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Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses
Published on: August 30, 2018
An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference
Anett Engel1, Lena Ruhe1, Neeraj Singh2
1Department of Studies, Collaborations and Innovation Management, Labor Berlin - Charité Vivantes Services GmbH, Berlin, Germany.
Objectives:
To develop an isotope dilution-liquid chromatography-tandem mass spectrometry-(ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for quantification of methotrexate in human serum and plasma.
Methods:
Quantitative nuclear magnetic resonance (qNMR) was used to determine absolute methotrexate content in the standard. Separation was achieved on a biphenyl reversed-phase analytical column with mobile phases based on water and acetonitrile, both containing 0.1% formic acid. Sample preparation included protein precipitation in combination with high sample dilution, and method validation according to current guidelines. The following were assessed: selectivity (using analyte-spiked samples, and relevant structural-related compounds and interferences); specificity and matrix effects (via post-column infusion and comparison of human matrix vs. neat samples); precision and accuracy (in a five-day validation analysis). RMP results were compared between two independent laboratories. Measurement uncertainty was evaluated according to current guidelines.
Results:
The RMP separated methotrexate from potentially interfering compounds and enabled measurement over a calibration range of 7.200-5,700 ng/mL (0.01584-12.54 μmol/L), with no evidence of matrix effects. All pre-defined acceptance criteria were met; intermediate precision was ≤4.3% and repeatability 1.5-2.1% for all analyte concentrations. Bias was -3.0 to 2.1% for samples within the measuring range and 0.8-4.5% for diluted samples, independent of the sample matrix. RMP results equivalence was demonstrated between two independent laboratories (Pearson correlation coefficient 0.997). Expanded measurement uncertainty of target value-assigned samples was ≤3.4%.
Conclusions:
This ID-LC-MS/MS-based approach provides a candidate RMP for methotrexate quantification. Traceability of methotrexate standard and the LC-MS/MS platform were assured by qNMR assessment and extensive method validation.
