Methotrexate and 7-hydroxymethotrexate: serum level monitoring by high-performance liquid chromatography

We have developed a reverse-phase high-performance liquid chromatographic assay with ultraviolet detection at 303 nm for methotrexate (MTX) and 7-hydroxymethotrexate (7-OH-MTX) in serum, plasma, and cerebrospinal fluid. The procedure involves the concentration of MTX and 7-OH-MTX in the aqueous phase by an n-butanol-ether extraction, resulting in a sensitivity of 10-20 nM in serum. Serum levels of MTX correlated well with those obtained by a competitive dihydrofolate reductase binding assay (r = 0.989). The clinical utility of the assay was demonstrated by measuring MTX and 7-OH-MTX serum levels in 42 patient samples.