Yi-Hui Lin 1 Jui-Feng Chiang 2 Maw-Rong Lee 3 Ren-Jye Lee 3 Wei-Kung Ko 4 Shou-Mei Wu 1, 5, 6 1 Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Department of Psychiatry, Chang-Hua Christian Hospital, Chang-Hua, Taiwan 3 Department of Chemistry, National Chung-Hsing University, Taichung, Taiwan 4 Jing-Ho Mental Hospital, Kaohsiung, Taiwan 5 Faculty of Fragrance and Cosmetics, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Received November 13, 2007 Revised December 20, 2007 Accepted December 25, 2007 Research Article Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography for analysis of morphine and its four metabolites in human urine A cation-selective exhaustive injection and sweeping micellar EKC (CSEI-Sweep-MEKC) was established to analyze morphine and its four metabolites, including codeine, normor- phine (NM), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). After SPE, the urine samples were analyzed by this CE method. The phosphate buffer (75 mM, pH 2.5) containing 30% methanol was first filled into an uncoated fused-silica capillary (40 cm, 50 mm id), then a high-conductivity buffer (120 mM phosphate, 10.3 kPa for 99.9 s) followed. The pretreated urine sample was loaded by electrokinetic injection (10 kV, 600 s). The stacking and separation were performed by using phosphate buffer (25 mM, pH 2.5) containing 22% methanol and 100 mM SDS at 220 kV, and detected at 200 nm. During method validation, calibration plots were linear (r  0.998) over a range of 30–3000 ng/mL for morphine, NM, and codeine, 100–2000 ng/mL for M6G, and 80–3200 ng/mL for M3G. The LODs (S/N = 5, sampling 600 s at 10 kV) were 10 ng/mL for morphine, NM, and codeine, 35 ng/mL for M6G, and 25 ng/mL for M3G. This stacking CE method could increase 2500-fold sensitivity of codeine, when comparing with CZE. Five addicts’ urine specimens were analyzed. Their results were compared with those of LC-MS-MS, and showed good coincidence. This method could be feasible for monitoring morphine and its metabolites in forensic interest and pharmacokinetic investigations. Keywords: Abused drugs / CSEI-Sweep-MEKC/ Morphine / Morphine metabolites / Urine DOI 10.1002/elps.200700825 2340 Electrophoresis 2008, 29, 2340–2347 1 Introduction Morphine is a potent opioid analgesic, but also an illicit abused drug. About 90% of morphine is converted into metabolites (Fig. 1), primary through conjugation with uri- dine diphosphoglucuronic acid in 3-position to form mor- phine-3-glucuronide (M3G) and in 6-position to form mor- phine-6-glucuronide (M6G) [1]. The principle metabolite M3G has no opioid action but seems to be a functional antagonist of morphine. M6G is produced in smaller amounts when comparing with M3G, however, it binds to the opioid receptors and possesses analgesic properties [2]. Human studies have indicated that when given in equia- nalgesic doses, M6G is less likely to induce nausea and vomiting than morphine [3]. In addition, codeine and nor- morphine (NM) are minor metabolites which are formed via O-methylation and N-demethylation, respectively. Codeine can acts as an analgesic, but NM only shows a minor extent and is not usually found in plasma [4]. According to the gov- ernment statistics in Taiwan, the numbers of morphine addicts are always in high ranking. It is important to estab- lish a specific and sensitive method for high-throughput screening and other associated studies. Many immunoassay and chromatography methods have been developed for the screening of morphine and/or its metabolites in biological specimens [5–20]. Immunoassay is still subject to cross-reaction and lacks of specificity [9, 12]. HPLC and GC methods [11–20] showed good sensitivity and specificity, but precolumn derivatizations or complicated sample pretreatments were always inevitable. CE has many advantages including high efficiency, low consumption of Correspondence: Professor Shou-Mei Wu, Faculty of Fragrance and Cosmetics, College of Pharmacy, Kaohsiung Medical Univer- sity, No. 100, Shih-Chung 1st Rd., Kaohsiung 80708, Taiwan E-mail: shmewu@kmu.edu.tw Fax: 1886-7-3159597 Abbreviations: CSEI-Sweep-MEKC, cation-selective exhaustive injection and sweeping micellar EKC; FPIA, fluorescence polari- zation immunoassay; HCB, high-conductivity buffer; IS, internal standard; M3G, morphine-3-glucuronide; M6G, morphine-6-glu- curonide; NM, normorphine; RE, relative error  2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com