Thennati R, Shahi PK, Chakra A, Patel H, Shah V and Bhokari A
The systemic circulating levels of Doxercalciferol and its active metabolite form were in the pg/mL range which represents a significant bioanalytical challenge for therapeutic monitoring. Hence Liquid chromatography with tandem mass spectrometry (LC–MS/MS) with derivatization technique was considered the most appropriate standard for the selective and sensitive determination of this molecule in biological matrices and a sensitive, selective, precise and well accurate liquid chromatography tandem mass spectro-metric (LC–MS/MS) assay method was developed for simultaneous determination of Doxercalciferol and its metabolite in human plasma at pg/ml lower limit of quantitation and in order to improve the accuracy and reliability of assay method for determination of parent compound, and metabolite, a derivatization procedure was adopted. Extraction procedure was optimized with solid phase extraction technique for better recovery, selectivity and low matrix effect. Prior to detection, doxercalciferol and metabolite were ionized using an ESI source in the multiple reaction monitoring (MRM) mode. The proposed method was extensively validated over the concentration range of 1 to 75.29 pg/mL for doxercalciferol and 1.5 to 75.07 pg/mL for its metabolite as per FDA guidelines and the results met the acceptance criteria and validated method was successfully applied for estimation of drug and metabolite concentration in the healthy male volunteers, bioequivalence and pharmacokinetic study of Doxercalciferol, 2.5 μg dose capsules under fasting condition.