Enantioselective liquid chromatography–mass spectrometry assay for the determination of ifosfamide and identification of the N-dechloroethylated metabolites of ifosfamide in human plasma

A sensitive and specific liquid chromatography–mass spectrometry (LC–MS) method has been developed and validated for the enantioselective determination of ifosfamide [(R)-IF and (S)-IF] in human plasma and for the detection of the N-dechloroethylated metabolites of IF, 2-N-dechloroethylifosfamide [(R)-2-DCl-IF and (S)-2-DCl-IF] and 3-N-dechloroethylifosfamide [(R)-3-DCl-IF and (S)-3-DCl-IF]. IF, 2-DCl-IF and 3-DCl-IF were extracted from plasma using solid-phase extraction and resolved by liquid chromatography on a column containing a Chirabiotic T chiral stationary phase. The enantioselective separations were achieved using a mobile phase composed of 2-propanol:methanol (60:40, v/v) and a flow rate of 0.5 ml/min. The observed enantioselectivities (α) for IF, 2-DCl-IF and 3-DCl-IF were 1.20, 1.17 and 1.20, respectively. The calibration curve was linear in the concentration range of 37.50–4800 ng/ml for each ifosfamide enantiomer (r2 > 0.997). The lower limit of detection (LLOD) was 5.00 ng/ml. The inter- and intra-day precision ranged from 3.63 to 15.8% relative standard deviation (R.S.D.) and 10.1 to 14.3% R.S.D., respectively, and the accuracy ranged from 89.2 to 101.5% of the nominal values. The method was applied to the analysis of plasma samples obtained from a cancer patient who received 3.75 g/m2/day dose of (R,S)-ifosfamide as a 96-h continuous infusion.