Analysis of methotrexate and its eight metabolites in cerebrospinal fluid by solid-phase extraction and triple-stacking capillary electrophoresis

Abstract

We establish a triple-stacking capillary electrophoresis (CE) separation method to monitor methotrexate (MTX) and its eight metabolites in cerebrospinal fluid (CSF). Three stacking methods with different mechanisms were combined and incorporated into CE separation. Complete stacking and sharp peaks were achieved. Firstly, the optimized buffer (60 mM phosphate containing 15% THF and 100 mM SDS) was filled into the capillary, which was followed by the higher conductivity buffer (100 mM phosphate, 2 psi for 45 s). The analytes extracted from CSF were injected at 2 psi for 99.9 s, which provided long sample zones and pH junction for focusing. Finally, the stacking step was performed by sweeping, and separation was achieved by micellar electrokinetic chromatography. The results of the linear regression equations indicated high linearity (r ≥ 0.9981) over the range of 0.5-7 μM. In intra- and inter-batch results, all data of RSD and RE were below 11%, indicating good precision and accuracy of this method. The LODs (S/N = 3) were 0.1 μM for MTX, 7-hydroxymethotrexate (7-OHMTX) and MTX-polyglutamates (MTX-(Glu)(n, n = 2-5)), 0.2 μM for MTX-(Glu)(6), and 0.3 μM for 2,4-diamino-N(10)-methylpteroic acid (DAMPA) and MTX-(Glu)(7). Our method was implemented for analysis of MTX and its metabolites in the CSF, and could be used for evaluation of its curative effects of acute lymphoblastic leukemia patients. The data were also confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results showed good coincidence.

Analyses of anticancer drugs by capillary electrophoresis: a review

“…Cheng et al . () reported triple‐stacking capillary electrophoresis for analysis of methotrexate and its eight different metabolites in cerebrospinal fluid. The buffer system chosen was 60 m m phosphate, pH 3.0 containing 15% Tetrahydrofuran (THF) and 100 m m SDS.…”

“…At this stage, the samples were ready to load onto the CE machine. Cheng et al (2010) reported SPE and triple-stacking CE for analysis of methotrexate and its eight different metabolites in cerebrospinal fluid. The blood samples were denatured by adding perchloric acid followed by vortex and centrifugation.…”

“…In addition to the above discussed biological samples, anticancer drugs have been analyzed in other biological sources such as CSF in humans and different parts of plants. Cheng et al (2010) reported triple-stacking capillary electrophoresis for analysis of methotrexate and its eight different metabolites in cerebrospinal fluid. The buffer system chosen was 60 mM phosphate, pH 3.0 containing 15% Tetrahydrofuran (THF) and 100 mM SDS.…”

Recent advances in enrichment techniques for trace analysis in capillary electrophoresis

“…Cheng et al combined three stacking techniques, including LVSS, dynamic pH junction, and sweeping for analysis of methotrexate (MTX) and its eight metabolites in whole blood by MEKC, and about 40‐fold sensitivity increase was attained compared with that the single CZE mode. Cheng et al also adopted such triple‐stacking CE mode to monitor MTX and its eight metabolites in cerebrospinal fluid (CSF). The LODs (hydrodynamic injection 2 psi, 99.9 s) were observed to be 0.1 μM for MTX, 7‐OHMTX, MTX‐(Glu) n , n = 2–5 and 0.2 μM MTX‐(Glu) 6 , and 0.3 μM for DAMPA and MTX‐(Glu) 7 .…”

Identification of impurities in methotrexate drug substances using high‐performance liquid chromatography coupled with a photodiode array detector and Fourier transform ion cyclotron resonance mass spectrometry

“…We used the HPLC-PDA/FTICR-MS method to screen for impurities in MTX drug substances produced by different manufacturers. The proposed formulae of the parent ions, the observed and calculated masses, and the measurement error for the 15 detected impurities (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) and the MS n product ions are summarized in Table 2. The MS n spectra and their assignments are shown in the online Supporting information.…”