Habib Bagheri Faezeh Khalilian Laleh Enayati Ahangar Department of Chemistry, Sharif University of Technology, Tehran, Iran Original Paper Liquid–liquid–liquid microextraction followed by HPLC with UV detection for quantitation of ephedrine in urine Liquid – liquid – liquid microextraction (LLLME) in combination with HPLC and UV detection has been used as a sensitive method for the determination of ephedrine in urine samples. Extraction process was performed in a homemade total glass vial without using a Teflon ring, usually employed. Ephedrine was first extracted from 3.5 mL of urine sample (pH 12) into a microfilm of toluene/benzene (50:50). The ana- lyte was subsequently back extracted into an acidic microdrop solution (pH 2) sus- pended in the organic phase. The extract was then injected into the HPLC system directly. An enrichment factor of 137 along with a good sample clean-up was obtained under the optimized conditions. The calibration curve showed linearity in the range of 0.01 – 50 mg/L with regression coefficient corresponding to 0.998. The LODs and LOQs, based on a S/N of 3 and 10, were 5 and 10 lg/L, respectively. The method was eventually applied for the determination of ephedrine in urine sample after oral administration of 5 mg single dose of drug. Keywords: Ephedrine / High performance liquid chromatography-ultraviolet detection / Liquid – liquid – liquid microextraction / Urine sample / Received: April 5, 2008; revised: July 17, 2008; accepted: July 17, 2008 DOI 10.1002/jssc.200800193 1 Introduction Ephedrine is a sympathomimetic amine commonly used as a stimulant, appetite suppressant, concentration aid, and decongestant. It is also applied to treat hypotension associated with regional anaesthesia [1 – 5]. The action mechanism of ephedrine relies on its direct and indirect actions on the adrenergic receptor system, which is part of the sympathetic nervous system [6, 7]. As shown in Fig. 1, ephedrine is similar in structure to the synthetic deriv- atives amphetamine and methamphetamine. Chemi- cally, it is an alkaloid derived from various plants in the genus Ephedra [8]. Botanicals containing this compound are marketed for weight loss and bodybuilding [9 – 11]. Consumption of these supplements has been associated with adverse cardiovascular and central nervous system events, even death, in some persons [12]. The medical commission of the International Olympic Committee (IOC) included the ephedrines in the list of forbidden sub- stances in 1990 [13]. Thus, a quantification method for this drug in biological fluids (e.g., urine, serum, and sal- iva) is of great interest for doping control purpose. Ephe- drine becomes widely distributed by the circulatory sys- tem, and is eliminated by first order kinetics via the uri- nary system, with the analysis of the urine being the pri- mary tool for quantitation of ephedrine in doping cases [14]. The IOC has set a permitted level of 10 lg/mL. In lower levels, the presence of ephedrine in urine will not be regarded as a positive doping case. Antidoping con- trols are routinely carried out on the urines. Therefore, a fast, simple, and reliable analytical method is needed for identifying the abuse of such compounds by athletes. Several analytical techniques, including GC [15 – 21] and HPLC [22, 23] ion-pair HPLC [24 – 26], and CE [27 – 29], have been used for the determination of this drug in bio- logical samples. However, GC methods involve a derivati- zation step and CE suffers from low sensitivity in terms Correspondence: Dr. Habib Bagheri, Department of Chemistry, Sharif University of Technology, P.O. Box 11365-9516, Tehran, Iran E-mail: bagheri@sharif.edu Fax: +98-21-66012983 Abbreviations: LLE, liquid – liquid extraction; LLLME, liquid – liquid – liquid microextraction; SPME, solid-phase microextrac- tion i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 3212 H. Bagheri et al. J. Sep. Sci. 2008, 31, 3212 – 3217 Figure 1. Chemical structure of ephedrine.