Anal. Chem. 1997, 69, 99-104
Enhanced Throughput with Capillary Electrophoresis via Continuous-Sequential Sample Injection Matthew E. Roche,† Robert P. Oda,† Dwaine Machacek,‡ George M. Lawson,‡ and James P. Landers*,†
Clinical Capillary Electrophoresis Facility and Toxicology Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905
A significant fraction of the total analysis time (injectionto-injection) in capillary electrophoresis is dedicated to a rigorous between-run capillary rinsing/regeneration procedure. This is of particular importance with CE-based assays developed for high-throughput analysis of clinical samples. In this study, we examine the necessity of a between-run rinsing step when detergent is present in the separation buffer. Using three model analyte systems (hypoglycemic drug standards, urinary estrogen standards, fasted normal human urine), the reproducibility for migration time (MT), peak area (PA), and peak height (PH) with separation in a borate/phosphate buffer containing 75 mM sodium cholate was found to be acceptable in the absence of between-run capillary cleansing. Continuous-sequential injection of hypoglycemic drug standards over the course of 39 consecutive runs without between-run capillary regeneration showed acceptable reproducibility. The average percent coefficient of variance values associated with MTrel(MeOH), PA, and PH over 39 consecutive runs were 2.22, 3.27, and 3.51%, respectively. In excess of 100 continuous-sequential injections could be performed in this manner without any significant effects on electroosmotic flow or reproducibility. Exclusivity of these results was ruled out with the continuous-sequential injection of the urinary estrogen and the human urine analyte systems under the same conditions, both of which yielded comparable results. The ability to circumvent or eliminate capillary rinsing procedures when detergent is a component of the separation buffer presents the possibility of decreasing the total analysis time (injection-to-injection) with certain analyte systems, the result of which will be to enhance sample throughput by 2-3-fold on single-capillary instrumentation. Capillary electrophoresis (CE) is a technique capable of rapid, automated, reproducible, and high-resolution separation of complex mixtures of analytes of diverse size and nature.1,2 Capillary †
Clinical Capillary Electrophoresis Facility. Toxicology Laboratory. (1) Gordon, M. J.; Huang, X.; Pentoney, S. L., Jr.; Zare, R. N. Science 1988, 241, 224-228. (2) Karger, B. L.; Cohen, A. S.; Guttman, A. J. Chromatogr. 1989, 492, 585614. ‡
S0003-2700(96)00514-8 CCC: $14.00
© 1996 American Chemical Society
zone electrophoresis (CZE) is the most universally used mode of capillary electrophoresis for analysis under native conditions. In CZE, net positively and negatively charged analytes form discrete zones on either side of neutral analyte migration. In order to obtain adequate resolution/sensitivity and good separation efficiencies, it is important to select a buffer with good buffering capacity, low UV absorption at the desired wavelength, and low ionic mobility. Micellar electrokinetic chromatography (MEKC) is becoming a commonly used mode of CE. With this mode, surfactant added to the low ionic strength buffer at concentrations above the critical micelle concentration (cmc) provides a partitioning component to the electrophoretic separation. Consequently, both charged and uncharged (or neutral) analytes can be resolved, charged analytes based on inherent electrophoretic mobility and ionic interaction with the micelle and neutral analytes via micellization based on hydrophobic character. Recently, CE has emerged as an effective analytical tool for the determination of a number of clinically relevant analytes.3 Within this arena, MEKC has been shown to be most useful for the analysis of drugs or other small molecules in biological fluids.4 Capillary electrophoretic-based separations have been shown to be applicable to the analysis of pharmaceuticals,5-7 identification of illicit drug substances,8,9 detection and quantitation of intoxicants (e.g., salicylate, tricyclic antidepressants), and the monitoring of a number of different classes of drugs, including cardiac agents, such as antiarrhythmics, or positive inotropic compounds (i.e., digitalis glycosides), aminoglycoside antibiotics, xanthines, antineoplastics, and antiepileptics.10 In capillary electrophoresis it is common practice for a rinsing or capillary cleaning step to be performed between sample injections. The cleaning usually consists of a 0.1 M NaOH rinse and a water rinse followed by a rinse with separation buffer to recondition the capillary. A caveat associated with the use of CE for analyzing a large number of samples is the need for serial analysis as compared to (3) Landers, J. P. Clin. Chem. 1995, 41, 495-509. (4) Thormann, W.; Molteni, S.; Caslavska, J; Schmutz, A. Electrophoresis 1994, 15, 3-12. (5) Swaile, D. F.; Burton, D. E.; Balchuyas, A. T.; Sepanik, M. J. J. Chem. Sci. 1988, 26, 406-409. (6) Altria, K.D.; Luscombe, D.C. J. Pharm. Biomed. Anal. 1993, 11, 415-20. (7) Thormann, W.; Meier, P.; Marcolli, C.; Binder, F. J. Chromatogr. 1991, 545, 445-460. (8) Weinberger, R.; Lurie, I. S. Anal. Chem. 1991, 63, 823-827. (9) Lurie, I. S. In Analysis of addictive and misused drugs; Adamovics, J. A., Ed.; Marcel Dekker: New York, 1995; pp 151-219. (10) Schmutz, A.; Thormann, W. Electrophoresis 1994, 15, 51-61.
Analytical Chemistry, Vol. 69, No. 1, January 1, 1997 99
other analytical methods (e.g., batch immunoassays or gel electrophoresis) where parallel sample analysis is possible. This becomes a critical issue with high throughput clinical analysis. One of two approaches can be explored to overcome this drawback: (1) reduce the analysis time to increase throughput with a single-capillary instrument, or (2) develop multicapillary11 or microfabricated chip12 electrophoresis systems. The first of these two options is clearly the simplest. However, once the capillary length, applied voltage, capillary temperature, and other separation parameters are optimized for resolution of the analytes of interest, options for further reducing the analysis time are scarce. If one critiques the methodology associated with capillary electrophoretic separations, it becomes clear that extensive between-run capillary rinsing/regeneration procedures are associated with most CE-based analyses. For example, electrophoresis time for serum protein analysis is less than 1.5 min but requires 4.5 min of between-run rinsing to clean and re-equilibrate the capillary.13 Clearly, methodologies and analyte systems that allow for reduction or even elimination of between-run rinsing (without significantly affecting reproducibility) would function to decrease the total analysis time and increase throughput. In the present study, we investigated whether the presence of a surfactant in the separation buffer, e.g., as with MEKC, would circumvent the need for between-run capillary rinsing. The reproducibility associated with separation of three model analyte systems (sulfonylurea drug standards, urinary estrogen standards, normal urine) using a pH 8.5 borate/phosphate/sodium cholate buffer system was assessed. The results indicate that the presence of detergent in the separation buffer prevents analyteinduced alteration of the capillary wall that can lead to changes in EOF and reproducibility. Hence, this presents the possibility that, with certain MEKC buffer systems and certain analyte systems, between-run rinsing procedures may be considered an option rather than a necessity. Consequently, throughput may be enhanced significantly by use of continuous-sequential injection of sample. EXPERIMENTAL SECTION Materials. Boric acid, borax (sodium tetraborate), sodium cholate, sodium dodecyl sulfate (SDS), glyburide (Gb), sodium hydroxide, tolbutamide (Tb), tolazamide (Ta), 17β-estradiol (E1), estrone (E2), and estriol (E3) were obtained from Sigma (St. Louis, MO). Chlorpropamide (Cp) was obtained from Pfizer, Inc. (New York). Acetohexamide (Ah) was obtained from ICN Pharmaceuticals, Inc. (Costa Mesa, CA). Sodium phosphate, HPLC-grade acetonitrile, and HPLC-grade methanol were purchased from Fisher Scientific (Fairlawn, NJ). Glipizide was obtained from Research Biochemicals, Inc. (Natick, MA), N-acetyl4-(2,3-dichlorophenylureido)benzenesulfonamide internal standard was obtained from Aldrich Chemical Co. (Milwaukee, WI). Capillaries were 50 µm i.d. (375 µm o.d.) bare silica capillary tubing purchased from Polymicro Technologies Inc. Human urine was obtained by a random first morning midstream collection from a patient who had fasted overnight and was stored at 4 °C when not in use. (11) Clark, S. M.; Mathies, R. A. Anal. Biochem. 1993, 215, 163-170. (12) Schmalzing, D.; Nashabeh, W.; Yao, X. W.; Mhatre, R.; Regnier, F. E.; Afeyan, N. B.; Fuchs, M. Anal. Chem. 1995, 67, 606-612. (13) Clark, R.; Katzmann, J. A., Wiegert, E. Namyst-Goldberg, C.; Sanders, L.; Oda, R. P.; Katzmann, J. A.; Kyle, R. A. Landers, J. P. J. Chromatogr. 1996, 744, 205-213.
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Buffer and Sample Preparation. Separation buffers were prepared from 500 mM stock solutions. Borate buffer was prepared by mixing 500 mM boric acid with the appropriate amount of 125 mM sodium tetraborate until a pH of 8.5 was obtained. The phosphate buffer was prepared by solubilizing the necessary amount of disodium phosphate to make a 500 mM solution. All buffers were made with Milli-Q (Millipore, Bedford, MA) water and filtered through a 0.2 µm filter (Gelman) before use. Sulfonylurea drug standards and the internal standard were prepared at a concentration of 1 mg/mL in methanol (HPLC grade); they were kept at 4 °C when in use and at -20 °C when not in use. Sulfonylurea drugs were brought to dryness and solubilized in 1 mM borate/phosphate buffer, 15 mM sodium cholate with 30% methanol prior to injection. Estrogen standards were prepared in 100% methanol at a concentration of 250 µg/ mL each; they were kept at 4 °C when in use and at -20 °C when not in use. Instrumentation. MEKC separation was carried out on a Beckman P/ACE System 5510 interfaced with an IBM Value Point 486 computer utilizing System Gold software (V. 8.1, Beckman Instruments, Fullerton, CA) for control of the instrument and data collection. Migration times for individual peaks as well as peak area and height were obtained through the System Gold version 8.0 software. Capillary Electrophoresis Separation Conditions. Polyimide-coated, fused-silica capillary (Polymicro Technologies) was 50 µm i.d. and 37 cm in length (30 cm to the detector). Polarity was such that the inlet was the anode and the outlet was the cathode. The capillary temperature was maintained at 22 °C. Detection was by absorbance at 200 nm. Standard CE Analysis. For MEKC, a new bare silica capillary (50 µm × 37 cm) was conditioned by a 20 column volume rinse each with 0.1 M NaOH, water, and separation buffer (in order). For a typical analysis, the following method was used after determining the length of time required to fill the capillary by high pressure (20 psi): a three capillary volume rinse with separation buffer, 2 s pressure injection (0.5 psi) of sample, separation at 25 or 28 kV (constant voltage), a three capillary volume rinse with 0.1 M NaOH, a momentary dip in glass-distilled water, followed by a three capillary volume rinse with separation buffer. The rinses with separation buffer were carried out with “fresh” separation (unelectrophoresed) buffer and the electrophoresis buffer was replenished every five to eight runs. Continuous-Sequential Injection CE Analysis. Sample injection was only initiated with a capillary that had been conditioned and extensively equilibrated with the separation buffer as described above. Once this had been accomplished, the following method was used: sample was injected for 2 s by pressure (0.5 psi), separation at 28 kV (constant voltage), the voltage terminated, sample injected, and the voltage reinitiated. The use of vials containing fresh running buffer was programmed into the method following every seven separations. After 13 continuous-sequential injections, the capillary was rinsed with 0.1 M NaOH (1 min) and separation buffer (1 min). Evaluation of Reproducibility. Evaluation of the relative migration time, integrated peak area, and peak height reproducibility with drug standards using sodium cholate containing buffer was carried out with 39 consecutive injections of a mixture containing either the hypoglycemic drugs at individual concentra-
tions of 143 µg/mL or the estrogen standards at individual concentrations of 250 µg/mL. Human urine was injected directly without dilution or cleanup. Percent coefficient of variance (% CV) values were calculated for migration time (MT), peak area (PA), and peak hieght (PH). RESULTS AND DISCUSSION One of the more common criticisms of capillary electrophoresis is the poor sample throughput that results from the necessity of doing sample analysis serially. Despite the advantages associated with CE (high-resolution, rapid, automated analysis), this fact alone precludes CE from being competitive with such methodologies as slab gel electrophoresis or batch immunoassays, where parallel sample analysis is easily accomplished. In the introduction, we alluded to the fact that many CE-based analyses are associated with extensive rinsing procedures. This typically is associated with either a high- (e.g., 0.1 M NaOH) or low-pH (e.g., 0.1M H3PO4) rinse to remove any wall-absorbed material, followed by a rinse with separation buffer to re-equilibrate the capillary surface for subsequent analysis. The extensive nature of many rinsing procedures can be substantiated from the literature. A random selection and review was carried out with 40 reports from the literature detailing CE-based separations, and the amount of time associated with between-run rinsing examined. Most researchers listed initial capillary conditioning parameters, while others only cited that the capillary was rinsed/washed between separations, not detailing the amount of time involved. In those CZE/MEKC reports providing details on between-run rinsing,14-30 the times ranged from 5 to 15 min with an average between-run rinse time of ∼9 min. This constitutes a significant fraction of the total analysis time, particularly when electrophoresis is rapid (
