284 Research Article Drug Testing and Analysis Received: 11 January 2010 Revised: 3 March 2010 Accepted: 23 March 2010 Published online in Wiley Interscience: 20 April 2010 (www.drugtestinganalysis.com) DOI 10.1002/dta.130 A rapid and most sensitive liquid chromatography/tandem mass spectrometry method for simultaneous determination of alverine and its major metabolite, para hydroxy alverine, in human plasma: application to a pharmacokinetic and bioequivalence study Chinmoy Ghosh, a,b∗ Vijay Jha, a Ramesh Ahir, a Sujal Shah, a C.P. Shinde, b and Bhaswat S. Chakraborty a A rapid and highly sensitive method for the determination of alverine (ALV) and its metabolite, para hydroxy alverine (PHA), in human plasma using LC-MS/MS in positive ion electrospray ionization (ESI) in multiple reactions monitoring (MRM) mode was developed and validated. The procedure involves a simple solid phase extraction (SPE). Chromatographic separation was carried out on a Hypersil GOLD C 18 column (50 mm × 4.6 mm, 5 μm) with an isocratic mobile phase and a total run time of 1.5 min. The standard calibration curves showed excellent linearity within the range of 0.060–10.051 ng/mL for ALV and 0.059–10.017 ng/mL for PHA (r ≥ 0.990). This method was successfully applied to a pharmacokinetic study after oral administration of alverine citrate 120 mg capsule in Indian healthy male volunteers. Copyright c  2010 John Wiley & Sons, Ltd. Keywords: alverine; para hydroxyl alverine; matrix effects; bio-equivalence; LC-MS/MS Introduction Alverine (ALV) citrate (Spasmonal, Norgine, Harefield, Middle- sex, UK) is thought of as a smooth muscle relaxant, [1,2] and is currently used as an antispasmodic in irritable bowel syn- drome and dysmenorrhoea. ALV was shown to inhibit spon- taneous electrical activity and nervous control of the proximal colon of the rabbit in vivo. [3] It was also found to decrease the sensitivity of the intestinal mechanoreceptors in response to chemical stimulation in anaesthetized cats [4] and reduce 5-hydroxytryptamine 1A receptor-mediated rectal hypersensitivity in the rat. [5] . ALV may increase Ca influx during action potentials due to inhibition of the inactivation of L-type Ca channels, but may also suppress evoked activity by inhibiting the sensitivity of con- tractile proteins to Ca 2+ . However, the exact mechanisms of ALV’s inhibitory action are still not clear, due to the lack of information on its effects on isolated smooth muscle in vitro. ALV is a frequently used antispasmodic for which there was hardly any specific and sensitive LC-MS method available. [6] The published method [6] was developed in API 5000 mass spectrome- try attached with ultra performance liquid chromatography (UPLC) as a solvent delivery module. In the reported method 0.100 ng/mL was the lower limit of quantification (LLOQ) value with 4.0 min of analysis time. In the present research a method was developed and validated that can be used for quantification (as well as identification) of ALV alone or in combination with its major metabolite in human plasma using liquid chromatography-mass spectrometry (LC-MS) technique up to low picogram level. The method was developed by using API 4000 MS/MS along with high performance liquid chromatography (HPLC), having the LLOQ value of 0.060 ng/mL and 0.015 ng/mL as limit of detection (LOD). The total analysis time of single injection was 1.5 min, which was almost three times faster than that of the reported one. So the novelty of this method is that, by using the almost same extraction technique as the reported one and the comparatively less sensitive instrument than the one reported, the most sensitive method with shortest analysis time was developed and validated. Therefore, it was worthwhile to develop a method, which can detect up to such lower limits with fast analysis at our laboratory. This bio-analytical method was validated in human plasma in multiple reactions monitoring (MRM), which can be applied to bioequivalence and pharmacokinetic studies of ALV. The principal goal of the present work was to develop an accurate, sensitive, and rapid LC-MS method with a dynamic linearity range that can cover the plasma concentrations following a single oral dose of ALV. The method had been validated by evaluating the precision, accuracy, ∗ Correspondence to: Chinmoy Ghosh, Research Scientist, Contract Research Organization, Cadila Pharmaceuticals Limited, 1389, Trasad road, Dholka-387 810, Dist – Ahmedabad, Gujrat, India. E-mail: chinmoy ghosh@yahoo.com a Contract Research Organization, Cadila Pharmaceuticals Limited, 1389-Trasad Road, Dholka, Gujarat, India b School of Studies in Chemistry, Jiwaji University, Gwalior, M.P., India Drug Test. Analysis 2010, 2, 284–291 Copyright c  2010 John Wiley & Sons, Ltd.