Analytica Chimica Acta 557 (2006) 229–235 Estimation of tegaserod in human plasma by high-performance liquid chromatography–tandem mass spectroscopy and its application to bioequivalence study Sonu Sundd Singh ∗ , Harshvardhan Patel, Kuldeep Sharma Zydus Research Centre, Ahmedabad, India Received 3 September 2005; received in revised form 17 October 2005; accepted 19 October 2005 Abstract A sensitive and novel HPLC–tandem mass spectrometric method has been developed for the estimation of tegaserod in human plasma using atorvastatin as internal standard. The methodology involved solid phase extraction (SPE) of the analyte from human plasma, using the Water’s Oasis ® HLB 1 cm 3 (30 mg) cartridges. The chromatographic separation was achieved within 5 min on XTerra MS C18, 100 mm × 2.1 mm i.d., 3.5 m analytical column with an isocratic mobile phase containing a mixture of acetonitrile and 0.2% formic acid in water (50:50, v/v) flowing through it at the rate of 0.3 ml/min. Multiple reaction monitoring (MRM) transitions; m/z 302.2, 173.2 and 559.0, 439.7 were measured in positive mode for tegaserod and internal standard, respectively. A detailed validation of the method was performed as per USFDA guidelines. The standard curves were linear in the range 0.2–20.0 ng/ml with the mean correlation coefficient more than 0.99. The absolute recovery for tegaserod was more than 70%. The method was simple, sensitive, precise, accurate and suitable for routine bioequivalence and pharmaco-kinetic studies. The method was successfully applied to the bioequivalence study of tegaserod tablets in healthy, male human subjects. © 2005 Elsevier B.V. All rights reserved. Keywords: Tegaserod; Human plasma; Validation; Bioequivalence 1. Introduction Tegaserod is a 5-hydroxy-tryptamine 4-receptor partial ago- nist, approved in the year 2002 to cure patients suffering from constipation predominant irritable bowel syndrome (IBS), a complex gastrointestinal disorder [1]. Altered bowel motility, visceral hypersensitivity, psychological disorder, neurotrans- mitter imbalance, infection and inflammation contribute to the pathophysiology of IBS. The absolute oral bioavailability [1] of tegaserod maleate under fasted conditions is around 10.0% and the T max occurs at 1.0–1.3 h. Food reduces the bioavailability of tegaserod. Tegaserod is a basic and lipophilic molecule and the initial rise in pH after meal intake reduces its solubility, which in turn reduces its bioavailability. About two-third of the drug administered is excreted unchanged in feces and the remain- der is eliminated via urine as inactive metabolites. In the ∗ Corresponding author. Tel.: +91 9327072300. E-mail address: sonusundd@rediffmail.com (S.S. Singh). stomach, tegaserod is hydrolyzed by acid, followed by hep- atic oxidation and conjugation to the major metabolite; 5- methoxy-indole-3-carboxylic acid glucuronide which is inac- tive. Tegaserod also undergoes direct glucuronidation to iso- meric N-glucuronides. About 98% of the drug is bound to plasma proteins, predominantly with -acid glycoprotein. The pharmaco-kinetics of tegaserod maleate does not exhibit gender difference. So far, there are a few reports on the method of estimation of tegaserod. The estimation of tegaserod has been carried out by HPLC at 1–10 M concentration and its metabolites were identified by LC–MS [2]. Tegaserod has also been estimated in human serum by differential pulse voltammetry [3] with a detection limit of 3.0 × 10 -10 M. The objective of the present investigation was to develop a simple and sensitive LC–MS/MS method for the estimation of tegaserod in human plasma. The method was meant to be applied to a bioequivalence study of tegaserod formulation on healthy male human subjects. To con- firm the suitability of the method, a detailed method validation was performed as per USFDA guidelines [4] prior to the initia- tion of the study. 0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.10.044