An isotope dilution-liquid-chromatography-tandem mass spectrometry-based candidate reference measurement procedure...

A candidate reference measurement procedure (RMP) based on isotope dilution (ID) liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated to accurately measure serum and plasma concentrations of mycophenolic acid (MPA).

Quantitative nuclear magnetic resonance (qNMR) spectroscopy was utilized for determining the absolute content of the reference material, guaranteeing its traceability to SI units. Separation of MPA from potential interferences, whether known or unknown, was accomplished by using a C18 column. For sample preparation, a protocol for protein precipitation followed by a high dilution step was established. Assay validation and measurement uncertainty determination were conducted following the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement guidelines.

The RMP demonstrated high selectivity and specificity without any indication of a matrix effect, enabling the quantification of MPA in the range between 0.240 and 18.0 mg/L. The intermediate precision and repeatability (n=60, measurements) were found to be <3.7 % and <3.0 %, respectively, across all concentration levels and independent from the matrix. The relative mean bias ranged from -1.9 to 2.1 % for serum and from -0.5 to 1.8 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were found to be <6.4 % and <3.0 % (k=2), respectively.

We are pleased to present a new LC-MS/MS-based candidate RMP for MPA in human serum and plasma which offers a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.