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Bioanalytical applications of solid-phase microextraction
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Trends in Analytical Chemistry, Vol. 26, No. 1, 2007
Bioanalytical applications of solid-phase microextraction Florin Marcel Musteata, Janusz Pawliszyn The development of solid-phase microextraction (SPME) has experienced significant growth since its introduction as a new approach to sample preparation in the early 1990s. In comparison to existing technologies, such as liquid-liquid or solid-phase extraction, SPME offers many advantages, including simplicity, speed, solventless extraction, and a convenient format for the analyst. The objective of this review is to discuss the most recent developments and future challenges in the application of SPME to in vivo and in vitro bioanalytical problems. We discuss applications of fiber SPME for determination of binding constants and free concentrations, inside living organisms or in conventional samples. We show that the ability to perform direct and selective extraction of analytes from complex samples greatly extends the applications of SPME into the field of bioanalysis. ª 2006 Elsevier Ltd. All rights reserved. Keywords: Bioanalysis; Complex sample; Solid-phase microextraction; SPME
1. Introduction Florin Marcel Musteata, Janusz Pawliszyn* Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada
*
Corresponding author. Tel.: +1 519 888 4641; Fax: +1 519 746 0435; E-mail: [email protected]
36
Bioanalytical chemistry is playing an increasingly central role in the fields of academic and industrial science. It overlaps with a diverse range of disciplines, including biotechnology, biopharmaceuticals, and diagnostics [1]. Bioanalytical chemistry can be defined as the development and application of chemical measurements and instrumentation to problems in biology, biochemistry, and medical science. For the pharmaceutical industry, bioanalytical chemistry is often synonymous with measurements in biological samples, typically in support of investigations of drug metabolism and pharmacokinetics [2]. Biological materials and pharmaceutical products are very complex mixtures. They often contain proteins, salts, acids, bases and numerous organic compounds that may be similar to the analyte of interest. Furthermore, the analytes often exist at low concentration in these samples. Despite significant advances in the development of highly efficient analytical
instruments for the end-point determination of analytes in biological samples and pharmaceutical products, a pre-treatment step is usually necessary to extract and isolate the analytes of interest from complex matrices. The goal of sample preparation is to eliminate interfering compounds from the matrix using a minimum number of steps, resulting in a reproducible methodology. Many of the current sample-preparation challenges are addressed by solid-phase microextraction (SPME), which was specifically developed to provide rapid sample preparation both in the laboratory and on-site (where the investigated system is located). With this technique, a small amount of extracting phase, which is dispersed on a solid support, is exposed to the sample for a well-defined period of time [3]. Based on the total time of contact between the sample and the extraction phase, two extraction methods are used: (i) equilibrium extraction, when the partition equilibrium is reached; and, (ii) pre-equilibrium extraction, when the sample makes contact with the extraction phase for a short period. Several variations of SPME are based on the geometry of the extraction phase, such as coated fibers, vessels, stir bars, disks, and coatings on the inside of tubes. In terms of extraction from the sample and subsequent delivery to an analytical instrument, the most convenient approach is the fiber-SPME design. Although SPME was initially applied only for the analysis of organic compounds from rather clean samples (air, water), it is now increasingly being used in bioanalysis (in vitro and in vivo) for the determination of proteins, polar alkaloids, pharmaceuticals and surfactants, because of its successful coupling with liquid
0165-9936/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2006.11.003
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chromatography (LC), capillary electrophoresis (CE) and mass spectrometry (MS) devices [4–6]. SPME is an excellent alternative to classical methods for separating drugs and biomolecules from biological samples. SPME is simpler and faster, and provides markedly cleaner extracts than methods based on liquid-liquid extraction or solid-phase extraction. In the past 10 years, the number of reports on the applications of SPME in bioanalysis increased from 10 to more than 300. In the relatively few years of its use, SPME has become a mature technique and a useful alternative to contemporary techniques in various scientific and research fields. Not surprisingly, SPME was one of the six ‘‘great ideas of the decade’’ as illustrated in a recent survey of Analytical Chemistry [7]. This review will present recent advances and future trends in SPME method development for the analysis of endogenous and exogenous compounds in biological samples, with a focus on the determination of free concentrations and binding constants.
2. Analysis of biological samples Analysis of drugs in biological samples and pharmaceutical products is growing in importance because of the need to understand therapeutic and toxic effects of drugs and the continuing development of more selective and more effective drugs [3]. Interest in the field of drug analysis is focusing on improving methodologies, with regard to how quickly, accurately and sensitively the
chemicals can be detected. The field is highly dependent on the development of new analytical instruments and techniques [8]. Knowledge of drug levels in body fluids, such as serum, saliva, and urine, allows the optimization of pharmacotherapy and provides the basis for studies on patient compliance, bioavailability, pharmacokinetics and genetics, organ function and the influences of co-medication [9]. The quantitative and qualitative analysis of drugs and metabolites is extensively applied in pharmacokinetic studies and therapeutic drug monitoring. Drugs of abuse, illicit drugs and intoxications by drugs and poisons are often analyzed in clinical and forensic toxicology. Although most biological samples are currently analyzed in vitro, many attempts are now directed towards in vivo analysis. It has been shown that the composition of the volatile extracts collected from detached or damaged plants can differ significantly from the mixture emitted by the live, undamaged specimen [10]. In vivo research is more suited to observing an overall effect than in vitro research, which is better suited to deducing molecular mechanisms of action. In vitro research aims to describe and understand the effect of an experimental variable on a subset of an organismÕs components. In vitro research has the advantage that there are fewer variables that can confound an experiment and the results are clearly visible. In vivo research has the advantage that the experimental system is a more complex biological system, and provides a better indication of what will happen in the real world. Table 1 includes some of the
Table 1. Selected recent applications of SPME in bioanalysis Analytes
Biological sample
Type of investigation
Extraction time
Extraction phase(s)
Ref.
Ibuprofen, Warfarin, Verapamil, Propranolol, Caffeine Diazepam and metabolites
Human plasma
Determination of plasma protein binding
10 min
Polypyrrole Polydimethylsiloxane
[27]
Whole blood (beagle vein, in vivo)
Quantitative analysis of total and free concentration Quantitative analysis Quantitative analysis
30 s 2 min
Hydrophilic polypyrrole Polyethylene glycol
[17]
30 min 45–60 min
[14] [13]
[15]
7-aminoflunitrazepam Angiotensin 1 Angiotensin 2 Diazepam Isosorbide dinitrate Chlorhexidine and its degradation products
Human urine Whole human blood
Ibuprofen Naproxen Angiotensin 2 Neurotensin Diazepam and metabolites Benzodiazepines
Human urine
Whole blood (beagle vein, in vivo) Human urine
Valproic acid
Human plasma
Human serum albumin Human saliva
Determination of binding parameters Quantitative analysis of total and free concentration Method development
15 min
Specific antibodies Exchange diol silica (restricted access) Alkyl diol silica (restricted access) Carbowax
5 min
Antibodies
[35]
Quantitative analysis
30 min
Polypyrrole
[6]
Quantitative analysis
5–60 min
[32]
Determination of free concentrations
5 min
Alkyl diol silica (restricted access) Polydimethylsiloxane
8 min
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[38]
[26]
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most recent applications of SPME in the analysis of biological samples. 2.1. In vitro assays In vitro applications of SPME developed to date include the analysis of drugs from serum, plasma, whole blood, milk, urine, saliva and hair, by headspace, direct immersion SPME and in-tube SPME. Generally, the analysis of biological fluids is encumbered by the presence of dissolved biopolymers. For example, 7–8% of human plasma comprises proteins. The composition of plasma can be subject to considerable differences due to pathological and non-pathological influences. For example, plasma albumin can be decreased to about 50% of the normal level in hepatic diseases, and the concentration of lipoprotein-bound triglycerides depends on dietary status. The main issues to be considered for qualitative and quantitative analysis in plasma and other biological samples include [11]: (i) a change in selectivity, because of interferences from endogenous substances; (ii) analyte binding to biopolymers; and, (iii) high viscosity of the sample. Analyte binding to biopolymers results in a decrease in the sensitivity for methods based on SPME. However, this also presents a unique advantage of SPME over other sample-preparation methods, because a direct assay of the free concentration can be performed without separating the phases. The viscosity of plasma and blood in vitro is about three times higher than the viscosity of water. Because the diffusion coefficients are inversely related to viscosity, the diffusion of analytes in the plasma is approximately three times slower than in a predominantly aqueous phase. When the extraction speed is controlled by diffusion in the sample, an increase in the equilibration time is expected. By increasing the extraction temperature, the distribution constant of the drug between the fiber coating and the sample decreases. At the same time, the analyte diffusion rate increases because of lower viscosity and higher diffusion coefficient. Consequently, SPME methods can be optimized by selecting extraction temperatures that result in satisfactory sensitivity in an acceptable period of time [12]. Applications of SPME in bioanalysis can be divided into eight main groups, according to the type of analyte: toxicological analysis; forensic analysis; drugs of abuse; clinical chemistry; analysis of pharmaceuticals in biological samples; biochemical analysis; semiochemical analysis; and, analysis of natural products [5]. A general problem with the applications of SPME in bioanalysis continues to be the lack of availability of commercial SPME coatings for polar and ionic 38
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analytes, such as endogenous peptides, pharmaceutical drugs and their metabolites. As a solution, many researchers have decided to develop their own extraction phases. In a recent application, a novel restricted access material with cation-exchange properties was evaluated for the extraction of two peptides, angiotensin 1 and 2, from whole blood. The ion-exchange diol silica (XDS) material exhibited enhanced efficiency for the extraction of peptides when compared with the conventional alkyl diol silica with reversed phase extraction centers. This new coating for SPME offered the possibility of combining all initial sample-preparation steps into one, even for complex biological samples, such as whole blood or plasma. Compounds extracted from whole blood were separated by narrow-bore HPLC and quantified by electrospray ionization-MS (ESI-MS). Total analysis time, including sample preparation, was less than 90 min. In addition, a single fiber could be used more than 150 times before a noticeable decrease in extraction capacity was observed [13]. SPME probes for drug analysis have been prepared with benzodiazepine-specific antibodies covalently immobilized on the surface of the fiber [14]. For extraction, immobilized antibody probes were exposed to a sample containing the drug for 30 min. Extracted drugs were subsequently desorbed from the probes in methanolic desorption solution, which was dried, reconstituted in a small volume of injection solution and analyzed by LC-tandem MS. The antibodies were characterized both before and after immobilization, to facilitate the optimal selection of antibodies for such analyses. The probes were evaluated for a range of benzodiazepines at sub-ng/mL concentrations. This would allow for quantitative analysis of samples at concentrations below those measurable by many other methods for analysis of benzodiazepines. Commercially available Carbowax fibers have also been successfully used for the direct extraction of chlorhexidine from saliva during a pharmacokinetic study (Fig. 1, [15]). Such methods of analysis based on SPME are economical and much faster compared to classical approaches. 2.2. In vivo studies In recent years, there has been considerable research into the development of techniques to monitor levels of biologically active compounds in living systems in their natural environment. In vivo sampling can eliminate errors and reduce the time associated with sample transport and storage, and can therefore result in more accurate, more precise analytical data [16]. An ideal in vivo sampling technique should be portable, solventfree and offer integration of the sampling, samplepreparation and analysis steps. Reliable and accurate analytical methods are indispensable for in vivo research.
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Figure 1. Chromatograms of saliva samples at different times after administration of chlorohexidine: (a) 0.25 h; (b) 1 h; (c) 4 h; (d) 8 h (Reproduced from [15] with permission of Elsevier, ª 2005).
Conversely, the development of techniques appropriate for in vivo analysis poses significant difficulties, due to the low and unceasingly changing concentrations of target analytes in complex biological samples. In vivo analysis is a special application area where SPME is gaining ground because of its unique characteristics, which include: on-site sampling; ease of extraction; and, analysis of the whole extracted amount. In any microextraction or membrane technique, compounds of interest are not exhaustively removed from the investigated system. On the contrary, conditions can be conceived where only a small proportion of the total compound is removed, thus avoiding the disruption of the normal balance of the chemical components. Early in vivo investigations with SPME focused on fragrances emitted by insects, fungi and bacteria. These investigations were extended to biogenic volatile organic compounds emitted by animals and plants. In a more recent application, SPME technology was used for the in vivo analysis of intravenous drug concentrations in a living animal. A novel SPME probe was developed and its effectiveness was demonstrated by acquiring the free and total concentration pharmacokinetic profiles of diazepam, nordiazepam and oxazepam. The method was validated by comparison to conventional sampling methods [17]. For the majority of in vivo applications of SPME, the sensitivity and the precision provided by SPME are comparable or better than those of the techniques traditionally employed for the same samples. Moreover, some applications would not be feasible using other sample-preparation methods, since they would cause severe damage to the live organisms or would demand their sacrifice [10].
3. Determination of free concentrations and binding constants The first step in all biological activities is essentially an interaction between separate molecular constituents, the ligand and the receptor (usually a protein), to form a molecular complex. Such interactions play a vital role in all basic life sciences, including biochemistry, biophysics, pharmacology, physiology, immunology, endocrinology, neurobiology, molecular biology and cell biology [18]. The freely dissolved concentration of the ligand is an important parameter in environmental chemistry, pharmacology and toxicology. In the environment, for example, the free concentration is the driving force for the transport, distribution and bioaccumulation of a chemical. In pharmacology and toxicology, it is generally accepted that only freely dissolved molecules can pass through the cell membranes and thus be effective in organisms [19]. Drug binding to specific plasma transport proteins (albumin, a1-acid glycoprotein, lipoproteins) is an integral step of many other types of intermolecular interactions in a cellular or organ environment. If one aspires to carry out a thorough study of biological responses to molecular stimuli, the strength of the binding of a ligand to its receptor must be investigated. All of these binding constants and interactions can be investigated by measuring either the concentrations of the bound or the free form. 3.1. Free concentrations Several methods have been developed to measure the free concentration of compounds in a sample, most of which involve the physical separation of the free fraction and the bound fraction followed by a conventional analysis step. Examples of separation techniques include equilibrium dialysis, ultrafiltration and gel filtration. These techniques are usually time-consuming, can suffer http://www.elsevier.com/locate/trac
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loss of analyte to membranes, or can create a shift in the binding equilibrium during the separation [19,20]. Recently developed chromatographic methods [21] allow only for the assay of the fraction of drug bound to proteins, a value that is known to fluctuate with drug and protein concentration, and the mobile phase that is used (50 mM ammonium acetate pH 7 with 4% or 20% isopropanol) is very different from physiological conditions. The same is true for methods based on ESI-MS [22], which are effective only when certain buffer solutions are used and when the combination ratio between the receptor and the ligand is 1:1. When this combination ratio is different, the equations developed cannot be applied, and the interpretation of the resulting mass spectra becomes very difficult. While chromatographic methods used for the assay of free concentrations and binding constants assume a very fast equilibrium (less than a few seconds) between the ligand and the receptor, ultrafiltration and ultracentrifugation techniques assume a slow equilibrium (more than 30 min), so the free fraction can pass through a membrane without shifting the equilibrium in the other compartment. The elegant solution offered by the flow-dialysis techniques [23] suffers from the need to use radiolabeled ligands and is applicable only when the combination ratio is 1:1. Electrophoretic methods have been applied for combination ratios of both 1:1 [24] and 1:n [25] between the receptor and the ligand, but they are restricted to certain buffer solutions and do not allow for precise control of the temperature. While ESI-MS, CE and chromatographic methods are only effective when the samples are dissolved in certain buffer solutions, the SPME method can be used for extraction from any media, and at any concentration range, through the selection of a suitable extraction phase. SPME is proposed as a new technique for extraction and concentration of target compounds from a complex matrix in order to determine their free concentrations [19,20,26–31]. Because of the small size of the extractive phase and partial extraction approach, this technique allows for the simultaneous analysis of the analyte and the matrix. Compared to other methods, SPME offers several advantages: small sample size; short analysis time; possibility of automation; and, direct study of complex samples (e.g., whole blood). The study of binding equilibria is no longer restricted to certain buffer solutions, but can be performed in any ‘‘natural’’ environment, by optimizing the experimental conditions. With the introduction of new extracting phases (restricted access materials [13,32], molecularly imprinted polymers [33,34], and fibers with immobilized antibodies [14,35,36]), SPME offers improved accuracy and selective separation of small ligand molecules from 40
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larger receptor molecules (matrix, proteins) in any sample. The SPME technique can be applied by exposing the fiber in the headspace above the sample or directly in the sample (direct immersion). The advantage of headspace SPME is that the matrix in the sample cannot interfere with the fiber, but it is applicable only for volatile compounds in a non-volatile matrix. While most applications of SPME are developed to achieve the highest possible extraction efficiency, Kopinke et al. [37] and Vaes et al. [29] have independently introduced a specific application of SPME to measure free concentrations based on negligible extraction of the free concentration. Because this application causes negligible depletion of the free concentration, it has been named negligible depletion SPME (nd-SPME). Nevertheless, measurement of free concentrations with SPME can be performed both in negligible or significant depletion conditions. As an example, the free concentration of lidocaine in plasma was successfully measured without applying nd-SPME [28]. Many theoretical approaches have been designed for the determination of free concentrations by SPME. One of the simplest methods is based on calculating the free concentration as the ratio between the amount of analyte extracted and the fiber constant [38]. Briefly, in the presence of an SPME fiber, an amount m (moles) of a drug is extracted from the solution and this amount, which is on the fiber, will be in equilibrium with the free concentration (Fig. 2). The free concentration of drug remaining in the solution is then given by: Cfree ¼
m fc
ð1Þ
where fc is the fiber constant and represents the product of the partition coefficient of the drug (between fiber and solution without binding matrix) and the volume of the fiber (for liquid coatings) or the active surface of the fiber (for solid coatings). By using special materials for the extracting phase, the large receptor molecules are prevented from being co-extracted. Such an approach is equally applicable for negligible and non-negligible depletions, for any combination ratio between the ligand and the receptor, and is independent of the analysis method. The quantification of the amount of ligand extracted may be performed by any method that can be coupled to SPME, including LC, gas chromatography (GC), MS, CE, or radiometry. The method was successfully applied for the in vivo and in vitro determination of free concentrations during pharmacokinetic studies [15,17]. Whole-blood concentration and free concentrations, easily obtained with SPME, are of utmost importance in therapeutics, as they correlate with the pharmacological effect and are more significant than plasma concentrations. Fig. 3 presents
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SPME fiber
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SPME fiber
Ligand bound to receptor – [R(L)b] Free receptor Free ligand - Cf - [R]
Ki
K fs
+ Ligand absorbed onto ADS-SPME fiber - m
Vortex
Figure 2. Schematic representation of experimental set-up for the determination of free concentrations and binding constants (Reproduced from [38] with permission of American Chemical Society, ª 2005).
30.00 Conc. (ng/mL)
Free Conc. (ng/mL)
10.00
25.00 20.00
8.00 6.00 4.00
Diazepam
2.00 0.00
15.00
Nordiazepam 1.5
2.5
3.5
4.5
5.5
6.5
7.5
8.5
Oxazepam
Time (h)
10.00 5.00 0.00 0
1
2
3
4
5
6
7
8
9
Time (h) Figure 3. Comparative pharmacokinetic profile for the free concentration of diazepam, nordiazepam and oxazepam in beagies (obtained by in vivo microextraction) (Reproduced from [17] with permission of AACC, ª 2006).
the free-concentration profile for diazepam and two if its metabolites in beagle whole blood, and reveals a fast in vivo conversion of diazepam to oxazepam, exhibiting similar free-concentration values. The free concentrations were determined by calibration with standard solutions of benzodiazepines in phosphate-buffered saline, pH = 7.4, while the total concentrations were determined by calibration with standard solutions in whole blood. Such results for total concentration are meaningful when the blood composition does not change significantly during a pharmacokinetic study. Conversely, reliable measurements of the free concentration can be obtained even when the concentration of plasmatic proteins changes, because the amount of analyte extracted by SPME is inherently related to the free concentration. This is an important advantage, as the free concentration is a valuable parameter in pharmacological applications. 3.2. Study of ligand-receptor binding The investigation of binding parameters has received significant attention since its importance was recognized
at the beginning of the twentieth century, and dozens of research papers are published yearly on this topic. Different aspects of ligand-receptor interactions have been reviewed, including their molecular nature, biological functions, and pharmacological significance, as well as the methodological approaches applied and their potential shortcomings [18,39]. When several ligand molecules can be bound by a receptor molecule, multiple equilibria are established. These can be described by different types of equilibrium constants, which reflect different perspectives in visualizing the equilibria. The multiple equilibria for the binding of a ligand (L) by a receptor (R) with a number of binding sites (b) may be formulated in terms of a stoichiometric analysis or on the basis of a site-oriented analysis [18,40]. In the case of step-wise stoichiometric equilibria, the number of ligand molecules bound per receptor molecule (B) can be expressed as a function of the free ligand concentration (Cf), the total-ligand concentration (Ct), the stoichiometric binding constants (ki), and the receptor concentration (Cm): http://www.elsevier.com/locate/trac
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B¼ ¼
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Ct Cf Cm k1 Cf þ 2k1 k2 C2f þ . . . þ bðk1 k2 . . . kb ÞCbf 1 þ k1 Cf þ k1 k2 C2f þ . . . þ ðk1 k2 . . . kb ÞCbf
ð2Þ
For the site-oriented approach, each site is considered to have a fixed, invariant affinity. Accordingly, stoichiometric binding constants (ki) are replaced with site binding constants (Ki), and the number of moles of ligand bound per moles of receptor is: B¼
b b X Ct Cf X K i Cf ¼ Bi ¼ Cm 1 þ K i Cf i¼1 i¼1
ð3Þ
A special case that is often encountered involves a system with two classes of binding sites, each with identical invariant affinities that differ from the identical invariant affinities of the other class [18,25,41]. Under these circumstances, Equation (3) can be reduced to: B¼
Ct Cf b1 K 1 Cf b2 K 2 Cf ¼ þ Cm 1 þ K 1 Cf 1 þ K 2 Cf
ð4Þ
where b1 and b2 are the number of sites in the respective classes (b1 + b2 = b, the total number of binding sites), and K1 and K2 are the respective site binding constants. Equations (3) and (4), although frequently used because of their convenience, often lead to non-real (complex) solutions, especially when the binding constants increase with increased occupancy of the receptor, as is the case with many enzymes or carrier proteins (e.g., hemoglobin). Regardless of the chosen mathematical model, the binding parameters are determined from pairs of Cf and Ct for a certain Cm. While Cm and Ct are usually known when a standard solution is prepared, Ct is usually determined with Equation (1). This method was successfully applied to obtain the binding curves of diazepam (a polar drug with a high binding constant – more than 105 L/mol) and isosorbide dinitrate (non-polar, with a low binding constant – less than 105 L/mol) to human serum albumin. The binding constants were subsequently used to calculate receptor, free and total ligand concentrations in synthetic samples [38]. A similar approach proved successful for the determination of the binding constant of chlorohexidine to salivary proteins [15]. When the amount of sample (receptor) is limited, the method of multiple extractions or multiple additions permits the generation of selected regions of the binding curve with a single small volume of receptor solution. Unlike other methods that assume a short or long equilibration time for the binding of ligands to receptors, SPME methods allow one to observe whether equilibrium has been reached during a certain period of time, for each ligand and receptor pair. The test is performed by determining the period of time after which the amount of ligand extracted from a solution with a 42
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binding matrix reaches a constant value. This period of time represents either the extraction equilibration time or the binding equilibration time, whichever is longer, and can subsequently be used for all experiments. The mathematical model based on Equations (1)–(4) works equally well with negligible and non-negligible extractions. In the case of non-negligible extractions, reliable studies may be performed even for drugs with a high binding constant, since some of the ligand bound to the receptor will move into the extraction phase, increasing the sensitivity. In this case, a fiber with a high fc for the target ligand should be used. This is an important advantage over dialysis, where the affinity of the acceptor buffer solution cannot be easily changed. 3.3. Determination of drug–plasma-protein binding Determining the amount of drug binding to plasma proteins is an essential step in both drug discovery and in clinical phases of drug development [42–45]. Plasmaprotein binding (PPB) affects the amount of drug available for diffusion into target tissues, such as the brain [46–48], the calculation of in vivo hepatic clearance [49], and the interpretation of the drugÕs bioavailability [43]. Due to the important clinical implications of PPB data and the role of PPB in characterizing a drugÕs behavior and proper dosing, there is increased need to make this measurement as early as possible in the discovery process, in order to understand drug disposition and to optimize individual drug therapy. Although the main drug-binding proteins are albumin and alpha 1-acid glycoprotein, plasma contains many other proteins, so there is a high probability that many small molecules will exhibit some levels of binding. To determine the extent of PPB, the molecule should be tested directly in a protein-binding assay using plasma or serum. This is a critical step in characterizing the distribution of a small molecule with respect to the plasma compartment [21,46,50]. The determination of PPB by SPME is based on determining the free concentration of drug in the presence of plasma proteins [27]. When extraction from a volume (V) of plasma containing binding proteins is performed, an amount (mplasma) of drug will be extracted by the fiber coating. Considering that the initial concentration of the drug is C0 plasma, the total final concentration of the drug in plasma is then given by: mplasma Ctotal ¼ C0 plasma ð5Þ V The free concentration of the drug in plasma is readily calculated with Equation (1): mplasma Cfree plasma ¼ ð6Þ fc Finally, the PPB percentage is calculated from the total and free concentrations of the drug:
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PPB% ¼
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Ctotal Cfree plasma 100 Ctotal
ð7Þ
This method of calculation can be easily extended to determine the PPB in diluted plasma samples, when the assay sensitivity is significantly increased for highly bound drugs. It has long been recognized that plasma pH has a significant influence on the extent of binding to plasmatic proteins. Plasma pH can change considerably during storage and even during short-term incubation. Conversely, most binding studies do not employ any means for controlling the pH of plasma samples. Usually, the binding of acid drugs is minimally influenced by experimental conditions and pH, whereas the binding of basic drugs is more susceptible to changes in the experimental conditions. This large variation of binding values, in the case of basic drugs, may be explained if the influence of pH on the degree of ionization is considered. At pH = 7.4, acid drugs are almost completely ionized and a small change in pH will not affect the degree of ionization. In the case of basic drugs, a small change in pH causes a significant change in the ionized fraction of the drug. For analytical methods based on SPME, changes in plasma pH can also induce changes in the amount of analyte extracted. Accordingly, control of the plasma pH is vital for accurate determinations of the target analyte. Table 2 presents a summary of the different methods that can be used to stabilize the pH of plasma, with the corresponding pH values. Obviously, pH values closest to physiological conditions are obtained in the case of incubation with 10% CO2 or 1:10 dilution with isotonic phosphate-buffered saline (PBS). Incubation in 10% CO2 atmosphere offers an environment that closely mimics physiological conditions and should be used whenever possible. Otherwise, the 1:10 dilution method with isotonic PBS should be employed, in which case the con-
Table 2. Changes in plasma pH with experimental conditions (Reproduced from [27] with permission of Wiley, ª 2006) Experimental conditions
pH value
Raw plasma samples (up to 2 weeks at 4C) Plasma diluted 1:1 with isotonic PBS buffer Plasma diluted 1:10 with isotonic PBS buffer Plasma incubated 1h with 10% CO2 Physiological pH of plasma
8.00–8.40 7.60–7.70 7.40–7.50 7.50–7.60 7.35–7.42
centration of the drug must be monitored to ensure it is at least 10 times lower than that of the protein concentrations in the diluted plasma. Representation of drug binding as a percentage of the bound fraction leads to substantial compression of results for strongly bound drugs, and to broadening of results for weakly bound drugs. The results obtained with different techniques could be compared more easily if PPB values obtained for a specific total concentration are transformed to free concentrations. Fig. 4 presents the results of a recent PPB study based on SPME, for a total drug concentration of 1 lM. All experimental values are close to the diagonal of the graph, indicating a good correlation with average values in the literature [27].
4. Calibration of SPME for bioanalytical applications To date, several calibration approaches have been developed for SPME. Equilibrium extraction is the most frequently used method, which involves using a known distribution constant or an external calibration curve to correlate the amount of analyte extracted by the SPME fiber to its concentration in the sample: n ¼ K fs V f C0
ð8Þ
warfarin
Experimental pD
9.5
ibuprofen
8.5
Standard approach Dilution 1:1 Dilution 1:10 CO2 headpressure
verapamil propranolol
7.5 caffeine
Literature max
6.5 5.5 5.5
Literature min
6.5
7.5
8.5
9.5
Average pD from literature Figure 4. Correlation of experimentally obtained pD values with average values from literature, at a total drug concentration of 1 lM (Reproduced from [27] with permission of Wiley, ª 2006).
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where n is the number of moles of analyte extracted, Kfs is the distribution coefficient of the analyte between the fiber coating and the sample matrix, C0 is the concentration of a given analyte in the sample, and Vf is the volume of the fiber. To shorten equilibrium extraction times, and/or address the displacement effects that occur when porous coatings are used, extraction can be interrupted before equilibrium. Even though extraction equilibrium is not reached, there is still a linear relationship between the amount of analyte extracted onto the fiber and the analyte concentration in the sample matrix, provided that the agitation, the extraction time, and the extraction temperature remain constant [30]: n ¼ K fs V f C0 ð1 ea:t Þ
ð9Þ
where t is the extraction time, and a is a time constant, representing the speed at which an equilibrium can be reached. This method eliminates the use of conventional calibration curves. Fast on-site analysis and long-term monitoring are thus possible [3]. While performing derivatization at the same time as extraction, if the reaction is the rate-limiting step, the first-order reactionrate constant (Kr1) can be used for calibration: n ¼ K rl C0
ð10Þ
In addition to these ‘‘classic’’ calibration methods, the newly developed method of ‘‘kinetic calibration’’ appears to be particularly useful for in vivo determinations. When an SPME coating that is preloaded with a standard compound is exposed to an agitated sample matrix, desorption of the compound from the fiber occurs. The desorbed compound diffuses through the boundary layer into the bulk of the sample matrix. The amount (Q) of standard remaining on the coating after time t can be described as: Q ¼ q0 ea:t
ð11Þ
where q0 is the initial amount of standard present on the fiber. The constant (a) in Equation (9) for the absorption has the same definition as constant (a) in Equation (11) for the desorption, and should have the same value for both the absorption and the desorption of an analyte, under the same experimental conditions (sample bulk velocity and temperature). The isotropy of absorption and desorption in SPME allows for the calibration of absorption (n) using desorption (Q): n Q þ ¼1 n0 q0
ð12Þ
where n0 is the amount of analyte extracted at equilibrium [17]. This is especially important for the calibration of on site and in vivo analyses, because control of the agitation conditions of the matrix is sometimes difficult and direct 44
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spiking of standards into the matrix is typically not possible. In addition to convenient applications for the determination of total concentrations of drugs and biomolecules, SPME was also introduced as a new technique for the determination of free concentrations (Fig. 2). In this case, calibration is usually based on the fiber constant, which represents the product of the partition coefficient of the analyte (between fiber and sample) and the volume of the fiber (for liquid coatings) or the active surface of the fiber (for solid coatings). The fiber constant may be easily determined by extracting the analyte from standard solutions in water or buffer when the total drug concentration is considered to be equal to the free concentration.
5. Conclusions SPME is a simple, solvent-free, reliable microextraction technique that has continued to revolutionize sampling and sample preparation since its discovery a decade ago. The small dimensions of SPME devices and their solventfree feature enable convenient sampling for bioanalytical applications, such as the analysis of biological samples, the measurement of free concentrations and the determination of binding constants. Furthermore, analysis of extracted compounds can be performed with highly specific instruments, such as GC-MS or LC-tandem MS. To date, SPME has been successfully applied around the world to a wide range of bioanalytical investigations, clearly demonstrating that the technique provides an excellent alternative to current sample-preparation methods. The development of biocompatible extraction phases for SPME has led to significant advances in bioanalysis: all sample preparation steps can be combined into a single one, even for complex biological samples such as whole blood or plasma. Furthermore, biocompatible devices permit the direct extraction of target analytes from the flowing blood of living organisms. Future research in this area should focus on applications for soft tissues, automation of sampling and analysis, and utilization of highly specific extraction phases. Direct extraction of target analytes from complex biological samples often results in co-extraction of interference compounds. In the case of solid (porous) coatings, co-extraction is an important issue because the interferant may displace the analyte from the extraction phase. In addition, analysis of a complex extract requires a suitable separation method, such as GC or LC, which increases the total processing time. An important aspect of the future application and growth of SPME is the development of new extraction coatings. An obvious choice is the application of extraction phases that are specific for the target compound. SPME
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devices based on molecularly imprinted polymers or antibodies would possess unsurpassed specificity and would be especially useful at very low concentrations of target analyte. Such devices could be interfaced directly with mass spectrometers, resulting in very fast analytical methods.
References [1] S.R. Mikkelsen, E. Corton, BioAnal. Chem, John Wiley & Sons, New Jersey, USA, 2004. [2] C.K. Larive, Anal. Bioanal. Chem. 382 (2005) 855. [3] J. Pawliszyn (Editor), Sampling and Sample Preparation for Field and Laboratory: Fundamentals and New Directions in Sample Preparation, Comprehensive Anal. Chem, Vol. 37, Elsevier, Amsterdam, The Netherlands, 2002. [4] M. Abdel-Rehim, M. Bielenstein, T. Arvidsson, J. Microcolumn Sep. 12 (2000) 308. [5] G. Theodoridis, E.H.M. Koster, G.J. de Jong, J. Chromatogr., B 745 (2000) 49. [6] G. Vas, K. Vekey, J. Mass Spectrom. 39 (2004) 233. [7] G. Theodoridis, J. de Jong Gerhardus, Adv. Chromatogr. 43 (2005) 231. [8] T. Kumazawa, X.-P. Lee, K. Sato, O. Suzuki, Anal. Chim. Acta 492 (2003) 49. [9] H. Kataoka, Trends Anal. Chem. 22 (2003) 232. [10] F. Augusto, A. Luiz Pires Valente, Trends Anal. Chem. 21 (2002) 428. [11] S. Ulrich, J. Chromatogr., A 902 (2000) 167. [12] M.E.C. Queiroz, F.M. Lancas, LC-GC Europe 18 (2005) 145. [13] F.M. Musteata, M. Walles, J. Pawliszyn, Anal. Chim. Acta 537 (2005) 231. [14] H.L. Lord, M. Rajabi, S. Safari, J. Pawliszyn, J. Pharm. Biomed. Anal. 40 (2006) 769. [15] F.M. Musteata, J. Pawliszyn, J. Pharm. Biomed. Anal. 37 (2005) 1015. [16] J. Pawliszyn, Aust. J. Chem. 56 (2003) 155. [17] F.M. Musteata, M.L. Musteata, J. Pawliszyn, Clin. Chem. 52 (2006) 708. [18] I.M. Klotz, Ligand-Receptor Energetics: A Guide for the Perplexed, Wiley, New York, USA, 1997 170 pp. [19] M.B. Heringa, J.L.M. Hermens, Trends Anal. Chem. 22 (2003) 575. [20] M.B. Heringa, D. Pastor, J. Algra, W.H.J. Vaes, J.L.M. Hermens, Anal. Chem. 74 (2002) 5993. [21] Y. Cheng, E. Ho, B. Subramanyam, J.-L. Tseng, J. Chromatogr., B 809 (2004) 67.
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Bioanalytical applications of solid-phase microextraction Florin Marcel Musteata, Janusz Pawliszyn The development of solid-phase microextraction (SPME) has experienced significant growth since its introduction as a new approach to sample preparation in the early 1990s. In comparison to existing technologies, such as liquid-liquid or solid-phase extraction, SPME offers many advantages, including simplicity, speed, solventless extraction, and a convenient format for the analyst. The objective of this review is to discuss the most recent developments and future challenges in the application of SPME to in vivo and in vitro bioanalytical problems. We discuss applications of fiber SPME for determination of binding constants and free concentrations, inside living organisms or in conventional samples. We show that the ability to perform direct and selective extraction of analytes from complex samples greatly extends the applications of SPME into the field of bioanalysis. ª 2006 Elsevier Ltd. All rights reserved. Keywords: Bioanalysis; Complex sample; Solid-phase microextraction; SPME
1. Introduction Florin Marcel Musteata, Janusz Pawliszyn* Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada
*
Corresponding author. Tel.: +1 519 888 4641; Fax: +1 519 746 0435; E-mail: [email protected]
36
Bioanalytical chemistry is playing an increasingly central role in the fields of academic and industrial science. It overlaps with a diverse range of disciplines, including biotechnology, biopharmaceuticals, and diagnostics [1]. Bioanalytical chemistry can be defined as the development and application of chemical measurements and instrumentation to problems in biology, biochemistry, and medical science. For the pharmaceutical industry, bioanalytical chemistry is often synonymous with measurements in biological samples, typically in support of investigations of drug metabolism and pharmacokinetics [2]. Biological materials and pharmaceutical products are very complex mixtures. They often contain proteins, salts, acids, bases and numerous organic compounds that may be similar to the analyte of interest. Furthermore, the analytes often exist at low concentration in these samples. Despite significant advances in the development of highly efficient analytical
instruments for the end-point determination of analytes in biological samples and pharmaceutical products, a pre-treatment step is usually necessary to extract and isolate the analytes of interest from complex matrices. The goal of sample preparation is to eliminate interfering compounds from the matrix using a minimum number of steps, resulting in a reproducible methodology. Many of the current sample-preparation challenges are addressed by solid-phase microextraction (SPME), which was specifically developed to provide rapid sample preparation both in the laboratory and on-site (where the investigated system is located). With this technique, a small amount of extracting phase, which is dispersed on a solid support, is exposed to the sample for a well-defined period of time [3]. Based on the total time of contact between the sample and the extraction phase, two extraction methods are used: (i) equilibrium extraction, when the partition equilibrium is reached; and, (ii) pre-equilibrium extraction, when the sample makes contact with the extraction phase for a short period. Several variations of SPME are based on the geometry of the extraction phase, such as coated fibers, vessels, stir bars, disks, and coatings on the inside of tubes. In terms of extraction from the sample and subsequent delivery to an analytical instrument, the most convenient approach is the fiber-SPME design. Although SPME was initially applied only for the analysis of organic compounds from rather clean samples (air, water), it is now increasingly being used in bioanalysis (in vitro and in vivo) for the determination of proteins, polar alkaloids, pharmaceuticals and surfactants, because of its successful coupling with liquid
0165-9936/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2006.11.003
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chromatography (LC), capillary electrophoresis (CE) and mass spectrometry (MS) devices [4–6]. SPME is an excellent alternative to classical methods for separating drugs and biomolecules from biological samples. SPME is simpler and faster, and provides markedly cleaner extracts than methods based on liquid-liquid extraction or solid-phase extraction. In the past 10 years, the number of reports on the applications of SPME in bioanalysis increased from 10 to more than 300. In the relatively few years of its use, SPME has become a mature technique and a useful alternative to contemporary techniques in various scientific and research fields. Not surprisingly, SPME was one of the six ‘‘great ideas of the decade’’ as illustrated in a recent survey of Analytical Chemistry [7]. This review will present recent advances and future trends in SPME method development for the analysis of endogenous and exogenous compounds in biological samples, with a focus on the determination of free concentrations and binding constants.
2. Analysis of biological samples Analysis of drugs in biological samples and pharmaceutical products is growing in importance because of the need to understand therapeutic and toxic effects of drugs and the continuing development of more selective and more effective drugs [3]. Interest in the field of drug analysis is focusing on improving methodologies, with regard to how quickly, accurately and sensitively the
chemicals can be detected. The field is highly dependent on the development of new analytical instruments and techniques [8]. Knowledge of drug levels in body fluids, such as serum, saliva, and urine, allows the optimization of pharmacotherapy and provides the basis for studies on patient compliance, bioavailability, pharmacokinetics and genetics, organ function and the influences of co-medication [9]. The quantitative and qualitative analysis of drugs and metabolites is extensively applied in pharmacokinetic studies and therapeutic drug monitoring. Drugs of abuse, illicit drugs and intoxications by drugs and poisons are often analyzed in clinical and forensic toxicology. Although most biological samples are currently analyzed in vitro, many attempts are now directed towards in vivo analysis. It has been shown that the composition of the volatile extracts collected from detached or damaged plants can differ significantly from the mixture emitted by the live, undamaged specimen [10]. In vivo research is more suited to observing an overall effect than in vitro research, which is better suited to deducing molecular mechanisms of action. In vitro research aims to describe and understand the effect of an experimental variable on a subset of an organismÕs components. In vitro research has the advantage that there are fewer variables that can confound an experiment and the results are clearly visible. In vivo research has the advantage that the experimental system is a more complex biological system, and provides a better indication of what will happen in the real world. Table 1 includes some of the
Table 1. Selected recent applications of SPME in bioanalysis Analytes
Biological sample
Type of investigation
Extraction time
Extraction phase(s)
Ref.
Ibuprofen, Warfarin, Verapamil, Propranolol, Caffeine Diazepam and metabolites
Human plasma
Determination of plasma protein binding
10 min
Polypyrrole Polydimethylsiloxane
[27]
Whole blood (beagle vein, in vivo)
Quantitative analysis of total and free concentration Quantitative analysis Quantitative analysis
30 s 2 min
Hydrophilic polypyrrole Polyethylene glycol
[17]
30 min 45–60 min
[14] [13]
[15]
7-aminoflunitrazepam Angiotensin 1 Angiotensin 2 Diazepam Isosorbide dinitrate Chlorhexidine and its degradation products
Human urine Whole human blood
Ibuprofen Naproxen Angiotensin 2 Neurotensin Diazepam and metabolites Benzodiazepines
Human urine
Whole blood (beagle vein, in vivo) Human urine
Valproic acid
Human plasma
Human serum albumin Human saliva
Determination of binding parameters Quantitative analysis of total and free concentration Method development
15 min
Specific antibodies Exchange diol silica (restricted access) Alkyl diol silica (restricted access) Carbowax
5 min
Antibodies
[35]
Quantitative analysis
30 min
Polypyrrole
[6]
Quantitative analysis
5–60 min
[32]
Determination of free concentrations
5 min
Alkyl diol silica (restricted access) Polydimethylsiloxane
8 min
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[38]
[26]
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most recent applications of SPME in the analysis of biological samples. 2.1. In vitro assays In vitro applications of SPME developed to date include the analysis of drugs from serum, plasma, whole blood, milk, urine, saliva and hair, by headspace, direct immersion SPME and in-tube SPME. Generally, the analysis of biological fluids is encumbered by the presence of dissolved biopolymers. For example, 7–8% of human plasma comprises proteins. The composition of plasma can be subject to considerable differences due to pathological and non-pathological influences. For example, plasma albumin can be decreased to about 50% of the normal level in hepatic diseases, and the concentration of lipoprotein-bound triglycerides depends on dietary status. The main issues to be considered for qualitative and quantitative analysis in plasma and other biological samples include [11]: (i) a change in selectivity, because of interferences from endogenous substances; (ii) analyte binding to biopolymers; and, (iii) high viscosity of the sample. Analyte binding to biopolymers results in a decrease in the sensitivity for methods based on SPME. However, this also presents a unique advantage of SPME over other sample-preparation methods, because a direct assay of the free concentration can be performed without separating the phases. The viscosity of plasma and blood in vitro is about three times higher than the viscosity of water. Because the diffusion coefficients are inversely related to viscosity, the diffusion of analytes in the plasma is approximately three times slower than in a predominantly aqueous phase. When the extraction speed is controlled by diffusion in the sample, an increase in the equilibration time is expected. By increasing the extraction temperature, the distribution constant of the drug between the fiber coating and the sample decreases. At the same time, the analyte diffusion rate increases because of lower viscosity and higher diffusion coefficient. Consequently, SPME methods can be optimized by selecting extraction temperatures that result in satisfactory sensitivity in an acceptable period of time [12]. Applications of SPME in bioanalysis can be divided into eight main groups, according to the type of analyte: toxicological analysis; forensic analysis; drugs of abuse; clinical chemistry; analysis of pharmaceuticals in biological samples; biochemical analysis; semiochemical analysis; and, analysis of natural products [5]. A general problem with the applications of SPME in bioanalysis continues to be the lack of availability of commercial SPME coatings for polar and ionic 38
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analytes, such as endogenous peptides, pharmaceutical drugs and their metabolites. As a solution, many researchers have decided to develop their own extraction phases. In a recent application, a novel restricted access material with cation-exchange properties was evaluated for the extraction of two peptides, angiotensin 1 and 2, from whole blood. The ion-exchange diol silica (XDS) material exhibited enhanced efficiency for the extraction of peptides when compared with the conventional alkyl diol silica with reversed phase extraction centers. This new coating for SPME offered the possibility of combining all initial sample-preparation steps into one, even for complex biological samples, such as whole blood or plasma. Compounds extracted from whole blood were separated by narrow-bore HPLC and quantified by electrospray ionization-MS (ESI-MS). Total analysis time, including sample preparation, was less than 90 min. In addition, a single fiber could be used more than 150 times before a noticeable decrease in extraction capacity was observed [13]. SPME probes for drug analysis have been prepared with benzodiazepine-specific antibodies covalently immobilized on the surface of the fiber [14]. For extraction, immobilized antibody probes were exposed to a sample containing the drug for 30 min. Extracted drugs were subsequently desorbed from the probes in methanolic desorption solution, which was dried, reconstituted in a small volume of injection solution and analyzed by LC-tandem MS. The antibodies were characterized both before and after immobilization, to facilitate the optimal selection of antibodies for such analyses. The probes were evaluated for a range of benzodiazepines at sub-ng/mL concentrations. This would allow for quantitative analysis of samples at concentrations below those measurable by many other methods for analysis of benzodiazepines. Commercially available Carbowax fibers have also been successfully used for the direct extraction of chlorhexidine from saliva during a pharmacokinetic study (Fig. 1, [15]). Such methods of analysis based on SPME are economical and much faster compared to classical approaches. 2.2. In vivo studies In recent years, there has been considerable research into the development of techniques to monitor levels of biologically active compounds in living systems in their natural environment. In vivo sampling can eliminate errors and reduce the time associated with sample transport and storage, and can therefore result in more accurate, more precise analytical data [16]. An ideal in vivo sampling technique should be portable, solventfree and offer integration of the sampling, samplepreparation and analysis steps. Reliable and accurate analytical methods are indispensable for in vivo research.
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Figure 1. Chromatograms of saliva samples at different times after administration of chlorohexidine: (a) 0.25 h; (b) 1 h; (c) 4 h; (d) 8 h (Reproduced from [15] with permission of Elsevier, ª 2005).
Conversely, the development of techniques appropriate for in vivo analysis poses significant difficulties, due to the low and unceasingly changing concentrations of target analytes in complex biological samples. In vivo analysis is a special application area where SPME is gaining ground because of its unique characteristics, which include: on-site sampling; ease of extraction; and, analysis of the whole extracted amount. In any microextraction or membrane technique, compounds of interest are not exhaustively removed from the investigated system. On the contrary, conditions can be conceived where only a small proportion of the total compound is removed, thus avoiding the disruption of the normal balance of the chemical components. Early in vivo investigations with SPME focused on fragrances emitted by insects, fungi and bacteria. These investigations were extended to biogenic volatile organic compounds emitted by animals and plants. In a more recent application, SPME technology was used for the in vivo analysis of intravenous drug concentrations in a living animal. A novel SPME probe was developed and its effectiveness was demonstrated by acquiring the free and total concentration pharmacokinetic profiles of diazepam, nordiazepam and oxazepam. The method was validated by comparison to conventional sampling methods [17]. For the majority of in vivo applications of SPME, the sensitivity and the precision provided by SPME are comparable or better than those of the techniques traditionally employed for the same samples. Moreover, some applications would not be feasible using other sample-preparation methods, since they would cause severe damage to the live organisms or would demand their sacrifice [10].
3. Determination of free concentrations and binding constants The first step in all biological activities is essentially an interaction between separate molecular constituents, the ligand and the receptor (usually a protein), to form a molecular complex. Such interactions play a vital role in all basic life sciences, including biochemistry, biophysics, pharmacology, physiology, immunology, endocrinology, neurobiology, molecular biology and cell biology [18]. The freely dissolved concentration of the ligand is an important parameter in environmental chemistry, pharmacology and toxicology. In the environment, for example, the free concentration is the driving force for the transport, distribution and bioaccumulation of a chemical. In pharmacology and toxicology, it is generally accepted that only freely dissolved molecules can pass through the cell membranes and thus be effective in organisms [19]. Drug binding to specific plasma transport proteins (albumin, a1-acid glycoprotein, lipoproteins) is an integral step of many other types of intermolecular interactions in a cellular or organ environment. If one aspires to carry out a thorough study of biological responses to molecular stimuli, the strength of the binding of a ligand to its receptor must be investigated. All of these binding constants and interactions can be investigated by measuring either the concentrations of the bound or the free form. 3.1. Free concentrations Several methods have been developed to measure the free concentration of compounds in a sample, most of which involve the physical separation of the free fraction and the bound fraction followed by a conventional analysis step. Examples of separation techniques include equilibrium dialysis, ultrafiltration and gel filtration. These techniques are usually time-consuming, can suffer http://www.elsevier.com/locate/trac
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loss of analyte to membranes, or can create a shift in the binding equilibrium during the separation [19,20]. Recently developed chromatographic methods [21] allow only for the assay of the fraction of drug bound to proteins, a value that is known to fluctuate with drug and protein concentration, and the mobile phase that is used (50 mM ammonium acetate pH 7 with 4% or 20% isopropanol) is very different from physiological conditions. The same is true for methods based on ESI-MS [22], which are effective only when certain buffer solutions are used and when the combination ratio between the receptor and the ligand is 1:1. When this combination ratio is different, the equations developed cannot be applied, and the interpretation of the resulting mass spectra becomes very difficult. While chromatographic methods used for the assay of free concentrations and binding constants assume a very fast equilibrium (less than a few seconds) between the ligand and the receptor, ultrafiltration and ultracentrifugation techniques assume a slow equilibrium (more than 30 min), so the free fraction can pass through a membrane without shifting the equilibrium in the other compartment. The elegant solution offered by the flow-dialysis techniques [23] suffers from the need to use radiolabeled ligands and is applicable only when the combination ratio is 1:1. Electrophoretic methods have been applied for combination ratios of both 1:1 [24] and 1:n [25] between the receptor and the ligand, but they are restricted to certain buffer solutions and do not allow for precise control of the temperature. While ESI-MS, CE and chromatographic methods are only effective when the samples are dissolved in certain buffer solutions, the SPME method can be used for extraction from any media, and at any concentration range, through the selection of a suitable extraction phase. SPME is proposed as a new technique for extraction and concentration of target compounds from a complex matrix in order to determine their free concentrations [19,20,26–31]. Because of the small size of the extractive phase and partial extraction approach, this technique allows for the simultaneous analysis of the analyte and the matrix. Compared to other methods, SPME offers several advantages: small sample size; short analysis time; possibility of automation; and, direct study of complex samples (e.g., whole blood). The study of binding equilibria is no longer restricted to certain buffer solutions, but can be performed in any ‘‘natural’’ environment, by optimizing the experimental conditions. With the introduction of new extracting phases (restricted access materials [13,32], molecularly imprinted polymers [33,34], and fibers with immobilized antibodies [14,35,36]), SPME offers improved accuracy and selective separation of small ligand molecules from 40
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larger receptor molecules (matrix, proteins) in any sample. The SPME technique can be applied by exposing the fiber in the headspace above the sample or directly in the sample (direct immersion). The advantage of headspace SPME is that the matrix in the sample cannot interfere with the fiber, but it is applicable only for volatile compounds in a non-volatile matrix. While most applications of SPME are developed to achieve the highest possible extraction efficiency, Kopinke et al. [37] and Vaes et al. [29] have independently introduced a specific application of SPME to measure free concentrations based on negligible extraction of the free concentration. Because this application causes negligible depletion of the free concentration, it has been named negligible depletion SPME (nd-SPME). Nevertheless, measurement of free concentrations with SPME can be performed both in negligible or significant depletion conditions. As an example, the free concentration of lidocaine in plasma was successfully measured without applying nd-SPME [28]. Many theoretical approaches have been designed for the determination of free concentrations by SPME. One of the simplest methods is based on calculating the free concentration as the ratio between the amount of analyte extracted and the fiber constant [38]. Briefly, in the presence of an SPME fiber, an amount m (moles) of a drug is extracted from the solution and this amount, which is on the fiber, will be in equilibrium with the free concentration (Fig. 2). The free concentration of drug remaining in the solution is then given by: Cfree ¼
m fc
ð1Þ
where fc is the fiber constant and represents the product of the partition coefficient of the drug (between fiber and solution without binding matrix) and the volume of the fiber (for liquid coatings) or the active surface of the fiber (for solid coatings). By using special materials for the extracting phase, the large receptor molecules are prevented from being co-extracted. Such an approach is equally applicable for negligible and non-negligible depletions, for any combination ratio between the ligand and the receptor, and is independent of the analysis method. The quantification of the amount of ligand extracted may be performed by any method that can be coupled to SPME, including LC, gas chromatography (GC), MS, CE, or radiometry. The method was successfully applied for the in vivo and in vitro determination of free concentrations during pharmacokinetic studies [15,17]. Whole-blood concentration and free concentrations, easily obtained with SPME, are of utmost importance in therapeutics, as they correlate with the pharmacological effect and are more significant than plasma concentrations. Fig. 3 presents
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SPME fiber
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SPME fiber
Ligand bound to receptor – [R(L)b] Free receptor Free ligand - Cf - [R]
Ki
K fs
+ Ligand absorbed onto ADS-SPME fiber - m
Vortex
Figure 2. Schematic representation of experimental set-up for the determination of free concentrations and binding constants (Reproduced from [38] with permission of American Chemical Society, ª 2005).
30.00 Conc. (ng/mL)
Free Conc. (ng/mL)
10.00
25.00 20.00
8.00 6.00 4.00
Diazepam
2.00 0.00
15.00
Nordiazepam 1.5
2.5
3.5
4.5
5.5
6.5
7.5
8.5
Oxazepam
Time (h)
10.00 5.00 0.00 0
1
2
3
4
5
6
7
8
9
Time (h) Figure 3. Comparative pharmacokinetic profile for the free concentration of diazepam, nordiazepam and oxazepam in beagies (obtained by in vivo microextraction) (Reproduced from [17] with permission of AACC, ª 2006).
the free-concentration profile for diazepam and two if its metabolites in beagle whole blood, and reveals a fast in vivo conversion of diazepam to oxazepam, exhibiting similar free-concentration values. The free concentrations were determined by calibration with standard solutions of benzodiazepines in phosphate-buffered saline, pH = 7.4, while the total concentrations were determined by calibration with standard solutions in whole blood. Such results for total concentration are meaningful when the blood composition does not change significantly during a pharmacokinetic study. Conversely, reliable measurements of the free concentration can be obtained even when the concentration of plasmatic proteins changes, because the amount of analyte extracted by SPME is inherently related to the free concentration. This is an important advantage, as the free concentration is a valuable parameter in pharmacological applications. 3.2. Study of ligand-receptor binding The investigation of binding parameters has received significant attention since its importance was recognized
at the beginning of the twentieth century, and dozens of research papers are published yearly on this topic. Different aspects of ligand-receptor interactions have been reviewed, including their molecular nature, biological functions, and pharmacological significance, as well as the methodological approaches applied and their potential shortcomings [18,39]. When several ligand molecules can be bound by a receptor molecule, multiple equilibria are established. These can be described by different types of equilibrium constants, which reflect different perspectives in visualizing the equilibria. The multiple equilibria for the binding of a ligand (L) by a receptor (R) with a number of binding sites (b) may be formulated in terms of a stoichiometric analysis or on the basis of a site-oriented analysis [18,40]. In the case of step-wise stoichiometric equilibria, the number of ligand molecules bound per receptor molecule (B) can be expressed as a function of the free ligand concentration (Cf), the total-ligand concentration (Ct), the stoichiometric binding constants (ki), and the receptor concentration (Cm): http://www.elsevier.com/locate/trac
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B¼ ¼
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Ct Cf Cm k1 Cf þ 2k1 k2 C2f þ . . . þ bðk1 k2 . . . kb ÞCbf 1 þ k1 Cf þ k1 k2 C2f þ . . . þ ðk1 k2 . . . kb ÞCbf
ð2Þ
For the site-oriented approach, each site is considered to have a fixed, invariant affinity. Accordingly, stoichiometric binding constants (ki) are replaced with site binding constants (Ki), and the number of moles of ligand bound per moles of receptor is: B¼
b b X Ct Cf X K i Cf ¼ Bi ¼ Cm 1 þ K i Cf i¼1 i¼1
ð3Þ
A special case that is often encountered involves a system with two classes of binding sites, each with identical invariant affinities that differ from the identical invariant affinities of the other class [18,25,41]. Under these circumstances, Equation (3) can be reduced to: B¼
Ct Cf b1 K 1 Cf b2 K 2 Cf ¼ þ Cm 1 þ K 1 Cf 1 þ K 2 Cf
ð4Þ
where b1 and b2 are the number of sites in the respective classes (b1 + b2 = b, the total number of binding sites), and K1 and K2 are the respective site binding constants. Equations (3) and (4), although frequently used because of their convenience, often lead to non-real (complex) solutions, especially when the binding constants increase with increased occupancy of the receptor, as is the case with many enzymes or carrier proteins (e.g., hemoglobin). Regardless of the chosen mathematical model, the binding parameters are determined from pairs of Cf and Ct for a certain Cm. While Cm and Ct are usually known when a standard solution is prepared, Ct is usually determined with Equation (1). This method was successfully applied to obtain the binding curves of diazepam (a polar drug with a high binding constant – more than 105 L/mol) and isosorbide dinitrate (non-polar, with a low binding constant – less than 105 L/mol) to human serum albumin. The binding constants were subsequently used to calculate receptor, free and total ligand concentrations in synthetic samples [38]. A similar approach proved successful for the determination of the binding constant of chlorohexidine to salivary proteins [15]. When the amount of sample (receptor) is limited, the method of multiple extractions or multiple additions permits the generation of selected regions of the binding curve with a single small volume of receptor solution. Unlike other methods that assume a short or long equilibration time for the binding of ligands to receptors, SPME methods allow one to observe whether equilibrium has been reached during a certain period of time, for each ligand and receptor pair. The test is performed by determining the period of time after which the amount of ligand extracted from a solution with a 42
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binding matrix reaches a constant value. This period of time represents either the extraction equilibration time or the binding equilibration time, whichever is longer, and can subsequently be used for all experiments. The mathematical model based on Equations (1)–(4) works equally well with negligible and non-negligible extractions. In the case of non-negligible extractions, reliable studies may be performed even for drugs with a high binding constant, since some of the ligand bound to the receptor will move into the extraction phase, increasing the sensitivity. In this case, a fiber with a high fc for the target ligand should be used. This is an important advantage over dialysis, where the affinity of the acceptor buffer solution cannot be easily changed. 3.3. Determination of drug–plasma-protein binding Determining the amount of drug binding to plasma proteins is an essential step in both drug discovery and in clinical phases of drug development [42–45]. Plasmaprotein binding (PPB) affects the amount of drug available for diffusion into target tissues, such as the brain [46–48], the calculation of in vivo hepatic clearance [49], and the interpretation of the drugÕs bioavailability [43]. Due to the important clinical implications of PPB data and the role of PPB in characterizing a drugÕs behavior and proper dosing, there is increased need to make this measurement as early as possible in the discovery process, in order to understand drug disposition and to optimize individual drug therapy. Although the main drug-binding proteins are albumin and alpha 1-acid glycoprotein, plasma contains many other proteins, so there is a high probability that many small molecules will exhibit some levels of binding. To determine the extent of PPB, the molecule should be tested directly in a protein-binding assay using plasma or serum. This is a critical step in characterizing the distribution of a small molecule with respect to the plasma compartment [21,46,50]. The determination of PPB by SPME is based on determining the free concentration of drug in the presence of plasma proteins [27]. When extraction from a volume (V) of plasma containing binding proteins is performed, an amount (mplasma) of drug will be extracted by the fiber coating. Considering that the initial concentration of the drug is C0 plasma, the total final concentration of the drug in plasma is then given by: mplasma Ctotal ¼ C0 plasma ð5Þ V The free concentration of the drug in plasma is readily calculated with Equation (1): mplasma Cfree plasma ¼ ð6Þ fc Finally, the PPB percentage is calculated from the total and free concentrations of the drug:
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PPB% ¼
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Ctotal Cfree plasma 100 Ctotal
ð7Þ
This method of calculation can be easily extended to determine the PPB in diluted plasma samples, when the assay sensitivity is significantly increased for highly bound drugs. It has long been recognized that plasma pH has a significant influence on the extent of binding to plasmatic proteins. Plasma pH can change considerably during storage and even during short-term incubation. Conversely, most binding studies do not employ any means for controlling the pH of plasma samples. Usually, the binding of acid drugs is minimally influenced by experimental conditions and pH, whereas the binding of basic drugs is more susceptible to changes in the experimental conditions. This large variation of binding values, in the case of basic drugs, may be explained if the influence of pH on the degree of ionization is considered. At pH = 7.4, acid drugs are almost completely ionized and a small change in pH will not affect the degree of ionization. In the case of basic drugs, a small change in pH causes a significant change in the ionized fraction of the drug. For analytical methods based on SPME, changes in plasma pH can also induce changes in the amount of analyte extracted. Accordingly, control of the plasma pH is vital for accurate determinations of the target analyte. Table 2 presents a summary of the different methods that can be used to stabilize the pH of plasma, with the corresponding pH values. Obviously, pH values closest to physiological conditions are obtained in the case of incubation with 10% CO2 or 1:10 dilution with isotonic phosphate-buffered saline (PBS). Incubation in 10% CO2 atmosphere offers an environment that closely mimics physiological conditions and should be used whenever possible. Otherwise, the 1:10 dilution method with isotonic PBS should be employed, in which case the con-
Table 2. Changes in plasma pH with experimental conditions (Reproduced from [27] with permission of Wiley, ª 2006) Experimental conditions
pH value
Raw plasma samples (up to 2 weeks at 4C) Plasma diluted 1:1 with isotonic PBS buffer Plasma diluted 1:10 with isotonic PBS buffer Plasma incubated 1h with 10% CO2 Physiological pH of plasma
8.00–8.40 7.60–7.70 7.40–7.50 7.50–7.60 7.35–7.42
centration of the drug must be monitored to ensure it is at least 10 times lower than that of the protein concentrations in the diluted plasma. Representation of drug binding as a percentage of the bound fraction leads to substantial compression of results for strongly bound drugs, and to broadening of results for weakly bound drugs. The results obtained with different techniques could be compared more easily if PPB values obtained for a specific total concentration are transformed to free concentrations. Fig. 4 presents the results of a recent PPB study based on SPME, for a total drug concentration of 1 lM. All experimental values are close to the diagonal of the graph, indicating a good correlation with average values in the literature [27].
4. Calibration of SPME for bioanalytical applications To date, several calibration approaches have been developed for SPME. Equilibrium extraction is the most frequently used method, which involves using a known distribution constant or an external calibration curve to correlate the amount of analyte extracted by the SPME fiber to its concentration in the sample: n ¼ K fs V f C0
ð8Þ
warfarin
Experimental pD
9.5
ibuprofen
8.5
Standard approach Dilution 1:1 Dilution 1:10 CO2 headpressure
verapamil propranolol
7.5 caffeine
Literature max
6.5 5.5 5.5
Literature min
6.5
7.5
8.5
9.5
Average pD from literature Figure 4. Correlation of experimentally obtained pD values with average values from literature, at a total drug concentration of 1 lM (Reproduced from [27] with permission of Wiley, ª 2006).
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where n is the number of moles of analyte extracted, Kfs is the distribution coefficient of the analyte between the fiber coating and the sample matrix, C0 is the concentration of a given analyte in the sample, and Vf is the volume of the fiber. To shorten equilibrium extraction times, and/or address the displacement effects that occur when porous coatings are used, extraction can be interrupted before equilibrium. Even though extraction equilibrium is not reached, there is still a linear relationship between the amount of analyte extracted onto the fiber and the analyte concentration in the sample matrix, provided that the agitation, the extraction time, and the extraction temperature remain constant [30]: n ¼ K fs V f C0 ð1 ea:t Þ
ð9Þ
where t is the extraction time, and a is a time constant, representing the speed at which an equilibrium can be reached. This method eliminates the use of conventional calibration curves. Fast on-site analysis and long-term monitoring are thus possible [3]. While performing derivatization at the same time as extraction, if the reaction is the rate-limiting step, the first-order reactionrate constant (Kr1) can be used for calibration: n ¼ K rl C0
ð10Þ
In addition to these ‘‘classic’’ calibration methods, the newly developed method of ‘‘kinetic calibration’’ appears to be particularly useful for in vivo determinations. When an SPME coating that is preloaded with a standard compound is exposed to an agitated sample matrix, desorption of the compound from the fiber occurs. The desorbed compound diffuses through the boundary layer into the bulk of the sample matrix. The amount (Q) of standard remaining on the coating after time t can be described as: Q ¼ q0 ea:t
ð11Þ
where q0 is the initial amount of standard present on the fiber. The constant (a) in Equation (9) for the absorption has the same definition as constant (a) in Equation (11) for the desorption, and should have the same value for both the absorption and the desorption of an analyte, under the same experimental conditions (sample bulk velocity and temperature). The isotropy of absorption and desorption in SPME allows for the calibration of absorption (n) using desorption (Q): n Q þ ¼1 n0 q0
ð12Þ
where n0 is the amount of analyte extracted at equilibrium [17]. This is especially important for the calibration of on site and in vivo analyses, because control of the agitation conditions of the matrix is sometimes difficult and direct 44
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spiking of standards into the matrix is typically not possible. In addition to convenient applications for the determination of total concentrations of drugs and biomolecules, SPME was also introduced as a new technique for the determination of free concentrations (Fig. 2). In this case, calibration is usually based on the fiber constant, which represents the product of the partition coefficient of the analyte (between fiber and sample) and the volume of the fiber (for liquid coatings) or the active surface of the fiber (for solid coatings). The fiber constant may be easily determined by extracting the analyte from standard solutions in water or buffer when the total drug concentration is considered to be equal to the free concentration.
5. Conclusions SPME is a simple, solvent-free, reliable microextraction technique that has continued to revolutionize sampling and sample preparation since its discovery a decade ago. The small dimensions of SPME devices and their solventfree feature enable convenient sampling for bioanalytical applications, such as the analysis of biological samples, the measurement of free concentrations and the determination of binding constants. Furthermore, analysis of extracted compounds can be performed with highly specific instruments, such as GC-MS or LC-tandem MS. To date, SPME has been successfully applied around the world to a wide range of bioanalytical investigations, clearly demonstrating that the technique provides an excellent alternative to current sample-preparation methods. The development of biocompatible extraction phases for SPME has led to significant advances in bioanalysis: all sample preparation steps can be combined into a single one, even for complex biological samples such as whole blood or plasma. Furthermore, biocompatible devices permit the direct extraction of target analytes from the flowing blood of living organisms. Future research in this area should focus on applications for soft tissues, automation of sampling and analysis, and utilization of highly specific extraction phases. Direct extraction of target analytes from complex biological samples often results in co-extraction of interference compounds. In the case of solid (porous) coatings, co-extraction is an important issue because the interferant may displace the analyte from the extraction phase. In addition, analysis of a complex extract requires a suitable separation method, such as GC or LC, which increases the total processing time. An important aspect of the future application and growth of SPME is the development of new extraction coatings. An obvious choice is the application of extraction phases that are specific for the target compound. SPME
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devices based on molecularly imprinted polymers or antibodies would possess unsurpassed specificity and would be especially useful at very low concentrations of target analyte. Such devices could be interfaced directly with mass spectrometers, resulting in very fast analytical methods.
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