Quantification of Alfacalcidol Tablets Dissolution Content by Chemical Derivatization and LC-MS

Application of liquid chromatography-mass spectrometry (LC-MS) in analyzing the content of alfacalcidol tablets dissolution faces big challenges due to the low amount of alfacalcidol in each tablet and the low ionization efficacy of the compound with electrospray ionization (ESI) or atmospheric-pressure chemical ionization (APCI). Here, extraction, derivatization, and LC-MS quantitation method have been developed and validated for measuring alfacalcidol tablets dissolution content. After alfacalcidol dissolution solution was extracted with dichloromethane to remove surfactant and inorganic salts, alfacalcidol was then derivatized via a Cookson reagent, 4-phenyl-1, 2, 4-triazoline-3, 5-dione (PTAD), under ambient conditions. Alfacalcidol derivative was successfully analyzed by LC-MS. Limit of detection (LOD) of the derivatized alfacalcidol was improved 100 times (0.01 μg/mL) compared with the nontreated compound (1 μg/mL). The new method was then validated following International Conference on Harmonization (ICH) guidance. The method shows a good linearity with r(2) > 0.99. Interday and intraday reproducibility was 3.3% and 7.9%, respectively. This procedure can be used in quantification of alfacalcidol tablets dissolution content and corresponding pharmaceutical quality control.