Analysis of drugs in Saliva and other body fluids: Microextraction - Essay Example

? Analysis of drugs in Saliva and other body fluids: Microextraction Contents Introduction 2 Saliva and bodily fluids as a target for analysis 3 Solid Phase Microextraction (SPME) 4 Advantages of SPME 4 Drawbacks and Disadvantages 7 Advances in SPME 9 Conclusions 11 References 12 Introduction: With the advent of a tremendous increase in diagnostic procedures and biochemical evaluation of biological fluids, the typical lag time for analysis has been significantly reduced. Using novel methods in separation science, analytes in bodily fluids can be qualified and quantified within a matter of a few minutes. The most widely used techniques include HPLC, GC-MS, LC-MS methods of separation and analyses. In addition to these, chromatographic variants such as electrophoresis (capillary and SDS-PAGE) and TLC are frequently used. Liquid-liquid or solid-liquid extractions, shakeout and partitioning have been the age-old procedures to achieve separation. Figure depicting Single drop Microextraction: (Sarafraz Yazdi, 2010) In this article, it would be to discuss an uncommon technique in separation chromatography: Microextraction (ME). This novel technique has gained popularity in the past decade, and can be categorized as a modification and combination of the Gas and partitioning chromatographies. Solid-phase microextraction is a comparatively distinct and a unique method in separation chemistry, which is based on the principle of chromatography. This technique introduced in the late 90s, can be used in the analysis of a solution having a very small amount of analyte. (Hinshaw, 2003) The figure above represents the schematic representation of the single drop microextraction technique, which is in wide use today. This will be discussed in the course of this essay. Saliva and bodily fluids as a target for analysis: The physiological fluid saliva, which is secreted by the salivary glands in the oral cavity, could be used as a valuable diagnostic tool for detection of drugs and their subsequent separation. A normal healthy adult produces about 500-1500 mL of saliva per day (Chiappin et al., 2007). After drug administration, its clearance from systemic circulation into saliva may be a result of factors such as ultrafiltration, passive diffusion and/or active transport. Some of the major advantages associated with saliva as a diagnostic aid, are its non-invasive collection protocol and ease of obtaining samples. A majority of drugs and metabolites can be easily and readily detected in saliva, even after hours of administration. Immunoglobulins, proteinaceous molecules, lipids and psychotropic metabolites could be quantified. The extents to which drugs manifest in saliva, in comparison with urine, are much less because the glomeruli in the kidney concentrate the urine. This results in an increase of the drug-urine concentration. Additionally, the volume of urine, produced daily is significantly higher in volume than saliva, adding to the ease of collection and further study. (Chiappin et al., 2007) (Aps and Martens, 2005) Although these minor drawbacks exist, investigation of saliva for presence of endogenous compounds could be conveniently handled. Solid Phase Microextraction (SPME): The principle of Solid Phase Microextraction (SPME) is based on the premise that the analyte in a sample is taken up and adsorbed on to an SPME adsorption layer. This process of adsorption continues to a point of equilibrium, following which, the solute molecules are transferred along with the SPME layer into a secondary system. Washing the adsorbed sample with the appropriate solvent brings about this secondary step. This system, which usually comprises of solvents of varying polarities, is responsible for desorbing the captured molecules. Once disassociated from the complex, the analyte molecules are quantitated using the existing robust techniques such as MS/MS or LC-MS. Essentially, the principle of microextraction rests on that of the GC. (Bojko et al., 2011) Constructive modifications of this method include liquid phase, capillary and dispersive liquid-liquid microextractions, among other minor derivatives. The dispersive ME method is a very specialized method, and the principle, protocol and advantages will be briefly discussed during the course of this article. Advantages of SPME: I would now like to discuss the advantages that the standalone microextraction process offers, over other commonly used analytical procedures for extraction. As requested, 5 recent scientific articles have been chosen and the major benefits have been highlighted, in comparison to some of the widely used chromatography techniques. Typically analytical procedures such as the GC or HPLC involve using a relatively larger volume of sample, when the analysis is restricted to using these techniques. The typical injection volume for satisfactory separation could range from a few ?l to mL. When in combination with ME, an analyte volume of 1-2 ?L could be used, wherein the extraction solvent usually never exceeds 10-12 ?L. ME does not require a set-up of complex instruments or carrier gases. Typically, a small fibrous matrix, which is coated on the inner side of a short and narrow tube, is utilized. Alternatively, this matrix could be coated on the outer surface of a tubular carrier, which is employed for sample collection. A precoated injection syringe, for example could be used to withdraw a biological sample from systemic circulation. (Junting et al., 1998) This will be discussed in one of the following paragraphs. The figure below (Kataoka et al., 2009) shows some of the commonly employed column or capillary packing methods. Contrary to GC or HPLC separations, where expensive carrier gases such as ultra-pure Helium or bone-dry grades of nitrogen are imperative for smooth running of the instrument, ME can be carried out under atmospheric air conditions. For the initial separation procedures, there is no stringent pressure requirement, as opposed to some other chromatographies, where vacuum pumps are mandatory. Figure depicting packing of columns or capillaries (Kataoka et al., 2009) Derivatization is a commonly employed step in GC analysis, in which non-volatile components are reacted with TMS, a special solvent, which renders the non-volatile compound capable of ionization when injected into the column. This extra step of preparing derivatives can be skipped in quite a few cases in ME and becomes necessary only when very specialized samples are being handled. The sensitivity, accuracy and precision with ME techniques are extremely high, and when combined with analytical equipment such as the MS, a high degree of precision is achieved and maintained. (Kataoka et al., 2009, Yamada and Kakehi, 2011) Some body fluids such as urine or blood are typically associated with a large number of components- blood cells, a high level of proteins, salts, and metabolites of drugs that the subject has been administered. When the ME technique is used for simpler fluids such as saliva or sweat, a majority of these interfering materials are absent. Considering the fact that quite a few drugs or their metabolites manifest themselves in salivary secretions, and the ease of saliva collection, this method proves to be convenient. (Ai et al., 2010) Some inherent components of saliva, which include enzymes such as alpha amylase, proteins and mucin, can be easily denatured by heat treatment. This avoids their interference while the compound of interest is being extracted. (Bojko et al., 2011) (Yamada and Kakehi, 2011) A significant number of biologicals are thermolabile. Hence, the GC clearly is not the right choice, as temperatures in the column and detector can rise up to as high as 300 degrees celcius, which, denature the compounds. HPLC determinations are usually made in dissolving the sample in a buffer, or solvents such as acetonitrile, water or methanol. Hydrolysis is a frequently encountered problem in such a situation, since HPLC runs are anywhere from a few seconds to some hours. ME techniques utilize a small column of solvent and the analyte is exposed to the solvent for a very limited period of time, thus limiting the incidences of degradation due to heat or hydrolysis. Partitioning processes, such as liquid-liquid shakeouts involve fairly large volumes of organic solvents, and emulsion formation is often encountered. The lag time for the emulsion to break and the two phases to separate could involve losing precious time. ME techniques, as the name suggests, utilizes solvents on a micro-scale and this eases the handling of separation. In my opinion, these are the reasons why I think microextraction is an important process. Drawbacks and disadvantages: After having discussed the advantages, I would like to point out some of the drawbacks associated with the Microextraction method. Similar to coating a capillary for electrophoretic determinations, the capillary used for ME have to be uniformly coated with the adsorbent. These packed tubes are referred to as cartridges. Although the cartridges are not expensive, they may have to be changed regularly due to leaching of the coating material, or changes in the thickness of the adsorbing layer, as a result of repeated usage. This usually gives us improper binding affinities of the analyte, which can almost always be immediately detected by a loss of signal when analyzed by MS. (Abdel-Rehim, 2011) ME is not the most preferred method if the analyte has poor solubility in the matrix, which in our discussion would refer to saliva. Since the size of the droplet is extremely minute at about 10 ?L, the actual sample that gets extracted could be below the levels of detection when subject to further analysis. In some samples, the affinity of the analyte to the adsorptive surface is so strong, that a larger volume of eluting or desorbing solvent is required, with some degree of agitation or heating. During this process, air bubbles are created which affect the equilibrium and dynamics of the reaction, and may damage the cartridge. (Ganjali et al., 2010) Similarly, recovery of the analyte, which gets entrapped within the lumen and fibers of the adsorptive surface, could pose some challenges. Advances in SPME: After looking at the major advantages and disadvantages offered by the microextraction method, I would now like to discuss the advances in this field and the scope of future improvement. Apart from the basic routine separations that the microextraction process is routinely used for, it could be extended to more specialized analyses, which would be tricky to carry out with traditional methods. The term in vivo, refers to processes occurring within a living organism. In pharmacokinetic studies, drug metabolites are analyzed, once urine or blood samples are obtained from the participants after varying intervals of time. Such studies could be made more dynamic by performing real-time analyses. This is carried out by direct insertion of a hypodermic needle into a vein. The needle is coated with the adsorbing surface, which has affinity to the analyte under study. This can be integrated with a GC or LC-MS for continuous monitoring. Thus, the need for withdrawing blood at regular intervals is eliminated. (Nuhu et al., 2011) Similarly, this technique could be employed for the separation and quantification of volatile components in breath exhalations, such as ethanol, and correlated to its concentration in systemic circulation. (Groschl et al., 2008) High throughput screening methods such as the 96 well plate assay used in ELISA, spectrophotometric or colorimetric determinations could be linked to a detector, using the microextraction principle. This could be suitable used for end-point determinations. For example, the amount of liberated glucose or a chromophore such as p-nitrophenol could be determined accurately when these products formed in situ are adsorbed on the membrane. This gives the user the flexibility of continuous monitoring, without having to stop an ongoing reaction by removing the plate from an inaccessible location, such as an incubator. Microextraction could also be extended to enzyme kinetic studies, where the amounts of free enzyme or substrate can be accurately determined. Open tubular capillary ME utilizing multiple capillaries is an innovation in this field of separation. In this technique, multiple columns are used, each bearing a different adsorptive surface. This procedure could be resorted to, if 2 or 3 components in a sample are adsorbed non-selectively on to the fibers. Upon adsorption, this complex is dissociated using appropriate solvents and re-subjected to additional tubular capillary microcolumns, varying in composition of the fibrous coat. (Kataoka et al., 2009) (Nuhu et al., 2011) (Pena-Pereira et al., 2010) The residual solvent after one run can be utilized for subsequent separation and purification. It is a known fact that an increase in surface area increases the rate of reaction. In common chromatographic language, the number of theoretical plates is increased exponentially when the surface area within a column is increased, and this results in better resolution and cleaner separations. In ME, when the surface area within the capillary tubes is increased, the binding equilibrium is attained much faster. (Kataoka et al., 2009) The insertion of either fine stainless wires, or rods of rigid heterocyclic polymers offer dual benefits- a reduction in the void volume of the capillary, and an increase in the surface area. Solid drop based liquid microextraction (SDLME) is a breakthrough in this field, where a hanging droplet of the solution to be separated, is brought into contact with the extraction phase. Gentle agitation with a magnetic stir bar ensures maximum separation of the analyte from the test droplet into the organic solvent. A continuous process could be developed too, where in the droplet has been concentrated to a high level of the compound of interest, and is constantly exposed to the extraction medium. A frequent change of the solvent will ensure removal of the analyte. On concentration of the solvent, the analyte can be recovered. In this method, some of the contributing parameters, which could lead to an experiment failure and thus need to be monitored constantly, are surface tension, partition coefficients, density, agitation speeds (rate of stirring) and temperature. (Sarafraz Yazdi, 2010) We can compare this method to a liquid-liquid shakeout in a separatory funnel. We are well aware that the volume of solvent used for extraction, and subsequent washings are significant. Often, problems such as formation of a third immiscible layer, or emulsification are encountered. Following the extraction procedure, combining and concentration of the solvents is a time consuming exercise, and this could be eliminated by the SDLPME method. (Vuckovic et al., 2010) Conclusions: The microextraction process, which is an offshoot of the GC or HPLC methods of analyses, is a scientific breakthrough in separation science. The principle of this process has made it possible to miniaturize the entire working, without the need of a large quantity of solvent. The use of this process is not limited to separation, but can also be extended to in vivo and pharmacokinetic studies, where the analyte of choice could be captured, concentrated and recovered. Due to the short preparation and process times, the method is useful in isolating thermally unstable or easily hydrolysable compounds. After weighing the benefits and limitations offered by the microextraction process, and the ease with which it could be linked to detectors of the HPLC, GC or atomic absorption spectrophotometers, I believe that this method is a revolution in separation science. Easy portability and elimination of pressurized devices and extreme temperature conditions should make it a choice of a lot of researchers and scientists for their future design of methods for separation studies. References: ABDEL-REHIM, M. 2011. Microextraction by packed sorbent (MEPS): a tutorial. Anal Chim Acta, 701, 119-28. AI, J., SMITH, B. & WONG, D. T. 2010. Saliva Ontology: an ontology-based framework for a Salivaomics Knowledge Base. BMC Bioinformatics, 11, 302. APS, J. K. & MARTENS, L. C. 2005. Review: The physiology of saliva and transfer of drugs into saliva. Forensic Sci Int, 150, 119-31. BOJKO, B., MIRNAGHI, F. & PAWLISZYN, J. 2011. Solid-phase microextraction: a multi-purpose microtechnique. Bioanalysis, 3, 1895-9. CHIAPPIN, S., ANTONELLI, G., GATTI, R. & DE PALO, E. F. 2007. Saliva specimen: a new laboratory tool for diagnostic and basic investigation. Clin Chim Acta, 383, 30-40. GANJALI, M. R., SOBHI, H. R., FARAHANI, H., NOROUZI, P., DINARVAND, R. & KASHTIARAY, A. 2010. Solid drop based liquid-phase microextraction. J Chromatogr A, 1217, 2337-41. GROSCHL, M., KOHLER, H., TOPF, H. G., RUPPRECHT, T. & RAUH, M. 2008. Evaluation of saliva collection devices for the analysis of steroids, peptides and therapeutic drugs. J Pharm Biomed Anal, 47, 478-86. HINSHAW, J. 2003. Solid-Phase Microextraction. LC.GC Europe. Europe: Serveron Corp. JUNTING, L., PENG, C. & SUZUKI, O. 1998. Solid-phase microextraction (SPME) of drugs and poisons from biological samples. Forensic Sci Int, 97, 93-100. KATAOKA, H., ISHIZAKI, A., NONAKA, Y. & SAITO, K. 2009. Developments and applications of capillary microextraction techniques: a review. Anal Chim Acta, 655, 8-29. NUHU, A. A., BASHEER, C. & SAAD, B. 2011. Liquid-phase and dispersive liquid-liquid microextraction techniques with derivatization: recent applications in bioanalysis. J Chromatogr B Analyt Technol Biomed Life Sci, 879, 1180-8. PENA-PEREIRA, F., LAVILLA, I. & BENDICHO, C. 2010. Liquid-phase microextraction approaches combined with atomic detection: a critical review. Anal Chim Acta, 669, 1-16. SARAFRAZ YAZDI, A. 2010. Liquid-phase Microextraction. Trends in Analytical Chemistry, 29, 14. VUCKOVIC, D., ZHANG, X., CUDJOE, E. & PAWLISZYN, J. 2010. Solid-phase microextraction in bioanalysis: New devices and directions. J Chromatogr A, 1217, 4041-60. YAMADA, K. & KAKEHI, K. 2011. Recent advances in the analysis of carbohydrates for biomedical use. J Pharm Biomed Anal, 55, 702-27. Read More