Direct quantification of anorethidrani disuccinate and determination of sterol metabolites by chemical derivatization combined with LC-MS/MS: Application to a Phase I pharmacokinetic study in humans

摘要

Anorethidrani disuccinate (ACP) is a domestically designed A-decarbonized steroid that is currently being investigated in Phase I clinical trials for the treatment of solid tumors. Only the parent drug exhibited antitumor activity; its sterol metabolite M2 showed obvious antiestrogenic effects. We have developed a rapid, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct quantification of ACP and a chemical derivatization method that can be used to quantify M2 derivatized with glycidyl trimethyl ammonium chloride (GTMA). A simple protein precipitation procedure was performed to quantify ACP. Injections were obtained within 3.5 min on an Eclipse Plus Phenyl-Hexyl column (50 mm x 2.1 mm i.d., 1.8 mu m) with gradient elution; the calibration curve was linear over the range of 2.00-8000 ng/mL. For quantification of M2 in plasma, analytes were extracted by protein precipitation and converted to their GTMA derivatives at 60 degrees C for 2 h at pH 12; the analytes and coelutants were separated on a Luna C8(2) column (50 mm x 2.0 mm i.d., 5.0 mu m). The precision (RSD) and accuracy (RE) of the infra- and interday determinations were within 10%. The derivatization procedure is a novel method for sterol determination by LC-MS/MS. The results confirmed the usefulness of this method for characterizing the pharmacokinetic profiles of ACP and its major metabolite M2 in a Phase I pharmacokinetic study.