On-line Precolumn Derivatization for HPLC Determination of Methotrexate Using a Column Packed Oxidant

Abstract

A high‐performance liquid chromatographic method involving on‐line precolumn oxidative cleavage and fluorimetric detection was developed for the determination of methotrexate in plasma. Plasma samples were subjected to protein precipitation followed by solvent purification and then injection into the chromatographic system. Cerium (IV) trihydroxyhydroperoxide (CTH) was introduced as a packed oxidant before analytical column for the conversion of methotrexate into highly fluorescent 2,4‐diaminopteridine derivatives. The oxidative cleavage of methotrexate occurs during the flow of 0.04 M phosphate buffer (pH 3.5) containing the drug through CTH column with a flow‐rate of 0.2 mL/min at 40 °C. The separation was performed on a reversed‐phase column (ODS/TM) using a mobile phase consisting of phosphate buffer (0.05 M, pH 6.6) and acetonitrile (90:10 v/v). The fluorescent products were monitored fluorimetrically at emission and excitation wavelengths of 463 and 367 nm, respectively. Validation of accuracy and precision were satisfactory for both within‐ and between‐run assays. All coefficients of variance were less than 4% and mean relative errors were within 1.11% to 7.83%. The average recovery was found to be 93.74% to 98.11%. The proposed method is highly sensitive, specific and applicable to biological fluids. © 1997 by John Wiley & Sons, Ltd.

Direct injection liquid chromatography/positive ion electrospray ionization mass spectrometric quantification of methotrexate, folinic acid, folic acid and ondansetron in human serum

“…Fluorescence polarization immunoassay [9], radioimmunoassay [10], capillary zone electrophoresis [11] have been used to quantify methotrexate. There have been numerous HPLC methods that utilize laborious and time-consuming sample preparation procedures with either UV or derivative fluorescence detection [12][13][14][15][16][17][18][19]. In addition, hyphenated techniques, such as LC-MS/MS have also been applied to the determination of methotrexate in human plasma [20,21] mouse brain tissues [22] and environmental samples for occupational expose monitoring [23][24][25].…”

Determination of methotrexate and its major metabolite 7-hydroxymethotrexate in mouse plasma and brain tissue by liquid chromatography-tandem mass spectrometry

“…Most often these methods utilize either liquid-liquid extraction or solid phase extraction for sample clean-up with either UV or derivative fluorescence detection [8][9][10][11][12][13][14][15][16]. Since most of these methods were developed for human samples, relatively large volumes [i.e.…”

A validated LC–MS/MS method for rapid determination of methotrexate in human saliva and its application to an excretion evaluation study

“…There have been numerous HPLC methods that utilize laborious and timeconsuming sample preparation procedures with either UV or derivative fluorescence detection [11][12][13][14][15][16][17][18]. Hyphenated techniques, such as LC-MS/MS have also been applied to the determination of methotrexate in human plasma [1,5,13,15,17,18] mouse brain tissues [19] and environmental samples for occupational expose monitoring [20][21][22].…”