Determination of methotrexate and its metabolites in human plasma by electromembrane extraction in conductive vials followed by LC-MS/MS

Abstract

• An EME system for extraction of methotrexate and its metabolites was developed. • EME was performed and optimized in prototype equipment based on conductive vials. • The developed system may be used for future EME of polar zwitterionic analytes. In the current study, an electromembrane extraction (EME) method for the simultaneous determination of methotrexate (MTX) and its metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA), was developed for human plasma samples. MTX and its metabolites are characterized as polar and zwitterionic, and the extraction mode (anionic/cationic) together with type of supported liquid membrane (SLM) was therefore initially studied. Extraction efficiency in anionic mode was found to be entirely suppressed by matrix components in plasma, whereas high efficiency could be achieved in cationic mode using an deep eutectic solvent-based supported liquid membrane of 6-methylcoumarin:thymol (1:2 molar ratio) with 0.5% (w/w) di(2-ethylhexyl) phosphate. EME was performed in prototype equipment based on conductive vials, and parameters of extraction time, voltage, shaking rate, sample and acceptor volumes, and plasma dilution factor, were optimized to get the highest possible sensitivity in subsequent LC-MS/MS determination. In the final method, plasma was diluted 2-fold with phosphoric acid to a volume of 500 µL, and extracted for 30 min at 10 V using 10 µL SLM, 200 µL 100 mM formic acid as acceptor solution, and a shaking rate of 900 RPM. Under these conditions, the average current was 10 µA per extraction cell. Recoveries of 23–53% were achieved, with linear ( R 2 ≥ 0.9952) calibration curves, accuracy in the range 98–121%, relative standard deviation (RSD) of 2.8–27.6%, and matrix effects of 103–111%. The greenness of the proposed method was also evaluated by the AGREEprep metric, and compared to other methods from literature. The findings of this paper demonstrate that EME can provide selective and robust extraction of polar and zwitterionic substances in a complex biological fluid, using very green extraction chemistry.