Determination of methotrexate and 7-hydroxymethotrexate by liquid chromatography for routine monitoring of plasma levels.
A high-performance liquid chromatographic (HPLC) method was designed to meet analytical and metrological requirements for routine blood level monitoring of methotrexate (MTX) and its main metabolite 7-hydroxymethotrexate (7OMTX). The metabolite, unavailable as a pure substance, was measured by reference to MTX calibration according to their respective ultraviolet absorbances. Acetonitrile deproteinization and chloroform clean-up provided plasma samples devoid of long-retained contaminants. The precision of the HPLC measurements, reproducibility of clean-up recovery, matrix effects and linearity were assessed by analysis of variance and linear regression in an appropriate experimental design, within a range from 0.205 to 16.7 mg/l of MTX and from 0.084 to 6.83 mg/l of 7OMTX. The clean-up recovery from plasma was 88% for MTX and 72% for 7OMTX, owing to retention on the protein precipitate. The assay was linear, the measurement precision was 3.3% for MTX and 6.2% for 7OMTX and the clean-up reproducibility was 4% for MTX and 3.6% for 7OMTX. By reference to automated fluorescence polarization immunoassay, the HPLC method resulted in plasma MTX values 10% lower, probably owing to the higher specificity of HPLC. Unsystematically sequenced plasma samples from 35 children following 24-h MTX infusions provided estimated half-decay times of 16 and 19 h for MTX and 7OMTX, respectively, and 7OMTX:MTX concentration ratios of 7 at 48 h and of 5 at 72 h from starting infusions.
Cociglio
M
M
Laboratoire de Pharmacologie Clinique, Centre Hospitalier Universitaire, Höpital Saint-Charles, Montpellier, France.
Hillaire-Buys
D
D
Alric
C
C
D000097:Acetonitriles; D000704:Analysis of Variance; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D016231:Fluorescence Polarization Immunoassay; D006801:Humans; D016014:Linear Models; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000097:blood* / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000097:Acetonitriles; C011864:7-hydroxymethotrexate; D008727:Methotrexate; C032159:acetonitrile