Quantitative Analysis Of Picamilon And Hydrochlorothiazide In Human Plasma And Applications In Pharmacokinetic Studies

Objective:To develop and validate two different sensitive, rapid and specific LC-MS/MS methods for determination of picamilon and hydrochlorothiazide in human plasma respectively, and application to the clinical pharmacokinetic studies. Method:For determination of the plasma concentration of picamilon, an aliquot of 100μL plasma was treated by protein precipitation, then separated on a Venusil ASB C18 column with a mobile phase consisting of methanol-10 mmol·L-1 ammonium acetate-formic acid (55:45:0.1, v/v/v) at a flow rate of 0.65 mL/min. The triple quadrupole tandem mass spectrometry was applied via positive atmospheric pressure chemical ionization (APCI) source for detection. For determination of the plasma concentration of hydrochlorothiazide, an aliquot of 200 pL plasma was treated by liquid-liquid extraction, then separated on a Venusil ASB C18 column with a mobile phase consisting of methanol-5 mmol·L-1 ammonium acetate-1% aqueous ammonia,(55:45:0.1, v/v/v) at a flow rate of 0.60 mL/min. The triple quadrupole tandem mass spectrometry was applied via negative electrospray ionization (ESI) source for detection. Quantification was performed using selected reaction monitoring (SRM) of the transitions m/z 209→m/z (78+106) and m/z 152→m/z (93+110) for picamilon and the internal standard paracetamol, the transitions m/z 296→m/z (205+269) and m/z 358→m/z 322 for hydrochlorothiazide and the internal standard methyclothiazide, respectively. Pharmacokinetic parameters for picamilon and hydrochlorothiazide were calculated using a non-compartmental approach. Results:The linear calibration curves were obtained in the concentration range of 1.00-5000 ng/mL for picamilon, and 0.500-300 ng/mL for hydrochlorothiazide. The intra- and inter-day precision (RSD) over the entire concentration range were below 15%. Accuracy determined at three QC levels was within±15% as terms of relative error (RE). Each plasma sample was chromatographed within 5 min. The methods were applied to characterize the pharmacokinetic profiles of picamilon and hydrochlorothiazide in healthy volunteers. Conclusion:The two methods described above were selective, sensitive, and are suitable for the pharmacokinetic study of picamilon and hydrochlorothiazide in humans.