Analysis of methotrexate and 7-hydroxymethotrexate by high-performance liquid chromatography and preliminary clinical...

The simultaneous analysis of methotrexate (MTX) and its putatively nephrotoxic metabolite, 7-hydroxymethotrexate (7OH-MTX), by high-performance liquid chromatography (HPLC) and ultraviolet spectrophotometric detection is described. Serum extraction employs SEP-PAK C-18 cartridges. Recovery ranges from 78.3 to 84.9% for MTX and 67.6 to 76.1% for 7OH-MTX. Between-day precision studies of serum (controls), containing 2.76 microM MTX and 4.40 microM 7OH-MTX, yielded coefficients of variation of 8.6 and 8.9%, respectively. Reconstitution of the dried residue in 5 mM HCl increases the retention times of 7OH-MTX and MTX, thereby enhancing their separation from extraneous serum peaks. A comparison of MTX levels determined by HPLC and a competitive protein binding assay yielded consistently lower results by HPLC. However, in comparing HPLC to EMIT, two relationships were observed: below 100 microM MTX the methods were in agreement, whereas above 100 microM MTX HPLC again provided lower values. Preliminary pharmacokinetic studies on two patients with osteogenic sarcoma are reported. After receiving 218.2 mg/kg and 148.5 mg/kg MTX in a 6-h infusion, their beta half-lives for MTX were 2.6 and 2.0 h, while their gamma half-lives were 26.2 and 42.9 h, respectively. The 7OH-MTX beta half-lives were 5.8 and 4.0 h, and the gamma half-lives were 10.2 and 15.8 h. Plasma concentration ratios of 7OH-MTX to MTX were 28.5 and 18.1 at 24 h after MTX infusion. 7OH-MTX was detected in the 15-min sample after the beginning of the MTX infusion.