BIO ANALYTICAL LC-MS METHOD DEVELOPMENT AND VALIDATION FOR RAVULIZUMAB IN RAT PLASMA USING ECULIZUMAB (INTERNAL STANDARD)
DOI:
https://doi.org/10.48047/Keywords:
Ravulizumab, EculizumabBioavailability, Validation, Accuracy, LC-MS/MS.Abstract
The objective of the study was to develop and validate simple, selective, specific Liquid
Chromatography - Tandem Mass Spectrometry (LC-MS/MS) method for the determination of
ravulizumab and eculizumab(Internal Standard) in rat plasma. The accuracy and precision data must
fulfill the requirements for the quantification of analytes in biological matrices to produce data for
bioavailability, bioequivalence, etc. The separation of the analyte was carried out on Waters, XBridge, C18, 5μm column having 4.6×50 mm internal diameter and the mobile phase containing
acetonitrile and 0.1 % formic acid (90:10 v/v) at a flow rate of 0.6 mL/min. The retention times of
ravulizumab and eculizumab(Internal Standard) were 2.886 min and 4.439 min simultaneously and
the total run time was 7.0 min. Monitoring of the fragmentation of m / z 473.54 → 157.6 performed
during MS/MS detection of ravulizumab and eculizumab(I.S.) on the mass spectrometer. The overall
recovery of ravulizumab and eculizumabwas 92.5 % and 89.9 % respectively. The matrix effect of
ravulizumab and eculizumabwas 5.51 and 1.33 % respectively. The method was validated over the
concentration range of 10ng/mL to 5000ng/mL. Multiple Reaction Monitoring (MRM) mode was
used as an operating mode in the mass spectrometer. Ion spray was kept in positive mode for the
detection of Analyte and IS during the production of ions. The method was validated for linearity,
accuracy, precision, specificity, selectivity, inter and intraday precision, LQC, HQC.
