Sensitive determination of methotrexate in monkey plasma by high-performance liquid chromatography using on-line solid-phase extraction.
A high-performance liquid chromatographic method was developed for the sensitive determination of methotrexate (MTX) in monkey plasma using direct injection and on-line solid-phase extraction. After application of a 100-microliters aliquot of plasma to a pre-treatment column, the column was washed with 0.02 M phosphate buffer (pH 7) to eliminate plasma proteins and endogenous substances, and subsequently the adsorbed MTX was eluted. The MTX fraction was transferred to an analytical column by a column-switching (heart-cutting) technique, and MTX was analyzed using ion-pair chromatography with tetrabutylammonium bromide. More than 50 injections could be performed onto one pretreatment column. The accuracy, precision, reproducibility and linearity were satisfactory over a wide range of MTX concentrations (5-1000 ng/ml). The quantitation limit was 5 ng/ml with a signal-to-noise ratio of 5. The method was suitable for the pharmacokinetic study of MTX in monkey.
Okuda
T
T
Takeda Analytical Research Laboratories, Osaka, Japan.
Motohashi
M
M
Aoki
I
I
Yashiki
T
T
D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003902:Detergents; D000882:Haplorhini; D008727:Methotrexate / Q000097:blood* / Q000493:pharmacokinetics; D000644:Quaternary Ammonium Compounds; D013056:Spectrophotometry, Ultraviolet
D001798:Blood Proteins; D003902:Detergents; D000644:Quaternary Ammonium Compounds; C009405:tetrabutylammonium; D008727:Methotrexate