UPLC-MS/MS Analysis of Methotrexate in Plasma and Serum for Clinical Research

Importance of Methotrexate Monitoring in Clinical Research

Methotrexate is a cornerstone therapeutic in oncology and immunology, yet its narrow therapeutic index and variable metabolism demand precise measurement in plasma and serum. Traditional immunoassays suffer from cross-reactivity, especially after rescue therapy with glucarpidase, leading to inaccurate results. A rapid, selective, and sensitive UPLC-MS/MS approach addresses these challenges, improving pharmacokinetic understanding and patient safety.

Objectives and Study Overview

This study aimed to develop and validate a high-throughput UPLC-MS/MS method for quantifying methotrexate in human plasma and serum. Key objectives included:

• Achieving chromatographic separation from main metabolites (7-hydroxymethotrexate and DAMPA).
• Establishing a wide dynamic range suitable for low-dose and high-dose regimens.
• Ensuring minimal sample volume and straightforward preparation.
• Demonstrating method robustness post-glucarpidase administration.

Methodology and Sample Preparation

Sample extraction involved protein precipitation of 50 µL of plasma or serum with deuterated methotrexate internal standard in methanol. After vortexing and centrifugation, the supernatant was diluted with water for direct injection. Calibration covered 0.025 to 10 µmol/L, with quality controls spanning 0.1 to 75 µmol/L. Dilution integrity was tested at high concentrations.

Instrumentation

Analysis was carried out on a UPLC system equipped with a high-strength silica C18 column (2.1×30 mm, 1.8 µm). Isocratic elution (77:23 aqueous ammonium acetate/formic acid and methanol) at 0.4 mL/min achieved separation within five minutes. Detection employed a tandem quadrupole mass spectrometer with positive electrospray ionization and multiple reaction monitoring for both analyte and internal standard.

Main Results and Discussion

Chromatography successfully resolved methotrexate from its metabolites, with no carry-over detected up to 100 µmol/L. The lower limit of quantification was 0.0025 µmol/L with ≤20% RSD. Precision studies over five days showed total RSD ≤5.5% across QC levels. Linearity was confirmed from 0.0175 to 13.0 µmol/L. Matrix effect evaluation indicated ion enhancement in plasma and serum, effectively compensated by the internal standard. Plasma and serum values were equivalent within ±7% difference. Common endogenous and exogenous interferents did not impact accuracy beyond acceptable limits. External quality assurance samples demonstrated overall mean deviation of –5.7% from reference values.

In patients undergoing glucarpidase rescue, the UPLC-MS/MS method revealed a rebound in methotrexate concentration not detected by immunoassay, underlining the method’s superior selectivity and its importance in clinical decision-making.

Benefits and Practical Applications

• High selectivity minimizes false elevations due to metabolites.
• Wide dynamic range supports standard and high-dose protocols.
• Fast run time and minimal sample requirement enable high throughput.
• Robust against common matrix interferences and dilution steps.

Future Trends and Potential Applications

Advancements may include integration with automated sample preparation platforms and incorporation into multiplexed panels for simultaneous drug monitoring. Application of high-resolution mass spectrometry and microflow UPLC could further reduce sample volume and increase sensitivity. Broader use in pharmacokinetic modeling and personalized dosing regimens is anticipated.

Conclusion

The validated UPLC-MS/MS method provides reliable, precise, and selective measurement of methotrexate in plasma and serum. Its rapid analysis time and minimal sample needs make it ideal for clinical research settings, particularly when monitoring patients receiving high-dose therapy and rescue agents.

References

• Ahmed M T, Hasan H J. J Cancer Sci Ther. 2013;5(3):106–12.