Sensitive and selective liquid chromatography–electrospray ionization tandem mass spectrometry analysis of hydrochlorothiazide in rat plasma

A sensitive and selective method for the determination of hydrochlorothiazide (HCTZ) concentrations in rat plasma was developed using high performance liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS). An aliquot of plasma (50 μl) was mixed with the solution of internal standard, hydrofluorothiazide (HFTZ), and extracted with tert-butyl methyl ether. The reconstituted extract was applied to the LC–MS/MS system with a reversed phase C8 column and eluted with distilled water/acetonitrile (85/15, v/v). To enhance negative ionization of HCTZ and HFTZ in the multiple reaction monitor (MRM), the solution consisting of acetonitlile/1% (v/v) ammonia solution (95/5, v/v) was delivered after column separation. This additional technique, so-called the post-column addition, increased sensitivity of HCTZ and HFTZ about 500- and 200-fold, respectively. The calibration curve showed good linearity (r=0.999) over the range of 4–1000 ng/ml. Acceptable accuracy (100.8–113.1%) and precision (0.28–16.4%) were confirmed in the intra- and the inter-day analyses. It is indicated that this LC–MS/MS method is useful for pharmacokinetic studies of HCTZ in small animals, because it enabled the serial determination of plasma level of HCTZ in rats.