Control of methotrexate concentration at chemotherapeutical treatment with HPLC on short columns

A method of methotrexate determination in blood serum is offered which is applicable to the control of its concentration at chemotherapeutical treatment. The sample is prepared in the following way: lipids are extracted with hexane, and proteins settle in the presence of lithium perchlorate, acetic acid and acetonitrile. The supernatant is injected into a chromatograph, and the sample components are separated on the 2 x 75 mm С 18 RP column in the gradient elution mode. The aqueous solution of 0.2 M lithium perchlorate (pH 3) and acetonitrile are used as mobile phases. Spectral ratios and direct coupling of LC to MS are used for identification of the methotrexate peak on the chromatogram and confirmation of its homogeneity. This method has been tested for correction of the methotrexate dose during treatment of children with acute lymphoblastic leukemia.