automated liquid handling, no centrifugation, minimal waste
PAHs in Spices (HMCart-DI-SPME) [54] 0.71 LOD: 0.09–0.88 µg kg⁻¹LOQ: 0.27–2.67 µg kg⁻¹Recovery: 87.5–108.5%Precision (RSD): <13% Simplified sample prep, low solvent consumption, high precision
Hormones in Biological Matrices (SPME/DLLME) [55] 0.68 (estimated) Sensitivity: ng L⁻¹ to pg L⁻¹ rangeSelectivity: High via MIPs/DES Miniaturization, green solvents (DES, SUPRAS), high selectivity sorbents
Therapeutic Drug Monitoring (Microextraction) [3] Varies by specific method Meets required sensitivity & precision for clinical analysis Small sample volumes, reduced solvent use, high throughput

The data in Table 1 refutes the notion of an inherent greenness-performance trade-off. The automated DLLME method for triazole fungicides, for instance, achieves an impressive AGREEprep score of 0.76 while also delivering low limits of detection and high accuracy [53]. This synergy is accomplished by integrating several green principles directly into the analytical workflow. The method uses bio-based solvents, automates the entire sample preparation process to enhance precision and throughput, and employs a salting-out strategy for phase separation, eliminating the need for energy-intensive centrifugation [53].

Detailed Experimental Protocols

Protocol 1: Automated High-Throughput DLLME for Triazole Fungicides

This protocol exemplifies how automation and green solvent substitution can create a method that is fast, environmentally friendly, and highly precise [53].

3.1.1 Research Reagent Solutions

Table 2: Essential Materials for Automated DLLME

Reagent/Material Function Green Alternative & Rationale
Octanoic Acid Extractant Natural fatty acid; biodegradable, low toxicity, renewable [53].
γ-Valerolactone (GVL) Dispersant Bio-based solvent derived from lignocellulosic biomass; low toxicity [53].
Saturated NaCl Solution Demulsifier Enables rapid phase separation without energy-intensive centrifugation [53].
Automated Liquid Handling Workstation Process Automation Ensures high precision, increases throughput (4 samples simultaneously), reduces operator error and exposure [53].

3.1.2 Procedure

  • Setup: Place samples in deep-well plates and position the plate, along with reservoirs containing octanoic acid (extractant), GVL (dispersant), and saturated sodium chloride solution, on the deck of the automated liquid handling workstation.
  • Extraction: The workstation pipettes 1 mL of sample, 100 µL of octanoic acid, and 500 µL of GVL into a well. It then mixes the contents by repeated aspiration and dispensing to form a fine emulsion, facilitating efficient analyte extraction.
  • Phase Separation: Add 1 mL of saturated sodium chloride solution to destabilize the emulsion. The aqueous and organic phases separate rapidly without centrifugation.
  • Analysis: The workstation collects the sedimented organic phase (containing the concentrated analytes) and injects it into a UHPLC-MS/MS system for analysis. Ten triazole fungicides can be separated and detected within 5.5 minutes [53].

Protocol 2: In Vivo SPME for Unstable Metabolites in Biological Tissues

This protocol highlights the unique capability of SPME to capture labile analytes in vivo, thereby improving analytical accuracy by preventing post-sampling degradation [56].

3.2.1 Research Reagent Solutions

  • SPME Probes: Biocompatible probes with hydrophilic-lipophilic balanced (HLB) coatings (e.g., 2 mm length).
  • Function: The porous structure of the coating excludes macromolecules like proteins and enzymes, thus quenching metabolism and stabilizing unstable small molecules immediately upon extraction [56].

3.2.2 Procedure

  • Probe Conditioning: Prior to the first use, condition the SPME probe by immersing it in an appropriate organic solvent (e.g., methanol) and then in water.
  • *In Vivo/In Situ Extraction: Gently implant the SPME probe into the target tissue (e.g., the brain of an anesthetized rodent or a cell culture medium). Allow the extraction to proceed for a predetermined time (typically 15-30 minutes) to reach equilibrium.
  • Probe Removal and Rinsing: Retract the probe and immediately rinse it with deionized water to remove any adherent matrix components (e.g., salts, blood cells).
  • Analyte Desorption: Desorb the extracted analytes by immersing the probe in a suitable vial containing a desorption solvent (compatible with the subsequent LC-MS analysis) for a set time with agitation.
  • Analysis: Inject the desorption solvent into the LC-MS system. The negligible depletion aspect of SPME allows for repeated sampling from the same living system, enabling temporal monitoring [56].

G Start Start SPME Protocol Condition Condition SPME Probe Start->Condition Implant Implant Probe In Vivo/In Situ Condition->Implant Extract Extract Analytes Implant->Extract Remove Remove and Rinse Probe Extract->Remove Desorb Desorb Analytes into Solvent Remove->Desorb Analyze LC-MS Analysis Desorb->Analyze End End Protocol Analyze->End

Diagram 1: In Vivo SPME Workflow for Unstable Analytes

The Scientist's Toolkit: Key Research Reagent Solutions

The successful implementation of green microextraction methods relies on a suite of specialized reagents and materials. The table below details critical solutions that facilitate the balance between greenness and performance.

Table 3: Key Research Reagent Solutions for Green Microextraction

Category Specific Examples Function & Green Advantage
Green Solvents Deep Eutectic Solvents (DES), Supramolecular Solvents (SUPRAS), Fatty Acids (e.g., Octanoic acid), γ-Valerolactone (GVL) Replace toxic traditional solvents (e.g., chlorinated, acetonitrile). Offer low toxicity, high biodegradability, and often renewable origins [53] [55].
Advanced Sorbents Molecularly Imprinted Polymers (MIPs), Hydrophilic-Lipophilic Balanced (HLB) coatings, Magnetic Nanoparticles (MNPs) Provide high selectivity for target analytes, reducing matrix effects and improving accuracy. MNP-integrated sorbents enable easy retrieval with a magnet, simplifying workflows [56] [42] [55].
Automation & High-Throughput Tools Automated Liquid Handling Workstations, 96-Well Plate Formats (for SPME, LPME) Maximize sample throughput (Principle 6 of GSP), minimize human error, enhance reproducibility, and improve operator safety [31] [53].
Green Derivatization/ Separation Aids Saturated Sodium Chloride Solution Acts as a demulsifier in DLLME, avoiding the need for high-energy centrifugation and speeding up phase separation [53].
DihydrolycorineDihydrolycorine, CAS:6271-21-2, MF:C16H19NO4, MW:289.33 g/molChemical Reagent
DihydroresveratrolDihydroresveratrol|Resveratrol Metabolite|CAS 58436-28-5

The journey toward greener analytical laboratories does not require a compromise in data quality. As demonstrated by the AGREEprep assessments and detailed protocols, a strategic approach that embraces green solvents, advanced materials, and process automation can yield microextraction methods that are superior in both ecological impact and analytical performance. By adopting the frameworks and solutions outlined in this application note, researchers and drug development professionals can confidently advance their analytical practices, ensuring they meet the dual imperatives of scientific excellence and environmental stewardship. Future work will continue to focus on the development of even more selective sorbents and the full integration of these green microextraction workflows with advanced analytical instrumentation.

The AGREEprep (Analytical Greenness Metric for Sample Preparation) tool is a comprehensive software-based metric that evaluates the environmental sustainability of sample preparation methods based on the 10 principles of Green Sample Preparation (GSP). For researchers developing microextraction techniques, AGREEprep provides a quantitative score between 0 and 1, where higher scores indicate greener methods, along with an intuitive pictogram that visually highlights strengths and weaknesses across all assessment criteria [5]. This tool has become increasingly vital for validating the greenness of sample preparation methods in analytical chemistry, particularly as journals and regulatory bodies place greater emphasis on sustainable practices.

In pharmaceutical research and drug development, where analytical methods are routinely employed for drug quantification, impurity profiling, and bioanalysis, AGREEprep offers a standardized approach to evaluate and improve the environmental footprint of sample preparation workflows. Understanding how to interpret AGREEprep scores and implement corrective actions for underperforming criteria is essential for developing truly sustainable analytical methods that balance analytical performance with environmental considerations [3].

Understanding the AGREEprep Scoring System

The AGREEprep assessment is structured around ten fundamental principles of green sample preparation, each representing a specific aspect of environmental sustainability and practical efficiency. The tool generates a circular pictogram with ten colored segments corresponding to these principles, with the overall score displayed in the center [5]. The principles are summarized in the table below.

Table 1: The Ten Principles of Green Sample Preparation Assessed by AGREEprep

Principle Number Assessment Criteria Key Focus Areas
1 Favoring in situ sample preparation On-site analysis, minimal transport
2 Using safer solvents and reagents Solvent toxicity, hazardous chemicals
3 Targeting sustainable, reusable, renewable materials Sorbent reusability, biodegradable materials
4 Minimizing waste Waste volume, hazardous waste
5 Minimizing sample, chemical, and material amounts Miniaturization, solvent volumes
6 Maximizing sample throughput Automation, parallel processing
7 Integrating steps and promoting automation Workflow integration, automated systems
8 Minimizing energy consumption Energy-intensive equipment, process efficiency
9 Choosing greenest post-sample preparation configuration Solvent-free techniques, direct coupling
10 Ensuring operator safety Exposure risks, protective equipment

Each criterion is scored from 0 to 1, with the extremes representing the worst and best performance, respectively. The software allows users to assign different weights to each criterion based on their relative importance, though default weights are typically used unless specific justifications exist [5]. The final aggregate score provides a quick reference for the method's overall greenness, while the colored segments enable researchers to immediately identify which specific principles require improvement.

Systematic Troubleshooting of Low AGREEprep Scores

A low overall AGREEprep score indicates significant room for improvement across multiple principles of green sample preparation. The following structured approach guides researchers through diagnosing issues and implementing effective corrective actions, with a focus on microextraction techniques common in pharmaceutical analysis.

Principle 1 (In Situ Preparation) and Principle 9 (Post-Preparation Configuration)

Common Deficiencies: Low scores in these interconnected principles often occur when methods require extensive sample transport or offline processing before analysis. Techniques that are not directly compatible with the analytical instrument, or those requiring intermediate steps that increase resource consumption, typically perform poorly here [5].

Corrective Actions:

  • Implement direct coupling: Interface the sample preparation device directly with the analytical instrument (e.g., direct SPME-GC coupling, online SPE-LC systems) to eliminate intermediate steps and reduce sample handling [57].
  • Develop field-based methods: Adapt methods for on-site analysis using portable or handheld instruments when possible, particularly for preliminary screening applications [5].
  • Optimize transfer lines: For hyphenated techniques, ensure efficient transfer of analytes from extraction to separation systems to maintain analytical performance while gaining green benefits.

Principles 2 (Safer Solvents) and 4 (Waste Minimization)

Common Deficiencies: These principles are frequently the primary contributors to low AGREEprep scores in traditional extraction methods. The use of chlorinated solvents, large solvent volumes, and methods generating significant hazardous waste result in poor performance [9] [58].

Corrective Actions:

  • Solvent substitution strategy: Systematically evaluate and replace hazardous solvents with safer alternatives. For instance, in Single-Drop Microextraction (SDME), toluene provided better droplet stability than volatile alternatives, though its greenness should be balanced with performance requirements [58].
  • Implement microextraction techniques: Techniques like Dispersive Liquid-Liquid Microextraction (DLLME) and SDME typically consume only microliters of extraction solvent, dramatically reducing waste generation compared to conventional Liquid-Liquid Extraction (LLE) [9] [58].
  • Explore alternative solvents: Investigate newer classes of green solvents, including deep eutectic solvents (DESs), switchable hydrophilicity solvents (SHSs), and supramolecular solvents (SUPRASs), which may offer favorable toxicological and environmental profiles [58].

Principles 3 (Sustainable Materials) and 5 (Minimizing Amounts)

Common Deficiencies: Methods relying on single-use, non-renewable materials or those requiring large amounts of samples and reagents score poorly here. Conventional Solid-Phase Extraction (SPE) using disposable cartridges is particularly vulnerable to low scores in these principles [3].

Corrective Actions:

  • Sorbent selection and reuse: Prioritize sorbents derived from renewable sources (e.g., bio-based materials) and establish validated regeneration protocols to extend sorbent lifetime. For Solid-Phase Microextraction (SPME), proper fiber conditioning can enable multiple reuses [3].
  • Method miniaturization: Reduce sample sizes to the minimum required for adequate sensitivity. For example, a HS-SPME-GC-QTOF-MS method for analyzing biogenic volatile organic compounds was successfully optimized using only 0.20 g of sample, significantly improving its greenness profile [57].
  • Micro-sampling techniques: Implement capillary microsampling or other micro-sampling approaches for biological samples in pharmaceutical studies to reduce material consumption while maintaining data quality.

Principles 6 (Throughput) and 7 (Integration and Automation)

Common Deficiencies: Manual, time-consuming methods with low sample throughput negatively impact these principles. Techniques requiring extensive manual manipulation or those with long extraction times typically achieve low scores [5].

Corrective Actions:

  • Process parallelization: Develop methods that enable simultaneous processing of multiple samples. For example, multi-well plate formats for microextraction techniques can significantly increase throughput [3].
  • Automated systems: Implement robotic systems or autosamplers capable of performing extractions unattended. The use of a GC autosampler for automated HS-SPME significantly improved the practicality and throughput of a method for analyzing tree emissions [57].
  • Workflow integration: Combine extraction and cleanup steps into a single, continuous process to reduce total handling time and improve efficiency.

Principle 8 (Energy Consumption)

Common Deficiencies: Methods employing energy-intensive equipment or processes, such as lengthy heating, sonication, or centrifugation steps, score poorly in this category. Large instrumentation with high power requirements also negatively impacts this principle [57].

Corrective Actions:

  • Energy-efficient alternatives: Replace energy-intensive steps with more efficient processes. For example, vortex-assisted extraction may replace sonication in some applications with comparable efficiency but lower energy demand.
  • Room temperature processes: Develop methods that operate effectively at ambient temperature. The SDME method for nitro compounds demonstrated that efficient extraction could be achieved without heating by optimizing other parameters like extraction time and solvent selection [58].
  • Instrument selection: When possible, select analytical instruments with better energy efficiency ratings or those designed for lower power consumption.

Principle 10 (Operator Safety)

Common Deficiencies: Methods using highly toxic, carcinogenic, or mutagenic reagents without adequate safety controls result in low scores for this principle. Techniques generating significant vapors or aerosols also raise safety concerns [5].

Corrective Actions:

  • Hazard elimination: Substitute hazardous reagents with safer alternatives. For example, replacing chlorobenzene with less toxic solvents in SDME methods improves both operator safety and environmental impact [58].
  • Engineering controls: Implement closed-system designs that minimize operator exposure to hazardous materials during sample preparation.
  • Safety procedures: Develop clear standard operating procedures (SOPs) that include safety precautions, personal protective equipment (PPE) requirements, and emergency procedures for handling accidents or spills.

Case Study: Corrective Actions for SDME Method

A research group developing a Direct Immersion Single-Drop Microextraction (DI-SDME) method for nitro compounds in environmental water faced challenges with droplet stability and extraction efficiency, which impacted several AGREEprep principles [58]. Their systematic troubleshooting approach provides an excellent example of corrective action implementation.

Table 2: Troubleshooting Example for SDME Method Optimization

AGREEprep Principle Initial Issue Corrective Action Outcome
P2: Safer Solvents Unstable microdroplets with "green" solvents Tested and selected toluene for stability Stable extraction with minimal solvent volume (μL)
P4: Waste Minimization Solvent loss during extraction Optimized solvent volume and extraction time Consistent droplet volume recovery
P5: Minimizing Amounts Suboptimal sample volume Systematic optimization of sample size Maintained sensitivity while reducing sample volume
P8: Energy Consumption Considered heating to improve efficiency Optimized extraction time at room temperature Efficient extraction without additional energy input

Through this systematic optimization, the method achieved excellent sensitivity with LODs ranging from 0.01 to 0.09 μg/L in deionized water, while maintaining a strong green profile as confirmed by AGREEprep assessment [58].

Complementary Assessment Frameworks

While AGREEprep specifically focuses on the sample preparation step, comprehensive method evaluation should incorporate complementary assessment tools that address other aspects of method performance:

  • BAGI (Blue Applicability Grade Index): This tool evaluates the practicality of analytical methods, including factors such as sample throughput, cost, instrumentation requirements, and operational simplicity. A method with a BAGI score above 60.0 is generally considered practical [35]. Using AGREEprep and BAGI together ensures methods are both green and practically applicable.

  • White Analytical Chemistry (WAC): This holistic approach evaluates the analytical method across three domains: Analytical Performance (Red), Environmental Impact (Green), and Practical & Economic Efficiency (Blue) [3]. The ideal "white" method balances all three aspects, avoiding over-optimization of one at the expense of others.

Table 3: Complementary Assessment Tools for Comprehensive Method Evaluation

Assessment Tool Focus Area Key Evaluation Criteria Optimal Score/Rating
AGREEprep Sample preparation greenness 10 principles of green sample preparation Closer to 1.0
BAGI Method practicality Throughput, cost, instrumentation, operational simplicity >60.0
WAC Balanced method performance 12 principles covering analytical, environmental, and practical aspects Balanced high scores across all three domains

Experimental Protocol: Systematic AGREEprep Optimization

This protocol provides a step-by-step approach to diagnose and improve low AGREEprep scores for microextraction methods in pharmaceutical analysis.

Initial Assessment Phase

  • Input method parameters into the AGREEprep software (available at: https://mostwiedzy.pl/AGREE), including sample size, solvent types and volumes, energy consumption, waste generation, and operational details [5].
  • Record the overall score and individual principle scores from the generated pictogram.
  • Identify critical deficiencies by noting principles with scores below 0.5, which represent significant opportunities for improvement.

Root Cause Analysis

  • Map low-scoring principles to specific methodological steps. For example, if Principle 2 (Safer Solvents) scores low, identify which specific solvents are problematic and their roles in the method.
  • Evaluate technological constraints that may limit improvements for certain principles. Some instrument limitations may be unavoidable but should be documented.
  • Prioritize improvement areas based on the potential impact on the overall score and feasibility of implementation.

Implementation of Corrective Actions

  • Begin with solvent-related issues (Principles 2, 4, 5), as these often offer the most significant improvement opportunities with minimal impact on analytical performance.
  • Address material sustainability (Principle 3) by investigating reusable sorbents or renewable materials.
  • Optimize for throughput and automation (Principles 6, 7) after establishing the core extraction parameters.
  • Reassess energy consumption (Principle 8) once other parameters are optimized.

Validation and Documentation

  • Verify method performance after each significant modification to ensure maintained or improved analytical figures of merit (sensitivity, precision, accuracy).
  • Re-run AGREEprep assessment with modified parameters to quantify improvement.
  • Document the optimization process, including failed attempts, to build institutional knowledge and support future troubleshooting efforts.

G Start Low AGREEprep Score Identified Assess Input Method Parameters into AGREEprep Software Start->Assess Identify Identify Principles with Scores < 0.5 Assess->Identify P2 Principle 2: Safer Solvents Identify->P2 Score < 0.5 P4 Principle 4: Waste Minimization Identify->P4 Score < 0.5 P5 Principle 5: Minimizing Amounts Identify->P5 Score < 0.5 P3 Principle 3: Sustainable Materials Identify->P3 Score < 0.5 P6 Principle 6 & 7: Throughput & Automation Identify->P6 Score < 0.5 P8 Principle 8: Energy Consumption Identify->P8 Score < 0.5 SolventAction Replace Hazardous Solvents Implement Microextraction P2->SolventAction WasteAction Reduce Volumes Reuse Materials P4->WasteAction AmountAction Miniaturize Sample Size Optimize Parameters P5->AmountAction MaterialAction Use Renewable Sorbents Establish Reuse Protocols P3->MaterialAction ThroughputAction Parallel Processing Automate Workflows P6->ThroughputAction EnergyAction Room Temp Processes Energy-Efficient Equipment P8->EnergyAction Validate Validate Analytical Performance SolventAction->Validate WasteAction->Validate AmountAction->Validate MaterialAction->Validate ThroughputAction->Validate EnergyAction->Validate Reassess Re-run AGREEprep Assessment Validate->Reassess Document Document Process and Outcomes Reassess->Document

AGREEprep Troubleshooting Workflow: This diagram illustrates the systematic process for diagnosing and addressing low AGREEprep scores, beginning with initial assessment and proceeding through targeted corrective actions based on specific underperforming principles.

The Scientist's Toolkit: Essential Reagents and Materials

Table 4: Key Research Reagent Solutions for Green Microextraction Method Development

Reagent/Material Function in Microextraction Greenness Considerations Example Applications
Deep Eutectic Solvents (DES) Extraction solvent for various microextraction techniques Biodegradable, low toxicity, renewable sourcing DLLME, SDME for organic compounds [58]
Switchable Hydrophilicity Solvents (SHS) Solvents with tunable solubility for extraction and recovery Reduced waste, recyclability Switching solvent-based microextraction [58]
Supramolecular Solvents (SUPRAS) Self-assembled nanostructured solvents for efficient extraction Reduced solvent consumption, enhanced extraction SUPRAS-based microextraction [58]
Bio-based Sorbents Solid-phase extraction materials from renewable sources Sustainable, biodegradable alternatives to synthetic sorbents SPME, MSPE [3]
Reusable SPME Fibers Solid-phase microextraction with multiple uses Reduced material waste through regeneration HS-SPME, DI-SPME [57]
Magnetic Nanoparticles Dispersive solid-phase extraction with magnetic recovery Reusable materials, efficient separation MSPE for various analytes [35]

Beyond Greenness: Integrating AGREEprep with BAGI and White Analytical Chemistry

The push for sustainable laboratory practices has made green analytical chemistry a cornerstone of modern method development, particularly in sensitive fields like therapeutic drug monitoring. The AGREEprep metric tool has emerged as a specialized standard for evaluating the greenness of sample preparation methods, scoring them against ten principles of green sample preparation [3]. However, an over-reliance on this single metric carries a significant risk: it may promote methods that are environmentally sound but lack the analytical performance or practical robustness required for reliable application in critical areas like drug development and bioanalysis [3].

This application note argues for a holistic assessment framework that balances greenness with other essential criteria. Using microextraction techniques as a case study, we demonstrate that AGREEprep is a necessary, but insufficient, tool for method evaluation. True sustainability in a laboratory context—often termed whiteness—is achieved only when a method maintains an optimal balance between its environmental impact, its analytical effectiveness, and its practical and economic feasibility [3].

The Limits of AGREEprep and the Rise of Holistic Frameworks

The AGREEprep tool is an invaluable benchmark for environmental sustainability. It evaluates sample preparation methods based on ten criteria, including the use of safer solvents, waste minimization, energy consumption, and operator safety [3]. A perfect greenness score on its 0-1 scale indicates an exemplary green method. However, this single score does not guarantee that the method is fit-for-purpose.

The Pitfall of One-Dimensional Assessment

Exclusive focus on greenness can lead to trade-offs that are unacceptable in a regulated environment. For instance, a method might achieve a high AGREEprep score by drastically reducing solvent consumption, but in doing so, it might compromise its sensitivity or fail to achieve the necessary detection limits for trace-level analytes in complex biological matrices [3]. In therapeutic drug monitoring, where patient care depends on accurate, precise, and sensitive measurements, such compromises are not viable [3].

White Analytical Chemistry: An Integrated Solution

To address this, the concept of White Analytical Chemistry was proposed. WAC demands a balance across three equally important pillars, represented by the RGB color model [3]:

  • Red Principles: Represent analytical performance, including the scope of application, limits of detection and quantification, precision, and accuracy.
  • Green Principles: Represent the environmental impact, as assessed by tools like AGREEprep, covering toxicity, waste, and energy consumption.
  • Blue Principles: Represent practical & economic factors, including cost-efficiency, method simplicity, and throughput [3].

A method is considered "white" when it achieves high scores in all three areas, demonstrating a harmonious balance. The following table summarizes the core principles of these complementary frameworks.

Table 1: Core Principles of AGREEprep and White Analytical Chemistry (WAC)

Framework Core Principle Focus Area Key Metrics
AGREEprep Green Sample Preparation Environmental Impact 10 criteria including solvent toxicity, waste generation, energy use, and operator safety [3].
White Analytical Chemistry (WAC) Balanced Method Performance Holistic Quality 12 principles divided into three pillars [3]:
Red (Analytical) Performance Scope, LOD/LOQ, Precision, Accuracy [3].
Green (Environmental) Sustainability Toxicity, Waste, Energy, Direct Impacts [3].
Blue (Practical/Economic) Usability Cost-efficiency, Instrumentation, Method Simplicity & Time [3].

Case Study: Microextraction Techniques in Bioanalysis

Microextraction techniques are a prime example of where a holistic assessment is critical. While they are generally greener than traditional extraction methods, their performance and practicality vary significantly.

Comparative Greenness and Performance of SPME Formats

A 2024 study directly compared different Solid-Phase Microextraction formats, revealing clear trade-offs. While Thin-Film SPME demonstrated superior extraction efficiency for a wide range of odorants compared to traditional SPME fibers and Stir Bar Sorptive Extraction, this performance comes from a larger sorption area and advanced coatings [59]. From a green perspective, a larger device might consume more material, but its superior efficiency could lead to better sensitivity, reducing the need for repeated analyses and thus balancing the environmental score.

Table 2: Comparison of Solid-Phase Microextraction Formats [59]

SPME Format Key Feature Extraction Efficiency Advantages Limitations
SPME Fiber Fused silica fiber with thin coating Moderate (varies by coating) Versatile, fully automatable [59]. Limited sorbent volume, lower sensitivity for some analytes [59].
Stir Bar Sorptive Extraction Thick PDMS layer on a magnetic bar High for non-polar compounds High capacity for apolar analytes [60]. Poor recovery of polar analytes, long equilibration times [59].
Thin-Film SPME Carbon mesh support with sorbent layer Highest (for tested odorants) Large surface area, high sensitivity, efficient for polar compounds [59]. -

Holistic Assessment of a Green Method: Single-Drop Microextraction

A 2025 study developed a method for nitro compounds in water samples using Direct Immersion-Single-Drop Microextraction followed by GC-ECD. The method was explicitly evaluated for its greenness using AGREEprep and other tools, demonstrating its environmental credentials [9]. However, the authors also rigorously validated its analytical performance, which is crucial for a holistic view:

  • Limits of Detection: Ranged from 0.01 to 0.11 μg/L across different water matrices, proving high sensitivity [9].
  • Solvent Consumption: Used only 1 μL of toluene per extraction, minimizing waste [9].
  • Practical Application: Successfully determined nitro compounds in real environmental and forensic rinse water, demonstrating practical robustness [9].

This method exemplifies the WAC ideal: it pairs an excellent green profile with validated analytical performance and demonstrated practical applicability.

Experimental Protocol: A Holistic Assessment Workflow

This protocol provides a step-by-step guide for holistically evaluating a microextraction method, integrating AGREEprep with White Analytical Chemistry principles.

Phase 1: Analytical Method Implementation

Procedure:

  • Method Selection: Choose a microextraction technique (e.g., DI-SDME, TF-SPME) suitable for your target analytes [9] [59].
  • Parameter Optimization: Systematically optimize key parameters (e.g., extraction solvent, time, temperature, pH, ionic strength) to maximize analyte recovery [9].
  • Analysis: Perform the extraction and analyze the samples using your chosen chromatographic system (e.g., GC-ECD, GC-MS) [9].

Phase 2: Holistic Method Evaluation

1. Performance (Red Principles) Assessment: - Determine the Limit of Detection and Limit of Quantification. - Calculate precision as repeatability (intra-day) and reproducibility (inter-day), expressed as %RSD. - Establish accuracy through recovery studies (%)

and analysis of certified reference materials [3]. - Define the calibration range and linearity [9].

2. Greenness (Green Principles) Assessment: - Input the parameters from the optimized method (e.g., solvent type and volume, waste produced, energy consumption, operator safety measures) into the AGREEprep software. - Record the final score and the pictogram provided by the tool [3].

3. Practicality (Blue Principles) Assessment: - Estimate the cost per analysis (consumables, instrumentation). - Evaluate the throughput (sample preparation and analysis time). - Assess method simplicity and the need for specialized equipment or training [3].

4. Data Integration and "Whiteness" Scoring: - Use a scoring rubric (e.g., 0-1 points for each of the 12 WAC principles) to translate the results from steps 1-3 into a unified WAC score [3]. - Visualize the outcome using a three-color radar chart (Red, Green, Blue) to instantly reveal the method's balance and overall "whiteness".

The following workflow diagram illustrates this integrated assessment process.

G Start Start Method Assessment Phase1 Phase 1: Implement & Optimize Analytical Method Start->Phase1 Phase2 Phase 2: Holistic Evaluation Phase1->Phase2 Sub_Red Red Principles: Analytical Performance Phase2->Sub_Red Sub_Green Green Principles: Environmental Impact Phase2->Sub_Green Sub_Blue Blue Principles: Practicality Phase2->Sub_Blue R1 LOD/LOQ Sub_Red->R1 R2 Precision (%RSD) Sub_Red->R2 R3 Accuracy (Recovery) Sub_Red->R3 R4 Linearity & Scope Sub_Red->R4 Integrate Integrate Scores into White Analytical Chemistry (WAC) Framework Sub_Red->Integrate G1 AGREEprep Tool Sub_Green->G1 Sub_Green->Integrate B1 Cost per Analysis Sub_Blue->B1 B2 Throughput & Time Sub_Blue->B2 B3 Simplicity Sub_Blue->B3 Sub_Blue->Integrate End Holistically Assessed Method Integrate->End

Diagram 1: Holistic Method Assessment Workflow

The Scientist's Toolkit: Essential Reagents and Materials

Table 3: Key Research Reagent Solutions for Microextraction Methods

Item Function/Application Example in Context
HLB/PDMS Thin-Film A sorbent for Thin-Film SPME; provides high efficiency for a broad range of analytes, including polar compounds [59]. Used for the non-targeted analysis of odorants in complex food samples like beer [59].
Toluene An organic solvent immiscible with water, used as an extraction phase in liquid-phase microextraction [9]. Served as a stable microdroplet solvent for the Direct Immersion-SDME of nitro compounds [9].
n-Butyl Acetate A potential green solvent candidate for microextraction, immiscible with water [9]. Tested for SDME, though it showed significant droplet volume loss in one study [9].
AGREEprep Software A free, user-friendly metric tool that calculates a greenness score for sample preparation methods based on 10 principles [3]. Used to generate an easy-to-read pictogram score for the greenness of an SDME method [9].
Gas Chromatograph with Electron Capture Detector An analytical instrument highly selective for analytes containing nitro groups or halogens [9]. Used for the separation and sensitive detection of nitroaromatic compounds after SDME [9].

AGREEprep is a fundamental tool for quantifying the environmental footprint of analytical methods. However, this application note demonstrates that it should be the starting point for evaluation, not the endpoint. For methods used in critical decision-making contexts—especially in drug development and therapeutic monitoring—a holistic standard is required. By integrating the red of analytical performance, the green of sustainability, and the blue of practical feasibility, researchers can develop methods that are not only kinder to the planet but also robust, reliable, and truly fit-for-purpose. Adopting the White Analytical Chemistry framework ensures that the pursuit of greenness does not come at the cost of scientific integrity or patient health.

The development of modern analytical methods necessitates a balanced consideration of environmental impact and practical applicability. Green Analytical Chemistry (GAC) principles have driven the reduction of hazardous waste and energy consumption in laboratories [16]. However, a method's sustainability is incomplete without assessing its practicality for routine use. White Analytical Chemistry (WAC) addresses this by integrating three dimensions: green (environmental impact), red (analytical performance), and blue (practicality and economic efficiency) [3] [61]. This framework ensures methods are not only environmentally sound but also analytically robust and practically viable.

Within the WAC framework, two specialized metric tools have emerged for focused assessment. AGREEprep (Analytical Greenness Metric for Sample Preparation) is dedicated to evaluating the environmental impact of the sample preparation step, often the most resource-intensive part of the analytical workflow [17]. Complementary to this, the Blue Applicability Grade Index (BAGI) assesses the practicality of the entire analytical method, including factors like cost, time, and operational simplicity [62]. This application note details the synergistic use of AGREEprep and BAGI for a holistic sustainability and applicability assessment of microextraction techniques, providing detailed protocols for researchers and scientists.

Theoretical Foundation of AGREEprep and BAGI

AGREEprep: The Greenness Metric for Sample Preparation

AGREEprep is a software-based metric tool designed to evaluate the greenness of sample preparation methods. It is based on the 10 principles of Green Sample Preparation (GSP), which include minimizing waste, using safer solvents, and maximizing energy efficiency [17]. The tool generates a score between 0 and 1, providing a quantitative measure of environmental impact. Its output is an intuitive pictogram where each section corresponds to one of the ten GSP principles, offering an at-a-glance view of a method's green strengths and weaknesses [3] [17]. AGREEprep is particularly valuable because it allows for the assignment of different weights to each criterion, enabling users to tailor the assessment to their specific environmental priorities [3].

BAGI: The Blue Applicability Grade Index

BAGI is a metric tool developed to evaluate the practicality and economic efficiency of an analytical method, representing the "blue" component of WAC [62]. It assesses ten key attributes related to the method's practical aspects, such as:

  • Type of analysis (e.g., quantitative vs. qualitative)
  • Sample throughput
  • Degree of automation
  • Availability of reagents and materials
  • Cost-efficiency and operational simplicity [61] [62]

Each criterion is scored, and the cumulative result is presented as a numerical score (with a benchmark of >60.0 indicating a genuinely practical method) and a colored asteroid-shaped pictogram [62]. This visual output immediately highlights a method's strong and weak points from a practical standpoint.

The Workflow for a Combined WAC Assessment

The following diagram illustrates the logical workflow for conducting a synergistic assessment using AGREEprep and BAGI, leading to a comprehensive White Analytical Chemistry (WAC) profile.

workflow Start Start: Developed Analytical Method AGREEprep AGREEprep Assessment Start->AGREEprep BAGI BAGI Assessment Start->BAGI Integrate Integrate Results AGREEprep->Integrate BAGI->Integrate WAC Holistic WAC Profile Integrate->WAC

Protocols for Assessment

Protocol 1: AGREEprep Assessment of a Microextraction Technique

This protocol provides a step-by-step guide for evaluating the greenness of a sample preparation method using the AGREEprep metric.

Objective: To perform a quantitative and qualitative greenness assessment of a sample preparation method based on the 10 principles of Green Sample Preparation. Materials and Software:

  • AGREEprep software (free, open-source)
  • Detailed description of the sample preparation method

Procedure:

  • Data Collection: Gather all relevant data for the sample preparation method. Essential parameters include:
    • Sample and Reagent Amounts: Mass or volume of all samples, solvents, and reagents used.
    • Waste Generation: Total mass of waste produced per sample, including all consumables.
    • Energy Consumption: Device power (kW) and operational time, used to calculate total kWh per sample.
    • Operator Safety: Document any hazards (e.g., toxic reagents, high pressure/temperature) and safety measures.
    • Procedure Details: Note the number of steps, potential for automation, sample throughput per hour, and whether the procedure is performed in-situ, on-site, or ex-situ.

  • Software Input:

    • Launch the AGREEprep software.
    • Input the collected data into the corresponding fields for the ten criteria. The software interface is designed to guide this process.
    • Assign weights to each of the ten criteria if specific environmental priorities exist. If not, use the default weighting.

  • Result Interpretation:

    • The software will generate an overall score between 0 and 1 and a circular pictogram divided into 10 sections.
    • Overall Score: A score closer to 1 indicates a greener sample preparation process.
    • Pictogram Analysis: Each segment of the pictogram corresponds to one of the 10 GSP principles. A darker green color in a segment indicates better performance for that specific principle. Analyze the pictogram to identify areas with the largest environmental impact (lighter segments) for potential improvement.

Protocol 2: BAGI Assessment of an Analytical Method

This protocol outlines the procedure for evaluating the practicality of a full analytical method using the BAGI metric.

Objective: To obtain a numerical score and visual pictogram representing the practicality ("blueness") of an analytical method. Materials and Software:

  • BAGI software (free, open-source web or desktop application)
  • Detailed description of the entire analytical method, from sample preparation to detection.

Procedure:

  • Data Collection: Compile information across the ten BAGI attributes [62] [63]:
    • Attribute 1: Type of analysis (e.g., qualitative, quantitative, confirmatory).
    • Attribute 2: Number and class of analytes determined simultaneously.
    • Attribute 3: Analytical technique and instrumentation required.
    • Attribute 4: Number of samples that can be prepared simultaneously.
    • Attribute 5: Type of sample preparation (e.g., none, on-site, microextraction).
    • Attribute 6: Sample throughput (samples per hour).
    • Attribute 7: Commercial availability and cost of reagents and materials.
    • Attribute 8: Need for a preconcentration step.
    • Attribute 9: Degree of automation in sample prep and analysis.
    • Attribute 10: Amount of sample required.

  • Software Input:

    • Access the BAGI web application at bagi-index.anvil.app or use the desktop version.
    • For each of the ten criteria, select the option that best describes your method from the drop-down menus.

  • Result Interpretation:

    • The software generates a final score and an asteroid-shaped pictogram with ten sections.
    • Final Score: A score above 60.0 indicates a method with good practicality. The maximum score is 100.0 [62].
    • Pictogram Analysis: Each section of the asteroid corresponds to one criterion. Colors range from dark blue (high practicality, 10 points) to white (low practicality, 2.5 points). This visual output instantly reveals the method's practical strengths and weaknesses.

Case Studies and Data Presentation

Case Study: PFAS Determination in Food and Water

A 2025 review evaluated 34 microextraction methods for determining per- and polyfluoroalkyl substances (PFAS) using BAGI. The scores ranged from 50.0 to 77.5, indicating significant room for improving practicality despite the existence of relatively robust methods [64]. The best-performing methods were further assessed with AGREEprep to provide a comprehensive WAC profile, demonstrating the synergistic application of both tools.

Case Study: Lab-in-Syringe Automated Microextraction

A 2024 study evaluated 32 different Lab-in-Syringe (LIS) automated microextraction systems using BAGI [63]. These systems, which perform sample preparation inside a syringe barrel, are designed for high practicality. The evaluation resulted in BAGI scores all above 60.0, confirming their excellent practicality. High scores were driven by attributes such as a high degree of automation, good sample throughput, and the use of commonly available reagents.

Comparative Data Table

The following table summarizes quantitative data from the assessment of various microextraction techniques, illustrating the relationship between their greenness and practicality scores.

Table 1: Greenness and Practicality Scores of Selected Microextraction Techniques

Analytical Technique Application Area AGREEprep Score (Greenness) BAGI Score (Practicality) Key Strengths
Lab-in-Syringe (LIS) Systems [63] Various automated microextractions Data Not Provided > 60.0 (Range: 62.5 - 87.5) High automation, good throughput
SULLME Method [16] Antiviral compounds 0.56 (via AGREE) Data Not Provided Use of green solvents, miniaturization
Novel PFAS Methods [64] Food and Water Assessed for top performers 50.0 - 77.5 Multi-analyte determination
SDME-GC-ECD [9] Nitro compounds in water Assessed via AGREE/AGREEprep Data Not Provided Minimal solvent use

Essential Research Reagent Solutions

The following table lists key materials and reagents commonly used in the development of green and practical microextraction methods, along with their functions.

Table 2: Key Reagents and Materials in Green Microextraction

Reagent / Material Function in Microextraction Green & Practical Consideration
Deep Eutectic Solvents (DES) [9] Extraction solvent Lower toxicity and biodegradability vs. traditional organic solvents.
Switchable Hydrophilicity Solvents (SHS) [9] Extraction solvent Ability to switch phases allows for recycling and reduces waste.
Magnetite-functionalized Sorbents [63] Solid-phase extraction material Enables easy retrieval from sample, simplifying automation and saving time.
Novel SPME Fibers [61] Solid-phase microextraction High selectivity and reusability, though some specialized fibers can impact BAGI cost/reagent availability score.
Toluene / n-Octanol [9] Conventional extraction solvent Effective but with higher toxicity and environmental impact; targets for replacement with greener solvents.

The synergistic use of AGREEprep and BAGI provides a robust framework for the holistic evaluation of analytical methods. This approach moves beyond a singular focus on greenness to a balanced assessment that includes practical applicability, which is crucial for the adoption of methods in routine analysis and industry.

For effective implementation:

  • Adopt Early: Integrate these tools during the method development phase, not as an afterthought, to guide choices toward sustainable and practical outcomes.
  • Iterate and Improve: Use the pictograms to identify weaknesses and conduct iterative improvements. For example, if AGREEprep shows a poor score for waste generation, strategies for waste reduction or treatment should be explored. If BAGI indicates low practicality due to low throughput, automation should be considered.
  • Standardize Reporting: Including AGREEprep and BAGI pictograms and scores in scientific publications and method documentation offers a standardized, transparent way to communicate the overall value of an analytical method to the scientific community.

This combined assessment strategy empowers scientists to develop methods that are not only kinder to the environment but also efficient, cost-effective, and ready for real-world application.

White Analytical Chemistry (WAC) represents an evolution in analytical method assessment, conceived to address the critical need for a balanced compromise between environmental sustainability, analytical performance, and practical/economic feasibility [3]. The core principle of WAC is that a truly excellent method must excel simultaneously in all three domains; superior performance in one area cannot compensate for deficiencies in another. This holistic framework was introduced to mitigate the risk that efforts to improve a method's greenness might inadvertently compromise its analytical reliability or practical implementation, a consideration of paramount importance in fields like therapeutic drug monitoring (TDM) where result accuracy is critical [3].

The WAC concept is operationalized through the RGB 12 algorithm, which distributes twelve principles into three primary color categories [3]. The Red (R) principles are dedicated to Analytical Performance, encompassing the scope of application, limits of detection and quantification (LOD/LOQ), precision, and accuracy. The Green (G) principles embody Green Chemistry criteria, focusing on reagent toxicity, waste generation, energy consumption, and direct environmental/human impacts. The Blue (B) principles address Practical and Economic considerations, including cost-efficiency, method throughput, operational simplicity, and operator skill requirements. The "whiteness" of a method is a function of how harmoniously it satisfies these twelve principles, much like combining red, green, and blue light produces white light [3].

For the specific evaluation of the sample preparation stage—often the least green step in an analytical procedure—the AGREEprep metric serves as a powerful, specialized tool [17]. AGREEprep is an open-source software that quantitatively assesses sample preparation methods against the ten core principles of green sample preparation (GSP) [3]. It generates a score between 0 and 1 for each principle, culminating in an overall pictogram and final score that reflects the environmental friendliness of the sample preparation work-up. The ten principles of GSP assessed by AGREEprep include favoring in-situ preparation, using safer solvents, minimizing waste and energy consumption, and ensuring operator safety, among others [3]. By integrating the detailed, sample-focused output of AGREEprep into the broader, tripartite WAC framework, a researcher can construct a comprehensive and defensible "whiteness" profile for their analytical method.

Integrated WAC Assessment Protocol: AGREEprep and RGB 12

This protocol provides a step-by-step guide for conducting a holistic whiteness assessment of a microextraction method, integrating the greenness evaluation from AGREEprep into the full White Analytical Chemistry profile.

Stage 1: Data Collection for AGREEprep

  • Step 1.1: Compile all quantitative data related to the sample preparation procedure. Essential parameters include the type and volume of all solvents and reagents used, the mass of any sorbents or specialized materials, the number of samples processed per batch, and the total preparation time [17] [3].
  • Step 1.2: Document the energy consumption for the sample preparation step. Where feasible, use direct measurement with a wattmeter; if not possible, calculate based on instrument power ratings and operational duration [65].
  • Step 1.3: Classify all chemicals used according to their safety and environmental hazard profiles (e.g., using GHS or NFPA codes). Note any use of renewable or reusable materials [3].
  • Step 1.4: Record the total volume of waste generated, including all spent solvents, cleaning solutions, and consumables.

Stage 2: AGREEprep Analysis

  • Step 2.1: Input the collected data into the AGREEprep software, which is freely available online.
  • Step 2.2: For each of the ten assessment criteria, the software will calculate a sub-score. The user can assign different weights to each criterion based on their analytical goals, though the default weights are recommended for initial assessment [17] [3].
  • Step 2.3: The software outputs a circular pictogram with ten sections, each colored from white (score of 1) to dark green (score of 0), and a final overall score in the center. This output provides a direct, visual representation of the method's greenness during sample preparation [17].

Stage 3: RGB 12 Whiteness Assessment

  • Step 3.1: Evaluate Green (G) Principles. Use the overall score and sub-scores from the AGREEprep analysis to inform the scoring of the four Green (G) principles in the WAC RGB 12 algorithm [3]. These principles are:
    • G1. Toxicity of reagents: Score based on the safety and renewability of solvents/reagents used.
    • G2. Number and amount of reagents and waste: Score based on the miniaturization and efficiency of the procedure.
    • G3. Energy and other media: Score based on energy consumption and the level of automation.
    • G4. Direct impacts: Score based on the method's impact on humans, animals, and genetic naturalness.
  • Step 3.2: Evaluate Red (R) Principles. Score the method's analytical performance based on validation data [3]:
    • R1. Scope of application: Assess the linearity range, number of analytes, and sample type compatibility.
    • R2. LOD and LOQ: Evaluate the sensitivity of the method.
    • R3. Precision: Determine the repeatability and reproducibility.
    • R4. Accuracy: Assess via recovery studies and determination of relative error.
  • Step 3.3: Evaluate Blue (B) Principles. Score the method's practical and economic aspects [3]:
    • B1. Cost-efficiency: Calculate the cost per analysis.
    • B2. Time of analysis: Measure the sample throughput and total analysis time.
    • B3. Operational simplicity: Evaluate the number of procedural steps and need for operator intervention.
    • B4. Skills and knowledge required: Assess the level of training needed to perform the analysis.
  • Step 3.4: Calculate the Whiteness Score. Each of the twelve principles is scored, typically on a 0-4 point scale. The scores are then aggregated according to the RGB 12 algorithm to produce a final whiteness score and a visual RGB graph, representing the balance between the three pillars [3].

Stage 4: Interpretation and Comparison

  • Step 4.1: A method with a high whiteness score and a balanced RGB graph is considered ideal, indicating it is environmentally friendly, analytically powerful, and practically applicable.
  • Step 4.2: Compare the whiteness profile of the new method against existing standard methods for the same analysis to highlight relative advantages and disadvantages.

The following workflow diagram illustrates the integrated assessment process.

WAC_Assessment Start Start WAC Assessment Stage1 Stage 1: Data Collection for AGREEprep Start->Stage1 Stage2 Stage 2: AGREEprep Analysis Stage1->Stage2 AGREEprep_Output AGREEprep Pictogram & Score Stage2->AGREEprep_Output Stage3 Stage 3: RGB 12 Whiteness Assessment AGREEprep_Output->Stage3 G_Principles Score Green (G) Principles Stage3->G_Principles R_Principles Score Red (R) Principles Stage3->R_Principles B_Principles Score Blue (B) Principles Stage3->B_Principles Calc_Score Calculate Final Whiteness Score G_Principles->Calc_Score R_Principles->Calc_Score B_Principles->Calc_Score WAC_Profile Final WAC Profile Calc_Score->WAC_Profile Stage4 Stage 4: Interpretation & Comparison WAC_Profile->Stage4

Application Example: Menthol-Based Microextraction for β-Blockers in Urine

To illustrate the integrated WAC assessment, we evaluate a published biosolvent-based liquid-liquid microextraction (LLME) method for isolating propranolol and carvedilol from human urine [66].

Experimental Protocol

  • Sample Preparation: A 250 μL aliquot of human urine is placed in a microcentrifuge tube. Then, 150 μL of NaCl solution (30% w/w), 50 μL of internal standard solution, and 50 μL of analyte mixture are added [66].
  • Microextraction: 65 μL of molten menthol (bio-solvent) is added as the extraction solvent. The mixture is vortexed for 10 seconds and sonicated for 30 seconds to form a fine dispersion [66].
  • Phase Separation: The sample is centrifuged at 10,000 rpm for 2 minutes. The tube is immediately transferred to an ice bath to solidify the menthol phase. The aqueous supernatant is removed and discarded with a syringe [66].
  • Analysis: The solidified menthol extract is dissolved in 500 μL of methanol, and the resulting solution is transferred to an HPLC vial for chromatographic analysis [66].

Essential Research Reagents and Materials

Table 1: Key Reagent Solutions and Materials for Menthol-Based LLME

Item Function / Role in the Protocol
Menthol Serves as the bio-solvent (extraction medium). It is a naturally sourced, low-toxicity, and biodegradable alternative to conventional organic solvents [66].
NaCl Solution Used to adjust the ionic strength of the sample solution, which can enhance extraction efficiency by salting-out effects.
Methanol (HPLC-grade) Used to dissolve the solidified menthol extract prior to HPLC injection, making it compatible with the chromatographic system.
Propranolol & Carvedilol Standards Target β-blocker analytes for quantification.
Internal Standard (e.g., Ethyl Paraben) Used to correct for variations in extraction efficiency and instrument response, improving method precision and accuracy.

Quantitative Assessment Data

The following table summarizes the key performance and greenness data for the method, which serves as the basis for the WAC scoring.

Table 2: Quantitative Data for the Menthol-Based LLME Method [66]

Parameter Result / Value
Analytical Performance (Red)
Linear Range 50 - 2000 ng mL⁻¹
LOD (Propranolol) 11 ng mL⁻¹
LOD (Carvedilol) 17 ng mL⁻¹
Intra-day Precision (RSD%) < 11%
Accuracy (% Recovery) 87.2% - 110.2%
Greenness & Practicality
Sample Volume 250 μL
Extraction Solvent Volume 65 μL (Menthol)
Solvent Disperser Sonication (30 sec)
Total Preparation Time ~10-15 minutes

AGREEprep and WAC Scoring Analysis

Based on the data in Table 2, the method can be scored. The use of menthol, a biodegradable and safe bio-solvent, contributes to high scores in G1 (Toxicity) and G4 (Direct impacts). The very low volumes of solvents and samples lead to a high score in G2 (Reagents and waste). The minimal energy consumption for sonication and centrifugation supports a strong score for G3 (Energy). The AGREEprep tool would reflect this, likely yielding a high overall greenness score [66].

The satisfactory analytical figures of merit (linearity, LOD, precision, accuracy) support solid scores for all four Red (R) Principles. From a practical standpoint (Blue Principles), the method is cost-efficient due to low solvent consumption, relatively fast, and operationally simple, leading to high scores in B1 (Cost), B2 (Time), and B3 (Simplicity).

The final whiteness score, derived from the aggregation of all twelve principle scores, would be high, indicating a well-balanced and sustainable analytical method suitable for routine application in bioanalysis [66]. This demonstrates the power of the combined AGREEprep and WAC assessment to validate the holistic quality of a microextraction technique.

The Scientist's Toolkit for WAC Implementation

Successfully implementing a WAC-focused method development strategy requires a set of key tools and conceptual frameworks. Adherence to Good Evaluation Practice (GEP) rules is essential to ensure assessments are transparent, reliable, and meaningful [65].

Table 3: Essential Toolkit for WAC-Compliant Method Development and Assessment

Tool / Concept Function and Role
AGREEprep Software The primary tool for quantifying the greenness of the sample preparation step. It provides a user-friendly interface and generates an easily interpretable pictogram [17] [3].
RGB 12 Algorithm The scoring framework for the Whiteness assessment. It ensures a balanced evaluation across the 12 principles of Greenness, Analytical Performance, and Practicality [3].
Good Evaluation Practice (GEP) Rules A set of five general rules to guide the evaluation process, promoting the use of quantitative data, transparency, and critical interpretation to avoid misuse of metrics [65].
Multivariate Optimization (e.g., PBD, CCD) Statistical design-of-experiment approaches used during method development to efficiently identify optimal conditions while minimizing experimental runs, resources, and time, aligning with GAC principles [66].
Bio-Solvents (e.g., Menthol) Sustainable, often naturally derived solvents that reduce toxicity and environmental impact, directly improving scores in G1 and G4 principles [66].

The following diagram outlines the strategic path from initial method conception to a validated white method, integrating the components from the toolkit.

WAC_Strategy Conception Method Conception GEP Apply GEP Rules Conception->GEP Design Design Method using Bio-Solvents & Microtechniques GEP->Design Optimize Multivariate Optimization Design->Optimize Validate Analytical Validation Optimize->Validate Assess Integrated WAC Assessment Validate->Assess White_Method Validated White Method Assess->White_Method

The principles of Green Analytical Chemistry (GAC) have driven the development of microextraction techniques, which aim to minimize the environmental impact of chemical analysis [3]. To quantitatively assess this impact, the Analytical Greenness Metric for Sample Preparation (AGREEprep) was introduced in 2022 as a dedicated tool for evaluating sample preparation methods [3] [7]. This open-source software calculates a score between 0 and 1 based on ten principles of green sample preparation, with a score above 0.5 generally indicating an acceptably green method [7]. AGREEprep provides an easily interpretable pictogram, offering researchers a quick visual assessment of a method's environmental performance [7].

This application note provides a comparative analysis of AGREEprep scores across major microextraction categories, offering structured protocols and data visualization to guide researchers in selecting and developing environmentally sustainable sample preparation methods.

AGREEprep Assessment Criteria

The AGREEprep metric tool evaluates sample preparation methods against ten core principles [3]:

  • Favoring in situ sample preparation
  • Using safer solvents and reagents
  • Targeting sustainable, reusable, and renewable materials
  • Minimizing waste generation
  • Minimizing sample, chemical, and material amounts
  • Maximizing sample throughput
  • Integrating steps and promoting automation
  • Minimizing energy consumption
  • Choosing the greenest possible post-sample preparation configuration for analysis
  • Ensuring safe procedures for the operator

Each criterion is scored and weighted, contributing to the final overall score presented in the center of the characteristic circular pictogram [3] [7].

Comparative AGREEprep Scores for Microextraction Techniques

The following table summarizes the typical AGREEprep scores and key characteristics for major microextraction technique categories, as reported in recent literature for applications such as environmental, cosmetic, and bioanalysis [35] [3] [7].

Table 1: AGREEprep Score Comparison Across Microextraction Categories

Microextraction Category Specific Techniques Evaluated Typical AGREEprep Score Range Key Greenness Advantages Common Applications Cited
Liquid-Phase Microextraction (LPME) Dispersive Liquid-Liquid Microextraction (DLLME), Hollow-Fiber LPME (HF-LPME) [3] [7] 0.64 - 0.75 [7] Very low solvent consumption, high enrichment factors, minimal waste [67] UV filters in cosmetics [7], organic pollutants in water [67]
Solid-Phase Microextraction (SPME) Fiber-SPME, in-tube SPME [35] [3] 0.55 - 0.70 [35] Solvent-free operation, reusability of fibers [35] PFAS in food/water [35], volatile compounds in solids [67]
Magnetic Solid-Phase Extraction (MSPE) Magnetic d-SPE [35] [3] 0.60 - 0.68 [35] Efficient phase separation without centrifugation, potential sorbent reusability [35] PFAS analysis [35], therapeutic drug monitoring [3]
Dispersive Solid-Phase Extraction (d-SPE) Pipette-tip SPE, µ-SPE [35] 0.58 - 0.65 [35] Reduced solvent volumes vs. conventional SPE, faster procedures [35] Pre-concentration of analytes from complex matrices [35]
Stir Bar Sorptive Extraction (SBSE) SBSE with various sorbent coatings [35] ~0.62 [35] High sorbent loading capacity, solventless desorption possible [35] Extraction of organic contaminants from water [35]

Detailed Experimental Protocols

Protocol 1: AGREEprep Evaluation of a DLLME Method for UV Filters in Cosmetics

This protocol is adapted from methods evaluated in the literature for determining UV filters in cosmetic samples, which achieved high AGREEprep scores [7].

  • Reagents and Materials: Methanol (HPLC grade), acetone, ultrapure water, cosmetic sample (e.g., sunscreen), standard solutions of target UV filters (e.g., avobenzone, octinoxate), centrifuge tubes, syringe filters, HPLC system with UV detector.
  • Procedure:
    • Sample Preparation: Accurately weigh approximately 0.1 g of the cosmetic sample into a 10 mL centrifuge tube.
    • Dispersion: Add 1 mL of methanol and vortex for 1 minute to dissolve/disperse the sample.
    • Microextraction: Rapidly inject a mixture containing 1.0 mL of acetone (disperser solvent) and 150 µL of chloroform (extraction solvent) into the sample solution using a syringe. A cloudy solution forms immediately.
    • Phase Separation: Centrifuge the mixture at 4000 rpm for 5 minutes to sediment the dense organic phase at the bottom of the tube.
    • Analysis: Carefully remove the aqueous layer with a syringe. Evaporate the organic phase to dryness under a gentle nitrogen stream and reconstitute the residue with 100 µL of methanol. Filter the extract and analyze via HPLC-UV.
  • AGREEprep Input Justification: This method scores highly due to the minimal sample amount (Principle 5), very low volumes of solvents used, and consequently minimal waste generated (Principle 4). The procedure is straightforward with a high sample throughput (Principle 6) [7].

Protocol 2: AGREEprep Evaluation of an SPME Method for PFAS in Water

This protocol is based on SPME methods reviewed for the determination of per- and polyfluoroalkyl substances (PFAS) in water matrices [35].

  • Reagents and Materials: Commercially available SPME fiber (e.g., C18 or WAX coated), water samples, standard solutions of target PFAS, HPLC vials, LC-MS/MS system.
  • Procedure:
    • Sample Collection: Collect a 10 mL water sample in a vial containing a magnetic stir bar.
    • Extraction: Place the vial on a stir plate. Expose and immerse the SPME fiber into the sample. Extract for 20-30 minutes with constant stirring at a fixed speed.
    • Desorption: After extraction, retract the fiber and immediately transfer it to the LC-MS/MS injection port for thermal or solvent desorption (as per fiber compatibility), directly introducing the analytes into the analytical instrument.
    • Analysis: Perform the chromatographic separation and mass spectrometric detection of the target PFAS.
  • AGREEprep Input Justification: The SPME technique is renowned for its green credentials, as it is virtually solvent-free (Principle 2). The fiber is reusable for multiple extractions (Principle 3), and the procedure can be easily automated (Principle 7), leading to a high overall greenness score [35].

Protocol 3: AGREEprep Evaluation of an MSPE Method for Therapeutic Drug Monitoring

This protocol reflects magnetic solid-phase extraction methods used in bioanalysis, such as therapeutic drug monitoring (TDM) [3].

  • Reagents and Materials: Functionalized magnetic nanoparticles (e.g., C18-coated), plasma or serum sample, methanol, acetonitrile, external magnet, vortex mixer, HPLC system.
  • Procedure:
    • Sorbent Conditioning: Disperse a suitable amount (e.g., 10 mg) of magnetic sorbent in 1 mL of methanol in a vial, then separate and wash with water.
    • Sample Loading: Add the pretreated biological sample (e.g., 500 µL of plasma) to the vial containing the dispersed sorbent. Vortex for 2-3 minutes to allow analyte adsorption.
    • Magnetic Separation: Place the vial on an external magnet to separate the sorbent from the sample matrix. Decant and discard the supernatant.
    • Washing and Elution: Wash the sorbent with 1 mL of a mild solvent (e.g., 5% methanol in water). Remove the washing solvent. Elute the target analytes by adding 200 µL of a stronger solvent (e.g., pure acetonitrile) and vortexing. Separate the eluent using the magnet and collect it for analysis.
    • Analysis: Inject the eluent into the HPLC system for quantification.
  • AGREEprep Input Justification: MSPE scores well due to the efficient separation without energy-intensive centrifugation (Principle 8), the reusability of the magnetic sorbent (Principle 3), and the small volumes of samples and eluents used (Principle 5) [3].

Workflow Diagram for AGREEprep-Assisted Method Selection

The following diagram illustrates the decision-making workflow for selecting a microextraction technique based on analytical requirements and AGREEprep assessment, leading to a whiter analytical method.

G Start Define Analytical Need Need1 Sample Type? Aqueous / Biological / Solid Start->Need1 Need2 Target Analyte Properties? Polarity / Volatility Need1->Need2 Need3 Key Requirement? Sensitivity / Speed / Cost Need2->Need3 Select Select Candidate Microextraction Technique(s) Need3->Select Evaluate Perform AGREEprep Assessment Select->Evaluate Score Score > 0.5? Evaluate->Score Score->Select No Validate Validate Method Performance Score->Validate Yes WAC Achieve 'White' Method: Balance of Greenness, Performance & Practicality Validate->WAC

Diagram 1: Workflow for green method selection.

The Scientist's Toolkit: Essential Reagents and Materials

Table 2: Key Research Reagent Solutions for Microextraction

Item Name Function / Application Greenness & Practical Considerations
Functionalized Magnetic Nanoparticles Core material for MSPE; surface can be modified with C18, graphene, etc., for specific analyte retention [35] [3]. Enables rapid, centrifugation-free separation; potential for reusability aligns with green principles [3].
SPME Fibers Coated fibers for solvent-free extraction of volatiles and semi-volatiles via direct immersion or headspace sampling [35] [67]. Eliminates solvent use; commercially available and reusable, though fiber cost and fragility are practical considerations [35].
Dispersive Solvents (e.g., Acetone, Methanol) Used in DLLME to disperse the extraction solvent as fine droplets in the aqueous sample, creating a large surface area for extraction [7]. Required in small volumes, but their toxicity is a greenness drawback. Safer alternatives should be explored where possible [3].
Low-Density & Low-Toxicity Extraction Solvents Solvents like dodecane or ethyl acetate used in DLLME or HF-LPME [7]. Safer profiles compared to chlorinated solvents; reduces hazard and waste toxicity, improving AGREEprep scores [3] [7].
Fabric Phase Sorptive Extraction (FPSE) Membranes A sorbent phase coated on a flexible, permeable fabric substrate for high-efficiency extraction [3]. Combines the high capacity of SPE with the flexibility and kinetics of SPME; membranes are reusable and require minimal solvent for desorption [3].

The evolution of sample preparation has been marked by a significant shift from traditional, resource-intensive techniques toward innovative, miniaturized approaches. Conventional methods like solid-phase extraction (SPE) and liquid-liquid extraction (LLE) have long been foundational in analytical workflows but are characterized by high consumption of organic solvents, substantial waste generation, and multiple manual steps [35] [42]. In response, novel microextraction techniques have emerged, aligning with the principles of Green Analytical Chemistry (GAC) by dramatically reducing solvent usage, minimizing waste, and integrating extraction steps [42] [55].

This application note provides a systematic benchmarking of these novel microextraction methods against established traditional protocols and standardized procedures. Framed within a broader thesis research on the AGREEprep assessment tool, we utilize standardized greenness and practicality metrics to deliver a quantitative comparison. The evaluation is critical for researchers and drug development professionals who must select methods that balance analytical performance, environmental impact, and practical applicability in compliance with modern regulatory and sustainability goals [3] [7].

The following table summarizes the core characteristics of traditional versus microextraction techniques, highlighting the fundamental differences in their approach and impact.

Table 1: Fundamental comparison between traditional and microextraction techniques.

Feature Traditional Techniques (e.g., SPE, LLE) Novel Microextraction Techniques (e.g., SPME, DLLME, FPSE)
Solvent Consumption High (mL to L volumes) Very low (µL volumes) to solvent-free [42] [55]
Chemical Waste Generation Significant Drastically reduced [35]
Sample Volume Relatively large Small, enabling high-throughput analysis [3]
Automation Potential Possible but often complex Easier to automate, leading to higher throughput [35] [42]
Principle Alignment Conventional Green Analytical Chemistry (GAC) and Green Sample Preparation [35] [42]
Key Advantages Well-established, robust Miniaturization, greenness, cost-effectiveness, reduced operator exposure [42] [55]

Greenness and Practicality Assessment Frameworks

To move beyond qualitative claims, the analytical community has developed metric tools for quantitatively evaluating method greenness and practicality.

  • AGREEprep (Analytical Greenness Metric for Sample Preparation): This software tool focuses exclusively on the sample preparation step, evaluating it against 10 principles of green sample preparation [17] [7]. It generates a score from 0 to 1 (where 1 is ideal) and a pictorial output, making it easy to identify the environmental strengths and weaknesses of a method [3] [7].
  • BAGI (Blue Applicability Grade Index): A complementary tool that assesses the practicality of an analytical method across ten criteria, including sample throughput, cost, instrumentation, and ease of implementation. A score above 60.0 recommends a method as practical [35].
  • WAC (White Analytical Chemistry): A holistic concept that seeks a balance between the three pillars: analytical performance (Red), ecological impact (Green), and practical/economic efficiency (Blue) [3].

Benchmarking Data and Comparative Analysis

Quantitative Greenness and Practicality Scores

The following table compiles data from published assessments, providing a direct comparison of various methods based on AGREEprep and BAGI scores.

Table 2: Benchmarking scores of various methods for different applications.

Application Method Type Specific Technique AGREEprep Score BAGI Score Key Performance Metrics Reference
PFAS in Water/Food Traditional Solid-Phase Extraction (SPE) Not Published ~50 High sensitivity, high solvent use [35]
Microextraction Magnetic Solid-Phase Extraction (MSPE) Not Published ~75 Good precision, high throughput, low cost [35]
Microextraction Fabric Phase Sorptive Extraction (FPSE) Not Published ~80 High accuracy & precision, user-friendly [35]
UV Filters in Cosmetics Standard Method Solvent Dissolution (EN 17156:2018) 0.51 Not Published Determines 22 UV filters [7]
Microextraction US-VA-DLLME 0.64 Not Published Higher preconcentration, low LOD [7]
Microextraction Dynamic HF-LPME 0.62 Not Published High selectivity, low solvent volume [7]
Safranal in Saffron Standard Method ISO 3632 Not Published Not Published Benchmark for validation [68]
Microextraction SFOD Not Published Not Published LOD: 3 ng mL⁻¹, High EF [68]
Microextraction USAEME Not Published Not Published LOD: 20 ng mL⁻¹, Good recovery [68]

Performance and Practicality Analysis of Microextraction Techniques

The data reveals that microextraction techniques consistently demonstrate superior practicality and greenness compared to traditional methods. For instance, in PFAS analysis, MSPE and FPSE achieved high BAGI scores (>75), indicating excellent practicality due to their high throughput, cost-efficiency, and simplicity [35]. Furthermore, a comparative study on food odorants showed that Thin-Film SPME (TF-SPME) significantly outperformed both traditional SPME fibers and Stir Bar Sorptive Extraction (SBSE) in extraction efficiency across a range of polar and non-polar analytes [59].

For the analysis of UV filters in cosmetics, microextraction methods like US-VA-DLLME achieved higher AGREEprep scores (0.64) than the standardized European method (0.51), confirming their reduced environmental impact [7]. This aligns with the findings in therapeutic drug monitoring (TDM), where many microextraction techniques achieved high greenness scores while maintaining a satisfactory balance with analytical performance (the "Red" principles of WAC) [3].

Detailed Experimental Protocols

Protocol 1: Fabric Phase Sorptive Extraction (FPSE) for Complex Matrices

FPSE combines the flexibility of a fabric substrate with the high efficiency of sol-gel derived sorbents, making it suitable for complex biological and environmental samples [35] [42].

  • Step 1: Sample Preparation. Take a 10 mL aqueous sample (e.g., urine, surface water). Adjust the pH using a dilute acid or base solution as required for optimal analyte extraction. If dealing with a solid sample, perform a preliminary extraction into an aqueous medium.
  • Step 2: Extraction.
    • a. Introduce a pre-cut (e.g., 2 cm x 2 cm) FPSE membrane into the sample solution.
    • b. Place the container on an orbital shaker and agitate for a predetermined time (e.g., 30-60 minutes) to achieve extraction equilibrium.
  • Step 3: Washing. Remove the FPSE membrane from the sample using clean forceps. Briefly rinse it with a small volume (e.g., 1 mL) of deionized water to remove any adsorbed matrix components.
  • Step 4: Analyte Elution (Back-Extraction).
    • a. Place the FPSE membrane into a vial containing a small volume (e.g., 500 µL - 1 mL) of a suitable organic solvent (e.g., methanol, acetonitrile).
    • b. Agitate for 5-10 minutes to desorb the analytes into the solvent.
  • Step 5: Analysis. Recover the eluent solvent. An aliquot is then directly injectable into an LC-MS/MS or GC-MS system for analysis [35] [42].

Protocol 2: Ultrasound-Assisted Dispersive Liquid-Liquid Microextraction (UA-DLLME)

UA-DLLME is a rapid, efficient technique that leverages ultrasound to form a fine dispersion of extraction solvent in the aqueous sample [68] [7].

  • Step 1: Dispersion.
    • a. To a 10-15 mL aqueous sample in a conical glass tube, rapidly inject a mixture containing a few hundred µL of a water-immiscible extraction solvent (e.g., chlorobenzene) and a disperser solvent (e.g., 1 mL acetone) using a syringe.
    • b. Alternatively, substitute with a green solvent like a Deep Eutectic Solvent (DES) [55].
    • c. Subject the tube to ultrasound for 1-2 minutes, forming a cloudy solution.
  • Step 2: Phase Separation. Centrifuge the tube at 5000 rpm for 5 minutes. This results in the sedimentation of the fine droplets of the extraction solvent at the bottom of the tube.
  • Step 3: Solvent Collection.
    • a. Cool the tube in an ice bath for a few minutes to solidify the organic solvent droplet (if a low-density solvent with a freezing point above water is used).
    • b. For non-solidifying solvents, use a micro-syringe to carefully withdraw the sedimented phase.
  • Step 4: Analysis. Transfer the collected organic phase to an autosampler vial for instrumental analysis by HPLC-UV or GC-MS [68] [7].

The Scientist's Toolkit: Key Reagents and Materials

Table 3: Essential research reagents and solutions for microextraction protocols.

Item Function/Description Example Use Cases
Hydrophilic-Lipophilic Balance (HLB) Sorbent A copolymer sorbent for extracting a wide range of polar and non-polar analytes. TF-SPME for food odorants [59], FPSE [42].
Molecularly Imprinted Polymers (MIPs) "Artificial antibody" sorbents with high selectivity for pre-determined target analytes. Selective hormone extraction from biological matrices [55].
Deep Eutectic Solvents (DES) Green, biodegradable solvents formed from natural compounds (e.g., choline chloride and urea). Replacement for toxic organic solvents in DLLME [42] [55].
Magnetic Nanoparticles (e.g., Fe₃O₄) Sorbent cores functionalized with various coatings, enabling easy separation using an external magnet. Magnetic Solid-Phase Extraction (MSPE) [35] [42].
Polydimethylsiloxane/Divinylbenzene (PDMS/DVB) Fiber A common SPME fiber coating for extracting volatile and semi-volatile compounds. Headspace SPME for volatiles [59] [42].

The benchmarking data unequivocally demonstrates that novel microextraction techniques are not merely alternatives but are often superior to traditional and standardized protocols. They achieve this by offering an optimal balance of analytical performance, practicality, and ecological sustainability.

  • For drug development and bioanalysis (e.g., TDM, hormone analysis), techniques like FPSE, MEPS, and in-vivo SPME are recommended due to their compatibility with complex matrices, minimal sample volume requirements, and capability for high-throughput analysis [3] [55].
  • In environmental and food analysis, where sensitivity and greenness are paramount, MSPE, TF-SPME, and DLLME using green solvents (DES) provide robust solutions with low detection limits and minimal environmental footprint [35] [59].

The consistent use of metric tools like AGREEprep and BAGI is critical for objective method selection and development. Future work should focus on the broader incorporation of these tools into regulatory guidelines and the continued development of automated, integrated microextraction systems to further enhance the sustainability and efficiency of analytical chemistry.

Conceptual Workflows and Relationships

G Traditional Traditional Methods (SPE, LLE) Assessment Assessment Framework Traditional->Assessment High Waste High Solvent Use Micro Novel Microextraction Methods (SPME, DLLME, FPSE) Micro->Assessment Low Waste Miniaturized Greenness Greenness (AGREEprep) Assessment->Greenness Practicality Practicality (BAGI) Assessment->Practicality Performance Analytical Performance Assessment->Performance Decision Informed Method Selection Greenness->Decision Practicality->Decision Performance->Decision

Microextraction Method Assessment Workflow

G The Three Pillars of White Analytical Chemistry (WAC) WAC Red Pillar • Analytical Performance • Sensitivity (LOD/LOQ) • Precision & Accuracy • Application Scope WAC2 Green Pillar • Green Sample Prep • Low Waste & Toxicity • Energy Efficiency • Safe for Operator WAC3 Blue Pillar • Practicality & Cost • Sample Throughput • Automation Potential • Instrumentation

Three Pillars of White Analytical Chemistry

Conclusion

The AGREEprep metric tool provides an indispensable, standardized framework for quantitatively assessing and improving the environmental sustainability of microextraction techniques in biomedical research. By systematically applying its ten principles, laboratories can make informed decisions to minimize waste, reduce toxic solvent use, and lower energy consumption. The true power of AGREEprep is realized when it is used not in isolation, but as part of an integrated White Analytical Chemistry approach, alongside tools like BAGI for practicality and RGB for analytical performance. This holistic strategy ensures that methods are not only green but also robust, cost-effective, and fit-for-purpose in critical areas like therapeutic drug monitoring. Future progress hinges on the widespread adoption of these metrics by regulatory bodies, continued innovation in green solvents and automated systems, and a collaborative effort across academia and industry to phase out outdated, resource-intensive methods in favor of sustainable, white analytical solutions.

References