Determination of raltitrexed in human plasma by high performance liquid chromatography–electrospray ionization-mass spectrometry

A sensitive high performance liquid chromatography–electrospray ionization-mass spectrometry (HPLC–ESI-MS) assay was developed to determine raltitrexed in human plasma. After addition of benazeprilat as the internal standard (IS), methanol was used to produce a protein-free extract. Chromatographic separation was achieved with a Zorbax SB-C18 column (Narrow-Bore 2.1 mm × 150 mm, 5-μm) using a mobile phase of acetonitrile–water containing 0.1% formic acid and 2% methanol (21.9:78.1, v/v). Raltitrexed was determined with electrospray ionization-mass spectrometry. HPLC–ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at [M + H]+ m/z 459.1 for raltitrexed and [M + H]+ m/z 397.1 for IS. Calibration curves were linear over the range of 2.0–3000 ng/ml. The lower limit of quantification was 2.0 ng/ml. The intra- and inter-batch variability values were less than 6.7% and 10.3%, respectively. The mean plasma extraction recovery of raltitrexed was in the range of 85.2–91.1%. The method was successfully applied to determine the plasma concentrations of raltitrexed in eight Chinese colorectal cancer patients.