High-performance liquid chromatography method for serum methotrexate levels in children with severe steroid-dependent asthma

Monitoring of low-dose methotrexate (MTX), as used in severe steroid-dependent asthma, requires a sensitive and reproducible technique which has hitherto not been available. A high-performance liquid chromatographic method for the determination of MTX in serum is reported. The method involves deproteinization with acetone followed by addition of butanol and diethyl ether. The percentage recovery with this method compared with others was high (90 versus 70%). The samples were chromatographed on a reversed-phase ODS column and monitored at 313 nm. The retention time for MTX was 14.7 min. Pharmacokinetics of MTX was studied in five patients (age 3–15 years) with severe asthma who received a weekly oral dose of 10 mg/m2 body surface area. Following administration, the serum disappearance was monophasic with a half-life of 5 h. A metabolite, 7-hydroxymethotrexate was detected in serum after 2 h and reached a maximum concentration after 6 h. This new method will facilitate monitoring of asthmatic patients on methotrexate and allow for dose response and toxicity studies to be conducted.