Research Article Received: 24 July 2008, Revised: 27 August 2008, Accepted: 27 August 2008 Published online 19 December 2008 in Wiley Interscience (www.interscience.wiley.com) DOI 10.1002/bmc.1149 Biomed. Chromatogr. 2009; 23: 563–567 Copyright © 2008 John Wiley & Sons, Ltd. 563 John Wiley & Sons, Ltd. Rapid determination of rifaximin in rat serum and urine by direct injection on to a shielded hydrophobic stationary phase by HPLC Rapid determination of rifaximin in rat serum and urine R. Nageswara Rao, a * Dhananjay D. Shinde a and Sachin B. Agawane b ABSTRACT: A simple and rapid reversed-phase HPLC method for determination of rifaximin in rat serum and urine was developed. Separation of rifaximin from biological matrix was achieved by direct injection of rat serum and urine onto a restricted-access medium, Supelco LC-Hisep, a shielded hydrophobic stationary phase, using acetonitrile:water:acetic acid (18:82:0.1 v/v/v) as a mobile phase. The linear range was 0.10–20 mg/mL (r 2 > 0.999, n = 6), intraday and interday variation was <6.10%. The limits of detection and quantification were 0.03 (signal-to-noise ratio >3) and 0.10 mg/mL (signal-to-noise ratio >10), respectively. The method was successfully applied to pharmacokinetic studies of rifaximin after an oral administration to rats. Copyright © 2008 John Wiley & Sons, Ltd. Keywords: rifaximin; antimicrobials; traveler’s diarrhea; rat serum; restricted access media; Hisep Introduction Hepatic encephalopathy (HE) is a complication of advanced hepatic disease. Non-absorbed disaccharides such as lactulose and lactitol are widely used as first-line drugs for treatment of HE. However, long-term treatment of HE with disaccharides is not only ineffective but also the patients develop adverse affects. Non-absorbable antibiotics are generally used as an alternative to disaccharides for treatment of HE (Sama et al., 2004; Adachi, 2006). Rifaximin is a virtually non-adsorbed semi-synthetic derivative of rifamycin used in the therapy of traveler’s diarrhea, tuberculosis and enteric infections. It is a novel antimicrobial agent with broad spectrum activity against Gram-positive, Gram-negative, aerobic and anaerobic bacteria (Steffen et al., 2003). It has a promising role in the treatment not only of trave- ler’s diarrhea but also of selected forms of non-systematic bacte- rial diarrhea, other small bowel infections and overgrowth syndromes. One of the most attractive aspects of this drug is the limited potential for side effects and safety for use in persons of all ages with varying underline health problems. Thus it is of great interest to medical practitioners and further research is needed to define the mechanism of its action, pharmacodynam- ics and the presence of drug-induced alteration of bacterial viru- lence (Huang and Dupont, 2005). The use of available techniques for extraction of rifaximin from biological matrices has been limited due to its complexity and wide variety of endogenous substances. Sample clean-up procedures play a critical role in eliminating the overlapping of endogenous substances with the peaks of interest. Conven- tional reversed-phase columns cannot handle a large number of direct injections of biological samples, serum and urine since proteins undergo denaturation and subsequent precipitation, resulting in a clogged system. There is a great need for a simpler and faster analysis of drugs in biofluids (Pinkerton, 1991). Our interest was the development of a direct injection technique using HPLC. The restricted access media (RAM) columns com- prise shielded hydrophobic materials of polymeric bonded phases containing hydrophobic regions enclosed in a hydrophilic network. These columns are useful for direct injection of spiked plasma, serum and urine without any prior extraction of the drug (Shihabi, 1988; Koenigbauer, 1990). The bulk of the protein matrix will elute as an unretained band without affecting the retention of the analytes. The advantages of direct injection include: (i) easier sample preparation; (ii) shorter analysis time; (iii) reduced cost of analysis; and (iv) excellent recovery of analytes. In the literature, the most widely used direct injection techniques include: (1) micellar chromatography (Pilar et al., 1993); (2) column- switching methods (Brewster et al., 1992); and (3) RAM columns (Ma et al., 2002; Ueno and Aoki, 1996; Uno and Maeda, 1995; Andeejani et al., 1994; Ueno et al., 1992; Riva et al., 1991; Locke- meyer and Smith, 1990; Venturini, 1983). Among them, the use of RAM columns is the simplest. Several analytical methods previously reported for rifaximin include HPLC-UV assays (Lv et al., 2004; Wang et al., 2003; Descombe et al., 1994), HPLC coupled with electrochemical detector (ECD) (Gionchetti et al., 1999; Venturini et al., 2005), LC-MS method (Zhang et al., 2007) and radio-ligand binding technique. However, none of the methods refer to the use of a RAM column for deter- mination of rifaximin in biological matrices. In this paper, a direct serum and urine injection method for the determination of rifaximin is reported using a Hisep column. It has the advantages * Correspondence to: R. Nageswara Rao, Analytical Chemistry Division, Discovery Laboratory, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 007, India. E-mail: rnrao55@yahoo.com a Analytical Chemistry Division, Discovery Laboratory, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 007, India b Pharmacology Division, Discovery Laboratory, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 007, India Abbreviations used: ACN, acetonitrile; HE, hepatic encephalopathy; RAM, restricted-access medium.