P erspective For reprint orders, please contact [email protected]
Is SPME a destination or just another station for bioanalysis? This article reviews the solid-phase microextraction technique and its potential to revolutionize bioanalysis in terms of combining sampling, sample preparation and extraction in one step with no blood withdrawal. Possible hurdles facing the technology when implemented in a pharmaceutical setting will be covered and the author will provide an outlook on the future of solid-phase microextraction as a novel microsampling technique for bioanalysis. Bioanalysis is the term given to the process of drug quantification in biological matrices, and sits at the heart of the drug discovery and development process. Without reliable and high-quality bioanalytical measurements, it would be impossible to fully understand and evaluate the PD and PK properties of a new drug [1]. Over the years, the field of bioanalysis has been able to bridge the gap between a range of disciplines, gracefully evolving to encompass various techniques and adopt new tools to enable bioanalytical scientists to overcome challenging tasks [2]. Today’s bioanalytical focus on emerging technologies has led to the identification and use of improved instrumentation and miniaturized analytical devices. Despite this, current trends in drug quantification still employ multistep sample-handling procedures that involve off-site sample preparation, extraction and ana lysis [3]. Although there has been a paradigm shift towards using smaller sample volumes with recently established microsampling methods [4,5], bioanalysts have not been able to completely move away from, or avoid using, wet sampling with relatively large sample volumes [6]. The quest for finding a ‘magic bullet’ that can address both sampling and sample preparation issues has led to the discovery of a novel technique that has recently found its way into the in vivo world of bioanalysis; the technique claims to extract the analyte without any sample usage [6,7]. Better yet, it also combines sampling, sample preparation and extraction in one step. This exciting concept is known as solid-phase microextraction (SPME) [8]. Being familiar with the idea of SPE extraction, what sparked new attention was the micro feature of SPME.
What is SPME? It was soon realized that the micro aspect of the name referred to the nonexhaustive nature of the extraction mechanism, where a miniature extraction phase is used alongside a reduced sample volume [3]. Unlike SPE, which is performed with a large sorptive surface, the SPME technique is based on the use of a small fiber coated with an extraction phase that is exposed to the sample matrix for a defined period of time [8]. An equilibrium process takes place in which the analyte partitions between the SPME coating and the sample matrix. The amount of analyte extracted by SPME is directly proportional to the concentration of analyte present in the sample matrix [9]. This novel invention was first introduced in the 1990s by Pawliszyn at the University of Waterloo, Canada [10]. Since then it has been utilized in numerous applications, including environmental research, food and fragrance analysis, as well as forensic and military investigations [8,11,12]. More noteworthy to the pharmaceutical industry, the potential to quantify drug concentrations in PK studies involving small rodents and large animals has been demonstrated for SPME [9]. The design of this approach revolves around direct on-site analysis, facilitated by biocompatible fibers that can be inserted directly into the living organism without causing any toxic reactions or side effects [13]. The launch of biocompatible fibers housed inside hypodermic needles has been a major breakthrough for SPME, and is one that may bring about a revolution in the world of microsampling and bioanalysis [7]. It allows the insertion of the miniaturized device into the blood vessel and enables exposure to the systemic circulation, where the transport of analyte begins
10.4155/BIO.13.260 © 2013 Future Science Ltd
Bioanalysis (2013) 5(23), 2897–2901
Sheelan Ahmad Platform Technologies & Science, Drug Metabolism & Pharmacokinetics, GlaxoSmithKline Research & Development, Ware, Hertfordshire, SG12 0DP, UK Tel.: +44 1920 882557 E-mail: [email protected]
ISSN 1757-6180
2897
P erspective |
Ahmad
Key Terms Microsampling: Process that
enables drug quantification without sample collection or through the usage of very small sample volumes, without causing any significant effects to the subject.
Solid-phase micro extraction: Analyte
extraction technique based on coated fibers, enabling quantification without the need for blood withdrawal.
Biocompatible: Capability of
a material to have no toxic or adverse effect when inserted into a living organism; coating with biocompatible polymers prevents adhesion of macromolecules and cells, which in turn provides efficient sample clean up.
Direct immersion: Insertion of a device directly into the living system.
3Rs: Strategies laid down by the National Centre for Replacement, Refinement and Reduction of Animals in Research.
immediately [14]. Extraction is considered complete when equilibrium is reached and the fiber is retracted with no blood withdrawal [15]. What will it bring? The lack of blood withdrawal is a striking feature worth every attention. The impact it posses is huge if implemented correctly. SPME can offer numerous advantages over conventional sampling and extraction techniques that have occupied quantitative bioanalysis in the past to support drug-development studies. No blood removal means serial or repeat sampling from the same animal without the need for satellite groups, which in turn will lead to improved data quality, reduced animal use and permit cost savings [14,16]. It will also enable juvenile toxicology and pediatric studies to be conducted with additional cost reductions, in terms of sample shipping and storage at ambient temperature [17]. All these advantages were previously encountered by the DBS technology; however, with the recent hematocrit concerns of DBS, the SPME microsampling may act as a complementary technique to maintain and add to the benefits of DBS [17]. The biocompatible feature of newer SPME fibers not only allows for direct immersion into the blood stream – it also provides efficient sample clean-up by preventing the adhesion of biological macromolecules such as phospholipids, thereby eliminating possible interference caused by biological substances within complex matrices [13,18]. Combining this trait with highly sensitive MS instrumentation will permit successful achievement of LLOQ that plays an essential role in determining the therapeutic moiety of drugs within the pharmaceutical industry. The prospect map The foreseen avenues for SPME within the industry are plenty; potentially, it could be implemented at various stages of the drug discovery and development process. Starting with early discovery phases, SPME can be applied in 3D cell cultures to determine the ability of the drug to elicit a biological response inside an in vitro model, where the cellular function is examined prior to full commitment in the in vivo system [19]. More importantly, it forms an attractive tool for knockout studies in which transgenic animals are used to understand mutagenesis and validate genetic variations. Such studies have been limited almost exclusively to mouse models by virtue of the ease of genetic manipulation
2898
Bioanalysis (2013) 5(23)
and their close reflection of the human physiology [20]. The generation of these unique species is an expensive process and therefore it is crucial to determine as much information as possible from these genetically engineered animals [21]. The approach, however, has always suffered from strict regulations on the availability of blood volumes, but this will no longer be an issue with the direct, no blood withdrawal aspect of SPME. The SPME technique offers a powerful option for use in preclinical stage toxicity studies. As previously mentioned, it will maintain the microsampling benefits of the 3Rs and ethics in animal usage. It will allow for serial or repeat sampling from the same animal, eliminating the need to use composite bleeds from several animals, as well as facilitating the removal of satellite groups [22]. This not only helps to reduce animal usage in toxicology studies, it also enhances the quality of data generated by measuring full toxicokinetic profiles from each animal, providing more reliable results and eliminating inter-individual variability of composite sampling [22,23]. Another significant and perhaps unforeseen advantage is the reduction of the overall number of sample processing steps for the bioanalyst [3]. The technique does not require sample aliquoting, centrifuging or sample freezing/thawing. Automation of the desorption step will essentially mean that very little input will be required from the bioanalyst throughout the whole analytical process. Recently, the technique has been applied in the metabolomic area, facilitating effective in vivo metabolite monitoring [7], and also for in vitro studies to monitor the metabolomics of disease pathways using relevant cell lines [24]. Multiple blood-free sampling aids the process of capturing unstable metabolites within the living organism, reflecting the actual metabolite component in real-time – that is, a true snapshot of the metabolome [25]. The acquisition of rapid metabolism data known as ‘metabolism quenching’ will provide the pharmaceutical industry with information that can form the foundations of a comprehensive metabolomic database used for designing future personalized medications. Biomarker monitoring is another area worth shedding some light on. SPME has been used for the detection of volatile compounds that act as indicators of various disorders. Recent publications have described the use of SPME-GC–MS coupled with nanosensors for the successful identification of 42 volatile compounds [26]. These small molecules that correspond to lung cancer future science group
Is SPME a destination or just another station for bioanalysis? biomarkers were detected using patient breath sampling. Tumor growth biomarkers and potential regulators of angiogenesis have also been captured by SPME [13]. If these as yet uncommercialized prototype devices could be further developed into commercial tools with selective coatings specific for biomarkers, then SPME can add further prognostic values to the industry. What about peptide biomarkers and larger biopharmaceutical molecules such as antibodies and proteins? The development of biotherapeutics is now an integral part of the pharmaceutical industry where, immunoassays have traditionally dominated the field of quantifying such molecules. However, with current advances in LC–MS/MS, it has been possible to accurately measure peptides and proteins with sufficiently reduced LLOQ. So although it may not yet be so well established, with intricate design of SPME fibers, it may prove feasible to coat fibers with specific antibodies to act as binding beds for antigens. This will enable a highly specific ‘lock and key’ mechanism between the fiber and the target harvested from the complex biological matrix, all of which would take place within the living organism, avoiding the necessity to collect blood samples. SPME applications could potentially be further extended to tissue analysis. The device can penetrate organ tissues without causing much regional damage compared with microdialysis probes [27]. Solvent compatibility and the difficulty of coupling microdialysis with LC–MS remains unsolved; therefore, the use of SPME coupled with LC–MS will enable detection of low concentrations without the ion suppression associated with microdialysis [27]. Measurements of drug levels in the brain of conscious free-moving rodents have been performed without the requirement for organ removal [13,15,28]. This opens the door for quantitative PK and TK analysis of drugs with complete organ exposure or accumulation profiles, without the need to take terminal samples for subsequent tissue homogenizing and wet-sample analysis. The clinical suitability of SPME brings another remarkable element to the drug development table. Although sample volume is not a major concern when dealing with the majority of human subjects, there are numerous cases where avoidance of blood withdrawal is essential, specifically in pediatric studies or blood coagulation disorders [16,17]. To date, most human SPME applications have been clustered around breath and skin analysis. However, the quantification of future science group
| P erspective
analgesic drugs in human urine samples has also been reported [13,29,30]. A notable novel application anticipated for SPME within the clinical arena is the possibility of the technique to serve as a diagnostic tool in the operating theater. Monitoring of blood drug concentrations during surgery is a critical element of many clinical procedures. An example that illustrates this significance is anesthetic management of liver transplantation patients. The function of the hepatic system varies during liver transplantation surgeries; this has a direct impact on the metabolism of the combination of drugs used for general anesthetic [31]. SPME can provide a simple method to measure the concentration of parent drug and metabolites throughout the various stages of the transplant process. This in turn will enable dosage control of anesthetics during surgical procedures [30,31]. Coupling SPME with current separation and detection techniques has allowed for the quantification of low drug concentrations. Although GC–MS has been the main analytical technique utilized in conjunction with SPME [32], the development of new SPME-LC–MS interfaces have facilitated the on-site application of SPME ana lysis [3]. The growth of commercial SPME autosamplers used for analyte desorption has enabled high-throughput analysis. This has played a major role in saving time and overall simplification of the whole process [23]. Hurdles to adoption Before marching down the SPME route, it is vital to take into account all of the barriers that may face the adoption of the technique within the pharmaceutical industry. The first is the invasiveness of the device, which is expected to be met with some apprehension from regulatory authorities [33]. Historically the regulatory environment has been a challenging place for innovation [34]. However, authors and users of SPME must work closely with regulators to embrace and address any compliance concerns. In-depth investigations are necessary to ensure that no adverse effects are associated with the insertion of the device into the living organism. Biocompatibility and sterility are the key characteristics needed to prove the safety of the device. It is also crucial that quality bioanalytical SPME methods are validated using current GLP regulatory standards. The collection of high-quality data that can support the validity of SPME and show a favorable benefit to risk ratio will steer the regulatory mood towards a receptive climate and enable proof-of-concept [34,35]. www.future-science.com
2899
P erspective |
Ahmad Alongside regulatory approval, SPME requires acceptance by fellow scientists, including bioanalysts, animal technicians and physicians. To assist this process, adaptation of the current tool is imperative in order to create a user-friendly and convenient device [2,13]. Although commercially available, the existing SPME product has not been extensively tested within animal laboratories or clinics. Collaborations between academic experts, analysts, toxicology technicians, hospital users and manufacturers will be a vital step for the development of SPME into a medical tool that can be marketed to accommodate customers’ needs. It is also important to execute a systematic evaluation of the technology, paying particular attention to certain characteristics that may become road blockers if ignored. One of these is the length of in vivo equilibration time, which will influence the number of time points that could be attained, as well as the amount of discomfort caused if the fiber is left inside the living system for a long period of time [36]. Another is interfiber reproducibility, which will have an impact on the overall accuracy and precision of the data obtained. A further hurdle is the availability of appropriate fiber coatings that can enable extraction of drugs over a wide range of physiochemical properties [23]. Taking these points into consideration will underpin the analytical applicability of the technique
and establish solid foundations for regulatory recognition. Future perspective Taking this journey may require resources in terms of time, expertise and financial input, but the gain is far greater to all disciplines within the pharmaceutical and medical world. In the author’s opinion, the recent innovative techniques brought about and used by bioanalysts are gradually shaping the future of the pharmaceutical industry. SPME has the potential to become a tool that will follow the life of the drug from the time of its discovery through to the time it becomes available to patients [10,15,36]. On-site drug extraction without the need to sacrifice any matrix will bring about a revolution and must become a destination for bioanalysis to modernize and simplify current sampling and sample extraction procedures. The author is confident that this technique can reserve a front-row place within the analytical arena. Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Executive summary Potential benefits of solid-phase microextraction
Solid-phase microextraction (SPME) is a blood-free microsampling technique with many benefits awaiting further exploration within the pharmaceutical field.
Various forms of SPME could potentially be developed to serve several areas that employ analyte quantification, ranging from drug discovery and development through to clinical, and ending in operating theaters.
The application of SPME is pending in new avenues such as quantification of large biopharmaceutical molecules and biomarkers.
Challenging but achievable
The invasiveness of the device, the analytical limitations of the technique and sterility may become barriers if not extensively investigated.
There is a current unmet need for direct on-site drug analysis, and this could be met by adoption and development of SPME. If correctly implemented, the future of the technique will be bright.
References Papers of special note have been highlighted as: n of interest nn of considerable interest 1
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2
Abu-Rabie P. Direct analysis of DBS: emerging and desirable technologies. Bioanalysis 3(15), 1675–1678 (2011).
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Bojko B, Mirnaghi F, Pawliszyn J. Solid-phase microextraction: a multi-purpose microtechnique. Bioanalysis 3(17), 1895–1899 (2011). Li F, Ploch S, Fast D, Michael S. Perforated dried blood spot accurate microsampling: the concept and its applications in toxicokinetic
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sample collection. J. Mass Spectrom. 47(5), 655–667 (2012). 5
Smith C, Skyes A, Robinson S, Thomas E. Evaluation of blood microsampling techniques and sampling sites for the analysis of drugs by HPLC–MS. Bioanalysis 3(2), 145–156 (2011).
future science group
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Vuckovic D, Zhang X, Cudjoe E, Pawliszyn J. Solid-phase microextraction in bioanalysis: new devices and directions. J. Chromatogr. A 1217(25), 4041–4060 (2010). Excellent overview of the recent developments in the field of solid-phase microextraction, reflecting the suitability of the technique for bioanalysis. Lord HL, Zhang X, Musteata FM, Vuckovic D, Pawliszyn J. In vivo solid-phase microextraction for monitoring intravenous concentrations of drugs and metabolites. Nat. Protoc. 6(6), 896–924 (2011). Lord H, Pawliszyn J. Evolution of solid-phase microextraction technology. J. Chromatogr. A 885(1), 153–193 (2000). Duan C, Shen Z, Wu D, Guan Y. Recent developments in solid-phase microextraction for on-site sampling and sample preparation. Trends Analyt. Chem. 30(10), 1568–1574 (2011).
10 Augusto F, Luiz Pires Valente A. Applications
of solid-phase microextraction to chemical analysis of live biological samples. Trends Analyt. Chem. 21(6), 428–438 (2002). 11 Wang J, Tuduri L, Millet M, Briand O,
Montury M. Flexibility of solid-phase microextraction for passive sampling of atmospheric pesticides. J. Chromatogr. A 1216 (15), 3031–3037 (2009). 12 Laor Y, Koziel JA, Cai L, Ravid U. Chemical-
sensory characterization of dairy manure odor using headspace solid-phase microextraction and multidimensional gas chromatography mass spectrometry-olfactometry. J. Air Waste Manag. Assoc. 58(9), 1187–1197 (2008). 13 Bojko B, Cudjoe E, Pawliszyn J, Wasowicz M.
Solid-phase microextraction. How far are we from clinical practice? Trends Analyt. Chem. 30(9), 1505–1512 (2011). 14 Musteata FM, Pawliszyn J. In vivo sampling
with PK. J. Biochem. Biophys. Methods 70(2), 181–193 (2007). 15 Musteata FM, de Lannoy I, Gien B, Pawliszyn
J. Blood sampling without blood draws for in vivo pharmacokinetic studies in rats. J. Pharmaceut. Biomed. 47(4), 907–912 (2008). 16 Spooner N, Lad R, Barfield M. Dried blood
spots as a sample collection technique for the determination of pharmacokinetics in clinical
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studies: considerations for the validation of a quantitative bioanalytical method. Anal. Chem. 81(4), 1557–1563 (2009).
27 Zhou SN, Ouyang G, Pawliszyn J.
Comparison of microdialysis with solid-phase microextraction for in vitro and in vivo studies. J. Chromatogr. A 1196–1197, 46–56 (2008).
17 Spooner N. A dried blood spot update: still an
important bioanalytical technique? Bioanalysis 5(8), 879–883 (2013).
28 Zhang X, Es-haghi A, Cai J, Pawliszyn J.
Simplified kinetic calibration of solid-phase microextraction for in vivo pharmacokinetics. J. Chromatogr. A 1216(45), 7664–7669 (2009).
18 Kataoka H, Saito K. Recent advances in
SPME techniques in biomedical analysis. J. Pharmaceut. Biomed. 54(5), 926–950 (2011). 19 Hughes JP, Rees S, Kalindjian SB, Philpott
29 Brown SD, Rhodes DJ, Pritchard BJ.
A validated SPME-GC-MS method for simultaneous quantification of club drugs in human urine. Forensic Sci. Int. 171(2), 142–150 (2007).
KL. Principles of early drug discovery. Br. J. Pharmacol. 162(6), 1239–1249 (2011). 20 Grujic D, Susulic VS, Harper ME et al.
Adrenergic receptors on white and brown adipocytes mediate selective agonist-induced effects on energy expenditure, insulin secretion, and food intake. A study using transgenic and gene knockout mice. J. Biol. Chem. 272(28), 17686–17693 (1997). 21 Rosenberg MP, Bortner D. Why transgenic
and knockout animal models should be used (for drug efficacy studies in cancer). Cancer Metastasis Rev. 17(3), 295–299 (1998). 22 Ohno Y. ICH guidelines-implementation of
the 3Rs (refinement, reduction, and replacement): incorporating best scientific practices into the regulatory process. ILAR J. 43(Suppl.), S95–S98 (2002). 23 Souza Silva EA, Risticevic S, Pawliszyn J.
Recent trends in SPME concerning sorbent materials, configurations and in vivo applications. Trends Analyt. Chem. 43, 24–36 (2012). 24 Pyo JS, Ju HK, Park JH, Kwon SW.
Determination of volatile biomarkers for apoptosis and necrosis by solid-phase microextraction–gas chromatography/mass spectrometry: a pharmacometabolomic approach to cisplatin’s cytotoxicity to human lung cancer cell lines. J. Chromatogr. B 876(2), 170–174 (2008). 25 Vuckovic D, de Lannoy I, Gien B et al.
In vivo solid phase microextraction: capturing the elusive portion of metabolome. Angew. Chem. 123(23), 5456– 5460 (2011). 26 Ouyang G, Vuckovic D, Pawliszyn J.
Nondestructive sampling of living systems using in vivo solid-phase microextraction. Chem. Rev. 111(4), 2784 (2011).
www.future-science.com
| P erspective
n
Study focusing on the use of solid-phase microextraction to determine drug concentrations in human samples.
30 Zhang ZM, Cai JJ, Ruan GH, Li GK. The
study of fingerprint characteristics of the emanations from human arm skin using the original sampling system by SPME-GC/MS. J. Chromatogr. B 822(1), 244–252 (2005). 31 University Health Network, Toronto. The Use
of Solid Phase Micro-extraction (SPME) For Metabolomics and Concomitant Measurements of Rocuronium Bromide Levels in Liver Transplantation. University Health Network, Toronto, Canada (2013). 32 Boussahel R, Bouland S, Moussaoui KM,
Baudu M, Montiel A. Determination of chlorinated pesticides in water by SPME/GC. Water Res. 36(7), 1909–1911 (2002). 33 Sparrow SS, Robinson S, Bolam S et al.
Opportunities to minimise animal use in pharmaceutical regulatory general toxicology: a cross-company review. Regul. Toxicol. Pharmacol. 61(2), 222–229 (2011). 34 Viswanathan CT. Bringing new technologies
into regulatory space. Bioanalysis 4(23), 2763–2764 (2012). 35 Harwood AD, Landrum PF, Lydy MJ.
A comparison of exposure methods for SPME-based bioavailability estimates. Chemosphere 86(5), 506–511 (2011). 36 Yeung JCY, Vuckovic D, Pawliszyn J.
Comparison and validation of calibration methods for in vivo SPME determinations using an artificial vein system. Anal. Chim. Acta 665(2), 160–166 (2010).
2901
Is SPME a destination or just another station for bioanalysis? This article reviews the solid-phase microextraction technique and its potential to revolutionize bioanalysis in terms of combining sampling, sample preparation and extraction in one step with no blood withdrawal. Possible hurdles facing the technology when implemented in a pharmaceutical setting will be covered and the author will provide an outlook on the future of solid-phase microextraction as a novel microsampling technique for bioanalysis. Bioanalysis is the term given to the process of drug quantification in biological matrices, and sits at the heart of the drug discovery and development process. Without reliable and high-quality bioanalytical measurements, it would be impossible to fully understand and evaluate the PD and PK properties of a new drug [1]. Over the years, the field of bioanalysis has been able to bridge the gap between a range of disciplines, gracefully evolving to encompass various techniques and adopt new tools to enable bioanalytical scientists to overcome challenging tasks [2]. Today’s bioanalytical focus on emerging technologies has led to the identification and use of improved instrumentation and miniaturized analytical devices. Despite this, current trends in drug quantification still employ multistep sample-handling procedures that involve off-site sample preparation, extraction and ana lysis [3]. Although there has been a paradigm shift towards using smaller sample volumes with recently established microsampling methods [4,5], bioanalysts have not been able to completely move away from, or avoid using, wet sampling with relatively large sample volumes [6]. The quest for finding a ‘magic bullet’ that can address both sampling and sample preparation issues has led to the discovery of a novel technique that has recently found its way into the in vivo world of bioanalysis; the technique claims to extract the analyte without any sample usage [6,7]. Better yet, it also combines sampling, sample preparation and extraction in one step. This exciting concept is known as solid-phase microextraction (SPME) [8]. Being familiar with the idea of SPE extraction, what sparked new attention was the micro feature of SPME.
What is SPME? It was soon realized that the micro aspect of the name referred to the nonexhaustive nature of the extraction mechanism, where a miniature extraction phase is used alongside a reduced sample volume [3]. Unlike SPE, which is performed with a large sorptive surface, the SPME technique is based on the use of a small fiber coated with an extraction phase that is exposed to the sample matrix for a defined period of time [8]. An equilibrium process takes place in which the analyte partitions between the SPME coating and the sample matrix. The amount of analyte extracted by SPME is directly proportional to the concentration of analyte present in the sample matrix [9]. This novel invention was first introduced in the 1990s by Pawliszyn at the University of Waterloo, Canada [10]. Since then it has been utilized in numerous applications, including environmental research, food and fragrance analysis, as well as forensic and military investigations [8,11,12]. More noteworthy to the pharmaceutical industry, the potential to quantify drug concentrations in PK studies involving small rodents and large animals has been demonstrated for SPME [9]. The design of this approach revolves around direct on-site analysis, facilitated by biocompatible fibers that can be inserted directly into the living organism without causing any toxic reactions or side effects [13]. The launch of biocompatible fibers housed inside hypodermic needles has been a major breakthrough for SPME, and is one that may bring about a revolution in the world of microsampling and bioanalysis [7]. It allows the insertion of the miniaturized device into the blood vessel and enables exposure to the systemic circulation, where the transport of analyte begins
10.4155/BIO.13.260 © 2013 Future Science Ltd
Bioanalysis (2013) 5(23), 2897–2901
Sheelan Ahmad Platform Technologies & Science, Drug Metabolism & Pharmacokinetics, GlaxoSmithKline Research & Development, Ware, Hertfordshire, SG12 0DP, UK Tel.: +44 1920 882557 E-mail: [email protected]
ISSN 1757-6180
2897
P erspective |
Ahmad
Key Terms Microsampling: Process that
enables drug quantification without sample collection or through the usage of very small sample volumes, without causing any significant effects to the subject.
Solid-phase micro extraction: Analyte
extraction technique based on coated fibers, enabling quantification without the need for blood withdrawal.
Biocompatible: Capability of
a material to have no toxic or adverse effect when inserted into a living organism; coating with biocompatible polymers prevents adhesion of macromolecules and cells, which in turn provides efficient sample clean up.
Direct immersion: Insertion of a device directly into the living system.
3Rs: Strategies laid down by the National Centre for Replacement, Refinement and Reduction of Animals in Research.
immediately [14]. Extraction is considered complete when equilibrium is reached and the fiber is retracted with no blood withdrawal [15]. What will it bring? The lack of blood withdrawal is a striking feature worth every attention. The impact it posses is huge if implemented correctly. SPME can offer numerous advantages over conventional sampling and extraction techniques that have occupied quantitative bioanalysis in the past to support drug-development studies. No blood removal means serial or repeat sampling from the same animal without the need for satellite groups, which in turn will lead to improved data quality, reduced animal use and permit cost savings [14,16]. It will also enable juvenile toxicology and pediatric studies to be conducted with additional cost reductions, in terms of sample shipping and storage at ambient temperature [17]. All these advantages were previously encountered by the DBS technology; however, with the recent hematocrit concerns of DBS, the SPME microsampling may act as a complementary technique to maintain and add to the benefits of DBS [17]. The biocompatible feature of newer SPME fibers not only allows for direct immersion into the blood stream – it also provides efficient sample clean-up by preventing the adhesion of biological macromolecules such as phospholipids, thereby eliminating possible interference caused by biological substances within complex matrices [13,18]. Combining this trait with highly sensitive MS instrumentation will permit successful achievement of LLOQ that plays an essential role in determining the therapeutic moiety of drugs within the pharmaceutical industry. The prospect map The foreseen avenues for SPME within the industry are plenty; potentially, it could be implemented at various stages of the drug discovery and development process. Starting with early discovery phases, SPME can be applied in 3D cell cultures to determine the ability of the drug to elicit a biological response inside an in vitro model, where the cellular function is examined prior to full commitment in the in vivo system [19]. More importantly, it forms an attractive tool for knockout studies in which transgenic animals are used to understand mutagenesis and validate genetic variations. Such studies have been limited almost exclusively to mouse models by virtue of the ease of genetic manipulation
2898
Bioanalysis (2013) 5(23)
and their close reflection of the human physiology [20]. The generation of these unique species is an expensive process and therefore it is crucial to determine as much information as possible from these genetically engineered animals [21]. The approach, however, has always suffered from strict regulations on the availability of blood volumes, but this will no longer be an issue with the direct, no blood withdrawal aspect of SPME. The SPME technique offers a powerful option for use in preclinical stage toxicity studies. As previously mentioned, it will maintain the microsampling benefits of the 3Rs and ethics in animal usage. It will allow for serial or repeat sampling from the same animal, eliminating the need to use composite bleeds from several animals, as well as facilitating the removal of satellite groups [22]. This not only helps to reduce animal usage in toxicology studies, it also enhances the quality of data generated by measuring full toxicokinetic profiles from each animal, providing more reliable results and eliminating inter-individual variability of composite sampling [22,23]. Another significant and perhaps unforeseen advantage is the reduction of the overall number of sample processing steps for the bioanalyst [3]. The technique does not require sample aliquoting, centrifuging or sample freezing/thawing. Automation of the desorption step will essentially mean that very little input will be required from the bioanalyst throughout the whole analytical process. Recently, the technique has been applied in the metabolomic area, facilitating effective in vivo metabolite monitoring [7], and also for in vitro studies to monitor the metabolomics of disease pathways using relevant cell lines [24]. Multiple blood-free sampling aids the process of capturing unstable metabolites within the living organism, reflecting the actual metabolite component in real-time – that is, a true snapshot of the metabolome [25]. The acquisition of rapid metabolism data known as ‘metabolism quenching’ will provide the pharmaceutical industry with information that can form the foundations of a comprehensive metabolomic database used for designing future personalized medications. Biomarker monitoring is another area worth shedding some light on. SPME has been used for the detection of volatile compounds that act as indicators of various disorders. Recent publications have described the use of SPME-GC–MS coupled with nanosensors for the successful identification of 42 volatile compounds [26]. These small molecules that correspond to lung cancer future science group
Is SPME a destination or just another station for bioanalysis? biomarkers were detected using patient breath sampling. Tumor growth biomarkers and potential regulators of angiogenesis have also been captured by SPME [13]. If these as yet uncommercialized prototype devices could be further developed into commercial tools with selective coatings specific for biomarkers, then SPME can add further prognostic values to the industry. What about peptide biomarkers and larger biopharmaceutical molecules such as antibodies and proteins? The development of biotherapeutics is now an integral part of the pharmaceutical industry where, immunoassays have traditionally dominated the field of quantifying such molecules. However, with current advances in LC–MS/MS, it has been possible to accurately measure peptides and proteins with sufficiently reduced LLOQ. So although it may not yet be so well established, with intricate design of SPME fibers, it may prove feasible to coat fibers with specific antibodies to act as binding beds for antigens. This will enable a highly specific ‘lock and key’ mechanism between the fiber and the target harvested from the complex biological matrix, all of which would take place within the living organism, avoiding the necessity to collect blood samples. SPME applications could potentially be further extended to tissue analysis. The device can penetrate organ tissues without causing much regional damage compared with microdialysis probes [27]. Solvent compatibility and the difficulty of coupling microdialysis with LC–MS remains unsolved; therefore, the use of SPME coupled with LC–MS will enable detection of low concentrations without the ion suppression associated with microdialysis [27]. Measurements of drug levels in the brain of conscious free-moving rodents have been performed without the requirement for organ removal [13,15,28]. This opens the door for quantitative PK and TK analysis of drugs with complete organ exposure or accumulation profiles, without the need to take terminal samples for subsequent tissue homogenizing and wet-sample analysis. The clinical suitability of SPME brings another remarkable element to the drug development table. Although sample volume is not a major concern when dealing with the majority of human subjects, there are numerous cases where avoidance of blood withdrawal is essential, specifically in pediatric studies or blood coagulation disorders [16,17]. To date, most human SPME applications have been clustered around breath and skin analysis. However, the quantification of future science group
| P erspective
analgesic drugs in human urine samples has also been reported [13,29,30]. A notable novel application anticipated for SPME within the clinical arena is the possibility of the technique to serve as a diagnostic tool in the operating theater. Monitoring of blood drug concentrations during surgery is a critical element of many clinical procedures. An example that illustrates this significance is anesthetic management of liver transplantation patients. The function of the hepatic system varies during liver transplantation surgeries; this has a direct impact on the metabolism of the combination of drugs used for general anesthetic [31]. SPME can provide a simple method to measure the concentration of parent drug and metabolites throughout the various stages of the transplant process. This in turn will enable dosage control of anesthetics during surgical procedures [30,31]. Coupling SPME with current separation and detection techniques has allowed for the quantification of low drug concentrations. Although GC–MS has been the main analytical technique utilized in conjunction with SPME [32], the development of new SPME-LC–MS interfaces have facilitated the on-site application of SPME ana lysis [3]. The growth of commercial SPME autosamplers used for analyte desorption has enabled high-throughput analysis. This has played a major role in saving time and overall simplification of the whole process [23]. Hurdles to adoption Before marching down the SPME route, it is vital to take into account all of the barriers that may face the adoption of the technique within the pharmaceutical industry. The first is the invasiveness of the device, which is expected to be met with some apprehension from regulatory authorities [33]. Historically the regulatory environment has been a challenging place for innovation [34]. However, authors and users of SPME must work closely with regulators to embrace and address any compliance concerns. In-depth investigations are necessary to ensure that no adverse effects are associated with the insertion of the device into the living organism. Biocompatibility and sterility are the key characteristics needed to prove the safety of the device. It is also crucial that quality bioanalytical SPME methods are validated using current GLP regulatory standards. The collection of high-quality data that can support the validity of SPME and show a favorable benefit to risk ratio will steer the regulatory mood towards a receptive climate and enable proof-of-concept [34,35]. www.future-science.com
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Ahmad Alongside regulatory approval, SPME requires acceptance by fellow scientists, including bioanalysts, animal technicians and physicians. To assist this process, adaptation of the current tool is imperative in order to create a user-friendly and convenient device [2,13]. Although commercially available, the existing SPME product has not been extensively tested within animal laboratories or clinics. Collaborations between academic experts, analysts, toxicology technicians, hospital users and manufacturers will be a vital step for the development of SPME into a medical tool that can be marketed to accommodate customers’ needs. It is also important to execute a systematic evaluation of the technology, paying particular attention to certain characteristics that may become road blockers if ignored. One of these is the length of in vivo equilibration time, which will influence the number of time points that could be attained, as well as the amount of discomfort caused if the fiber is left inside the living system for a long period of time [36]. Another is interfiber reproducibility, which will have an impact on the overall accuracy and precision of the data obtained. A further hurdle is the availability of appropriate fiber coatings that can enable extraction of drugs over a wide range of physiochemical properties [23]. Taking these points into consideration will underpin the analytical applicability of the technique
and establish solid foundations for regulatory recognition. Future perspective Taking this journey may require resources in terms of time, expertise and financial input, but the gain is far greater to all disciplines within the pharmaceutical and medical world. In the author’s opinion, the recent innovative techniques brought about and used by bioanalysts are gradually shaping the future of the pharmaceutical industry. SPME has the potential to become a tool that will follow the life of the drug from the time of its discovery through to the time it becomes available to patients [10,15,36]. On-site drug extraction without the need to sacrifice any matrix will bring about a revolution and must become a destination for bioanalysis to modernize and simplify current sampling and sample extraction procedures. The author is confident that this technique can reserve a front-row place within the analytical arena. Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Executive summary Potential benefits of solid-phase microextraction
Solid-phase microextraction (SPME) is a blood-free microsampling technique with many benefits awaiting further exploration within the pharmaceutical field.
Various forms of SPME could potentially be developed to serve several areas that employ analyte quantification, ranging from drug discovery and development through to clinical, and ending in operating theaters.
The application of SPME is pending in new avenues such as quantification of large biopharmaceutical molecules and biomarkers.
Challenging but achievable
The invasiveness of the device, the analytical limitations of the technique and sterility may become barriers if not extensively investigated.
There is a current unmet need for direct on-site drug analysis, and this could be met by adoption and development of SPME. If correctly implemented, the future of the technique will be bright.
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