LC–MS/MS Method for Quantitation of Hydrochlorothia-zide and Nifedipine in Human Plasma

We have developed and validated a novel, sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC–ESI-MS/MS) for the simultaneous quantitation of hydrochlorothiazide (HCTZ) and nifedipine (NFP) from only 200 µL of human plasma. Diazepam (DZP) was used as an internal standard (IS). The analytes were extracted by a liquid-liquid extraction procedure with ethyl acetate-dichloromethane and separated on a reversed-phase Polaris 5 C18 Aanalytical column using a mobile phase composed of methanol containing 0.1% (v/v) formic acid and 5mM aqueous ammonium formate pH 6.0, delivered at a flow-rate of 300µL/min. Multiple reaction monitoring was performed in the negative ion mode using the transition m/z296.1→m/z205.2 (HCTZ), positive ion mode transitions m/z347.2→m/z 315.1 (NFP) and m/z285.0→m/z 193.2 (DZP) to quantify the drugs. Calibration curves in spiked plasma were linear (r2 ≥ 0.9953) from 5–2000 ng/mL for HCTZ and 5 – 400 ng/mL for NFP with a lower limit of quantification (LLOQ) of 5 ng/mL for both drugs. The intra- and inter- assay precisions (coefficient of variation) were less than 9% and the mean extraction recoveries were 98.1% (HCTZ), 99.5% (NFP) and 93.8% for the IS (DZP). The validated method was successfully applied to a limited population pharmacokinetics study of HCTZ and NFP among patients on hypertension care.