Simultaneous determination of azelastine and its major metabolite desmethylazelastine in human plasma using high performance liquid chromatography–tandem mass spectrometry

A selective and sensitive high performance liquid chromatography–tandem mass spectrometric method was developed for the analysis of azelastine and its major metabolite, desmethylazelastine, in human plasma. Azelastine-13C, d3 was used as internal standard. Azelastine, desmethylazelastine and the internal standard were extracted by a liquid–liquid extraction method and separation was performed under isocratic chromatographic condition. An abnormal signal loss issue for desmethylazelastine during method development was investigated and resolved. The developed method was precise and reproducible as shown by good intraday assay and interday assay precision (CV% ≤ 12.8%). The calibration curve was linear over a range of 10.0/10.0–1000/200 pg/mL for azelastine/desmethylazelastine. The method was successfully applied to a pilot bioequivalence study subsequently.

► LC–MS/MS method for simultaneous quantitation of azelastine and desmethylazelastine.
► Low LOQ of 10 pg/mL for both compounds were achieved.
► An abnormal signal loss issue for desmethylazelastine was investigated and resolved.