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J. Sep. Sci. 2003, 26, 734–738Petra Sˇevc ˇ?kov?,Zdene ˇkGlatzDepartment of Biochemistry,Faculty of Science, MasarykUniversity, Kotl?r ˇsk? 2, 611 37Brno, Czech RepublicSpecific determination of cysteine in human urineby capillary micellarelectrokinetic chromatographyThis paper describes a method for the analysis of cysteine in human urine using capil-lary micellar electrokinetic chromatography and on-column reaction with 2,29-dipyridyldisulfide. In this reaction cysteine is quantitatively transformed into a mixed disulfideconcomitantly with formation of an equimolar amount of 2-thiopyridone that is furtherseparated by capillary micellar electrokinetic chromatography and determined spec-trophotometrically at 343 nm. The concentration of cysteine is thus estimated indi-rectly from the result of 2-thiopyridone determination. The linear detection range forconcentration versus peak area for the assay is from 0.05 to 5 mM (correlation coeffi-cient 0.989) with a detection limit of 2.5 lM and a limit of quantitation of 8.5 lM. Theinter-day reproducibility of the peak area was 2.18% and the inter-day reproducibilityof the migration time 0.51%. The method is relatively rapid, simple, and can be easilyautomated. Moreover, its detection limit covers the concentration range at whichcysteine is present in biological samples such as human urine.Key Words: Thiol; Cysteine; 2,29-Dipyridyl disulfide; On-column detection; Capillarymicellar electrokinetic chromatography;Received: June 27,2002;revised: November 18,2002;accepted:December 2,2002DOI 10.1002/jssc.2003013721 IntroductionCysteine is an amino acid of high sulphur content synthe-sised by the liver. It is involved in a variety of important cel-lular functions, including protein synthesis, detoxication,and metabolic processes [1, 2]. The biological signifi-cance of cysteine is well established but it is only recentlythat its level in physiological fluids such as plasma andurine has been recognized as an important indicator for anumber of clinical disorders [3–5]. Typical examples arecystinosis and cystinuria. Nephropathic cystinosis is anautosomal-recessive lysosomal storage disease causedby defective transport of cystine out of lysosomes [6, 7].The stored cystine is poorly soluble and crystallizes withinthe lysosomes of many cell types, leading to widespreadtissue and organ damage. Cystinuria is an autosomalrecessive genetic defect affecting the transport of cystine,lysine, arginine, and ornithine in the renal tubules and gas-trointestinal tract [8, 9]. The impaired resorption results inhigh concentrations of cystine and cysteine in urine(usually >1 mM) and the patients suffer from kidney-stoneformation. Determination of urinary content of totalcysteine after reduction may consequently be taken ashelpful non-invasive methodof diagnosis.Although a number of analytical approaches have beenreported for the measurement of cysteine in urine such ascolorimetric [10], high-performance liquid chromatogra-phy with spectrophotometric [11, 12] or fluorimetric [13,14] detection, and flow-injection analysis with electroche-mical detection [15], none of these methods matches theoverall performanceofcapillaryelectrophoresis (CE).CE is a high-efficiency analytical technique that has had agreat impact as a tool in clinical and forensic practice inthe last ten years [16]. The advantages of CE with respectto other analytical techniques, viz. the very small samplevolume required, rapid analysis, high resolution power,and low cost, havemadethistechniqueideal forthe analy-sis of a numerous endogenous and exogenous sub-stances present in biological fluids including biologicallyimportant thiols. The different modes of CE have beencoupled to different detection techniques such as UV-absorbance, electrochemical, and laser-induced fluores-cence detection to determine homocysteine, cysteine,and/or glutathione in plasma, serum, urine, cell extract,microdialysate, etc. [17–32]. These methods usuallyrequire expensive derivatization reagents and/or specialequipment. Recently a method using thiol exchange reac-tion of 2,29-dipyridyl disulfide (Figure 1) for the specificCorrespondence: Zdene ˇk Glatz, Department of Biochemis-try, Faculty of Science, Masaryk University, Kotl?r ˇsk? 2,611 37 Brno, Czech Republic. Phone: +420 5 41129404.Fax: +420 5 41211214. E-mail: glatz@chemi.muni.cz.Abbreviations: 2,29-Dipyridyl disulfide, DPDS; thiol, RSH;glutathione, GSH; homocysteine, Hcys; cysteine, Cys; cy-stine, Cys-Cys; tri-n-butylphosphine, TBP; 2-thiopyridone, T.734i 2003WILEY-VCH VerlagGmbH & Co.KGaA,Weinheim1615-9306/2003/0806–0734$17.50+.50/0