title: The influence of 7-OH methotrexate metabolite on clinical relevance of methotrexate determination.
abstract: BACKGROUND
  A modified high performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of methotrexate (MTX) and its main metabolite 7-hydroxymethotrexate (7-OHMTX) and compared to the immunochemical fluorescence polarization immunoassay (FPIA2) determination of methotrexate.
  
  
  METHODS
  Methotrexate was determined by HPLC with UV detection at 303 nm after precipitation of proteins with trichloroacetic acid. Fluorescence polarization immunoassays (FPIA2) of methotrexate were performed on the TDx FLx Immunoassay Analyzer.
  
  
  RESULTS
  Our data indicate good correlation between methotrexate levels > 1 micromol/L determined by HPLC and FPIA2. (r = 0.94, Spearman correlation coefficient). However, concentrations of methotrexate < 1 micromol/L measured by fluorescence polarization immunoassay were overestimated.
  
  
  CONCLUSIONS
  The concentration of MTX < 1 micromol/L are overestimated due to the cross reactivity with metabolites 7-OHMTX and 2,4-diamino-N10-methylpteroic acid (DAMPA). The cross reaction may affect the therapy and lead to relapse in children with acute lymphoblastic leukemia.
authors: Department of Clinical Biochemistry and Pathobiochemistry, Charles University, 2nd Faculty of Medicine and Faculty Hospital Motol, Prague, Czech Republic. eva.klapkova@email.cz
authors: Eva
authors: E
authors: Klapkova
authors: Jiri
authors: J
authors: Kukacka
authors: Karel
authors: K
authors: Kotaska
authors: Iveta
authors: I
authors: Suchanska
authors: Romana
authors: R
authors: Urinovska
authors: Richard
authors: R
authors: Prusa
mesh_terms: D000293:Adolescent; D000964:Antimetabolites, Antineoplastic / Q000097:blood* / Q000493:pharmacokinetics; D001711:Biotransformation; D001859:Bone Neoplasms / Q000097:blood / Q000188:drug therapy; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016903:Drug Monitoring / Q000379:methods*; D016231:Fluorescence Polarization Immunoassay; D006801:Humans; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood* / Q000493:pharmacokinetics; D012516:Osteosarcoma / Q000097:blood / Q000188:drug therapy; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000097:blood / Q000188:drug therapy; D055815:Young Adult
chemical_list: D000964:Antimetabolites, Antineoplastic; C000203:deoxyaminopteroic acid; C011864:7-hydroxymethotrexate; D008727:Methotrexate