Determination of serum methotrexate and 7-hydroxymethotrexate concentrations. Method evaluation showing advantages of...

The use of large doses of methotrexate (MTX), greater than 3 g/m2, for the treatment of some malignant disorders requires careful monitoring of serum concentrations. A simple and sensitive method for the separation of MTX and 7-hydroxymethotrexate (7-OH-MTX) by reversed-phase high-performance liquid chromatography (HPLC) is described. The method involves deproteinizing the serum sample on a Sep-Pak C18 cartridge, followed by separation on a C18 column and detection at 313 nm. The extraction efficiency of free MTX from serum is 70% and the maximum sensitivity is 2.2 X 10(-8) M. A high degree of correlation was obtained between the HPLC method of serum MTX determination and an enzyme multiplied immunoassay technique. The HPLC method separates MTX from its analogues, or drugs which may be administered concomitantly with MTX. Concentrations of MTX and 7-OH-MTX achieved over a 24-h period during high-dose therapy, (500-1000 mg/m2), and over 48 h for very-high-dose methotrexate therapy (8-12 g/m2) are described. A significant observation is the presence of 7-OH-MTX in sera of patients 6 h after commencement of infusion. This method was also utilized for monitoring cerebrospinal fluid MTX concentrations.