Literature Thesis THE APPLICATION OF MICROEXTRACTION FOR DETERMINATION OF DRUGS IN BIOLOGICAL SAMPLES

Transcription

1 Literature Thesis THE APPLICATION OF MICROEXTRACTION FOR DETERMINATION OF DRUGS IN BIOLOGICAL SAMPLES Febri Annuryanti Supervisor: Dr. Henk Lingeman Master in Chemistry Analytical Sciences University of Amsterdam

2 MSc Chemistry Analytical Sciences Literature Thesis The Application of Microextraction for Determination of Drugs in Biological Samples by Febri Annuryanti April 2013 Supervisor: Dr. Henk Lingeman Daily Supervisor: Dr. Wim Th. Kok

3 Table of contents : Table of contents i Abbreviations ii I. Introduction 1 II. Liquid-phase Microextraction (LPME) Principle of LPME Classification of LPME Single-drop microextraction (SDME) Hollow fiber microextraction (HF-LPME) Carrier-mediated HF-LPME Dispersive liquid-liquid membrane (DLLME) Recovery and enrichment factor In SDME, HF-LPME, and carrier mediated HF-LPME In DLLME Influence factors on the LPME efficiency Organic solvent Volume of donor and acceptor solution Extraction time ph adjustment Agitation of the sample The addition of salt 15 III. Solid-phase Microextraction (SPME) Principle of SPME Classification of SPME Fiber SPME In-tube SPME Influence factors on the SPME efficiency Agitation method Sample ph Ionic strength Sample temperature Sample derivatization 22 IV. Discussion Recent applications of LPME for determination of drugs in biological 23 samples 4.2 Recent applications of SPME for determination of drugs in biological 37 samples V. Conclusion 43 VI. References 45 i Febri Annuryanti [ ]

4 Abbreviations LLE = Liquid-liquid extraction SPE = Solid-phase extraction HPLC = High-performance Liquid Chromatography GC = Gas Chromatography CE = Capillary Electrophoresis SPME = Solid-phase microextraction LPME = Liquid-liquid microextraction DI = Direct immersion HS = Head space HS-SDME = Head space single-drop microextraction SDME = Single-drop microextraction CF-SDME = Continous flow single-drop microextraction DLLME = Dispersive liquid-liquid microextraction PDMS = Polydimethylsiloxane PA = Polyacrylate PDMS-DVB = Polydimethylsiloxane-divinylbenzene CW-TPR = Carbowax-templated resin LC-MS = Liquid chromatography-mass spectrometry GC-FID = Gas-Chromatography-Flame Ionization Detector NPD = Nitrogen-phosphorus detector LOD = Limit of detection LOQ = Limit of quantification I.S. = Internal standard TSD = Thermionic specific detector ii Febri Annuryanti [ ]

5 Introduction Analysis of drugs in biological samples is becoming increasingly important due to the need to understand more about the therapeutic and the toxic effects of drugs [1,2]. Many advantages are obtained by knowing the drug levels in body fluids such as in plasma, serum, and urine [1-3]. The data of drug levels can be used to optimize pharmacotherapy and give the basis for studies on patient compliance [1-3], to perform routine drug monitoring [1,2], to compare the pharmacokinetics study for release of new drugs [6], to reveal the influence of co-medication and to monitor the organ function [2,3]. Furthermore, the screening of drug abuse in body fluids may be used to identify potential users of illegal drugs and to control drugs addicts following withdrawal therapy [1,2]. Although there is an advance development of analytical instrumentation for the determination of analytes in biological fluids, most of the instruments cannot handle the sample matrices directly because of sample complexity [1-2,4]. Biological samples may contain acids, bases, proteins, salts and other organic compounds that may have chemical properties similar to the analyte of interest [3,5,7]. Therefore, sample preparation becomes a crucial part of analysis in order to extract, isolate, and concentrate the analytes [1-3,7-8]. In addition to complex matrices, limited sample volumes and low analyte concentrations have to be considered during sample preparation [2,7]. In order to get an efficient sample pre-treatment, it is important to minimize sample loss so the analytes can be recovered in good yield [1,2], coexisting components can be removed efficiently [1,2], problems do not occur in chromatography system, the analysis cost is low and the procedure can be performed quickly [1,2]. Conventionally, sample preparation is carried out by liquid-liquid extraction (LLE) or by solid-phase extraction (SPE) and the final analysis is accomplished by Highperformance Liquid Chromatography (HPLC), Gas Chromatography (GC) or Capillary Electrophoresis (CE) [3,5-6,9-10]. However, both of LLE and SPE have various drawbacks 1 Febri Annuryanti [ ]

6 such as requires large amounts of organic solvents that are toxic and expensive [8,11], time-consuming [8], result in hazardous waste [11], tedious [8], laborious, and difficult to automate [4]. An ideal sample preparation technique should be easy to use, inexpensive, fast and compatible with a range of analytical instruments [2,4]. To overcome or reduce the drawbacks of LLE and SPE, miniaturizations have been reported on alternative sample preparation methods for drug analysis, namely solid-phase micro extraction (SPME) and liquid-phase micro extraction (LPME) [2,10,12]. This article presents the main principle of SPME and LPME, factors that affecting SPME and LPME, their application on determination of drugs in biological fluids, and further prospect of LPME for drug analysis in biological samples. 2 Febri Annuryanti [ ]

7 Liquid-Phase Microextraction (LPME) 2.1 Principle of LPME LPME is a new sample-preparation technique for the extraction of analytes. Basically, LPME is performed between a small amount of water immiscible solvent (known as acceptor phase) and an aqueous phase containing the analyte of interest (donor phase) [1,4,13,14]. The volume of acceptor phase is usually in the microliter or submicroliter region, while the donor phase between ml for biological samples [8,15,16]. Hence, high analyte enrichments are obtained because of the high sample volume-to-acceptor phase volume ratio [8]. LPME procedures can be divided into static and dynamic mode. In static mode, the extractant is suspended in a large volume of sample phase and the extraction of the analytes is passively carried out. In dynamic mode, extraction occurs by withdrawing aqueous sample into the extraction unit (usually a micro syringe) that already containing solvent. The aquase phase is then pushed out of the syringe and this procedure is repeated several times (typically 20 times) so a higher enrichment factors is obtained [4,17,18]. As a sample preparation, LPME has many advantages. It is rapid, effective, minimize exposure to toxic organic solvents and inexpensive [1,3]. The LPME concept is also compatible for analysis of drugs using HPLC, GC or CE [19]. 2.2 Classification of LPME In general, LPME can be divided into single-drop microextraction (SDME), hollowfiber microextraction, and dispersive liquid-liquid microextraction (DLLME) [4] Single-drop microextraction (SDME) SDME is the simplest form of LPME. It is based on the extraction of analytes into a small drop of organic solvent that is held at the tip of a micro syringe needle [20]. In a two-phase system, the organic solvent was placed into the aqueous sample and the analytes are extracted into the organic solvent based on passive diffusion. In a three-phase system, analytes are extracted from an aqueous sample into the organic 3 Febri Annuryanti [ ]

8 phase. Then, analytes are back extracted into a separate aqueous phase [1]. After extraction, the organic phase is retracted into the needle and the syringe is transferred for further analysis [4]. In practice, there are three main approaches to perform SDME, direct immersion (DI)-SDME, head space (HS)-SDME, and continuous flow (CF)-SDME [6,20]. DI-SDME is a static mode of LPME. It can be done in a two-phase or a threephase system (Fig. 1). It is based on the suspension of a single drop of organic solvent from the tip of a microsyringe needle immersed in the aqueous sample. In a two-phase system, the analytes can be directly injected into the GC-system after the extraction. While in a three-phase system, analytes can be injected into the HPLC system for analysis. The application of DI-SDME is normally restricted to medium polarity, nonpolar analytes and analytes whose polarities can be reduced before extraction. The main problem of DI-SDME is the instability of the droplet at high stirring rate and the option of acceptor phase is limited only for water-immiscible solvent [1,4,6]. Furthermore, fast stirring in DI-SDME may form air bubbles when it is applied to biological samples like plasma. This condition may emulsify organic solvents and increase the stability problem [6]. In most cases, DI-SDME involves a two-phase extraction mode. However, a three-phase extraction mode has also been reported for SDME [1]. In a three-phase 4 Febri Annuryanti [ ]

9 extraction, the ph of the donor phase is adjusted to ensure that the analyte is in its unionized form so it can be extracted into an organic phase whereas the ph of acceptor phase is kept below the ph of the donor phase to prevent back-extraction into the organic phase again. Subsequently, the acceptor phase can be transferred to an HPLC or CE system for final analysis [12]. In HS-SDME (Fig. 2) the analyte is extracted into a microdrop of appropriate solvent located in the head space of sample solution or in a flowing air sample stream, which is thermostated at a given temperature for a preset extraction time [4,20]. This method is most suitable for determination of volatile or semivolatile analytes [1,6]. In this mode, the analyte is distributed among three phases, the aqueous sample, head space and organic solvent, and the rate of this extraction is determined by mass transfer of aqueous phase [1,20]. The advantage of HS-SDME is it allows the use of both organic solvent and aqueous solvent as acceptor phase because the droplet does not directly contact with the sample solution. In addition, HS-SDME provides an excellent clean up for sample with complicated matrix [4,6,20]. The drawback of this method is the need of solvent with low vapor pressure and low viscosity [20]. CF-SDME (Fig. 3) is a dynamic mode of SDME and was first introduced by Liu and Lee in 2000 [20]. In this method, a polyetheretherketone (PEEK) connecting tube hold an organic drop at the outlet tip and immersed in a continuously flowing sample 5 Febri Annuryanti [ ]

10 solution. This PEEK connecting tube acts as the fluid delivery duct and solvent holder. This method produces a higher concentration factor than static mode of SDME because the solvent drop makes continuous and full contact with the sample solution [4,20]. Because of its high concentration factor that can be achieved, only a small volume of sample is needed for extraction [4]. The disadvantages of this method are the need of peristaltic pump and extra filtration since complex matrices affect the stability of solvent drop during the extraction [20] Hollow fiber liquid-phase microextraction (HF-LPME) HF-LPME is an alternative concept for LPME. This concept was introduced in 1999 by Pedersen-Bjergaard and Rasmussen to improve the stability and reliability of LPME [21]. This technique use single, low-cost, disposable porous hollow fiber made of propylene [22,23]. The advantages of HF-LPME are that the sample can be vibrated or stirred vigorously without any loss of the extracting liquid and the extracting liquid is not partly dissolved in the sample during extraction [24]. The small pore size of hollow fiber allows microfiltration of the samples to yield very clean extracts [25] and the use of disposable hollow fiber eliminates the possibility of carry over and ensures reproducibility [25-27]. Particularly, in the three-phase system when both extraction and back-extraction are included, excellent clean-up has been observed, even in complicated biological samples [28]. 6 Febri Annuryanti [ ]

11 In this system, the extracting liquid is not directly contact with the acceptor phase. The acceptor phase is contained within the lumen of porous hollow fiber, either as loop or a rod sealed at the bottom [21,29]. Prior to extraction, the hollow fiber is dipped in the immiscible organic solvent (like toluene, dihexyl ether or n-octanol) for several seconds to immobilize the organic solvent in its pores. Alternatively, a small volume of organic solvent can be injected into the lumen of hollow fiber and immobilized from the inside of hollow fiber [21]. The organic solvent forms a thin layer within the wall of the hollow fiber and the excess solvent outside the hollow fiber is removed by ultra-sonification [23]. Subsequently, the hollow fiber is then placed into a sample vial that contains the sample of interest. An extensive agitation or stirring of the sample can be applied to speed up the extraction process [23]. The organic solvents are used in HF-LPME should be immiscible with water, strongly immobilized in the pores of hollow fiber, provide an appropriate extraction selectivity, and has a low volatility, to ensure that it remains within the pores during extraction with no leakage to the biological samples [22,23]. Like SDME, HF-LPME may be accomplished both in a two-phase or a threephase system (Fig. 4) [21-23,27]. In a two-phase system, the analytes are extracted from the aqueous sample into an organic solvent immobilized in the pores and the lumen of hollow fiber [24-25]. This technique may be applied for analytes with high solubility in non-polar organic solvents. Since the pores and the lumen of hollow fiber are filled with an organic solvent immiscible with water, the final extract may be directly analyzed with GC, or may be evaporated and reconstituted in an aqueous solution for analysis with CE or HPLC [25]. In a three-phase system, the analytes are extracted from an aqueous sample through the thin film of the organic solvent into an aqueous acceptor solution. The thin film of organic phase serves as a barrier between the donor phase and the acceptor phase [25]. This extraction mode is limited to acidic or basic analytes with ionizable functionalities, where the analyte is in its neutral form in the donor phase [22,25]. For the extraction of acidic compounds, ph in the sample has to be adjusted in acidic region to promote their extraction into the organic phase, whereas the ph in the 7 Febri Annuryanti [ ]

12 acceptor solution should be high to promote high extraction efficiency from organic phase into the acceptor phase [22,25]. In contrast for basic compounds, the ph of sample solution should be in alkaline region and the acidic solution should be utilized within the lumen of fiber. Following extraction, the acceptor phase is directly analyzed by HPLC, CE, or MS without any further treatments [22,25] Carrier-mediated HF-LPME In two-phase and three-phase HF-LPME, the extraction is based on passive diffusion, in which the high partition coefficient plays an important role. However, some analytes, such as very hydrophilic drugs, have poor partition coefficients that prevent them from being extracted by passive diffusion. In order to enhance the extraction of hydrophilic drugs, HF-LPME may be accomplished in a carrier-mediated mode [22,23]. Carrier-mediated, as illustrated in Fig. 5, is an active transport mode of HF- LPME. In this method, a carrier is added to the sample solution or is dissolved in the impregnation solvent in the pores of the hollow fiber [25]. The carrier, which is relatively hydrophobic ion-pair reagent providing acceptable water solubility, forms ion-pairs with the analyte of interest followed by the extraction of ion-pair complexes into the organic phase in the pores of hollow fiber. In the contact region of organic phase and acceptor phase, the analytes are released from the ion-pair complexes into the acceptor solution, whereas an excessive counter-ions in the acceptor solution form ion-pairs with the carrier in the contact area. The new ion-pair complexes are then 8 Febri Annuryanti [ ]

13 back-extracted into the donor phase. In the sample solution, the carrier releases the transporter counter ion and forms an ion pair with a new analyte molecule, and the cycle is repeated [22,23]. A carboxylic acid with an appropriate hydrophobic moiety may be used as the carrier (such as octanoic acid) for basic analytes. In the extraction process, the ph of the sample is adjusted to ensure that the analytes are present in their ionized state in order to form the ion pair, and the ph of acceptor is adjusted to low value to ensure that the carrier is not trapped within the phase. Furthermore, the low ph value provides sufficient protons to serve as counter ion for the carrier [22,23] Dispersive liquid-liquid microextraction (DLLME) DLLME is another recent technique of LPME that was introduced by Assadi and co-workers in 2006 [4,16]. It is based on ternary solvent component system involving an aqueous sample, a polar water miscible solvent (disperser solvent) and a non-polar water immiscible solvent (extracting solvent) [6]. The selection of extracting solvents is based on their density, extraction capability of interest compounds and good chromatographic behavior. The density of extracting solvent should be higher than 9 Febri Annuryanti [ ]

14 water. Halogenated hydrocarbons such as chlorobenzene, carbon disulfide, carbon tetrachloride, tetrachloroethylene and chloroform are usually chosen as extracting solvents [20,30]. The choice of disperser solvent is determined by its ability to miscible in both extracting solvent and aqueous sample. Methanol, ethanol, acetonitrile and acetone are mostly used as disperser solvent [20,30]. Figure 6 shows the different step of DLLME. When the mixture of disperser and extraction solvent is injected into the sample solution, a cloudy solution is produced. This cloudy solution gives rise to the formation of fine droplets, which are dispersed throughout the aqueous sample. After the formation of cloudy solution, the surface area between extracting solvent and the sample solution becomes very large so the equilibrium state is achieved quickly and the extraction time is relatively short. The cloudy solution is then cooled and centrifuged to form a sediment phase in the bottom 10 Febri Annuryanti [ ]

15 of conical tube and used for further analysis. DLLME can be coupled with HPLC, GC and also with atomic absorption spectrometry [16,20]. 2.3 Recovery and enrichment factor In SDME, HF-LPME and carrier mediated HF-LPME In two-phase SDME and HF-LPME, the analytes are extracted from donor solution by passive diffusion from directly into the acceptor solution, described in equation (1). The extraction process in this system depends on the partition between the acceptor (organic) solution and the donor solution (K a/d ), defined by equation (2) [11,21]. A donor A acceptor (organic) solution (1) K a/d = C eq.acceptor / C eq.donor (2) where C eq.acceptor is the concentration of analyte in the acceptor (organic phase) solution at equilibrium and C eq.donor is the concentration of analyte in the sample at equilibrium. Based on Eq. (2) and a mass balance of the two-phase LPME system, the recovery of analyte (R) at equilibrium may be calculated by the following equation [21] : R = (K a/d. V a )/{(K a/d. V a ) + V d }. 100% (3) where V a is the volume of acceptor solution in the organic phase system (sum of organic solvent present in the porous wall of the hollow fiber and in the lumen of hollow fiber and V d is the volume of donor solution [21]. In three-phase SDME, HF-LPME and carrier mediated HF-LPME, the analytes are extracted from the aqueous phase by passive diffusion, through the organic phase, and further into the acceptor solution presents inside the lumen of hollow fiber. This process may be illustrated by following equation [11,21] : A donor A organic acceptor A acceptor(aqueous) acceptor (4) 11 Febri Annuryanti [ ]

16 The total extraction process is affected by both partition coefficient between the organic phase and the donor solution (K org/d ) and that between the acceptor solution and the organic phase (K a/org ), defined by equation (5) and (6) [21] : K org/d = C eq.org /C eq.d (5) K a/org = C eq.a /C eq.org (6) where C eq.org is the analyte concentration at equilibrium in the organic phase, C eq.d is the analyte concentration at equilibrium in the donor solution, and C eq.a is the analyte concentration at equilibrium in the acceptor solution. The partition coefficient between the acceptor solution and donor solution is calculated as the product of K org/d and K [11] a/org : K a/d = C eq.a /C eq.d = K org/d. K a/org (7) The recovery, R, in the three-phase LPME system may be calculated by equation [18] : R = (K a/d. V a ) / {(K a/d. V a ) + (K org/d. V org ) + V d }. 100 % (8) where V a is the volume of acceptor phase, V org is the volume of organic phase immobilized in the pores of the hollow fibre and V d is the volume of the donor solution. The analyte enrichment (E) in two-phase and three-phase LPME may be calculated by equation (9) and (10), respectively [18] : E = (V d. R) / (V org. 100) (9) E = (V d. R) / (Va.100) (10) In DLLME The recovery, R, in DLLME is defined as the percentage of total analyte amount (n o ) extracted to the sediment phase (n sed ) [16] : R = (n sed / n o ) x 100 = {(C sed x V sed )/(C o x V s )} x 100 (11) where C sed is the analyte concentration in sediment phase, C o is the initial concentration of analyte, V sed and V s are the volumes of sediment phase and sample solution, respectively. 12 Febri Annuryanti [ ]

17 2.4 Influence factors on the LPME efficiency There are some factors which affect the method optimization and extraction efficiency (recovery and enrichment) in liquid phase [11,12,20] : Organic solvent The selection of organic solvent is an essential step for an efficient extraction. The choice of organic solvents should be based on several considerations. Firstly, it should have good affinity for analyte of interest. Secondly, it should have a low solubility in water to prevent dissolution into the aqueous phase. Thirdly, the organic solvents should have low volatility so it will not evaporate during extraction. Fourthly, it should be stable during extraction time. Finally, the organic solvent should have excellent GC or LC behavior. In general, several water-immiscible solvents which have different solubility and polarity may be used as extraction solvent. 1-octanol, di-nethylether, n-hexane, o-xylene and toluene may be used as organic solvent in HF-LPME [11]. For DI-SDME and DLLME, the density of organic solvent plays an important role in the extraction process. In DI-SDME, the density of organic solvent should be lower than water. On the other hand, DLLME requires organic solvent which has density higher than water. Decane, 1-butanol, isooctane and n-octanol are usually used in SDME, whereas chlorobenzene, dichlorocarbene and tetrachloride carbon are used in DLLME [20] Volume of donor and acceptor solution The volume of donor and acceptor solutions directly affects the extraction efficiency. The biological sample volume usually between ml, while the volume of acceptor solution may vary depends on the method of extraction and on the analytical technique coupled to LPME. The volume of acceptor solution in SDME is typically in the range of µl because larger drops lead to instability of the microdrop. In HF-LPME, the extraction volume depends on the length of hollow fiber. 2-8 cm of hollow fiber are usually used in the range of µl. As for the DLLME, the volume of acceptor solution is in the range of µl. The extraction efficiency and enrichment factor can be increased by increasing the ratio of acceptor-to-donor phase. 13 Febri Annuryanti [ ]

18 However, enrichment factor will decrease when it exceeds a certain limit. Therefore, keeping a low extraction volume is necessary to obtain highest selectivity [11,20] Extraction time Most of extraction in LPME is a time-dependent process, in which the extraction efficiency is attained at the equilibrium condition. Accordingly, it is important to determine the extraction time profile of analyte in order to configure the equilibrium time. In SDME and HF-LPME, the equilibrium time usually between 30 and 60 minutes without lose of organic solvent [11]. Even though longer extraction times generally result in increased extraction efficiency, it is not always practical to apply extended extraction times. Sampling times shorter than the total chromatographic time is more likely to ensure high sample throughput [20]. Unlike in SDME and HF-LPME, the extraction time in DLLME is not very important. As the infinitely large surface area between extraction solvent and aqueous phase forms after the formation of cloudy solution, so the target analytes differ quickly into the extraction solvent. Therefore, DLLME is a time-independent, which is the most important advantage of this technique [11] ph adjustment ph adjustment can enhance the extraction efficiency, because the dissociation equilibria is influenced by the solubility of the acidic/basic target analytes. Many reports show that the ph changes in the donor solution resulted in higher analyte preconcentration of analytes in a two-phase and a three-phase LPME. Particularly in three-phase LPME, adjusting ph in the donor and acceptor phase is very critical, since it influences the distribution ratio, enrichment factor and recoveries of target analytes. To obtain high enrichment factor and high recoveries, the ph of donor solution should be adjusted so the analytes of interest are in their unionized form. In this form, the solubility of analyte in the donor solution will decrease and an efficient transfer into the organic phase will be obtained. On the other hand, the ph of acceptor solution should be adjusted to make the analytes of interest 14 Febri Annuryanti [ ]

19 in their ionized condition, in order to ensure efficient extraction of analytes into the acceptor solution and to prevent analytes trapped in the organic phase [11] Agitation of the sample The main purpose of agitation is to accelerate the extraction kinetics and enhance the extraction efficiency, since stirring allows the continuous exposure of the extraction surface to the aqueous sample. Hence, thermodynamic equilibrium can be achieved in a short time and induces the convection in membrane phase. Sample agitation can be done in two ways, by stirring or vibrating the sample. Vibrating the sample solution has more advantages than stirring the sample using magnetic stirrer, because it eliminates the possibility of analytes being contaminated by the magnetic stirrer. Furthermore, the use of magnetic stirrer in high stirring rate promotes bubble formation, solvent evaporation and instability of micro drops [11,20] The addition of salt Salt addition is widely used in microextraction to improve the analyte partitioning into the organic phase by salting-out effect. However, the effect of salt addition to extraction efficiency may vary from enhancing, not influence to decreasing, depending on the nature of target analytes. Caution should be given to the presence of high concentration of salt in sample solution that may change the physical properties of the extraction film. This condition will decrease the diffusion rate of analyte into the organic phase [11,20]. 15 Febri Annuryanti [ ]

20 Solid-Phase Microextraction (SPME) 3.1 Principle of SPME SPME is a sample preparation technique that was developed by Pawliszyn and coworkers in 1990 [31-33]. This technique is simple, rapid, highly sensitive, solvent free, inexpensive and easy to automate [1-2,34-35]. The basic principle of SPME is the partitioning of analyte between the sample phase and the coated fiber when the coated fiber is exposed to the sample for a well-defined period time [10,31]. The extraction is completed when the analyte concentration has reached distribution equilibrium between sample matrix and the fiber coating. Once equilibrium is reached, the extracted amount is constant and it is independent of further increase of extraction time. The equilibrium condition can be described as [12] : n = (K fs. V f. V s. C o ) / {(K fs. V f )+ V s } (11) where n is the mass of analyte absorbed by the coating; k fs is the partition coefficient of analyte between the coating and sample matrix; C o is the initial concentration of a given analyte in the sample; V f and V s are the volume of the coating and the sample, respectively. When the sample volume is very large, such as in river, production stream and ambient air (V s >> K fs. V f ), equation (11) can be simplified to [12] : n = K fs. V f. C o (12) It can be seen from equation (12) that the amount of extracted analyte is independent from sample volume, so it is no need to collect a defined amount of sample prior to analysis. The amount of extracted analyte will correspond directly to its concentration in the matrix. This condition is very useful for on-site applications [12]. After the completion of extraction process, the fiber with concentrated analyte are thermally desorbed in the case of GC or GC-MS, or injected via a sample loop in the case of HPLC [36]. 16 Febri Annuryanti [ ]

21 As shown in Figure 7, SPME device consists of coated fused silica fiber connected to stainless steel tubing that is used to increase the mechanical strength of the fiber assembly for repeated sampling. The stainless steel then contained in a specially design syringe. During extraction, the fiber is first withdrawn into the syringe needle then lowered into the vial by pressing down the plunger [10]. As seen in equation (11), the extraction efficiency is dependent on the partition coefficient of the analyte between the coating and sample matrix (K fs ) [37]. Therefore, the selection of fiber coating plays an important role in SPME [10]. The coating materials can be liquid polymer, solid sorbent or combination of both, where the extraction mechanism is quite different between liquid and solid polymer. In liquid coating, the extraction mechanism is absorption. In absorption mode, the magnitude of analyte diffusion coefficient allows the molecule to penetrate to the entire volume of the coating within a well-defined extraction time [37]. On the other hand, solid polymers as coating agent possess complex crystalline structure, lead to reduce analyte diffusion coefficient within the structure. In this polymer, the extraction only occurs on the surface of the coating or through an adsorption mechanism. Consequently, the extraction time for adsorption in solid polymer is shorter than the absorption mechanism in liquid polymer [37]. 17 Febri Annuryanti [ ]

22 The fiber coating selection of microextraction is based on the principle like dissolves like [10,34]. Most of non-polar analytes can be extracted using polydimethylsiloxane (PDMS), whereas polyacrylate (PA) is more suitable as extracting agent for polar compounds, such as phenols. Mixed phases such as Carboxen-PDMS, polydimethylsiloxane-divinylbenzene (PDMS-DVB) and divinylbenzene-carboxen-pdms are suitable for the extraction of volatile low-molecular mass [10,37]. The coating thickness is selected based on the efficiency required, the nature of the analyte, the extraction time, and the molecular mass of analyte. Faster partition equilibrium can be obtained by using thinner coating while small-molecular mass compounds can yield high extraction with relatively thick coatings [10,37]. 3.2 Classification of SPME SPME may be performed in two arrangements, fiber SPME and in-tube SPME Fiber SPME Fiber SPME is based on a modified syringe which contains stainless steel micro tubing within needle. Inside the syringe, there is a fused silica fiber tip coated with organic polymer. The techniques that usually used are headspace (HS) SPME and direct immersion (DI) SPME (Fig. 8) [1,2]. The selection of extraction technique depends on the nature of sample matrix, analyte volatility and affinity of sample between the matrix and coating [10]. HS-SPME is used for volatile sample or sample that can be made volatile by moderate heating [1,10]. In this technique, the fiber is placed above the sample so there is no direct contact between the fiber and the sample [2]. This design can protect the fiber coating from damage caused by extreme condition (very low or high ph) or large molecules that tend to foul the coating [1]. In addition, HS-SPME can minimize contamination on the surface of the fiber, gives cleaner extracts, greater sensitivity and longer fiber life time. Extraction process of HS-SPME involves three phases (coating, headspace and sample matrix), in which the limiting step is the transfer time of analytes from the sample matrix to the head space [35]. Because of the requirements of a high vapour pressure analytes, the transfer of the fiber to the GC as well as 18 Febri Annuryanti [ ]

23 desorption should be performed immediately after the extraction to minimize the risk of analyte loss during storage of the loaded fiber. DI-SPME is used for the extraction of low-to-medium volatility and high-tomedium polarity [1,37]. In this technique, the fiber is directly immersed in the liquid samples [2] and the mass transfer rate is determined by diffusion of the analyte in the coating provided that the sample is perfectly agitated [10]. As the sample directly contact with the fiber, strong acidic or alkaline condition should be avoided. An HF membrane can be used to protect the SPME fiber from insoluble component in the sample [1] In-tube SPME In-tube SPME is a new sample preparation technique using an open tubular capillary as an SPME device (Fig. 9). It can be coupled with HPLC, liquid chromatography-mass spectrometry (LC-MS) or GC and allows the convenient automation of the extraction process [1,2,6]. In in-tube SPME, aqueous sample that contains organic compounds can be directly extracted from the sample into the internally coated stationary phase of a capillary. Subsequently, analytes are desorbed using a stream of mobile phase. When the analytes are more strongly adsorbed to the capillary coating, a static desorption solvent can be used [1,2]. Finally, the desorbed compounds are injected into the column for further analysis. To prevent plugging of 19 Febri Annuryanti [ ]

24 the capillary column and flow lines, the sample solution need to be filtered before extraction. The extraction, desorption, and injection in in-tube SPME can be performed continuously using standard auto sampler. The automation of sampling handling process not only reduces analysis time, but also provides better sensitivity and precision than manual techniques. Despite the low extraction yields of in-tube SPME, this technique may provide reproducibility of extracted compound using an auto sampler. Moreover, all of the extracts may be introduced into the LC column after intube SPME [1]. 3.3 Influence factors on the SPME efficiency There are some variables which can influence the extraction efficiency in SPME Agitation of the sample Sample agitation is important in order to ensure rapid and efficient extraction. Agitation accelerates the transfer of analytes from matrix to the coating. Different agitation methods can be chosen, namely fast sample flow, rapid fiber movement, stirring and sonication. A suitable agitation method will result in shorter equilibration time and higher extraction amount of analyte [10,34,37]. 20 Febri Annuryanti [ ]

25 3.3.2 ph of sample The extraction efficiency in SPME is enhanced by fully converting the analytes into neutral forms because SPME coatings are more efficient to extract neutral forms of analytes [10]. The adjustment of ph sample can be done by adding buffers to the sample to prevent ionization of the sample. A high ph value and a lower ph value are efficient to improve the extraction of basic and acid compounds, respectively. For molecules possessing both acidic and basic functionalities, the optimum ph for extraction must be determined empirically. The determination of optimum ph of the sample should be between the stability ranges of the coatings and an extreme ph value should only be used in HS-SPME mode owing to the potential fiber deterioration when DI-SPME is used [37] Ionic strength The addition of salt influences the partition coefficient of analyte (K fs ). By adding salt into the sample solution, the ionic strength will increase and the aqueous solubility of sample will decrease (K fs increase). This salting out effect causes the analytes more easily to pass from the sample onto the coating. However, in some cases, the salt addition may improve the extraction efficiency for both the target and interfering compounds [37]. In this case, the effect of salt addition on the analyte extraction depends on the nature of target analyte and the salt concentration. Therefore, for a particular target analyte and sample matrix, experiments are needed to determine the effect of adding salt on extraction efficiency. Generally, the addition of salts is preferred for HS-SPME because fiber coating are prone to deteriorate during agitation in DI-SPME. The salts commonly used to increase extraction efficiency are (NH 4 ) 2 SO 4, Na 2 CO 3, K 2 CO 3 and NaCl [10,37] Sample temperature Extraction temperature should be considered during SPME. Increasing the sample temperature may help to release the sample into the headspace and increase analyte diffusion coefficient, which leads to an increase in the extraction rate or the mass transfer rate onto the fiber coating. Nevertheless, as the temperature increases, 21 Febri Annuryanti [ ]

26 the fiber coating begins to lose its ability to adsorb analytes and the distribution constant of sample between matrix and coating decreases. As a result, the sensitivity and the analyte recovery at equilibrium condition are decreased. Furthermore, an extremely high sample temperature may result in decomposition of some compounds and creation of other component artifacts. As increased sample temperature affects both diffusion coefficient and distribution constant, the optimum sample temperature will depend on the physicochemical properties of target analytes [37] Sample derivatization Derivatization is commonly used in SPME-GC applications. Analyte derivatization is used to transform an original compound into a product that has different physicochemical properties. This step is important for the analysis of nonvolatile, polar, and ionic species which are difficult to extract and tend to react with the injection port and analytical column. Some examples of derivatizing agents are trimethyloxonium tetrafluoroborate, pentafluorobenzaldehyde, and bis(trimethylsylil) trifluoroacetamide [37,38]. Two derivatization strategies that can enhance the extraction efficiency are as follows: a. Pre-extraction derivatization In this method, the derivatizing agent is added to the sample matrix. Pre-extraction derivatization is used for underivatized highly polar target analytes which do not have a high affinity toward the commercially polar fiber coating. Accordingly, the analytes must be first converted into less polar derivatives before SPME process [37]. b. Simultaneous extraction and derivatization This derivatization involves the loading of a derivatizing agent onto the fiber followed by fiber exposure to the sample matrix, allowing simultaneous derivatization and extraction processes to occur. Subsequently, the derivatized analogs desorbed into an analytical instrument for further analysis. The loading procedure for derivatizing agent needs to be optimized and factors such as reagent s vapor pressure, volatility and affinity toward the coating must be considered [37]. 22 Febri Annuryanti [ ]

27 Discussion 4.1. Recent applications of LPME for determination of drugs in biological samples Single-drop microextraction has become a very popular LPME technique because it is inexpensive, easy to operate and nearly solvent-free. There are few publications on SDME extraction for drugs analysis in biological samples. Yao et al. [39] developed a single drop LPME combined with HPLC-UV detector for the simultaneous analysis of local anesthetics, lidocaine, bupivacaine, and tetracaine. Organic solvent o-dibutyl phthalate was selected to extract local anesthetics in human urine sample because it is compatible with the mobile phase of HPLC. The mobile phase consisted of (A) a mixture of acetonitrile and triathylamine aqueous solution (11 mm)-0.1% phosphoric acid aqueous solution (10:90,v/v) and (B) a mixture of acetonitrile and triathylamine aqueous solution (20mM)- 0.1% phosphoric acid aqueous solution (50:50,v/v). 6 ml of urine is made alkaline to ph 11 with 1.0 M NaOH and then extracted using 1 µl of o- dibutyl phthalate. Higher enrichment factor (more than 86.0 fold) and significant sample clean up were achieved within 30 min under the optimized extraction condition (160 rpm of the stirring rate at 30 o C). No matrix effects occur during the extraction and the method was applied to urine sample from a patient who was treated with extradural anaesthesia of lidocaine, bupivacaine, and tetracaine. Figure 10 shows the chromatogram of urine sample analysis. The result reveals that the method is selective and sensitive enough to allow determination of lidocaine, bupivacaine, and tetracaine in urine. This method may be applicable for drug monitoring, forensic toxicology, and medico-legal practices. 23 Febri Annuryanti [ ]

28 In 2005, Gioti et al. [40] reported the analysis of hyperforin and hypericins (hypericin and pseudohypericin) in biological fluids using single-drop LPME in conjunction with HPLC-fluorescence detector. Those drugs are the extracts of St. John s Wort (Hypericum perforatum L.), which has been known for many medicinal properties such as hepatic disorders, gastric ulcers, anti-inflammatory, anti-microbial, anti-viral, anti-depressant, and anti-cancer agent. Many methods have been developed for the measurement of hypericins and hyperforin in a variety of biological media, but most of the methods employed hitherto that require non polar organic solvents where hyperforin is unstable. Hence, the author proposed a new option for analysis of hypericins and hyperforin in biological samples in order to reduce the steps required prior to analysis and increasing the sensitivity. In this method, urine samples were filtered before use in extraction to remove the suspended particles, while plasma samples were mixed with methanol to precipitate protein. The ph of the sample was adjusted to 6.0 prior to extraction. A mixture of n-octanol:chloroform (7:3 v/v) was chosen as organic drop to avoid drop dislodgement and improve extraction yield of hypericins and hyperforin. Extraction was held for 15 min with the stirring rate of Febri Annuryanti [ ]

29 rpm at 40 o C and no salt addition. After extraction, the organic solvent drop was transferred to a micro vial and made up to 30 µl with methanol. Using isocratic reversed-phase HPLC with methanol and phosphate buffer solution (ph 2.2) as mobile phase (95:5, v/v), a complete analysis of urine and plasma samples can be performed within 22 and 25 min, respectively. The author claims that the method is selective, flexible and amenable to improvements towards improving identifications and LOQs. Ebrahimzadeh et al. has determined fentanyl, a potent synthetic narcotic analgesic, in plasma, urine and waste water using SDME combined with HPLC-UV [41]. To diminish matrix effect, plasma and urine sample were diluted with water at 1:5 and 1:1 ratios, respectively. The procedure is based on three-phase SDME, where fentanyl was extracted from 3.6 ml samples solution containing 0.01 M NaOH into 100 µl n- octane, then back extracted into 5 µl of 1x10-3 M HClO 4. Others optimum experimental conditions were stirring rate of 1000 rpm for 30 min in pre-extraction and 700 rpm for 20 min in back extraction; extraction temperature at 30 0 C; and no salt addition. Within optimum condition, enrichment factor of 355 was obtained. Table 1 compares the proposed method with alternative methods for the extraction of fentanyl from biological fluids. Although the proposed method has higher LOD (0.1 ng/ml) than others, but reliable measurements of fentanyl can be performed with lower cost. Table 1. Comparison of figures of merit of the proposal method with other methods applied for the analysis of fentanyl (from Ref. 27) Method Sample LOD (ng/ml) r LDR (ng/ml) preparation Proposed LLLME GC-NPD LLE GC-MS SPE GC-MS HS-SPME GC-MS SDME < LC-MS/MS LLE HPLC-UV LLE He and Kang extracted a popular drug of abuse, methamphetamine and amphetamine from urine samples by coupling three-phase SDME with HPLC-UV [42]. The author used method from other previous studies with some modification in organic solvent volume and HPLC syringe. Instead of using Teflon ring in organic phase and a Teflon sleeve on the tip of syringe needle, they used larger volume of organic 25 Febri Annuryanti [ ]

30 solvent and larger HPLC syringe. Urine samples were diluted with pure water (1:1) before use to overcome unstable acceptor drop caused by interferences of coextractives in urine samples. The optimized extraction condition were 6.0 ml sample solution containing 0.5 M NaOH, 400 µl n-hexane as organic phase, 5 µl 0.02 M H 3 PO 4 as acceptor phase, 40 min pre-extraction followed by 40 min back-extraction with the simultaneous extraction. The enrichment factor was found 730 and 500 for methamphetamine and amphetamine, respectively. Figure 11 shows the chromatogram result. Unknown peaks are found, with one tiny peak overlapped with methamphetamine in spiked urine sample. The author reveals that the tiny peak could be neglected since the area only 2% of amphetamine. Moreover, better selectivity of the method could be improved by using mass spectrometer as detector. This method exhibited low detection limit (0.5 µg/l), a wide linear range ( µg/l) and a good repeatability (RSD < 5%). The wide linear range made of this method can be applied in the initial screening test and confirmatory test of drug abuse. 26 Febri Annuryanti [ ]

31 The use of DLLME for analyte extraction in biological samples is limited because of some reasons. First, the production of sediment phase for injection in analytical instrument is not possible due to the interaction of matrix sample with the organic solvents. Serial dilutions of sample may be used to procedure sediment phase, but this procedure may alter the inherent property of matrix. Secondly, DLLME is only applicable for the samples containing high concentration of analytes [16]. However, some experiments for determining drugs in biological fluids were done using DLLME. The application of DLLME combined with GC-FID was developed for separation and determination of tricyclic antidepressants drugs, amitryptiline and nortryptiline, in water samples by Yahdi and co-workers [43]. The performance of proposed method was evaluated by determining amitryptiline and nortryptiline in human plasma. Prior to extraction, amitryptiline and nortryptiline were liberated from protein plasma by adding 1.0 ml methanol to 0.5 ml plasma sample. The samples then centrifuged for 15 min at 1000 rpm ml of supernatant was transferred to vial tube and diluted with water to 5.0 ml. Subsequently, samples were basified using 1 M NaOH to the ph 12. A mixture of 1.0 ml methanol (disperser solvent) and 18.0 µl of carbon tetrachloride (extraction solvent) were injected rapidly into plasma samples and followed by gently shaken of the mixture. Afterward, the mixture was centrifuged for 10 min at 1000 rpm to form sediment phase. Finally, 2.0 µl of sediment phase was injected into GC. The limit of detections was 0.07 and 0.02 µg/ml for amytriptyline and nortryptiline, respectively. According to author, this method provides high recovery and enrichment factor within a very short time. Xiong et al. [44] proposed a DLLME combined with HPLC-UV for the determination of three psychotropic drugs (amitryptiline, clomipramine, and thioridazine) in urine samples. Prior to extraction, urine sample was centrifuged for 15 min at 4000 rpm. The supernatant was filtrated through a 0.45 µm filter and 10 M NaOH was added to adjust the ph to 10. Subsequently, 0.50 ml of acetonitrile (disperser solvent) and 20 µl of tetrachloride (extraction solvent) were rapidly injected into 5.0 ml of urine sample and formed a cloudy solution. The cloudy solution was gently shaken and followed by centrifugation at 4000 rpm for 3 min. A different 27 Febri Annuryanti [ ]

32 phenomenon was observed between the aqueous standard and urine samples after the centrifugation process. For aqueous standard, a small droplet of carbon tetrachloride was sediment in the bottom of the conical test tube. While for urine sample, white lipidic solid was sediment in the bottom of the conical tube. The white lipidic solid in urine sample might be due to co-sedimentation of the matrixes (such as carbamide and uric acid) in urine at high ph values. The white lipidic solid was dissolved in 200 µl of acetonitrile and then filtrated through a 0.45 µm membrane to discard the white floccules in the extract urine. Finally, extract was injected into HPLC for further analysis. The proposed method was applied to two urine samples collected from two female patients who taken some psychotropic drugs combinations including amitryptilline and clomipramine, respectively. The chromatogram result is shown in Figure 12. As can be seen, the presence of major endogenous components, coexisting drugs and their metabolites in urine sample has no obvious influence on the determination of target anaytes. This result reveals that the proposed method has a good selectivity for the analysis of the analytes and the method can be used in clinical situations. 28 Febri Annuryanti [ ]