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

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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.

Mechanism of LVSS-DypH-sweeping method

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Acknowledgments

We gratefully acknowledge the support of the National Science Council of Taiwan and Kaohsiung Medical University in funding this work. We also express our gratitude to the Center of Excellence for Environmental Medicine (KMU-EM-98-1-2) for partial financial support provided for this work.

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Authors and Affiliations

  1. Center of Excellence for Environmental Medicine, Kaohsiung Medical University, 100, Shi-chuan 1st Rd., Kaohsiung, 807, Taiwan

    Shou-Mei Wu

  2. School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, 100, Shi-chuan 1st Rd., Kaohsiung, 807, Taiwan

    Hui-Ling Cheng & Shou-Mei Wu

  3. Department of Pediatric, Kaohsiung Medical University Chung-Ho Memorial Hospital, 100, Tzyou 1st Rd., Kaohsiung, 807, Taiwan

    Shyh-Shin Chiou & Yu-Mei Liao

  4. College of Medicine, Graduate Institute of Biochemistry, Kaohsiung Medical University, 100, Shi-chuan 1st Rd., Kaohsiung, 807, Taiwan

    Chi-Yu Lu

  5. Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, 100, Shi-chuan 1st Rd., Kaohsiung, 807, Taiwan

    Yen-Ling Chen

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  1. Hui-Ling Cheng
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  2. Shyh-Shin Chiou
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  3. Yu-Mei Liao
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  4. Chi-Yu Lu
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Correspondence to Shou-Mei Wu.

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Cheng, HL., Chiou, SS., Liao, YM. et al. Analysis of methotrexate and its eight metabolites in cerebrospinal fluid by solid-phase extraction and triple-stacking capillary electrophoresis. Anal Bioanal Chem 398, 2183–2190 (2010). https://doi.org/10.1007/s00216-010-4152-3

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  • DOI: https://doi.org/10.1007/s00216-010-4152-3

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