Highly sensitive high-performance liquid chromatographic assay for methotrexate in the presence and absence of anti-methotrexate antibody fragments in rat and mouse plasma

摘要

Recently, Balthasar and Fung have proposed that anti-methotrexate antibody fragments may be employed to enhance the selectivity of intraperitoneal methotrexate (MTX) therapy. This current work presents a sensitive high-performance liquid chromatographic method for measuring plasma concentrations of total (i.e, bound and unbound) MTX and free (unbound) MTX in rat and mouse plasma, in the presence or absence of therapeutic anti-MTX antibody fragments. The assay involves pre-column derivatization of MTX by sodium hydrosulfite to 2,4-diamino-6-methylpteridine. The limit of quantitation for MTX by this assay was 1.25 ng in rat plasma, mouse plasma and mouse plasma ultrafiltrate, which corresponds to a concentration of 25 ng/ml for a 50 #mu#l sample. The limit of quantitation was found to be 2.5 ng in rat plasma ultrafiltrate (i.e,50 ng/ml in 50 #mu#l rat plasma ultrafiltrate). The method was shown to be quite accrate, as the mean assayed concentration of quality control samples was within 10% of theoretical values. We have applied the method to the investigation of MTX pharmacokinetics in mice and rats, following the administration of MTX alone or following simultaneous administration of MTX and anti-MTX Fab fragments. The method has been shown to be suitable for the assay of total and free methotrexate in the plasma of these species and will enable the testing of pharmacokinetic hypothese regarding the influence of anti-MTX Fab fragments on the disposition of MTX.