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.