Trinity College of Pharmaceutical Sciences, Peddapalli, Telangana 505172
In recent years, the pharmaceutical industry¹?² has faced increasing pressure to adopt more sustainable and eco-friendly practices. Traditional analytical methods, while accurate and robust, often consume large volumes of solvents and generate significant laboratory waste. Micro sampling and miniaturized analytical techniques are emerging as powerful tools in addressing these environmental concerns. These approaches offer multiple advantages, including reduced sample and solvent requirements, enhanced efficiency, and suitability for high-throughput applications. This review presents a comprehensive analysis of micro sampling techniques such as dried blood spot (DBS), volumetric absorptive micro sampling (VAMS), and capillary micro sampling, along with miniaturized analytical tools like microextraction methods, lab-on-a-chip systems, and paper-based analytical devices. The paper also discusses their applications, integration with advanced detection methods, and contributions toward greener pharmaceutical analysis.
The pharmaceutical industry has long relied on conventional analytical techniques for the development, validation, and quality control of drugs. However, these traditional methods typically involve high consumption of reagents and solvents, extensive sample preparation, and considerable energy usage. In response to increasing environmental awareness and regulatory pressures, there is a growing shift toward green analytical chemistry. This movement focuses on minimizing the environmental footprint of laboratory operations without compromising the reliability or sensitivity of analytical results.
Micro sampling and miniaturized techniques are key players in this paradigm shift. They offer unique opportunities to perform accurate, efficient, and environmentally responsible analyses by using smaller sample volumes and integrating multiple analytical functions into compact systems. This review aims to highlight the principles, developments, and applications of these methods, with a special focus on their role in promoting eco-friendly pharmaceutical analysis.
2. Microsampling Techniques
2.1 Dried Blood Spot (DBS) Sampling
Principle of DBS Sampling: Dried Blood Spot (DBS) sampling is based on collecting a small volume of peripheral blood—usually via finger or heel prick—onto a specially designed filter paper. The blood spreads across the paper and dries under ambient conditions, forming a stable matrix that can be stored and transported without refrigeration. A circular portion of the dried spot is later punched out and analytes are extracted using appropriate solvents for analysis, often by techniques like LC-MS/MS or HPLC.
The principle relies on the stabilization of analytes in dried form, allowing for delayed analysis while minimizing degradation and contamination risks. DBS is one of the oldest and most widely used microsampling techniques. It involves collecting a small volume of blood (typically 10–30 μL) on a filter paper, which is then dried and stored. This method has gained popularity due to its simplicity, low cost, and minimal invasiveness, especially in pediatric and geriatric populations.
Advantages of DBS: include ease of transport and storage, reduced biohazard risks, and minimal sample preparation. However, challenges such as hematocrit effect, analyte stability, and variability in spot size must be carefully managed through proper validation protocols.
Limitations of DBS Sampling
Despite its advantages, DBS sampling has several limitations:
2.2 Volumetric Absorptive Micro-sampling (VAMS)
Principle of VAMS Sampling
Volumetric Absorptive Microsampling (VAMS) operates on the principle of absorbing a precise and consistent volume of biological fluid—usually blood—into a porous, hydrophilic polymeric tip. When the tip contacts the sample, capillary action ensures the fluid is absorbed until the exact volume is reached, independent of hematocrit variations. The collected sample is dried and extracted for analysis using solvents suitable for techniques like LC-MS/MS or HPLC. This controlled volumetric uptake offers improved reproducibility, essential for accurate bioanalysis.
VAMS is a more recent innovation that addresses some of the limitations of DBS. It uses a porous, polymeric tip designed to absorb a fixed volume of blood regardless of hematocrit variations. Devices like Mitra® have made VAMS more accessible for clinical and pharmaceutical applications.
VAMS ensures better reproducibility and accuracy, and it has been increasingly used in pharmacokinetic and therapeutic drug monitoring studies. Its user-friendly design also supports remote sampling, which is valuable for decentralized clinical trials.
Advantages of VAMS
Limitations of VAMS
2.3 Capillary Microsampling
Principle of Capillary Microsampling:
Capillary microsampling relies on the passive movement of blood from small capillaries into narrow bore tubes or microcontainers following a lancet-induced finger or heel prick. This process uses capillary action to draw small, precise volumes of blood (typically 5–50 μL) without the need for suction or external force. Once collected, the samples can be analyzed directly or after minimal processing, supporting accurate quantification of analytes with minimal invasiveness—ideal for pediatric, preclinical, or frequent sampling scenarios.
Capillary microsampling involves collecting small blood volumes directly from capillaries using microtubes or capillary pipettes. This technique is especially suitable for serial sampling in small animals or vulnerable populations. Though it requires more technical skill during collection, capillary microsampling provides high-quality samples and is compatible with various bioanalytical methods, including LC-MS/MS.
Advantages of Capillary Microsampling
Limitations of Capillary Microsampling
Figure.2. Overview of microsampling techniques: (A) DBS, (B) VAMS, and (C) Capillary microsampling.
Table.No.1: Comparison Table of Microsampling Techniques
|
Feature |
Dried Blood Spot sampling {DBS} |
Volumetric Absorptive Micro-sampling {VAMS} |
Capillary microsampling |
|
Sample Volume |
10–30 μL |
10-20 μL |
5-50 μL |
|
Volumetric Accuracy |
Low (affected by hematocrit) |
High (fixed volume) |
Moderate (depends on technique) |
|
Hematocrit Effect |
Significant |
Minimal |
Moderate |
|
Analyte Stability |
Moderate |
High |
High |
|
Ease of Collection |
Easy |
Very Easy |
Moderate |
|
Biohazard Risk |
Low |
Low |
Moderate |
|
Cost |
Low |
Moderate to High |
Low |
|
Need for Equipment |
Minimal |
Requires specific device |
Requires micro collection tools |
|
Regulatory Acceptance |
Widely accepted |
Emerging |
Accepted with validation |
|
Use in Remote Sampling |
Possible |
Highly suitable |
Limited |
3. Miniaturized Analytical Techniques
Miniaturized analytical techniques refer to strategies that reduce the scale of traditional analytical procedures. These methods aim to use significantly smaller volumes of reagents and samples while enhancing throughput, accuracy, and sustainability. Miniaturization not only supports the goals of green chemistry but also facilitates field-deployable and point-of-care testing in pharmaceutical and clinical settings. This section elaborates on the most widely adopted miniaturized techniques: microextraction methods, microfluidic (lab-on-a-chip) systems, and paper-based analytical devices (PADs).
3.1 Microextraction Methods
Microextraction is a class of sample preparation methods that involves extracting analytes from complex matrices using minimal volumes of solvents or no solvents at all. These methods are environmentally friendly, cost-effective, and compatible with automation.
Solid Phase Microextraction (SPME): SPME uses a fiber coated with an extracting phase, which selectively binds analytes from a sample. The fiber is later desorbed thermally or with a solvent into a chromatographic system such as GC-MS or LC-MS. This technique eliminates the need for solvents and allows direct coupling with analytical instruments.
Figure.3. SPME: Analyte adsorption onto coated fiber and desorption for analysis.
Dispersive Liquid-Liquid Microextraction (DLLME): DLLME involves the rapid injection of a mixture of extraction and disperser solvents into an aqueous sample, forming a cloudy solution that facilitates efficient analyte transfer into the extraction solvent. It offers rapid extraction, low cost, and high enrichment factors.
Figure.4. DLLME: Disperser and extraction solvents added to water sample for analyte extraction.
Single-Drop Microextraction (SDME): In SDME, a single droplet of solvent is suspended at the tip of a microsyringe and exposed to the sample solution. Analytes diffuse into the droplet, which is then withdrawn and injected into the analytical system. This method requires very small amounts of solvent.
Figure.5. SDME: A single organic solvent droplet extracts analytes from an aqueous sample for GC analysis.
Microdialysis: Used extensively for in vivo applications, microdialysis utilizes a semipermeable membrane probe inserted into biological tissues or fluids to continuously sample unbound drug concentrations. This dynamic sampling method is ideal for pharmacokinetic studies and brain drug delivery research.
Figure.6. Microdialysis: Analyte diffusion into perfusion fluid via a semipermeable probe.
Advantages:
3.2 Microfluidics and Lab-on-a-Chip Systems
Microfluidic systems manipulate minute amounts of fluids (typically nanoliters to microliters) in interconnected channels etched into a substrate such as glass, silicon, or polymer. Lab-on-a-chip (LOC) technology integrates multiple laboratory functions—sample preparation, separation, reaction, and detection—on a single chip.
These systems enhance analytical speed, sensitivity, and reproducibility while dramatically lowering reagent use and waste generation.
Types of microfluidic applications include:
Figure.7. Microfluidic lab-on-a-chip system for sample-reagent mixing and on-chip detection.
Advantages:
3.3 Paper-Based Analytical Devices (PADs)
PADs are microfluidic systems fabricated on porous paper substrates. They utilize capillary action to move fluids, eliminating the need for external pumps. These devices can be designed for colorimetric, fluorescent, or electrochemical detection.
Common formats include:
Figure.8. PAD: Sample flows via capillary action through hydrophilic zones for detection.
Advantages:
Applications of PADs in pharmaceutical analysis:
By miniaturizing analytical processes, these innovative tools contribute significantly to sustainable pharmaceutical practices, offering faster analysis, smaller sample needs, and significantly less environmental impact.
4. Green Chemistry Aspects of Microsampling and Miniaturized Techniques
The integration of microsampling and miniaturized techniques in pharmaceutical analysis is a significant step forward in achieving the goals of green chemistry. These methods directly contribute to sustainability by reducing hazardous solvent usage, minimizing sample waste, and decreasing energy consumption. In this section, we examine how these innovations align with green analytical chemistry principles and how their environmental impact can be quantitatively assessed using green metrics.
4.1 Reduction in Solvent and Sample Use
Conventional analytical methods often require milliliter-scale solvent volumes for sample preparation, extraction, and analysis. In contrast, microsampling techniques like DBS, VAMS, and capillary sampling typically involve microliter volumes, which drastically cut down on solvent requirements during extraction and analysis. Similarly, miniaturized platforms like SPME, SDME, and microfluidics reduce solvent volumes while maintaining analytical sensitivity and precision. This not only lowers the cost of analysis but also significantly reduces the generation of chemical waste.
4.2 Waste Minimization and Safer Processes
Miniaturized devices and paper-based platforms often utilize biodegradable materials and require fewer reagents. Their inherent simplicity means fewer steps and less chance of exposure to hazardous chemicals. Moreover, since many of these techniques can be automated or integrated into closed systems, operator safety is improved, and laboratory air quality is better maintained.
4.3 Energy Efficiency and Portability
Traditional laboratory instruments, such as high-performance liquid chromatography (HPLC) and gas chromatography (GC), are energy-intensive. In contrast, miniaturized systems such as lab-on-a-chip devices operate at low power and often do not require sophisticated infrastructure. Paper-based devices, in particular, can function entirely without electricity, which is advantageous for remote or resource-limited settings.
4.4 Compatibility with Green Detection Technologies
Many miniaturized systems are compatible with greener detection techniques, such as smartphone-based imaging, electrochemical sensors, and LED-induced fluorescence. These methods replace bulky, power-hungry detectors with more sustainable alternatives while still offering sufficient sensitivity for pharmaceutical analysis.
4.5 Evaluation Using Green Analytical Metrics
Quantitative tools have been developed to evaluate the greenness of analytical procedures. The most widely accepted ones include:
Green Analytical Procedure Index (GAPI): This tool uses a pictogram with color-coded fields to visualize the greenness of an analytical method across different stages—from sample preparation to detection. Miniaturized and microsampling techniques generally score well due to their minimal reagent use and energy efficiency.
Analytical GREEnness (AGREE): This holistic metric evaluates an analytical procedure against all 12 principles of green analytical chemistry and gives a score (0 to 1), where higher values represent greener methods. Techniques like VAMS, SPME, and PADs often achieve high AGREE scores due to their minimal waste, small sample sizes, and reduced environmental impact.
Eco-Scale: This semi-quantitative tool assigns penalty points to steps in an analytical process based on factors like solvent toxicity, energy use, and safety. Lower penalty points indicate a greener process. Microsampling and miniaturized approaches typically receive fewer penalties.
4.6 Comparative Analysis
Table 2. Comparison of analytical techniques based on greenness parameters.
|
Technique |
Solvent usage |
Waste Generation |
Energy Demand |
GAPI/ AGREE Rating |
|
Traditional HPLC |
High |
High |
High |
Moderate to Low |
|
DBS Sampling |
Very Low |
Low |
Very Low |
High |
|
VAMS |
Very Low |
Low |
Low |
High |
|
SPME |
Minimal |
Very Low |
Moderate |
High |
|
PADs |
Negligible |
Biodegradable |
None |
Very High |
Microsampling and miniaturized techniques are not just analytical innovations—they are environmental solutions. By conserving resources and reducing laboratory footprints, they offer a practical, scalable pathway toward greener pharmaceutical analysis. These methods align well with global efforts to achieve sustainable laboratory practices and are likely to become integral to future regulatory and industrial frameworks.
5. Applications in Pharmaceutical Analysis
Microsampling and miniaturized analytical techniques have found wide-ranging applications in the pharmaceutical domain, particularly where sample volume, speed, and environmental impact are critical considerations. These tools are increasingly integrated into both early-stage research and routine quality control due to their efficiency and sustainability.
5.1 Pharmacokinetic and Bioavailability Studies
Microsampling methods such as DBS, VAMS, and capillary microsampling are extensively used in pharmacokinetic (PK) and bioavailability (BA) studies. They enable serial sampling in clinical and preclinical settings, allowing for detailed drug concentration-time profiling with minimal blood volume. This is particularly advantageous in pediatric studies and animal models where ethical and physiological limitations restrict blood withdrawal.
5.2 Therapeutic Drug Monitoring (TDM)
Therapeutic drug monitoring benefits from miniaturized techniques that provide high sensitivity and specificity with low sample volumes. VAMS and microfluidic platforms are increasingly used to monitor drug levels in chronic treatments, especially for narrow therapeutic index drugs such as antiepileptics and immunosuppressants.
5.3 Clinical Diagnostics and Point-of-Care Testing
Lab-on-a-chip and paper-based analytical devices (PADs) have enabled the development of portable, rapid, and user-friendly diagnostic tools. These are ideal for bedside testing or use in remote locations. They are applied for detecting infectious diseases, monitoring glucose or electrolytes, and screening for cardiovascular biomarkers.
5.4 Drug Stability and Degradation Testing
Miniaturized extraction methods like SPME and SDME are valuable in analyzing degradation products and impurities in stability studies. They allow rapid screening with minimal solvent use and are suitable for detecting trace-level analytes.
5.5 Quality Control and Counterfeit Drug Detection
Paper-based devices are being developed to identify falsified or substandard medications, especially in low-resource settings. These low-cost, simple tools provide qualitative and semi-quantitative assessments of active pharmaceutical ingredients (APIs), excipients, and contaminants.
5.6 Environmental and Residue Analysis
Microextraction techniques such as DLLME and SPME are applied in monitoring pharmaceutical residues in environmental matrices like wastewater and surface water. These methods are effective for trace detection and require minimal sample preparation, aligning with green chemistry goals.
The application landscape for microsampling and miniaturized techniques is broad and growing. Their adaptability to clinical, industrial, and environmental needs, along with their green profile, ensures they will continue to play a pivotal role in the evolution of pharmaceutical analysis.
6. Integration with Advanced Detection Techniques
The success of microsampling and miniaturized analytical approaches in pharmaceutical analysis is closely tied to the performance of the detection systems they are coupled with. These small-scale methods must be paired with highly sensitive and selective detection tools to ensure robust analytical outcomes. Recent innovations in detection technologies have greatly enhanced the utility and applicability of these miniaturized platforms.
6.1 Mass Spectrometry (MS)
Microsampling techniques, especially DBS and VAMS, are commonly interfaced with liquid chromatography-tandem mass spectrometry (LC-MS/MS) due to its high sensitivity, specificity, and ability to handle complex matrices. MS-based detection compensates for the low sample volumes and provides quantitative results suitable for pharmacokinetic, toxicological, and clinical studies.
6.2 Electrochemical Detection
Electrochemical sensors are highly compatible with microfluidic and paper-based devices. These sensors offer excellent sensitivity, rapid response times, and low power requirements. They are increasingly used in point-of-care testing for detecting small-molecule drugs, metabolites, and biomarkers.
6.3 Optical Detection Methods
Miniaturized platforms often integrate colorimetric, fluorometric, and chemiluminescent detection systems. Colorimetric detection, in particular, is ideal for PADs due to its simplicity and visual readout capability. Smartphone-assisted image analysis is also gaining traction as a low-cost, user-friendly detection method.
6.4 Spectroscopic Techniques
Techniques such as infrared (IR), ultraviolet-visible (UV-Vis), and Raman spectroscopy are being adapted to work with micro-scale platforms. Portable spectrometers now allow for rapid, non-destructive analysis on-site, supporting applications in drug identification, counterfeit detection, and environmental residue monitoring.
6.5 Integration with Digital and AI Tools
Advanced detection platforms are now being designed to transmit data wirelessly and interface with digital tools, enabling real-time monitoring and remote diagnostics. The incorporation of AI and machine learning in data interpretation further enhances the accuracy, repeatability, and predictive capabilities of these systems.
By integrating with advanced detection technologies, microsampling and miniaturized systems can deliver high analytical performance despite their compact design and minimal resource requirements. These synergies are essential for ensuring reliable data, particularly in personalized medicine, remote healthcare, and green analytical practices.
7. Challenges and Future Perspectives
Despite their numerous advantages, microsampling and miniaturized analytical techniques are not without limitations. Several challenges must be addressed to enable their broader adoption and integration into mainstream pharmaceutical analysis.
7.1 Analytical Sensitivity and Validation Standards
One of the major challenges is ensuring that miniaturized techniques provide sufficient sensitivity and reproducibility, especially when working with trace-level analytes. Standardization and validation protocols for microsampling methods, such as DBS and VAMS, must be clearly defined to ensure regulatory compliance and data comparability.
7.2 Hematocrit and Matrix Effects
In DBS and other blood-based microsampling methods, the hematocrit effect can significantly influence analyte distribution, leading to variability in results. This necessitates the development of correction algorithms or standardized sampling materials that mitigate such matrix effects.
7.3 Device Fabrication and Commercialization
While microfluidic chips and PADs offer great promise, large-scale, reproducible fabrication remains a technical and economic challenge. There is a need for cost-effective, scalable manufacturing processes that ensure device reliability and shelf life without compromising eco-friendliness.
7.4 Regulatory and Industry Acceptance
Adoption of novel microsampling and miniaturized techniques requires clear regulatory guidance and validation frameworks. Despite encouraging results from academic and pilot studies, many organizations hesitate to fully transition without harmonized international standards.
7.5 Data Handling and Integration
With the rise of point-of-care and remote sampling, managing and integrating data from diverse analytical platforms becomes complex. Ensuring data security, consistency, and interoperability is critical, especially in clinical trials and therapeutic monitoring.
7.6 Future Perspectives
Looking ahead, the convergence of microsampling with digital health, automation, and AI presents exciting opportunities. Smart microdevices, wearable sampling platforms, and integrated biosensors could enable continuous monitoring and real-time analysis, especially in personalized medicine. Further, coupling miniaturized systems with green detection technologies and sustainable materials will enhance their environmental and economic viability.
7.6.1 Automation and AI Integration
The future of pharmaceutical analysis lies in the automation of microsampling and miniaturized techniques. Integration with robotics and artificial intelligence (AI) can streamline workflows, enhance data accuracy, and support high-throughput analysis. AI algorithms can also aid in real-time interpretation and decision-making, improving both clinical and laboratory efficiency.
7.6.2 3D-Printed Micro devices
Advances in 3D printing are enabling the creation of customizable, low-cost microfluidic devices and sampling tools. These innovations support rapid prototyping and on-demand fabrication, making microsampling more accessible and adaptable to diverse analytical tasks.
7.6.3 Sustainability Trends in Analytical R&D
As sustainability becomes a strategic priority, future research and development will focus on biodegradable materials, energy-efficient devices, and solvent-free sample preparation. The integration of green metrics into method development will guide innovation toward more sustainable analytical systems.
While microsampling and miniaturized analytical techniques hold transformative potential, ongoing research, regulatory clarity, and technological innovation are key to overcoming current challenges. As these tools mature, they are poised to become central to the next generation of sustainable pharmaceutical analysis.
8. Regulatory Considerations and Limitations
The adoption of microsampling and miniaturized analytical techniques in pharmaceutical analysis, while promising, requires thorough consideration of regulatory frameworks. These methods must meet stringent criteria set by regulatory bodies such as the U.S. Food and Drug Administration (FDA), European Medicines Agency (EMA), and International Council for Harmonisation (ICH) to ensure data integrity, reliability, and patient safety.
8.1 Regulatory Acceptance of Microsampling
Microsampling methods like DBS and VAMS have gained significant attention in regulatory submissions, particularly in pharmacokinetic and bioequivalence studies. The FDA and EMA have released guidance documents encouraging their use, provided they are validated according to accepted bioanalytical method validation standards. However, full regulatory acceptance still requires extensive bridging studies to compare microsampling data with conventional venous blood sampling.
8.2 Validation Challenges
Validation of microsampling techniques presents unique challenges, especially in terms of accuracy, precision, stability, and carryover. Regulatory authorities expect complete demonstration of method reliability across varying hematocrit levels, extraction efficiencies, and analyte stability conditions. These requirements can complicate the method development process and increase the cost and time for implementation.
8.3 Device and Material Standardization
Another regulatory hurdle is the lack of standardized sampling devices and materials. For example, differences in filter paper properties or polymeric tip compositions may affect analyte recovery. Harmonization of materials and clear manufacturing guidelines are needed to reduce variability and facilitate global regulatory acceptance.
8.4 Data Integrity and Traceability
Ensuring data integrity is critical, particularly when microsampling devices are used in remote or decentralized settings. Regulatory agencies emphasize traceability, secure sample labeling, and data tracking systems to prevent misidentification or tampering.
8.5 Miniaturized Systems and Regulatory Gaps
Miniaturized analytical platforms such as lab-on-a-chip and PADs are still emerging in the regulatory landscape. While their analytical performance is often robust, regulatory pathways for their use in drug development or quality control are not yet well defined. Standardized validation protocols and performance benchmarks are needed to accelerate their adoption.
Regulatory considerations play a crucial role in the broader adoption of microsampling and miniaturized analytical techniques. While progress is being made, continued dialogue between researchers, manufacturers, and regulators is essential to overcome current limitations and establish these green innovations as part of mainstream pharmaceutical practice.
CONCLUSION
Microsampling and miniaturized analytical techniques represent a pivotal advancement in the pursuit of greener, more sustainable pharmaceutical analysis. These technologies offer significant benefits, including reduced sample and solvent usage, improved portability, and compatibility with remote and decentralized testing. Their integration with advanced detection systems, such as mass spectrometry, electrochemical sensors, and AI-based data processing, further enhances their applicability across clinical, regulatory, and industrial settings. However, several challenges persist. Issues related to method validation, hematocrit effects, device standardization, and regulatory acceptance must be addressed to ensure broader implementation. Regulatory agencies are gradually embracing these techniques, but harmonized validation protocols and long-term performance data are necessary for universal acceptance.
Looking ahead, innovations in automation, 3D printing, and eco-friendly materials are poised to elevate these technologies to new heights. Continued interdisciplinary collaboration and investment in green research and development will be crucial to overcoming existing barriers. In summary, microsampling and miniaturized techniques are not just innovations in analytical science—they embody a fundamental shift toward more environmentally responsible pharmaceutical practices. With sustained progress, they are likely to play a central role in shaping the future of analytical and clinical laboratories worldwide.
REFERENCES
Sravanthi. Gandu, V. Swapna, Micro Sampling and Miniaturized Techniques: A Green Revolution in Pharmaceutical Analysis, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 7, 3151-3164. https://doi.org/10.5281/zenodo.16356857
10.5281/zenodo.16356857
