High-throughput protein precipitation method with 96-well plate for determination of Doxepin and Nordoxepin in human plasma by LC-MS/MS.

The aim of this study was to establish high-throughput and sensitive liquid chromatography tandem mass spectrometry method for the determination of Doxepin and its major active metabolite Nordoxepin in human plasma. It has been designed for bioequivalence study for formulations containing 25 mg of Doxepin. Doxepin and Nordoxepin were extracted from human plasma samples by protein precipitation (PPT) with acetonitrile by using PPT 96-well plates. Analyte separation was done using Phenomenex Kinetex Biphenyl (100 x 2.1 mm, 2.6 mum) column using isocratic elution with a mobile phase of 20 mM ammonium formate (30%) and acetonitrile:methanol 3:7 V:V (70%) at a flow rate of 0.5 mL/min and an injection volume of 10 muL. The detection was performed on a triple quadrupole mass spectrometer by multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 280.4 --> 107.0, 283.4 --> 235.0 for Doxepin and Doxepin-D3 and 266.3 --> 106.9, 269.3 --> 235.0 for Nordoxepin and Nordoxepin-D3 in positive electrospray ionization mode, correspondingly. Total run time was 3.5 min. The method was validated over a concentration range of 50-10,000 pg/mL on SCIEX Triple Quad 4500 MS System for both analytes. The developed and validated method can be successfully applied for the bioequivalence/pharmacokinetic studies of Doxepin and Nordoxepin.