Research Article Received: 29 September 2008, Accepted: 30 October 2008 Published online 9 April 2009 in Wiley Interscience (www.interscience.wiley.com) DOI 10.1002/bmc.1177 Copyright © 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2009; 23: 732–739 732 John Wiley & Sons, Ltd. Simultaneous estimation of four proton pump inhibitors—lansoprazole, omeprazole, pantoprazole and rabeprazole: development of a novel generic HPLC-UV method and its application to clinical pharmacokinetic study Simultaneous estimation of four proton pump inhibitors D. Vijaya Bharathi, a,b * Kishore Kumar Hotha, a B. Jagadeesh, a Pankaj K. Chatki, a K. Thriveni, a Ramesh Mullangi c and A. Naidu b ABSTRACT: A highly selective, sensitive and accurate HPLC method has been developed and validated for the estimation of four proton-pump inhibitors (PPI), lansoprazole (LPZ), omeprazole (OPZ), pantoprazole (PPZ) and rabeprazole (RPZ), with 500 mL human plasma using zonisamide as an internal standard (IS). The sample preparation involved simple liquid–liquid extraction of LPZ, OPZ, PPZ and RPZ and IS from human plasma with ethyl acetate. The baseline separation of all the peaks was achieved with 0.1% triethylamine (pH 6.0):acetonitrile (72:28, v/v) at a flow rate of 1 mL/min on a Zorbax C 8 column. The total chromatographic run time was 11.0 min and the simultaneous elution of IS, OPZ, RPZ, PPZ and LPZ occurred at approximately 2.42, 4.45, 5.02 and 9.37 min, respectively. The method was proved to be accurate and precise at linearity range of 20.61–1999.79 ng/mL with a correlation coefficient (r) of ≥0.999. The limit of quantitation for each of the PPI studied was 20.61 ng/mL. The intra- and inter- day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The devel- oped assay method was applied to a pharmacokinetic study in human volunteers. Copyright © 2009 John Wiley & Sons, Ltd. Keywords: lansoprazole; omeprazole; pantoprazole; rabeprazole; proton pump inhibitors; human plasma; validation; HPLC; pharmacokinetics Introduction Proton pump inhibitors (PPIs), viz. lanoprazole (LPZ), omeprazole (OPZ), pantoprazole (PPZ) and rabeprazole (RPZ), are the first-line treatment for many patients with acid-peptic disorders, including erosive gastro-oesophagal reflux disease (GERD), nonerosive reflux disease (NERD) and duodenal gastric ulcers. PPIs are superior to histamine 2 -receptor antagonists (ranitidine, cimetidine, famoti- dine, etc.) in control of gastric acid and in the management of acid-mediated disorders. PPIs selectively and irreversibly inhibit the H + /K + -adenosine triphosphatase (ATPase) (proton pump) that performs the final step in the acid secretory process by stimula- tion of histamine or gastrin or acetylcholine receptors present on the parietal cells. All the PPIs (Fig. 1) are substituted benzimi- dazole derivatives and they function as prodrugs. They inhibit both basal and stimulated secretion of gastric acid, independ- ently of the nature of parietal cell stimulation (Huber et al., 1999; Sachs et al., 1995). Following oral administration they are absorbed and enter into the cannalicular lumen of parietal cells and are activated by conversion to the tetracyclic planar sulfena- mide. The activated sulfenamide bind covalently to cystine residue on the proton pump, thereby irreversibly inhibiting the H + /K + - ATPase and gastric acid secretion (Richardson et al., 1998). All PPIs are acid labile, hence orally they are administered as enteric- coated formulations. Following oral administration all PPIs are rapidly absorbed and the maximum concentrations in plasma are attained between 1 and 3 h. Relatively all PPIs have prolonged pharmacodynamic activity (48–72 h), when compared with their short plasma half-life (t ½ ) of ~1 h. The maximum concentration in plasma (C max ) and area under the time–concentration curve (AUC) does not correlate with acid suppression for all PPIs. All four PPIs are highly protein bound (>95%) and metabolized extensively in liver by CYP2C19 and 3A4 to varying degrees into less active or inactive metabolites and excreted through urine. The oral bioavailabilities of LPZ, OPZ, PPZ and RPZ were found to be 80–85, 30–40, 77 and 52%, respectively. Acid suppression studies comparing OPZ and LPZ, and OPZ and PPZ suggest * Correspondence to: D. V. Bharathi, Bioanalytical Department, Integrated Product Development, Dr. Reddy’s Laboratories Ltd, Bachupalli, Hyderabad- 500 072, India. E-mail: vijayabd@drreddys.com a Bioanalytical Department, Integrated Product Development, Dr Reddy’s Laboratories Ltd, Bachupalli, Hyderabad-500 072, India b Department of Chemistry, JNTU College of Engineering, Kukatpally, Hyderabad-500 072, India Jubilant Innovation, 96 Industrial Suburb, 2nd Stage, Yeshwanthpur, Bangalore-560022, India Abbreviations used: ATPase, H + /K + -adenosine triphosphatase; GERD, gastro- oesophagal reflux disease; LPZ, lanoprazole; NERD, nonerosive reflux disease; OPZ, omeprazole; PPI, proton pump inhibitors; PPZ, pantoprazole; RPZ, rabeprazole.