A sensitive assay for estimation of DM1 fortified with trastuzumab in human serum utilizing positive LC-MS/MS electrospray ionization and its application to clinical study

AIM: A sensitive LC-MS/MS method was developed and validated for the estimation of DM1 fortified with trastuzumab in human serum. MATERIALS AND METHODS: Quantification was performed in positive mode with protein precipitation extraction using DM4 as an internal standard. Chromatographic separation was achieved using gradient elution and column ACE 5 C18, 100 mm × 4.6 mm. The mobile phase consisted of 0.1% formic acid in 1 mM ammonium formate in water and acetonitrile:methanol (50:50%, v/v) with a total run time of 8.0 min. RESULTS: The calibration curve was linear over the range of 0.5-100 ng/mL. Inter-day and intra-day accuracy and precision, matrix effect, and recovery were well within the acceptance criteria. CONCLUSION: A method to quantify free DM1 in human serum was fully validated, and the validated assay was successfully applied to determine the clinical pharmacokinetics of DM1 in HER2-positive patients.