Copyright © 2007 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 214– 222 (2008) DOI: 10.1002/bmc 214 R. Nirogi et al. ORIGINAL RESEARCH ORIGINAL RESEARCH Copyright © 2007 John Wiley & Sons, Ltd. BIOMEDICAL CHROMATOGRAPHY Biomed. Chromatogr. 22: 214–222 (2008) Published online 15 October 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/bmc.926 Sensitive liquid chromatography tandem mass spectrometry method for the quantification of sitagliptin, a DPP-4 inhibitor, in human plasma using liquid–liquid extraction Ramakrishna Nirogi,* Vishwottam Kandikere, Koteshwara Mudigonda, Prashanth Komarneni, Raghupathi Aleti and Rajeshkumar Boggavarapu Biopharmaceutical Research, Suven Life Sciences Ltd, Serene Chambers, Road—5, Avenue—7, Banjara Hills, Hyderabad 500034, India Received 26 July 2007; revised 4 August 2007; accepted 6 August 2007 ABSTRACT: A sensitive high-performance liquid chromatography–positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of sitagliptin, a DPP-4 inhibitor, in human plasma. Following liquid–liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H] + ions, m/z 408–235 for sitagliptin and m/z 310–148 for the inter- nal standard. The assay exhibited a linear dynamic range of 0.1–250 ng/mL for sitagliptin in human plasma. The lower limit of quantification was 0.1 ng/mL with a relative standard deviation of less than 6%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for appli- cation in pharmacokinetic studies. Copyright © 2007 John Wiley & Sons, Ltd. KEYWORDS: sitagliptin; DPP-4 inhibitor; liquid chromatography–tandem mass spectrometry; human plasma; pharmacokinetic study *Correspondence to: R. Nirogi, Biopharmaceutical Research, Suven Life Sciences Ltd, Serene Chambers, Road—5, Avenue—7, Banjara Hills, Hyderabad 500034, India. E-mail: Ramakrishna_nirogi@yahoo.co.in Abbreviations used: GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide-1. INTRODUCTION Sitagliptin (Fig. 1), a dipeptidyl peptidase inhibitor (DPP-4 inhibitor), has recently been approved in the USA and Europe for the therapy of type 2 diabetes (Gallwitz, 2007; Lyseng-Williamson, 2007). DPP-4 inhibitors rep- resent a new therapeutic approach to the treatment of type 2 diabetes that functions to stimulate glucose- dependent insulin release and reduce glucagons levels, by inhibiting the inactivation of incretins, particularly glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP), thereby improving glycemic control (Herman et al., 2007). Sitagliptin is effective in lowering HbA1c and fasting as well as postprandial glucose in monotherapy and in combination with other oral antidiabetic agents. Sitagliptin is relatively rapidly absorbed (median t max 1– 4 h) after oral administration with an apparent terminal t 1/2 of approximately 8–14 h (Herman et al., 2005). Figure 1. Chemical structures for (a) sitagliptin and (b) IS (fluoxetine).