Measuring methotrexate polyglutamates in red blood cells: a new LC-MS/MS-based method

Area of Science:

• Pharmacokinetics and Drug Metabolism
• Analytical Chemistry
• Rheumatology

Background:

• Methotrexate (MTX) is a primary drug for rheumatoid arthritis, with efficacy linked to intracellular MTX polyglutamates (MTXPGn).
• Accurate quantification of MTXPGn is essential for optimizing MTX therapy.

Purpose of the Study:

• To develop and validate a sensitive and precise liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) assay for quantifying MTXPGn in red blood cells.
• To assess the assay's performance characteristics and applicability in clinical settings.

Main Methods:

• Development of a novel LC-ESI-MS/MS assay using stable-isotope-labeled internal standards for MTXPGn quantification.
• Analysis involved a 6-minute UPLC run with a C18 column and gradient elution, followed by detection via tandem mass spectrometry.
• Assay validation was conducted according to FDA guidelines, assessing linearity, precision, stability, recovery, and matrix effects.

Main Results:

• The assay demonstrated excellent linearity (R(2) > 0.99) from 1-1,000 nM for all MTXPGn.
• Intraday and interday precision were favorable (CVs 1-4% and 6-15%, respectively), with high recovery (98-100%) and minimal matrix effects (95-99%).
• The lower limit of quantification was 1 nM, and analysis of patient samples showed comparable MTXPGn levels to existing literature.

Conclusions:

• The developed LC-ESI-MS/MS method provides a sensitive, precise, and robust quantification of MTXPGn in red blood cells within the clinically relevant range.
• This assay is suitable for routine clinical laboratory use due to its simple sample preparation and efficient analysis.
• Accurate MTXPGn measurement can aid in personalized methotrexate dosing for rheumatoid arthritis patients.