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EXTENED ASMS ABSTRACT Capillary Electrophoresis/Mass Spectrometry: From One Meter Capillaries to Chip-Based Devices Jack Henion, Katja Heinig, and Timothy Wachs, Analytical Toxicology, New York State College of Veterinary Medicine, Cornell University, 927 Warren Dr., Ithaca, NY 14850, and Gary Schultz and Tom Corso, Advanced BioAnalytical Services, Inc., 15 Catherwood Rd., Ithaca, NY 14850. INTRODUCTION Capillary electrophoresis coupled with mass spectrometry (CE/MS) was first reported by R. Smith et al. in the last decade. Although one can argue that CE/MS should be a powerful complement to LC/MS techniques, this promise has not been fulfilled. As a so-called nanoscale technique most researchers have not been particularly successful with practical applications which convincingly demonstrate either qualitative or quantitative capabilities. Some of the problems are due to the rather low levels of sample that are typically injected (loaded) onto the capillary as well as the somewhat limited detection limits afforded by current MS techniques. In spite of the relatively good sensitivity that we typically have from MS, the demands of CE capillary loadings, etc. place stringent detection limit demands upon the CE/MS combination. This paper presents a brief review of the historical developments in CE/MS using atmospheric pressure ionization (API) techniques. It will review the successful strategies for interfacing CE to API MS followed by recent examples showing the CE/MS and CE/MS/MS determination of carnitine and its acyl analogs in biological samples including human plasma and urine. Finally, recent developments will be presented which focus on miniaturizing CE/MS techniques to chipbased devices. It is the authors’ belief that nanofabricated devices which can provide rapid, routine, serial and/or parallel separations on a chip-based substrate may contribute in the future to expanded use of CE/MS techniques in the pharmaceutical, agrochemical, and chemical industries. EXPERIMENTAL A Crystal CE model 310 (ATI Unicam, Madison, WI) was used for the electrophoretic separations. Uncoated fused-silica capillaries of 75 m i.d., 360 m o.d. and 80 cm length were filled with CE electrolyte solutions containing ammonium acetate (10-30 mmol/l) with acetic or formic acid in aqueous and organic (methanol, acetonitrile) solution. The separation voltage was 30 kV. A pressure between 0 and 50 mbar was applied at the injection (inlet) end during the electrophoretic separation. Injection was performed hydrodynamically or electrokinetically. The PE Sciex API 365 tandem mass spectrometer was coupled to the CE apparatus via an in-house built coaxial sheathflow interface, placed into the commercial ion spray interface housing. A 2 M resistor was used to decouple the electrospray voltage from the CE voltage. Pneumatically assisted electrospray (ion spray) with 2-propanol/water or methanol/water containing 0.1% acetic acid as sheath liquid at 4-10 l/min and nitrogen as nebulizer gas was used for all CE/MS experiments. The sheath liquid was delivered by a Harvard Apparatus syringe pump. All experiments were carried out in the positive ion mode. Full-scan, selected ion monitoring (SIM) or selected reaction monitoring (SRM) modes were used. The Q1 and Q3 quadrupoles were operated at unit mass resolution (0.7 Da at half-height. RESULTS AND DISCUSSION This report introduces the development and application of capillary electrophoretic methods with electrospray mass spectrometric detection for the determination of carnitine and acylcarnitine homologues in human blood, urine and plasma. The selected reaction monitoring mode provides high selectivity and sensitivity, although SIM and full-scan analyses are also possible for the detection of the targeted analytes in biological samples. Performing a CE/MS analysis provides the possibility to separate isomers that are not distinguishable by MS detection without prior separation. This work shows the feasibility for determining carnitine and acylcarnitines in human urine and plasma samples with acceptable selectivity, sensitivity, precision and accuracy. The simultaneous determination of all analytes with one sample preparation and separation method was not practical in this work. Nonaqueous CE electrolytes (methanol/acetonitrile as solvent mixture) provided good homologous peak resolution of carnitine and all acylcarnitines inclusive of C2 to C18 in a relatively short separation time, but could only accommodate relatively “clean” biological samples prepared by liquid-liquid extraction. Therefore, this approach can be applied if the determination of long-chain homologues is required. A 100% aqueous electrolyte was wellsuited for each sample matrix studied and showed good isomeric acylcarnitine separation efficiency (Figure 1). Although the C16 and C18 homologues could not be detected, this method is applicable to characterize important metabolic disorders, such as propionyl and isovaleryl acidemia and MCAD. Therefore, CE/MS might be used as an alternative for acylcarnitine determinations in clinical samples. 1 400000 110000 4b A 4a 300000 C 7 90000 8 4 200000 1 70000 8 100000 8.5 10 6 5 9 3 9 2 50000 intensity, cps 10 0 12 11 30000 4 370000 6 8 10 12 14 12 650000 B 320000 14 16 18 D 550000 270000 450000 220000 350000 170000 12 1 120000 12 250000 1 150000 70000 20000 50000 4 5 6 time, min 7 8 2 3 4 5 6 7 8 time, min Figure 1. Separation of synthetic carnitine/acylcarnitine standard mixtures in different electrolytes by SIM CE/MS. (A) aqueous buffer containing 0.8% formic acid, (B) buffer containing 3.2% acetic acid and 50% acetonitrile, (C) buffer containing 3.2% acetic acid and 50% methanol, (D) nonaqueous buffer (6.4% formic acid in methanol/acetonitrile 1:1); buffer: 10 mmol/l ammonium acetate; voltage: 30 kV; injection: 0.1 min 50 mbar standard mixture 1.5 g/ml; for peak assignment see Table 1, 4a) iso-butyrylcarnitine, 4b) n-butyrylcarnitine These developments provide a useful test platform to evaluate the analytical potential of chipbased CE/MS techniques. We have initiated studies to evaluate the potential to accomplish competitive results to those described above using chip-based substrates. Preliminary results from nanofabricated capillary channels in silicon substrates will be shown. A novel monolithic nanosprayer nozzle fabricated in a silicon chip in a radial array will be described with a focus on new strategies for high-throughput CE/MS analyses in the future. ACKNOWLEDGMENTS The authors wish to thank SmithKline Beecham for financial support of our research and PE Sciex for the loan of the API 365 mass spectrometer and financial support. We also thank ATI Unicam for the loan of the Crystal CE apparatus. Furthermore, we thank Dr. Carla Vogt for supplying the acylcarnitine standards and helpful information as well as Dr. Donald Chace of Neo Gen Screening for generously providing blood spot samples and internal standards.