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Sensitive liquid chromatography-tandem mass spectrometry method for quantification of hydrochlorothiazide in human plasma.

Ramakrishna NV, Vishwottam KN, Manoj S, Koteshwara M, Wishu S, Varma DP

Biopharmaceutical Research, Suven Life Sciences Ltd, Serene Chambers, Road 7, Banjara Hills, Hyderabad 500034, India. nvsrk@suven.com

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of hydrochlorothiazide (I), a common diuretic and anti-hypertensive agent. The analyte and internal standard, tamsulosin (II) were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reversed-phase column (Waters symmetry C18) with a mobile phase of 10 mm ammonium acetate-methanol (15:85, v/v). The protonated analyte was quantitated in negative ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 296.1 solidus in circle 205.0 and m/z 407.2 solidus in circle 184.9 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/mL for hydrochlorothiazide in human plasma. The lower limit of quantitation was 500 pg/mL, with a relative standard deviation of less than 9%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.5 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.

Published 5 December 2005 in Biomed Chromatogr, 19(10): 751-60.
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