Trace analysis of methotrexate and 7-hydroxymethotrexate in human plasma and urine by a novel high-performance liquid chromatographic method.
A new high-performance liquid chromatographic method for the quantitative determination of methotrexate (MTX) and its metabolite 7-hydroxymethotrexate (7-OHMTX) in blood plasma and urine was developed. The method utilized a solid-phase extraction procedure (Certify II cartridges) for the simultaneous isolation of MTX and 7-OHMTX. Chromatographic separation was achieved using a C18 reversed-phase column with isocratic elution. The eluent was irradiated with UV light of 254 nm, which converted MTX and 7-OHMTX by photolytic oxidation to fluorescent products. The limits of detection of MTX and 7-OHMTX in plasma were approximately 0.2 and 1 nmol/L, respectively. The intraday variability in the quantitation of MTX and 7-OHMTX was less than 8% down to 1 nmol/L and 4.6 nmol/L, respectively. Both MTX and 7-OHMTX could be detected in plasma from a patient being treated for rheumatoid arthritis 1 week after the last dose (10 mg orally).
Beck
O
O
Department of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden.
Seideman
P
P
Wennberg
M
M
Peterson
C
C
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005470:Fluorometry; D006801:Humans; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood / Q000652:urine; D008832:Microchemistry
C011864:7-hydroxymethotrexate; D008727:Methotrexate