Analysis of Methotrexate and 7hyphen;Hydroxymethotrexate by Highhyphen;Performance Liquid Chromatography and Preliminary Clinical Studies

摘要

The simultaneous analysis of methotrexate (MTX) and its putatively nephrotoxic metabolite, 7-hydroxymethotrexate (70H-MTX), by high-performance liquid chromatography (HPLC) and ultraviolet spectrophotometric detection is described. Serum extraction employs SEP-PAKreg; C-18 cartridges. Recovery ranges from 78.3 to 84.9percnt; for MTX and 67.6 to 76.1percnt; for 70H-MTX. Between-day precision studies of serum (controls), containing 2.76 mu;M MTX and 4.40 mu;M 70H-MTX, yielded coefficients of variation of 8.6 and 8.9percnt;, respectively. Reconstitution of the dried residue in 5 mM HC1 increases the retention times of 70H-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 EMITreg;, two relationships were observed: below 100 mu;M MTX the methods were in agreement, whereas above 100 mu;M 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.