Abstract

A SIMPLE AND RAPID METHOD FOR SIMULTANEOUS QUANTIFICATION OF DOXORUBICIN, EPIRUBICIN, CYCLOPHOSPHAMIDE AND 5-FLUOROURACIL IN HUMAN PLASMA BY LCMS/MS.

Gopinath Sambasivam*, Deepak Gopal Shewade, Biswajit Dubashi, Rajan Sundaram

Abstract

A reverse-phase HPLC method with detection by mass spectrometry is described for the simultaneous determination of doxorubicin, epirubicin, cyclophosphamide and 5 fluorouracil in human plasma. The chromatographic separation was carried out on a Waters Xterra® C18 column (3.9mmx150 mm, 5μm particle size) with a mobile phase of 0.1% formic acid in water and methanol (30:70 v/v). The flow rate was kept as 0.4ml/min with total run time of 7 minutes. A tandem quadrupole MS was operated in multiple reaction monitoring (MRM) in both positive and negative electrospray ionization (ESI) mode. Doxorubicin epirubicin and cyclophosphamide were detected in positive mode and 5- Fluorouracil was detected well in negative ionisation mode. Protein precipitation method by using 100% methanol was used to extract the drugs. To 200 μL of plasma along with internal standard, 600 μL of 100% methanol was added then vortexed for 1 minute and centrifuged at 13,500 rpm for 5min. After centrifugation 100 μL of the supernatant layer was used for analysis. The retention time of doxorubicin, epirubicin, 5-fluorouracil and cyclophosphamide were 2.32min, 2.35 min, 2.43 min and 4.64 min respectively. Ifosamide was used as internal standard and its retention time was 4.12 min. The developed method was validated in human plasma with a lowest limit of quantification of 5 ng/mL for all the drugs. The method was accurate with the intra-day and inter-day precision of 15% and the there was no endogenous contamination of any of the analytes. This method was robust, precise and accurate for the determination of doxorubicin epirubicin cyclophosphamide and 5 fluorouracil in human plasma using LC/MS/MS method.

Keywords: Doxorubicin, Epirubicin, Cyclophosphamide 5-Fluorouracil, LC-MS/MS.