Centrifuge Microextraction Coupled with On-Line Back-Extraction

In general, sampling and sample preparation steps account for over 80% of the .... was provided by the Department of Public Security of Hubei Province...
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Anal. Chem. 2006, 78, 6043-6049

Centrifuge Microextraction Coupled with On-Line Back-Extraction Field-Amplified Sample Injection Method for the Determination of Trace Ephedrine Derivatives in the Urine and Serum Huaifang Fang, Zhaorui Zeng,* and Lan Liu

Department of Chemistry, Wuhan University, Wuhan 430072, China

Although sample stacking has enjoyed some degree of success in electrophoretic separation techniques, there is still a major problem with complex matrix sample as it suffers tremendously from sample matrix effects. A novel method that combines two concentration techniques, centrifuge microextraction (CME) and on-line backextraction field-amplified sample injection (OLBE-FASI), is used to determine trace ephedrine derivatives in urine and serum by capillary zone electrophoresis. The CME, integrating the sample cleanup and preconcentration into a single step, is a promising sample preparation method for biological samples. The CME technique provided 9-14-fold enrichment within 10 min. The OLBE-FASI eliminated the need to perform solvent exchange and provided a further concentration of the analytes. Using CME coupled with OLBE-FASI, over a 3800-fold increase in sensitivity could be obtained as compared with the normal hydrodynamic injection without sample stacking. For a 1-mL urine sample, the linear range was 5/10200 ng/mL with the square of the correlation coefficients (r2) ranging from 0.9988 to 0.9994. Detection limits were from 0.15 to 0.25 ng/mL using a photodiode array UV detection at wavelength 192 nm. The possibility of this method to determine ephedrine derivatives in 20-µL serum samples was also demonstrated. The analysis of biological samples (e.g., blood, urine, saliva, tissue homogenates) by capillary electrophoresis (CE) is usually divided into five steps: sampling, sample preparation, separation, detection, and date analysis. In general, sampling and sample preparation steps account for over 80% of the whole analysis time, and the quality of these steps has a great effect on the success of analysis interested analytes from complex matrixes.1,2 The objectives of sample preparation for analysis of components in biological fluids are as follows: (i) removing macromolecular contaminants (mainly proteins, carbohydrates, and lipids), (ii) eliminating other interfering components (endogenous/exogenous or low-/highmolecular mass) from the sample, (iii) getting rid of (in)organic * To whom correspondence should be addressed. E-mail: zrzeng@ whu.edu.cn. Fax: +86-27-8764-7617. (1) Fu, X.; Liao, Y.; Liu, H. Anal. Bioanal. Chem. 2005, 381, 75-77. (2) Kataoka, H. Trends Anal. Chem. 2003, 22, 232-244. 10.1021/ac060360q CCC: $33.50 Published on Web 07/26/2006

© 2006 American Chemical Society

salts and particulate matter, (iv) concentrating the analyte of interest (if possible) and hence enhancing the detection sensitivity for CE analysis, and (v) matching the sample solvent to the appropriate CE buffer.3,4 Liquid-liquid extraction (LLE),5 solid-phase extraction (SPE),6 solid-phase microextraction (SPME),7 membrane-based extraction (MBE),8 and liquid-phase microextraction (LPME)9 are the general sample preparation techniques for biological samples. Among them, LLE and SPE are mainly used. However, these techniques have some fundamental limitations. For example, LLE is a laborious, relatively slow process and requires large amounts of high-purity solvents, which are expensive and harmful for the human body and the surrounding environment.3 The entire analysis of SPE can be lengthy, with a series of stages including washing, conditioning, eluting, and drying. Moreover, plugging of the cartridge or blocking of the pores by matrix components happens sometimes. Furthermore, SPE can also be expensive because the cartridges are normally disposed after one extraction.10 Therefore, developing a relatively simple, rapid, inexpensive, and virtually solvent-free sample preparation method is a challenging and attractive task. The quest for novel sample preparation techniques has never ceased, and one of the most recent trends is miniaturization of the traditional LLE method. The major ideas behind this were to allow efficient extraction along with reduced solvent volumes and time and with a high level of automation. To reduce solvent consumption, LLE has been performed in volumetric flasks11 or in centrifuge tubes12 to be coupled with gas chromatography. More recently, a novel technique termed liquid-phase microex(3) Gilar, M.; Bouvier, E. S. P.; Compton, B. J. J. Chromatogr., A 2001, 909, 111-135. (4) Veraart, J. R.; Lingeman, H.; Brinkman, U. A. Th. J. Chromatogr., A 1999, 856, 483-514. (5) Pedersen-bjergaard, S.; Rasmussen, K. E.; Halvorsen, T. G. J. Chromatogr., A 2000, 902, 91-105. (6) Lin, C. C.; Li, Y. T.; Chen, S. H. Electrophoresis 2003, 24, 4106-4115. (7) Lord, H.; Pawliszyn, J. J. Chromatogr., A 2000, 902, 17-63. (8) Jo ¨nsson, J. A° .; Mathiasson, L. J. Sep. Sci. 2001, 24, 495-507. (9) Pedersen-bjergaard, S.; Rasmussen, K. E. Trends Anal. Chem. 2004, 23, 1-10. (10) Kumazawa, T.; Lee, X. P.; Sato, K.; Suzuki, O. Anal. Chim. Acta 2003, 492, 49-67. (11) Gonza´lez-Casado, A.; Navas, N.; del Olmo, M.; Vı´lchez, J. L. J. Chromatogr. Sci. 1998, 36, 565-569. (12) Ramsey, J. J. Chromatogr. 1971, 63, 303-308.

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traction (LPME) has been developed.13-17 This relatively new technique can be performed by using a single drop of solvent13-15 or a small length of porous hollow fiber-protected solvent.16,17 It is similar to LLE but at a miniaturized scale and, therefore, requires much less solvent. An on-line concentration method represents one of the most facile ways for sample enrichment in CE, since the preconcentration step is performed within the same capillary used for analysis. This approach involves chromatographic and electrophoretic preconcentrations. Sample stacking,18 sweeping,19 and isotachophoresis20 are most frequently used. Sample stacking is an experimentally simple approach for on-column sample preconcentration and has been applied extensively to improve the sensitivity of CE. Various formats of sample stacking, including normal stacking,21-23 large-volume sample stacking,24,25 headcolumn field-enhanced sample stacking,26,27 pH-mediated stacking,28,29 and transient moving chemical reaction boundary method,30 have been developed to focus analytes, resulting in up to a 2000fold sensitivity enhancement. Furthermore, a combination of fieldenhanced sample injection and sweeping, referred to as cationselective exhaustive injection and sweeping, has achieved almost 1 million-fold enhancement in detector response for some cationic hydrophobic analytes.31 The concentration effect of sample stacking is directly proportional to the enhancement factor (γ) or ratio between the conductivity of the separation buffer and sample or water zones. The application of the methods to real biological and environmental samples is hindered by interfering ionic matrix constituents. Therefore, the use of suitable sample pretreatment techniques is essential for sample stacking. LLE,32,33 SPE,34,35 SPME,36,37 MBE,38 and LPME39 have been used for sample preparation before sample stacking. The present study is concerned with developing (13) Jeannot, M. A.; Cantwell, F. F. Anal. Chem.1996, 68, 2236-2240. (14) He, Y.; Lee, H. K. Anal. Chem. 1997, 69, 4634-4640. (15) Zhang, J.; Su, T.; Lee, H. K. Anal. Chem. 2005, 77, 1988-1992. (16) Pedersen-Bjergaard, S.; Rasmussen, K. E. Anal. Chem. 1999, 71, 26502656. (17) Jiang, X.; Oh, S. Y.; Lee, H. K. Anal. Chem. 2005, 77, 1689-1695. (18) Chien, R. L. Electrophoresis 2003, 24, 486-497. (19) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-468. (20) Gebauer, P.; Boe`ek, P. Electrophoresis 2002, 23, 3858-3864. (21) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, T. P. E. M. J. Chromatogr. 1979, 169, 1-10. (22) Chien, R. L.; Burgi, D. S. J. Chromatogr. 1991, 559, 141-152. (23) Burgi, D. S.; Chien, R. L. Anal. Chem. 1991, 63, 2042-2047. (24) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 1046-1050. (25) Burgi, D. S. Anal. Chem. 1993, 65, 3726-3729. (26) Zhang, C. X.; Thormann, W. Anal. Chem. 1996, 68, 2523-2532. (27) Liu, W.; Lee, H. K. Anal. Chem. 1998, 70, 2666-2675. (28) Xiong, Y.; Park, S. R.; Swerdlow, H. Anal. Chem. 1998, 70, 3606-3611. (29) Britz-McKibbin, P.; Otsuka, K.; Terabe, S. Anal. Chem. 2002, 74, 37363743. (30) Cao, C. X.; He, Y. Z.; Li, M.; Qian, Y. T.; Gao, M. F.; Ge, L. H.; Zhou, S. L.; Yang, L.; Qu, Q. S. Anal. Chem. 2002, 74, 4167-4174. (31) Quirino, J. P.; Terabe, S. Anal. Chem. 2000, 72, 1023-1030. (32) Breadmore, M. C.; Procha´zkova´, A.; Theurillat, R.; Thormann, W. J. Chromatogr., A 2003, 1014, 57-70. (33) Quirino, J. P.; Terabe, S. Anal. Chem. 1998, 70, 1893-1901. (34) Strausbauch, M. A.; Landers, J. P.; Wettstein, P. J. Anal. Chem. 1996, 68, 306-314. (35) Zhu, L.; Lee, H. K. Anal. Chem. 2001, 73, 3065-3072. (36) Herna´ndez-Borges, J.; Cifuentes, A.; Garcı´a-Montelongo, F. J.; Rodrı´guezDelgado, M.AÄ . Electrophoresis 2005, 26, 980-989. (37) Fang, H.; Liu, M.; Zeng, Z. Talanta 2006, 68, 979-986. (38) Pa´lmarsdo´ttir, S.; Thordarson, E.; Edholm, L. E.; Jo ¨1nsson, J. A° .; Mathiasson, L. Anal. Chem. 1997, 69, 1732-1737. (39) Zhu, L.; Tu, C.; Lee, H. K. Anal. Chem. 2001, 73, 5655-5660.

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an alternative sample preparation method that automated liquidliquid microextraction with the aid of centrifuge applied to very small volumes of biological sample. Recently, we have developed an on-line back-extraction fieldamplified sample injection method that can be applied to directly analyze the extractants by solvent microextraction.40 Solvent microextraction coupled with the method has been shown to be a fast, simple, inexpensive, and sensitive technique. However, solvent microextraction is susceptible to drop instability both at high stir rates and in samples with significant particulate matter. The use of the centrifuge in LLE often plays the role of providing a sample cleanup. To our knowledge, no report that studies the factors influencing the centrifuge extraction efficiency has been presented. Here, we developed a method termed “centrifuge microextraction”, which combined removal of macromolecular contaminants and other interfering components, desalting, and preconcentration into one single step. Several factors that influence the microextraction efficiency, such as centrifuge rate, extraction time, salt, and pH of the sample, were optimized. In this article, the CME coupled with OLBE-FASI method to determine trace ephedrine derivatives in human urine and serum is presented. EXPERIMENTAL SECTION Reagents. (1R,2S)-Ephedrine ((-)-E) was obtained from the National Institute for Control of Pharmaceutical and Biological products (Beijing, China); (1R,2R)-pseudoephedrine ((-)-PE) and (1S,2S)-pseudoephedrine ((+)-PE) were acquired from Aldrich (Milwaukee, WI). β-Cyclodextrin (β-CD) was purchased from Shanghai Chemical Plant (Shanghai, China). (S)-(+)-Methamphetamine ((+)-MA) was provided by the Department of Public Security of Hubei Province, China. Toluene was obtained from Shanghai Chemical Factory. All chemicals used were analytical grade. Deionized water from a Milli-Q water purification system (Bedford, MA) was used throughout. Apparatus. All separation experiments were performed on a Beckman P/ACETM MDQ instrument (Beckman Coulter, Fullerton, CA) equipped with a photodiode diode array detector (190600 nm), automatic injector, fluid-cooled column cartridge (1560 °C), and system Gold Date Station. The separations were performed in an uncoated fused-silica capillary of 50.2 cm × 50 µm i.d. (effective length, 40 cm; the inlet end of capillary was cut off 4 mm) (Yongnian Photoconductive Fiber Factory, Hebei, China). A centrifuge model TGL-16C with a 30° fixed angle rotor (rmin ) 25 mm, rmax ) 65 mm) (Shanghai Anting Instrument Factory, Shanghai, China) was used to perform CME. Electrophoretic Conditions. The electrolyte was 100 mM sodium dihydrogen phosphate buffer (pH 2.5 adjusted by phosphoric acid and filtered through a 0.45-µm pore size filter). The capillary was conditioned before use by successive washings with 0.1 mol/L sodium hydroxide (10 min), water (10 min), and running buffer (10 min), respectively. Between runs, the capillary was rinsed with water for 1 min and then the running buffer for 3 min, successively. The separation conditions were as follows: run buffer of 100 mM phosphate background electropholyte at pH 2.5 with 15 mM β-CD, separation voltage of 20 kV, and capillary temperature of (40) Fang, H.; Zeng, Z.; Liu, L.; Pang, D. Anal. Chem. 2006, 78, 1257-1263.

20 °C. The injection of the samples was electroinjection (3 kV × 90 S). The diode array UV detection was performed at 192 nm, where the detection exhibits the best sensitivity for ephedrine derivatives. Sample Preparation. The standard sample for optimization of the OLBE-FASI was prepared in toluene. Stock standard solutions with ephedrine (1000 µg/mL), pseudoephedrine (1000 µg/mL), and methamphetamine (1000 µg/mL) for CME were prepared in water and, when in use, diluted to the final concentration with blank urine or serum sample. A 1-mL volume of urine sample was adjusted with sodium hydroxide to the optimized pH and placed into a 2-mL polypropylene vial for CME. The human serum samples from normal subjects were obtained from Zhongnan Hospital of Wuhan University (Whuhan, China). Prior to CME, samples of serum were prepared by the following procedure: 20 µL of sample in the polypropylene vial was spiked with standard solution, 0.03 g of NaCl, and subsequently 10 µL of 12 M NaOH was added. The sample was mixed and then allowed to stand for 30 min so that protein was denatured completely by sodium hydroxide and sodium chloride. OLBE-FASI Optimization Procedure. The OLBE-FASI parameters were optimized before CME. The organic solvent sample, sealed with a water plug in the sample vial, was used for the OLBE-FASI. The inlet end capillary was cut off ∼4 mm to prevent the capillary tip from contacting with the organic solvent sample. The injection voltage, the injection time, and the matrix of water plug, such as organic solvents, were optimized in sequence. Centrifuge Microextraction Procedure. Centrifuge microextraction was carried out using a 2-mL polypropylene vial sealed with a cap to prevent solvent evaporation during the process. The 1-mL spiked urine sample was placed into the polypropylene vial containing sodium hydroxide and sodium chloride; after mixing, 50 µL of toluene was added. Then, sample was centrifuged. The target compounds were extracted into toluene (the acceptor solution), which was layered at the top of urine sample (the donor solution). After extraction, 20 µL of the acceptor solution was transferred to a Beckman Coulter Standard PCR vial (200 µL, P/N 144709) by a 25-µL syringe, sealed with a 50-µL water plug, and analyzed by CE after 3 min. RESULTS AND DISCUSSION Optimization of the Electrokinetic Injection. In principle, application of a higher voltage and a longer injection time period should result in more solute injected.22,26 In practice, the voltage is limited by Joule heating and should be moderate to avoid troubles associated with high temperature in the low-conductivity zones. It is favorable to prefer long injection time and use moderate voltages.22,40 According to our experimental results, 3 kV × 90 s was adopted for injection conditions. Optimization of Matrix of Water Plug. As noticed by Thormann,26 stacking efficiencies can be increased by harnessing differences in conductivity of the sample and buffer regions. It has been reported that the addition of an organic solvent to the sample solution can decrease the conductivity and viscosity of the sample matrix and, thus, result in signal enhancements due to increased electric field strengths and electrophoretic mobility in the sample matrix zone.26 In the current studies, methanol (10%, v/v), 1-propanol (10%, v/v), and acetonitrile (10%, v/v) were tested

Figure 1. Effect of acetonitrile content on the concentration efficiency of ephedrine derivatives (n ) 3). Concentrations of analytes, 0.50 µg/mL; sample plug, 20 µL; water plug, 50 µL of H2O; sample injection, 3 kV × 90 s; CE run buffer, 100 mM phosphate at pH 2.5 with 17.5 mM β-CD; separation voltage, 20 kV.

as additives in the water plug. In comparison, acetonitrile was better than other solvents tested and was adopted for the subsequent experiment. The influence of the acetonitrile content in the water plug was investigated with a constant volume of the water plug (50 µL). As presented in Figure 1, the volume fraction of acetonitrile in the water plug was found to drastically influence the sensitivity. Peak areas were increased as the percentages of acetonitrile increased from 0 to 15%, and then sensitivity decreased at higher percentages of acetonitrile (20%, v/v). Thus, a concentration of 15% (v/v) acetonitrile was chosen for the following experiment. For convenience, this acetonitrile-water plug is as the “water plug” in the subsequent discussion. CME Optimization. Several factors that influence the microextraction efficiency, such as extractant, extraction time, centrifugation rotor speed, salt, and pH of the sample, were investigated. The selection of extractant used in CME is based on its properties for the extraction of moderately polar drugs from a urine sample. Technically, a solvent with a density lower than water should be preferred in order to transfer the acceptor solution easily without it being contaminated by the urine sample. Five solvents nonmiscible with water, i.e., n-hexane, cyclohexane, isooctane, benzene, and toluene, were investigated with respect to their extraction efficiency for ephedrine derivatives from a 1-mL urine sample. The extraction efficiencies showed toluene was the best extractant. In addition to the good extraction efficiency, the high boiling point and low vapor pressure of toluene, allowing reproducible analysis due to small solvent losses by evaporation, make it the favorite extractant. Hence, toluene was selected as extraction solvent for CME. As the analytes of interest were basic substances, the urine sample was alkalized to deionize the analytes and consequently reduce the solubility within the sample solution. When the deionized form of the analyte was predominant in the donor phase, good solubility could be expected in the acceptor phase. To avoid diluting the samples, pH adjustment was performed by directly Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

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Figure 2. Effect of NaOH on the extraction efficiency of ephedrine derivatives (n ) 3). CME conditions: centrifugation rotor speed, 10 000 rpm; extraction time, 10 min; sample concentration, 50 ng/ mL; urine sample volume, 1 mL; extraction toluene, 50 µL; sample plug, 20 µL; water plug, 50 µL of H2O containing 15% (v/v) acetonitrile; sample electrokinetic injection, 3 kV × 90 s. CE conditions were as Figure 1.

dissolving sodium hydroxide in the urine sample. The extraction efficiency increased with the amount of sodium hydroxide from 0.04 to 0.16 g and then slightly decreased for 0.20 g (Figure 2). So 0.16 g of sodium hydroxide was favorable. Drug-protein interactions in the biological sample resulted in low recoveries compared with extraction of the drugs from a pure water matrix.3,7 So, denaturation of the proteins is very important to achieve high recovery for a drug in a biological sample. In our experiment, denaturation of the proteins in strongly alkaline solution resulted in release of bound compounds. The effect of ionic strength was studied using sodium chloride. As the amount of sodium chloride increased, the extraction efficiency increased and reached the maximum at 0.10 g; however, more sodium chloride was founded to be negative (Figure 3). This behavior can be explained by considering two simultaneously occurring processes. Initially, increasing salt concentration, the salting-out effect enhanced the analyte recovery. However, more salt in the basic solution might hinder the shift of the ionized form of ephedrine derivatives toward the neutral form, which has a higher solubility in the acceptor phase.37 Based on this, the urine sample prepared with 0.1 g of NaCl was selected for subsequent extractions. The use of the centrifuge often plays the role of removing macromolecular contaminants (mainly proteins, carbohydrates, and lipids) and any particles from the biological sample. In our experiment, various centrifugation rotor speeds were studied to determine the optimal conditions for CME. As can be seen, the peak area increased with the increase of rotor speed in the studied range of 4000-10 000 rpm (Figure 4). The reason may be as follows: during centrifugation, the solute forms a concentration gradient with a slope dependent on the relative strengths of opposing sedimentation and diffusion forces.41,42 Unlike conven(41) Ifft, J. B.; Voet, D. H.; Vinograd, J. J. Phys. Chem. 1961, 65, 1138-1145. (42) Hosken, B. D.; Cockrill, S. L.; Macfarlane, R. D. Anal. Chem. 2005, 77, 200-207.

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Figure 3. Effect of NaCl on the extraction efficiency of ephedrine derivatives (n ) 3). CME conditions: centrifugation rotor speed, 10 000 rpm; extraction time, 10 min; sample concentration, 50 ng/ mL; urine sample volume, 1 mL; NaOH in urine sample, 0.16 g; extraction toluene, 50 µL; sample plug, 20 µL; water plug, 50 µL of H2O containing 15% (v/v) acetonitrile; sample electrokinetic injection, 3 kV × 90 s. CE conditions were as Figure 1.

Figure 4. Centrifugation rotor speed on the extraction efficiency of ephedrine derivatives (n ) 3). CME conditions: extraction time, 10 min; sample concentration, 50 ng/mL; urine sample volume, 1 mL; NaOH in urine sample, 0.16 g; NaCl in urine sample, 0.10 g; extraction toluene, 50 µL; sample plug, 20 µL; water plug, 50 µL of H2O containing 15% (v/v) acetonitrile; sample electrokinetic injection, 3 kV × 90 s. CE conditions were as Figure 1.

tional liquid-liquid microextration, the centrifuge force will set up a high concentration gradient of analytes at the interface of the donor phase and acceptor phase, which will speed up the extraction rate. The natural ephedrine derivatives, with a density lower than that of water and higher than that of toluene, floated to the toluene and diffused into the acceptor phase on the basis of the concentration gradient. It was an important finding that high centrifuge force will speed up the extraction of analytes with a density lower than that of the donor phase. The CME provides a new perspective to fast extraction of drug from biological samples, since the densities of most drugs are lower than that of water. As the higher rotor speed was limited by our centrifuge, the final rotor speed of 10 000 rpm was chosen.

Table 1. CME-OLBE-FASI Enrichment Factors Obtained for Ephedrine Derivativesa compd

HI areab

FASI areac

CME-FASI aread

CME EFareae

FASI EFareaf

CME-FASI EFareag

(-)-PE (-)-E (+)-PE (+)-MA

1987 2061 1600 1954

6312 12197 5604 7863

76 044 107 087 63 058 108 311

12 9 11 14

318 592 350 402

3827 5196 3941 5543

a CE conditions are the same as shown in Figure 2. b Sample concentration, 10 µg/mL in buffer. Hydrodynamic injection (0.5 psi × 5 s). n ) 3. c Sample concentration, 0.1 µg/mL in toluene; sample plug, 20 µL; water plug, 50 µL of H2O containing 15% (v/v) acetonitrile; sample electrokinetic injection, 3 kV × 90 s; n ) 3. d Sample concentration, 0.1 µg/mL in urine. Operation conditions are the same as shown in Figure 6; n ) 3. e EF ) areaCME-FASI/areaFASI. f EF ) areaFASI / areaHI × 100. g EF ) areaCME-FASI / areaHI × 100.

Figure 5. Extraction time on the extraction efficiency of ephedrine derivatives (n ) 3). CME conditions: centrifugation rotor speed, 10 000 rpm; sample concentration, 50 ng/mL; urine sample volume, 1 mL; NaOH in urine sample, 0.16 g; NaCl in urine sample, 0.10 g; extraction toluene, 50 µL; sample plug, 20 µL; water plug, 50 µL of H2O containing 15% (v/v) acetonitrile; sample electrokinetic injection, 3 kV × 90 s. CE conditions were as Figure 1.

Figure 5 shows the influence of extraction time on the extraction efficiency. As can be seen, the peak area increased with enrichment time from 3 to 15 min for ephedrine derivatives and decreased when time was higher than 15 min. For methamphetamine, the highest extraction efficiency was achieved within 3 min. An extraction time of 10 min was selected as a compromise. During centrifugation, there are two primary forces at work that oppose each other: sedimentation of molecules as a result of the centrifugal forces being applied by the spinning rotor, and diffusion.43 Since the density of natural ephedrine derivatives is lower than that of water and higher than that of toluene, analytes floated to the top of the donor phase and diffused into the acceptor phase on the basis of a concentration gradient at first. When the diffusion rate of analytes into the acceptor phase equaled to the sedimentation rate of analytes out of it, the highest extraction efficiency was observed. With the density gradient gradually becoming distinct, more analytes in the acceptor phase sediment to the position between the donor phase and acceptor phase. Therefore, a diminution on extraction efficiency with time was observed (Figure 5). In the experiment, the time for methamphetamine to achieve the highest extraction efficiency was shorter than that for ephedrine derivatives, which might be attributes to the density and polarity of analytes. The polar analytes interacted more strongly with the water molecule than hydrophobic analytes, which would reduce the flotation velocity of polar analytes and hence increase the time to achieve the equilibration between sedimentation and diffusion. Methamphetamine, with the lowest density and weakest polarity, obtained the equilibration within a shorter time. Concentration of Ephedrine Derivatives by CME-OLBEFASI. The system utilizes two sequential enrichment steps, which are off-line CME and on-line OLBE-FASI. The enrichment factor (EF), defined as the ratio of analyte concentration after extraction (43) Johnson, J. D.; Bell, N. J.; Donahoe, E. L.; Macfarlane, R. D. Anal. Chem. 2005, 77, 7054-7061.

Figure 6. Electropherograms of blank urine (a) and spiked urine sample (b) after CME. CME conditions: centrifugation rotor speed, 10 000 rpm; extraction time, 10 min; sample concentration, 70 ng/ mL; urine sample volume, 1 mL; NaOH in urine sample, 0.16 g; NaCl in urine sample, 0.10 g; extraction toluene, 50 µL; sample plug, 20 µL; water plug, 50 µL of H2O containing 15% (v/v) acetonitrile; sample electrokinetic injection, 3 kV × 90 s. CE conditions were as Figure 1. Peak identification: (1) (-)-PE, (2) (-)-E, (3) (+)-PE, and (4) (+)MA.

and that before extraction, was used to evaluate the extraction efficiency. The maximum attainable enrichment factor (EFmax) for CME is the phase ratio of the donor phase to the acceptor phases. The EFmax for the present paper is 20 (1000 µL/50 µL). It should be noticed that 9-14-fold enrichment was achieved within 10 min (Table 1). Increasing the sample volume or decreasing the receiving phase volume could be adopted if a larger preconcentration factor is desired. The OLBE-FASI provided a further online concentration of the analytes. Using CME coupled with OLBEFASI, over 3800-fold increase in sensitivity could be obtained as compared with the normal hydrodynamic injection without sample stacking (Table 1). Validation of the CME-OLBE-FASI-CE-UV Method for Ephedrine Derivatives. To evaluate the proposed CME-OLBEAnalytical Chemistry, Vol. 78, No. 17, September 1, 2006

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Table 2. Linear Correlation, Linear Range, Correlation Coefficients (R), Limits of Detection, RSD, and Recovery of Ephedrine Derivatives with CME-OLBE-FASI-CE-UVa RSD (%) compd

linear correlation

linear range (ng/mL)

correlation coefficient (r2)

LOD (ng/mL)b

without IS

with ISc

(-)-PE (-)-E (+)-PE (+)-MA

y ) 803x - 1058 y ) 1172x - 6072 y ) 780x - 6413 y ) 1166x - 5907

5-200 5-200 10-200 5-200

0.9994 0.9988 0.9996 0.9989

0.20 0.15 0.25 0.15

8.7 11.5 10.8 17.7

5.3 6.4 8.9

recoveryd 97 102 114 105

a CME and CE conditions as in Figure 6; n ) 6. b S/N ) 3. c (+)-PE as internal standard. d Recovery ) (the amount found in the spiked sample - the amount found in the sample) × 100/the amount added. Sample concentration, 0.1 µg/mL.

Figure 7. Electropherograms of urine sample of a patient under E. herba treatment after CME. CME conditions were as in Figure 6. Sample plug, 20 µL; water plug, 50 µL of H2O containing 15% (v/v) acetonitrile; sample electrokinetic injection, 3 kV × 90 s. CE conditions were as Figure 1. Peak identification: (2) (-)-E and (3) (+)-PE.

FASI-CE-UV method, some parameters such as linearity, limit of detection (LOD), reproducibility, and relative recovery for trace ephedrine derivatives in urine were determined under the optimized conditions. The performance of this technique is shown in Table 2. For all the studied ephedrine derivatives, linearity was obtained in the range of 5/10-200 ng/mL with correlation coefficients (r2) ranging from 0.9988 to 0.9994. The relative standard deviations (RSDs, n ) 6) of all of the analytes were from 8.70 to 17.7% on the basis of peak areas. Utilizing (-)-PE as internal standard, the RSD results showed significant improvement (5.3-8.9%). The LODs ranged from 0.15 to 0.25 ng/mL, based on a signal-to-noise ratio of 3 (S/N ) 3). After exhaustively searching the literature, we found it provided the highest sensitivity reported for capillary electrophoresis to determine ephedrine derivatives in the urine. The recovery experiments for the four stimulant analytes were performed to investigate the accuracy of the CME-OLBE-FASI-CE-UV method. Ephedrine derivatives with known concentrations in urine were extracted as described and then injected for CE separation. Table 2 gives the recovery data for ephedrine derivatives at 0.1 µg/mL. A typical electropherogram obtained from the extraction of a urine sample is shown in Figure 6. Urine is a highly variable matrix as diet and liquid intake vary urine ionic strength and pH to a great degree. Testing of accuracy and precision in different urine matrixes should therefore be included in the validation of a CME method. Relative recoveries, defined as the ratio of CE peak areas of spiked three healthy individuals urine extracts to spiked 6048 Analytical Chemistry, Vol. 78, No. 17, September 1, 2006

Figure 8. Electropherograms of blank serum (a) and spiked serum sample (b) subjected to centrifuge microextraction. CME conditions: centrifugation rotor speed, 10 000 rpm; extraction time, 10 min; 20µL serum sample volume adjusted with 0.03 g of NaCl and 10 µL of 12 M NaOH; extraction toluene, 50 µL; concentration of analytes in (b), 2 µg/mL; sample plug, 20 µL; water plug, 50 µL of H2O containing 15% (v/v) acetonitrile; sample electrokinetic injection, 3 kV × 90 s. CE conditions were as Figure 1. Peak identification as in Figure 6.

urine extracts in previous experiment, were calculated to evaluate the effect of the matrix.15 The experiments were repeated three times. The relative recoveries for all target drugs were from 90 to 112%. This means that the matrix had little effect on CME. Finally, to demonstrate the applicability of the assay, it was used to analyze a urine sample of a patient under Ephedrae herba (mahuang) treatment. The concentrations of (1R,2S)-ephedrine and (1S,2S)-pseudoephedrine were determined to be 196 and 27 ng/mL, respectively. The electropherogram is shown in Figure 7. Application of the Method to a Small-Volume Serum Sample. Most analytical methods validated and adapted for pharmacokinetic investigations in adults use 200-1000 µL of plasma or serum. This volume is too large for studies in children and small animals, where the amount of blood collected should be kept to a minimum. The experiment conditions for the 20-µL serum sample differ a little from that for the urine: 20 µL of sample

with 0.03 g of NaCl and 10 µL of 12 M NaOH was adopted for CME. To achieve high recovery, the sample was mixed and then allowed to stand for 30 min before CME so that protein denatured completely. The other CME conditions were as with the urine sample. Typical electropherograms obtained with extracts of serum samples are shown in Figure 8. Data produced with blank serum from the volunteers who did not receive any drugs showed no peaks interfered with the investigated drugs (electropherogram a). Figure 8 (electropherogram b) shows that ephedrine derivatives presented in serum can be extracted and determined without interference. The result demonstrates the potential of trace analysis of serum sample with a small volume by OLBE-FASICZE after efficient cleanup with the CME technique.

throughput can be further improved by extracting several samples at the same time with a centrifuge. (d) The cross-contamination and carryover effects were totally eliminated, since fresh organic solvent and disposable polypropylene vials are used for each extraction. (e) The CME is highly cost-effective and environment friendly with the minimized human manipulations and very small volume of organic solvent used. (f) The procedure only needed sample volumes above several microliters, which is very convenient for small-volume samples. As many samples can be extracted at one time by a centrifuge, the parallel microextraction coupling with capillary array electrophoresis to provide the higher sample throughput method is under consideration.

CONCLUSION AND FUTURE RESEARCH The present work has demonstrated a high potential of CMEOLBE-FASI-CE to detect trace compounds in complex sample matrixes. It has a variety of merits such as the following: (a) the CME integrated the sample cleanup and preconcentration into a single step, and the OLBE-FASI eliminated the need to perform solvent-exchange, which simplified the operation greatly. (b) A high concentration efficiency could be obtained by CME-OLBEFASI (over 3800-fold increase in sensitivity). (c) The sample preparation time is only a few minutes; moreover, the sample

ACKNOWLEDGMENT This work was kindly supported by the National Natural Science Foundation of China (Grant 20375028) and the High Technology Research and Development Program of China (863 program) (2002AA2Z2004).

Received for review February 26, 2006. Accepted June 26, 2006. AC060360Q

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