Simultaneous determination of amiloride and hydrochlorothiazide in human plasma by liquid chromatography/tandem mass spectrometry with positive/negative ion-switching electrospray ionisation

Abstract

A new method for simultaneous determination of amiloride and hydrochlorothiazide by liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) operated in positive and negative ionization switching mode was developed and validated. Protein precipitation with acetonitrile was selected for sample preparation. The analytes were separated on a Phenomenex Curosil-PFP (250x4.6 mm, 5 microm) column by a gradient elution with a mobile phase consisting of 0.15% formic acid solution containing 0.23% ammonium acetate and methanol pumped at a flow rate of 1.0 mL.min(-1). Rizatriptan was used as the internal standard (IS) for quantification. The determination was carried out on a Waters Quattro-micro triple-quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode using the following transitions monitored simultaneously: positive m/z 230-->171 for amiloride, m/z 270-->158 for rizatriptan, and negative m/z 296-->205 for hydrochlorothiazide. The lower limits of quantification (LLOQs) were 0.1 and 1.0 ng.mL(-1) for amiloride and hydrochlorothiazide, respectively, which were lower than other published methods by using ultraviolet (UV), fluorimetric or mass spectrometric detection. The intra- and inter-day precision and accuracy were studied at three different concentration levels and were always better than 15% (n=5). This simple and robust LC/MS/MS method was successfully applied to the pharmacokinetic study of compound amiloride and hydrochlorothiazide tablets in healthy male Chinese volunteers.