Rapid determination of methotrexate in plasma by high-performance liquid chromatography with on-line solid-phase extraction and automated precolumn derivatization

Abstract

A high‐performance liquid chromatographic system, combining solid‐phase extraction and automated precolumn derivatization is described for the routine determination of methotrexate in human plasma. The sample extraction and elution onto the analytical column were performed automatically and concomitantly using the column‐switching technique and a protein‐coated precolumn. Cerium (IV) trihydroxyhydroperoxide (CTH) was introduced as a packed oxidant before the analytical column for the conversion of methotrexate into highly fluorescent products. The oxidative‐cleavage of methotrexate occurs during the flow of 0.04 M phosphate buffer (pH 3.5) containing plasma sample through CTH column with a flow rate of 0.5 mL/min at 40°C. The fluorescent products were transferred to the protein‐coated precolumn, which was then flushed with the same buffer for clean‐up and enrichment from plasma sample. The trapped substances were desorbed from the precolumn and eluted onto the ODS/TM analytical column by isocratical elution with a mobile phase containing 0.05 M phosphate buffer, pH 6.6 and acetonitrile (90–10, v/v) for subsequent separation. The fluorescent products were detected fluorimetrically at excitation and emission wavelengths of 367 and 463 nm, respectively. The complete analysis was achieved within 15 min per sample. The calibration graph was linear in the range of 50–500 ng/mL of methotrexate and there was no interference from endogenous components. Copyright © 1998 John Wiley & Sons, Ltd.

Simple and sensitive HPLC method for the fluorometric determination of methotrexate and its major metabolites in human plasma by post‐column photochemical reaction

“…MTX has been reported to be separable on reversed‐phase columns by elution with an aqueous mobile phase containing acetonitrile and phosphate buffer (Okuda et al , ; Belz et al , ; Emara et al , , ). The best separation of MTX, 7‐OH‐MTX, DAMPA and IS was achieved on a Capcell Pak C18 UG120 reversed‐phase column with 50 µmol/mL phosphate buffer (pH 5.3)–acetonitrile (9:1, v/v) as an eluent.…”

“…Several HPLC methods have been reported to determine the levels of MTX in human plasma following clean‐up procedures, such as liquid–liquid extraction (Emara et al , ) or solid‐phase extraction (Emara et al , ; McCrudden and Tett, ; Lu and Jun, ). In order to shorten the time required to analyze MTX, 7‐OH‐MTX and DAMPA in plasma, we studied a protein precipitation method usually using acetonitrile, methanol, perchloric acid and trichloroacetic acid as chemical reagents.…”

Dispersive Micro-Solid Phase Extraction for Sensitive Determination of Methotrexate from Human Saliva Followed by Spectrophotometric Method

“…The performance of the newly developed D-µ-SPE/spectrophotometric method is comparable with other techniques from the viewpoints of a straightforward technique, LLOQ, apparatus, and validation tests (Albertioni et al, 1995;Hirai et al, 1997;Emara et al, 1998;Turci et al, 2000;Kuo et al, 2006;Chen and Zhang, 2008;Moghbel et al, 2010;Jouyban et al, 2011;Begas et al, 2013;Rodin et al, 2013;Wang et al, 2014;Wei et al, 2014;Wu et al, 2015;Fabrizi et al, 2016;Asadian et al, 2017). For a number of MTX determination LC methods cited in other reports, sophisticated detection systems such as MS or fluorescence were used which are not available in routine bioanalytical laboratories.…”

Sensitivity Enhancement for Separation-Based Analytical Techniques Utilizing Solid-Phase Enrichment Approaches and Analyte Derivatization for Trace Analysis in Various Matrices

“…According to the obtained results, online oxidative cleavage with florescence detection was six to eight times more sensitive than UV detection. MTX in human plasma was also measured by HPLC with the integration of online pre-column derivatization and automated SPE utilizing packed oxidant and PC-RP-pre-columns, respectively (Figure 6) [197]. The goal of this work was to reduce analytical errors by developing a direct injection chromatographic technique for derivatization, extraction, separation, and quantification of the tested analyte in plasma.…”

“…The trapped compounds were desorbed from the pre-column, and isocratic was eluted onto the end-capped RP-C18 analytical column for further separation and quantification. Table 3 lists some analytical techniques with UV and FL detection systems for measuring MTX in various matrices in addition to their characteristic LODs as well as LOQs [177,193,194,[196][197][198][199][200][201][202][203][204][205][206][207][208]. All HPLC procedures that utilized the FL detector showed lower LODs and LOQs than the more frequently employed UV-Vis system.…”