ABSTRACT
A selective, sensitive and rugged liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay has been developed for the simultaneous determination of doxepin (Dox) and its pharmacologically active metabolite, nordoxepin (NDox) in human plasma. The analytes and their internal standards (IS) were extracted from 500 μL of human plasma by liquid-liquid extraction using methyl tert-butyl ether. Chromatographic separation was achieved on Hypurity C8 column (100 mm × 4.6 mm, 5 μm) using a mixture of acetonitrile-methanol (95:5, v/v) and 2.0 mM ammonium formate in 93:7 (v/v) ratio. Detection was accomplished by tandem mass spectrometry in the positive ionization and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions studied for Dox, NDox, and their corresponding ISs, propranolol and desipramine, were m/z 280.1-107.0, 266.0 -107.0, 260.1-116.1 and 267.1-72.1, respectively. A linear dynamic range of 15.0–3900 pg/mL for Dox and 5.00– 1300 pg/mL for NDox was established with mean correlation coefficient (r2) of 0.9991 and 0.9993, respectively. The extraction recovery ranged from 86.6％–90.4％ and 88.0％–99.1％ for Dox and NDox, respectively. The intra-batch and inter-batch precision (％ CV) across quality control levels was ≤ 8.3％ for both the analytes. Stability evaluated under different storage conditions showed no evidence of degradation and the ％ change in stability samples compared to nominal concentration ranged from 4.7％ to 12.3％. The method was successfully applied to a bioequivalence study of 6 mg doxepin hydrochloride orally disintegrating tablet in 41 healthy Indian subjects under fasting and fed conditions.
ABSTRACT
A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been described for the determination of asenapine (ASE) in presence of its inactive metabolites N-desmethyl asenapine (DMA) and asenapine-N-glucuronide (ASG). ASE, and ASE 13C-d3, used as in-ternal standard (IS), were extracted from 300 μL human plasma by a simple and precise liquid-liquid extraction procedure using methyl tert-butyl ether. Baseline separation of ASE from its inactive meta-bolites was achieved on Chromolith Performance RP8e(100 mm × 4.6 mm) column using acetonitrile-5.0 mM ammonium acetate-10% formic acid (90:10:0.1, v/v/v) within 4.5 min. Quantitation of ASE was done on a triple quadrupole mass spectrometer equipped with electrospray ionization in the positive mode. The protonated precursor to product ion transitions monitored for ASE and ASE 13C-d3 were m/z 286.1 → 166.0 and m/z 290.0 → 166.1, respectively. The limit of detection (LOD) and limit of quantitation (LOQ) of the method were 0.0025 ng/mL and 0.050 ng/mL respectively in a linear con-centration range of 0.050–20.0 ng/mL for ASE. The intra-batch and inter-batch precision (% CV) and mean relative recovery across quality control levels were ≤5.8% and 87.3%, respectively. Matrix effect, eval-uated as IS-normalized matrix factor, ranged from 1.03 to 1.05. The stability of ASE under different storage conditions was ascertained in presence of the metabolites. The developed method is much simpler, matrix free, rapid and economical compared to the existing methods. The method was suc-cessfully used for a bioequivalence study of asenapine in healthy Indian subjects for the first time.