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  • Title: Determination of methotrexate and 7-hydroxymethotrexate by liquid chromatography for routine monitoring of plasma levels.
    Author: Cociglio M, Hillaire-Buys D, Alric C.
    Journal: J Chromatogr B Biomed Appl; 1995 Dec 01; 674(1):101-10. PubMed ID: 8749257.
    Abstract:
    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.
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