Anal. Chem. 2006, 78, 1257-1263
On-Line Back-Extraction Field-Amplified Sample Injection Method for Directly Analyzing Cocaine and Thebaine in the Extractants by Solvent Microextraction Huaifang Fang, Zhaorui Zeng,* Lan Liu, and Daiwen Pang
Department of Chemistry, Wuhan University, Wuhan 430072, China
This paper describes a novel method that applies on-line back-extraction field-amplified sample injection (OLBEFASI) to the extractants by solvent microextraction (SME) in capillary zone electrophoresis (CZE). To our knowledge, it provides the first report that the water-immiscible solvent samples were directly analyzed by CZE. The waterimmiscible solvent sample, sealed with a water plug in the sample vial, was used for direct electroinjection. The water plug with a moderate content of organic solvent, lowconductivity, and the presence of a small amount of H+ provided the highest sensitivity for analyzing positively chargeable compounds, such as cocaine and thebaine. The linear range was at least 2 orders of magnitude, with the square of the correlation coefficient (r2) > 0.9999, and a separate calibration over the range 0.016-10 µg/ mL showed the linear range to be approaching 3 orders of magnitude. Detection limits were in the range of 2-10 ng/mL. Because the need to perform solvent exchange (from organic to aqueous solution) was eliminated, the OLBE-FASI method could be conveniently coupled with SME. In the present work, SME-OLBE-FASI-CE was validated for quantitative purposes, and applications to human urine were demonstrated. In recent years, capillary electrophoresis (CE) has been widely developed for analyzing drugs in biological fluids (urine, plasma, serum, and saliva) due to its many attractive features, such as high separation efficiency, reduced analysis time, a low consumption of reagents, and relatively simple method development.1,2 In the determination of drugs in biological samples, a major analytical challenge for CE is the limit of detection and quantification because of the low concentration levels, which may be of analytical interest. For example, the Substance Abuse and Metal Health Services Administration (SAMHSA) mandatory guidelines for federal workplace drug testing programs sets the initial test cutoff * To whom correspondence should be addressed. Fax: +86-27-8764-7617. E-mail:
[email protected]. (1) Bonato, P. S. Electrophoresis 2003, 24, 4078-4094. (2) Thormann, W.; Wey, A. B.; Lurie, I. S.; Gerber, H.; Byland, C.; Malik, N.; Hochmeister, M.; Gehrig, C. Electrophoresis 1999, 20, 3203-3236. 10.1021/ac0516537 CCC: $33.50 Published on Web 12/22/2005
© 2006 American Chemical Society
level for cocaine metabolites at 0.3 µg/mL.3 The relatively high detection limits for CE derive from the low amount of sample introduced into the capillary (usually picoliters to nanoliters) and, for spectrophotometric detection, the short optical path length (25-100 µm). Therefore, an increase in the amount of analyte introduced into the capillary is needed to reduce the detection limits. To increase the amount of analyte introduced into the capillary, several preconcentration methods that can be combined with the CE in a different manner are currently used. Depending on the concentration mechanism, these methods can be classified as chromatography- or electrophoresis-based preconcentration. Normal stacking,4 field-amplified sample injection (FASI),5-13 and isotachophoresis14 are the electrophoresis-based preconcentration techniques most frequently used. Among them, the technique of FASI is one of the simplest techniques and can provide a sensitivity enhancement over 1000-fold without adverse effects on peak shape and resolution.8 The basic theory of FASI is that when voltage is applied to the two ends of the capillary, sample ions prepared in a more diluted buffer or solvent will experience a higher electric field strength because of the low conductivity of sample matrix and move faster than the ions inside the background electrolyte (BGE). The sample ions will slow when they pass the boundary between the sample and the BGE compartments and stack into a zone much narrower than the original sample plug. The most important prerequisite for achieving high sensitivity associated with FASI is that samples have to be free of electrolytes. Thus, sample cleanup is required for biological samples, which normally contain high concentration of salts. (3) Department of Health and Human Services, National Clearinghouse For Alcohol and Drug Information; Mandatory Guidelines for Federal Workplace Drug Testing Programs; U.S. Government Printing Office: Washington, DC, 1994; http://www/health.org/GDLNS-94.htm (accessed December 1999). (4) Urba´nek, M.; Krˇiva´nkova´, L.; Bocˇek, P. Electrophoresis 2003, 24, 466485. (5) Chien, R. L.; Burgi, D. S. J. Chromatogr. 1991, 559, 141-152. (6) Chien, R. L. Anal. Chem. 1991, 63, 2866-2869. (7) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-496A. (8) Zhang, C. X.; Thormann, W. Anal. Chem. 1996, 68, 2523-2532. (9) Zhang, C. X.; Thormann, W. Anal. Chem. 1998, 70, 540-548. (10) Liu, W.; Lee, H. K. Anal. Chem. 1998, 70, 2666-2675. (11) He, Y.; Lee, H. K. Anal. Chem. 1999, 71, 995-1001. (12) Zhu, L.; Lee, H. K. Anal. Chem. 2001, 73, 3065-3072. (13) Macia`, A.; Borrull, F.; Aguilar, C.; Calull, M. Electrophoresis 2004, 25, 428436. (14) Gebauer, P.; Bocˇek, P. Electrophoresis 2002, 23, 3858-3864.
Analytical Chemistry, Vol. 78, No. 4, February 15, 2006 1257
Liquid-liquid extraction (LLE),15 solid-phase extraction (SPE),16 sweeping,17,18 and solid-phase microextraction (SPME)19 are chromatography-based preconcentration techniques most frequently used. Among them, LLE is the most commonly used technique for preconcentration and cleanup of biological samples; however, it is needed to evaporate the solvent to dryness after LLE and to reconstitute the dry residue in a suitable solvent for CE. These steps are tedious, time-consuming, and also prone to loss of analytes by evaporation and adsorption.15 In recent years, research activities have been oriented toward the development of efficient and miniaturized sample pretreatment methods. Solvent microextraction (SME) has been shown to be an attractive alternative method to conventional LLE.20-22 The SME can be performed by using a single drop of solvent23-29 or a small length of porous, hollow fiber-protected solvent.22,30 Compared to LLE, SME gives a comparable and satisfactory sensitivity and much better enrichment of analytes. Moreover, the consumption of solvent is significantly reduced by up to several hundred or several thousand times, and the method is extremely affordable, simple to operate, and fast. Just as LLE, the SME provides sample cleanup, because inorganic salts and biological macromolecules are normally insoluble in the organic solvents for SME. Thus, SME extractant normally provides an excellent medium for fieldamplified sample stacking. The SME technique has frequently been used in conjunction with gas chromatography (GC) and high-performance liquid chromatography (HPLC) to detect many compounds, such as cocaine,31,32 hydrophobic biomolecules,28 and chemical warfare agents.33 The combination of SME with CE poses some technical challenges, because the organic extractants, which are immiscible with water, are not injectable in conventional aqueous CE. Thus, there will be significant interest if analytes in an organic solvent can be directly on-line stacked to achieve enhancement of sensitivity without any loss of the high separative efficiency and resolution of CE. To expand the applications of the on-line preconcentration technique with capillary electrophoresis to the extractants by solvent microextraction, an on-line back-extraction field-amplified sample injection method is presented. Parameters affecting online concentration, such as the electroinjection time and voltage; (15) Pedersen-Bjergaard, S.; Rasmussen, K. E.; Halvorsen, T. G. J. Chromatogr., A 2000, 902, 91-105. (16) Lin, C. C.; Li, Y. T.; Chen, S. H. Electrophoresis 2003, 24, 4106-4115. (17) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-468. (18) Zhu, L.; Tu, C.; Lee, H. K. Anal. Chem. 2002, 74, 5820-5825. (19) Lord, H.; Pawliszyn, J. J. Chromatogr., A 2000, 902, 17-63. (20) Jeannot, M. A.; Cantwell, F. F. Anal. Chem. 1996, 68, 2236-2240. (21) Theis, A. L.; Waldack, A. J.; Hansen, S. M.; Jeannot, M. A. Anal. Chem. 2001, 73, 5651-5654. (22) Shen, G.; Lee, H. K. Anal. Chem. 2002, 74, 648-654. (23) He, Y.; Lee, H. K. Anal. Chem. 1997, 69, 4634-4640. (24) Jeannot, M. A.; Cantwell, F. F. Anal. Chem. 1997, 69, 235-239. (25) Jeannot, M. A.; Cantwell, F. F. Anal. Chem. 1997, 69, 2935-2940. (26) Wang, Y.; Kwok, Y. C.; He, Y.; Lee, H. K. Anal. Chem. 1998, 70, 46104614. (27) Ma, M.; Cantwell, F. F. Anal. Chem. 1998, 70, 3912-3919. (28) Keller, B. O.; Li, L. Anal. Chem. 2001, 73, 2929-2936. (29) Liu, J. F.; Jiang, G. B.; Chi, Y. G.; Cai, Y. Q.; Zhou, Q. X.; Hu, J. T. Anal. Chem. 2003, 75, 5870-5876. (30) Zhao, L.; Lee, H. K. Anal. Chem. 2002, 74, 2486-2492. (31) de Jager, L. S.; Andrews, A. R. J. J. Chromatogr., A 2001, 911, 97-105. (32) de Jager, L. S.; Andrews, A. R. J. Analyst 2001, 126, 1298-1303. (33) Palit, M.; Pardasani, D.; Gupta, A. K.; Dubey, D. K. Anal. Chem. 2005, 77, 711-717.
1258 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006
Figure 1. Chemical structures of the drugs investigated in this work.
the influence of a water plug added above the water-immiscible solvent sample; the matrix of the water plug; the amount of phosphoric acid; and several factors that influence the microextraction efficiency, such as extraction time, stirring rate, and pH of the sample, were optimized. In this article, the OLBE-FASI method coupled with SME to determine trace cocaine and thebaine in water and human urine is illustrated. EXPERIMENTAL SECTION Reagents. Cocaine and thebaine were obtained from the National Institute for the Control of Pharmaceutical and Biological Products of China. Methamphetamine (internal standard (IS)) was provided by the Department of Public Security of Hubei Province, China (their structures are shown in Figure 1). Chloroform 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 array detector (190-600 nm), automatic injector, a fluid-cooled column cartridge (15-60 °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) (Yongnian Photoconductive Fiber Factory, Hebei, China). A 10-µL syringe (Shanghai syringe the third plant, Shanghai, China), with a bevel needle tip (bevel 22°; needle o.d., 0.485 mm) was used for introducing organic solvent for SME. A magnetic stirrer, DF-101B (Leqing, China), with a 1 cm stir bar (7 mm × 5 mm PTFE stir bar) was employed for stirring the sample during SME. Electrophoretic Conditions. The electrolyte was 100 mM sodium dihydrogen phosphate buffer (pH 2.5 adjusted by phosphoric acid) and filtered through a 0.22-µm pore size filter. The capillary was conditioned before use by successive washings for 10 min with 0.1 mol/L sodium hydroxide and 10 min with water, followed by running buffer for 10 min. Between runs, the capillary was rinsed with water for 1 min and then the running buffer for 3 min, successively. Separation was performed at a constant voltage of 20 kV (anode at injection end). The temperature of the capillary cartridge was set at 25 °C. UV absorbance was monitored at 192 nm with diodearray detection (Beckman Instruments). Standard Solutions and Urine Sample Preparation. The standard sample for optimization of the OLBE-FASI was prepared in chloroform. Stock standard solutions with cocaine (1000 µg/ mL) and thebaine (1000 µg/mL) for SME were prepared in water and, when in use, diluted to the final concentration with water or the blank urine sample. A 9-mL volume of urine sample was adjusted with 1 M sodium hydroxide to the optimized pH, placed
Figure 2. Schematic illustration of the sample stacking mechanism of SME-OLBE-FASI-CE. O represents neutral analyte; x represents analyte’s conjugate acid.
into a 10-mL silanized vial, and agitated at a stirring rate of 2000 rpm for 2 min. The solution was then passed through a 0.22-µm pore size filter. An 8-mL volume of the urine sample was transferred to the extraction vial for SME. OLBE-FASI Optimization Procedure. The OLBE-FASI parameters were optimized before SME. The organic solvent sample, sealed with a water plug in the sample vial, was used for the OLBE-FASI. The injection voltage; injection time; and the matrix of water plug, such as organic solvents and acids, were optimized in sequence. SME Procedure. SME experiments were performed by taking all the required precautions.24,31,33 The 8-mL spiked water sample or urine sample was placed into a 10-mL silanized vial containing a small magnetic stirring bar, then 4 µL of chloroform was withdrawn into a 10-µL syringe, and the needle of the syringe was immersed into the sample solution. The plunger was depressed to generate a 2-µL solvent drop on the needle tip (with 2 µL of chloroform remaining in the syringe). Meanwhile, the solution was constantly stirred at 200 rpm to promote the diffusion of the drugs from the aqueous phase into the solvent drop. After 5 min of extraction, the drop was retracted into the needle, and the plunger was drawn back to 4 µL. The syringe was then taken out of the vial; the solvent was transferred to a Beckman Coulter Standard PCR vial (200 µL, P/N: 144709), sealed with a 40-µL water plug, and analyzed by CE after 3 min. RESULTS AND DISCUSSION Basic Mechanism of the OLBE-FASI Method. The organic solvent sample, sealed with a water plug in the sample vial, was used for the OLBE-FASI. It is crucial to keep the inlet end of the capillary in the water plug. The inlet end capillary was cut off about 1-2 mm to prevent the capillary tip from contacting with the organic solvent sample. The on-line back-extraction involves two phases, namely, the organic phase and the water plug. The analyte in the organic phase distributes between organic and water plug. This can be represented by the following equation,
Io h Iaq
(1)
where I is the analyte, o is the organic phase, and aq is the water plug above the organic phase.
When the analytes are extracted into the water plug, partial protonation occurs because of their weak alkalescence.
Iaq + H3O + h HI + + H2O
(2)
When the electrokinetic injection is initiated, an electric field in the water plug up to several hundred times higher than that employed in normal CE is established. The enhanced electric field enables positively charged analytes to move rapidly into the capillary. Once the analytes have migrated into the highconductivity buffer region, they will slow and concentrate into narrow bands. Since the analytes are ionized within the low pH buffer, they are prevented from re-entering the water plug. The dissociation equilibrium of the analytes in the water plug is then disrupted. According to Le Chaˆtelier’s principle, the position of chemical equilibrium will shift to the right in eq 2. The neutral analytes will be protonated continuously to replenish the HI+ in the water plug, and their concentration in the water plug will reduce; therefore, the analytes are transferred from the organic phase into the water plug. A great number of analyte molecules can be back-extracted from the organic sample and injected into capillary, and a very high preconcentration can be obtained for the analytes. Thus, the OLBE and FASI can be carried out under an enhanced electric field at the same time. On the basis of this, a scheme was designed for the stacking of positively chargeable compounds in water-immiscible solvent, as illustrated in Figure 2. Optimization of the Electrokinetic Injection. In principle, application of a higher voltage and a longer injection time period should result in more solute injected.5,8 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.5,6,34 The effects of the injection voltages on stacking were examined with the injection time at 80 s. As shown in Table 1, the peak areas of both substances increased with the applied voltage from 1 to 5 kV. Although higher voltages are very effective for a strong preconcentration of analytes, the use of high voltages is disad(34) Wu, S. M.; Ho, Y. H.; Wu, H. L.; Chen, S. H.; Ko, H. S. Electrophoresis 2001, 22, 2717-2722.
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Table 1. Influence of Injection Voltage on the Peak Area, Resolution, and Efficiencya injection voltage (kV) 1 2 3 4 5
peak area cocaine thebaine 6.88 × 103 1.35 × 104 1.61 × 104 2.60 × 104 3.64 × 104
5.47 × 103 1.12 × 104 1.46 × 104 2.30 × 104 3.08 × 104
resolution 3.94 3.07 2.52 2.18 1.58
efficiency (theoretical plates) cocaine thebaine 2.23 × 105 1.47 × 105 1.01 × 105 8.31 × 104 4.95 × 104
2.32 × 105 1.04 × 105 7.47 × 104 5.71 × 104 3.66 × 104
a The samples were prepared in chloroform. The concentration of cocaine and thebaine was 3 µg/mL. Sample plug, 4 µL; water plug, 40 µL of H2O. The injection voltage was from 1 to 5 kV with injection time at 80 s. CE run buffer: 100 mM phosphate (pH 2.5). Separation voltage: 20 kV; n ) 3.
Table 2. Influence of Injection Time on the Peak Area, Resolution, and Efficiencya
injection time (s) 20 40 60 80 100
peak area cocaine thebaine 9.74 × 103 1.72 × 104 2.12 × 104 2.60 × 104 3.17 × 104
7.63 × 103 1.27 × 104 1.67 × 104 2.30 × 104 2.70 × 104
resolution 3.75 2.50 2.25 2.18 1.57
efficiency (theoretical plates) cocaine thebaine 2.12 × 105 1.36 × 105 1.02 × 105 8.31 × 104 5.85 × 104
1.64 × 105 8.05 × 104 6.62 × 104 5.71 × 104 3.69 × 104
a The injection times were from 20 to 100 s with voltage at 4 kV. Other conditions as in Table 1; n ) 3.
vantageous for our system. Air bubbles were found at the interface between the organic phase and the water plug, which could be caused by excessive Joule heating in the low-conductivity zones. Moreover, the repeatability was worse when voltage was higher than 4 kV because of those air bubbles. Thus, 4 kV was chosen for the following experiment. Concerning the sample injection time, a more exhaustive study between 20 and 100 s of electrokinetic injection was performed. Table 2 reports the influence of sample injection time on the resolution between two analytes, peak efficiencies, and peak areas of cocaine and thebaine (concentration of analytes: 3 µg/mL). Increasing the injection time of the sample induced a higher peak area and better repeatability but lower peak efficiency and resolution values. Although some peak deformations occurred, the resolution was still above 2.00 for 80 s of electroinjection. From practical interest, 80 s of electrokinetic injection of the sample was chosen to achieve a better sensitivity. Optimization of Matrix of Water Plug. The water plug added to the sample vial is the key for this on-line preconcentration method. It plays two roles: first, it provides a short zone of low conductivity between the anode and the capillary inlet, across which an enhanced electric field is established. The enhanced electric field permits charged analytes to be injected at high velocity and condensed at the interface between the lowconductivity zone and the running buffer at the tip of the capillary. Second, hydronium ion in it acts as the H+ donor for cocaine and thebaine. It was proved by the following experiment: When the same volume of chloroform instead of water was added to the sample vial, although the chloroform has a lower conductivity, smaller and irreproducible peaks were observed. 1260 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006
Figure 3. Effect of acetonitrile content on the concentration efficiency of cocaine, thebaine (n ) 3). Concentrations of analytes, 1.0 µg/mL; sample plug, 4 µL; water plug, 40 µL of H2O; sample injection, 4 kV × 80 s; CE run buffer, 100 mM phosphate (pH 2.5); separation voltage, 20 kV.
Several groups have studied the effect of a water plug at the capillary inlet in sample stacking by electrokinetic injection.6-8 Chien noted that the water plug provides a higher electric field at the tip of the capillary, which will eventually improve the sample stacking procedure.6,7 In this paper, insertion of a water plug in the head column of the capillary was demonstrated to deteriorate the performance of OLBE-FASI. The reason may be that the insertion of a water plug in the capillary caused a drop in the electric field strength at the water plug in the sample vial, which played an important role in this on-line preconcentration method. Stacking efficiencies can be increased not only by harnessing differences in conductivity in the sample and buffer regions but also by altering the electrophoretic mobility within the sample matrix. As noticed by Chien5 and Thormann,8,9 the mobility of analytes could be increased by additives such as organic solvents, acids, and ions in the sample matrix. 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.8,9 In the current studies, methanol (10%, v/v), 1-propanol (10%, v/v), and acetonitrile (10%, v/v) were tested 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 (40 µL). As presented in Figure 3, the volume fraction of acetonitrile in the water plug was found to drastically influence the sensitivity. With the increase of acetonitrile percentages, peak areas increased. This effect is based on changes in conductivity; however, a concentration of acetonitrile higher than 20% was found to be unsuitable, because a failure of the separation sometimes occurred as a result of an air bubble formed in the capillary. The air bubble might result from the higher solubility of chloroform in the water plug and then diffused into the buffer. The percentage of acetonitrile employed in this work was 20% considering the sensitivity,
Table 3. Calibration Data for Cocaine and Thebaine in Chloroform with OLBE-FASI-CE-UVa RSD (%) (n ) 6) MT
compound
linear correlation
linear range (µg/mL)
correlation coefficient (r2)
LOD (µg/mL) (S/N ) 3)
intraday
interday
cocaine thebaine
y ) 37563x + 5256 y ) 45409x - 2860
0.016-10 0.080-10
0.9999 0.9999
0.002 0.010
0.51 0.60
1.88 2.01
a
peak area intraday interday 3.26 4.32
4.63 5.77
Sample plug, 4 µL; water plug as in Figure 5. Sample electrokinetic injection, 4 kV × 80 s. CE conditions as in Figure 3; n ) 3.
Figure 4. The effect of phosphoric acid on the concentration efficiency of cocaine and thebaine (n ) 3). Concentrations of analytes, 1.0 µg/mL; sample plug, 4 µL. The water plug was 40 µL of H2O containing 20% acetonitrile (v/v); sample injection, 4 kV × 80 s. CE conditions were as Figure 3.
precision, and accuracy. For convenience, this acetonitrile-water plug is as the “water plug” in the subsequent discussion. It has been demonstrated that the addition of phosphoric acid to the sample solvent may lead to an increase in the electrophoretic velocity of positively chargeable substances and improve the sensitivity detection during FASI injection because a larger amount of higher-mobility ions is introduced.5,8,9 Moreover, as the protonation of analytes increased under acidic conditions, eq 2 shifted to the right, and the concentration of the analytes (Caq + CHI+) in the water plug increased, as well. Different concentrations of phosphoric acid were added to the water plug. Indeed, peak areas were increased as the concentration of phosphoric acid increased from 0 to 75 µM, and then the sensitivity decreased at higher acid concentrations due to an increase in conductivity of the water plug (Figure 4), so a concentration of 75 µM was adopted for further experiments. Performance of the OLBE-FASI Method. To evaluate the practical applicability of the proposed preconcentration method, repeatability, linearity, and detection limit were investigated by utilizing standard solutions of cocaine and thebaine in chloroform (Table 3). The linear range was at least 2 orders of magnitude, and a separate calibration over the range 0.016-10 µg/mL showed the linear range to be approaching 3 orders of magnitude. This is exceptionally good for FASI-CE-UV detection. The FASI-CEUV usually has a narrow linear range (usually 1-2 orders of magnitude), which could be attributed to the stacking effect.5,35-37 Employing the on-line preconcentration method compared to a
Figure 5. Electropherograms of cocaine and thebaine dissolved in chloroform. Sample concentration, 0.4 µg/mL; sample plug, 4 µL. The water plug was 40 µL of H2O containing 20% (v/v) acetonitrile and 75 µM phosphoric acid. Sample electrokinetic injection, 4 kV × 80 s. CE conditions were as Figure 3.
nonstacking sample introduction (hydrodynamic injection, 0.5 psi × 5 s; concentration of analytes, 50 µg/mL analytes in BGE), about 200-fold enhancement in concentration sensitivity for analytes was attained. An excellent accuracy on retention times and peak areas was found, with relative standard deviations (RSD, n ) 6) being generally less than 2% and 6% without use of the internal standard. A typical electropherogram obtained from the chloroform standard sample is shown in Figure 5. To maintain good repeatability, fresh samples should always be used for each injection. The data presented here demonstrates the applicability of the method to qualitative and quantitative analysis. SME Optimization. Several factors that influence the microextraction efficiency, such as extraction time, stirring rate, and pH of the sample, were investigated. The analytical signal increased with the extraction time from 1 to 5 min; however, drop loss was encountered for longer extraction time, which may be due to the relatively high water solubility of the chloroform (0.795 (35) Burgi, D. S.; Chien, R. L. Anal. Chem. 1991, 63, 2042-2047. (36) Wen, J.; Cassidy, R. M. Anal. Chem. 1996, 68, 1047-1053. (37) Liu, Y. M.; Cheng, J. K. Electrophoresis 2002, 23, 556-558. (38) Caslavska, J.; Thormann, W. Electrophoresis 2004, 25, 1623-1631. (39) Clauwaert, K. M.; Van Bocxlaer, J. F.; Lambert, W. E.; De Leenheer, A. P. Anal. Chem. 1996, 68, 3021-3028. (40) Kikura-Hanajiri, R.; Kaniwa, N.; Ishibashi, M.; Makino, Y.; Kojima, S. J. Chromatogr., B 2003, 789, 139-150.
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Table 4. Linear Correlation, Linear Range, Correlation Coefficients (R), Limits of Detection, RSD, and Recovery of Cocaine and Thebaine with SME-OLBE-FASI-CE-UVa compound
linear correlation
linear range (µg/mL)
correlation coefficient (r2)
LOD (µg/mL)b
RSD (%)c
recoveryd
cocaine thebaine
y ) 0.63x + 0.056 y ) 2.33x -0.012
0.040-4 0.040-2
0.9990 0.9999
0.005 0.005
5.57 6.16
94.71 98.65
a SME conditions as in Figure 7. Sample plug, 4 µL; water plug as in Figure 5. Sample electrokinetic injection, 4 kV × 80 s. CE conditions were as Figure 3. n ) 3. b S/N ) 3. c Internal standard added. n ) 6. d Recovery ) (the amount found in the spiked sample - the amount found in the sample) × 100/the amount added. Sample concentration, 0.5 µg/mL.
Figure 6. Effect of pH on the extraction efficiency of cocaine and thebaine (n ) 3). SME conditions: stirring rate, 200 rpm; extraction time, 5 min; sample concentration, 1.0 µg/mL; sample volume, 8 mL; extraction chloroform volume, 4 µL; water plug as in Figure 5. Sample electrokinetic injection, 4 kV × 80 s. CE conditions were as Figure 3.
g/100 mL of water). Thus, we chose 5 min as the extraction time for the rest of the study. Previous studies showed that a stir rate below 240 rpm provided good extraction with a low rate of drop loss.31 According to our experimental results, a stirring rate 200 rpm was used for subsequent experiments. The sample pH is crucial for SME of acids and bases. It results in a large distribution ratio and ensures high enrichment factors for target analytes.27 Because all the examined drugs are basic (Figure 1), at a pH above the highest pKa value, it is expected that drugs would be in their neutral form and extractable into the organic drop. Figure 6 shows the effect of pH on extraction efficiency of cocaine and thebaine. A higher extraction yield was observed at pH 11, which was in agreement with that reported elsewhere.31 The optimum value of pH 11 was chosen for subsequent analysis. The effect of salt was also examined in this study by varying the amount of sodium chloride added (0-1%, w/v) to the aqueous solution containing the target analytes. The obtained results reveal that there was a slight decrease in extraction efficiency with the increase in sodium chloride concentration (data not shown). This behavior can be explained by considering two simultaneously occurring processes: the salting out effect, and a change of physical properties of the Nernst diffusion film, which reduced the diffusion rates of the analytes into the drop.22,26,29,33 Thus, SME was performed without salt addition on the water samples. Validation of the SME-OLBE-FASI-CE Method for Cocaine and Thebaine. The OLBE-FASI method coupled with SME 1262 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006
Figure 7. Electropherograms of cocaine and thebaine in water after SME. SME conditions: stirring rate, 200 rpm; extraction time, 5 min; sample volume, 8 mL; sample pH, 11; sample concentration, 1 µg/ mL of analytes and 0.25 µg/mL of IS; extraction chloroform volume, 4 µL; water plug as in Figure 5. Sample electrokinetic injection, 4 kV × 80 s. CE conditions were as Figure 3.
was performed to test trace cocaine and thebaine. The extraction process was operated as described in the section entitled SME Procedure. Although the 2 µL of chloroform in the syringe had no effect on achieving a better sensitivity, it improved the repeatability of the SME (RSD from 10% to 7% (n ) 5)). Table 4 lists the method validation for cocaine and thebaine in SME-FASICE analysis (40 µL of 50 µg/mL methamphetamine as internal standard). The results were based on the ratio of the analyte peak area to the internal standard peak area (Table 4). In this paper, the reliability of the method for the analysis of cocaine and thebaine in urine by standard addition was evaluated (Table 4). A typical electropherogram obtained from the extraction of a water sample is shown in Figure 7. An extra step to transfer the organic matrix into the aqueous matrix was eliminated. The method can be applied to directly analyze the extractants by SME or LLE, which would be very time-efficient. Application of the SME-OLBE-FASI-CE Method. To evaluate the practical applicability of the SME-OLBE-FASI-CE method, the developed system was applied to the analysis of cocaine and thebaine in urine samples. The extraction time of blank urine was equivalent to that of analyte-spiked urine. Figure 8 shows that the electropherogram for extracts (electropherogram b) is cleaner than the electropherogram of straight urine (elec-
Figure 8. Separation of blank and spiked urine sample. Electropherogram of blank urine sample with direct injection (a) and after SME (b); analyte-spiked urine sample with direct injection (c) and after SME (d). Concentration of analytes in c: 50 µg/mL. Concentration of analytes in d: 0.5 µg/mL. (a) Sample injection, 0.5 psi × 5 s; (b-d) sample electrokinetic injection, 4 kV×80 s. (b and d) Extraction chloroform volume, 4 µL; water plug as in Figure 5. CE conditions and SME were as Figure 7.
tropherogram a), especially where analytes eluted. Separation of a spiked urine sample with direct injection (electropherogram c) and after SME (electropherogram d) is also shown in Figure 8. As can be seen from the Figure 8 (electropherogram b and d), a sample cleanup effect was observed in SME. Desalting of the sample automatically occurred during SME because most salts are insoluble in chloroform. Thus, SME provided an ideal sample preparation procedure for the OLBE-FASI-CE method to analyze biological samples. Compared with direct electroinjection (4 kV × 80 s), the sensitivity enhancement factor was 120-340-fold for SME-OLBE-FASI-CE. Complete analysis for a urine sample was achieved in 25 min, using a 5-min extraction followed by separation on CE. As to consecutive analysis, one urine sample could be analyzed only in 15 min. Compared with previous reports for analyzing cocaine/thebaine in urine samples,38-40 the present method is fast, simple, inexpensive, and sensitive. CONCLUSION In the present study, an OLBE-FASI-CE method for direct analysis of positively chargeable analytes in water-immiscible
solvent was developed. Good linearity, sensitivity, and repeatability were obtained, all of which demonstrate the applicability of this method to trace analysis. Because the extractants by SME/LLE can be directly analyzed by CZE with the OLBE-FASI-CE method, the method developed may find applications for analyzing positively chargeable compounds in complex matrixes, such as biological fluids or environmental samples. The OLBE-FASI method opens a new perspective to SME coupled with CZE. ACKNOWLEDGMENT This work was kindly supported by the National Natural Science Foundation of China (Grant No. 20375028) and the High Technology Research and Development Program of China (863 Program) (2002AA2Z2004).
Received for review September 15, 2005. Accepted November 28, 2005. AC0516537
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