Analytica Chimica Acta 673 (2010) 194–199 Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca Novel, selective sample stacking microemulsion electrokinetic capillary chromatography induced by reverse migrating pseudostationary phase for the determination of the new ultra-short acting hypnotic “HIE-124” in mice serum Mohamed Hefnawy a,∗ , Mohammed Al-Omar a , Saeed Julkhuf a , Sabry Attia b , Ehab Abourashed c , Hussein El-Subbagh a a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia b Department of Pharmacology, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia c ElSohly Laboratories, Inc., University of Mississippi, 5 Industrial Park Drive, NCNPR, Oxford, MS 38655, USA article info Article history: Received 3 July 2009 Received in revised form 16 March 2010 Accepted 20 May 2010 Available online 27 May 2010 Keywords: HIE-124 Microemulsion electrokinetic capillary electrophoresis Reverse migrating pseudostationary phase Pharmacokinetics Serum abstract Microemulsion electrokinetic capillary chromatography (MEEKC) with sample stacking induced by reverse migrating pseudostationary phase (SRMP) technique in a suppressed electro-osmotic flow (EOF) strategy was investigated for analysing the new ultra-short hypnotic HIE-124 in mice serum. The pro- posed method utilized fused-silica capillary with a total length of 50 cm (effective length 40 cm), applied voltages for stacking and separation were 5.0 kV for 4.30 min and subsequently 25 kV, respectively, with a sample injection of 0.5 psi for 90 s. All the runs were carried out at 25 ◦ C and detected at 213 nm. The optimum microemulsion background electrolyte (BGE) solution consisted of 0.8% (v/v) ethyl acetate, 6.6% (v/v) butan-2-ol, 1.0% (v/v) acetonitrile, 2.0% (w/v) sodium dodecyl sulfate (SDS), and 89.6 mL with 25 mM phosphate buffer pH 8. When this preconcentration technique was used, the sample stacking and the separation processes took place successively with changing the voltage with an intermediate polarity switching step. The proposed method was validated carefully with respect to high specificity of the method, good linearity (r = 0.9994), fair wide linear concentration range (66–1500 ng mL -1 ), limit of detection and quantitation were 21.6 and 65.5 ng mL -1 , respectively. The mean relative standard devia- tion (RSD) of the results of intra- and inter-day precision and accuracy were less than 6.0%, and overall recovery higher than 95% of HIE-124 in mice serum. The developed method could be used for the trace analyses of HIE-124 in serum and was finally used for the pharmacokinetic study investigation of HIE-124 in mice serum. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Recently, the ultra-short acting hypnotic activity of ethyl 8-oxo- 5,6,7,8-tetrahydro-thiazolo[3,2-a][1,3]diazepin-3-carboxylate (HIE-124) (Fig. 1) was reported as a member of a novel class which might overcome many of the disadvantages and problems that are usually associated with the use of thiopental or benzodiazepines as intravenous anaesthetic agents [1]. HIE-124 has been shown to not only induce anaesthesia but also maintain the anaesthetic state during the surgical procedure [2]. In contrast to HPLC, CE is superior in terms of the speed, high resolution and tiny amounts of samples and reagents required. Whereas, CE suffers from poor sensitivity due to short optical path of capillary (i.d. 50–100 m), CE has become a more practical and powerful tool for trace analysis ∗ Corresponding author. Tel.: +966 1 467 7346; fax: +966 1 467 6220. E-mail address: mhefnawy2003@yahoo.com (M. Hefnawy). because many stacking methods have been developed to lower detection limit of CE [3–19]. The pre-concentration methods include field-amplified sample injection (FASI) advanced by Chien and Burgi [3,4], pH-mediated stacking idealized by Lunte et al. [5–7], dynamic pH junction stacking technique developed by Britz-Mckibbin et al. [8–10], transient isotachophoresis (TITP) shown by Gebauer et al. [11–13], sweeping of neutral or charged analytes in micellar electrokinetic capillary chromatography (MEKC) advanced by Quirino et al. [14,15] and Palmer et al. [16,17]. In the last few years MEEKC has become an important field of research in capillary electrophoresis (CE) because it can efficiently separate both charged and neutral solutes covering a wide range of water solubilities [18–23]. A search of the internet indicates that only one published method for the determination of HIE-124 in spiked human plasma by HPTLC and densitometric detection set at 265 nm has been developed by Abourashed et al. [24]. This method was suffered from some major drawbacks such as low sensitivity, laborious multiple extraction steps and time-consuming. 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.05.030