Direct injection liquid chromatography/positive ion electrospray ionization mass spectrometric quantification of methotrexate, folinic acid, folic acid and ondansetron in human serum

A rapid liquid chromatography/positive ion electrospray mass spectrometric assay (LC/ESI-MS) was developed for the quantitation of methotrexate, folinic acid, folic acid and ondansetron in human serum. The assay was based on 100 μL serum samples, following acetonitrile precipitation of proteins and filtration that enabled direct injection into the LC/MS system. All analytes and the internal standard, alfuzosin, were separated by using a Zorbax Eclipse XDB–C8 analytical column (2.1 mm × 150.0 mm i.d., particle size 3.5 μm) with isocratic elution. The mobile phase was composed of a mixture of water/acetonitrile containing 0.1%, v/v formic acid (75:25, v/v), pumped at a flow rate of 0.15 mL min−1. Quantitation of the analytes was performed with selected ion monitoring (SIM) in positive ionization mode using electrospray ionization interface. The assay was found to be linear in the concentration range of 0.01–25.00 μg mL−1 for methotrexate and 0.01–5.00 μg mL−1 for folic acid, folinic acid and ondansetron. Intermediate precision was found to be less than 4.2% over the tested concentration ranges. A run time of less than 7.0 min for each sample made it possible to analyze a large number of human serum samples per day. The method can be used to quantify methotrexate, folinic acid, folic acid and ondansetron in human serum covering a variety of clinical studies and it was applied to the analysis of human serum samples obtained from children with acute lymphoblastic leukemia.