MSACL 2023 Abstract

INTRODUCTION: Mass spectrometry (MS) in hyphenation with high-resolution separation techniques, most notably liquid chromatography (LC) and gas chromatography (GC), is the gold standard for reference measurement procedures in clinical diagnostics. The utilization of highly characterized, SI-traceable reference materials and stable isotope-labeled internal standards is of paramount importance for an assay to meet the quality requirements of highest order reference measurement procedures.

OBJECTIVES: By the example of cortisol, we will show how clinically relevant measurement services in endocrinology are made safe and reliable by the establishment of mass spectrometry based reference measurement procedures (RMP) and services.

METHODS: An isotope dilution liquid chromatography mass spectrometry (ID-LC-MS/MS) based candidate RMP was developed for the quantification of cortisol in serum and plasma matrices. To ensure traceability to SI units, either certified primary reference materials or in house quantitative nuclear magnetic resonance spectrometry (qNMR)-characterized material was utilized. A supported liquid extraction (SLE) sample preparation protocol was optimized to minimize matrix effects. To avoid co-elution of interferences, a two-dimensional LC (2D-LC) approach using two orthogonal stationary phases was adopted. An extensive measurement protocol over 5 days was applied to determine precision and accuracy. The measurement uncertainty was evaluated according to the "Guide to the Expression of Uncertainty of Measurement" (GUM).

RESULTS: The RMP allowed the quantification of cortisol in serum and plasma without interference from structurally-related compounds and evidence of matrix effects. Linear range was 0.800 ng/mL – 600 ng/mL. Intermediate precision was ≤ 2.6% and repeatability ranged between 0.6% - 1.9% for all analyte concentrations over the entire measuring range. The relative mean bias was between -1.5% - 0.5% for all levels and matrices. Total measurement uncertainties were found to be ≤ 2.8%; expanded uncertainties were ≤ 5.7% (k=2) for all levels.

CONCLUSION: This highly selective 2D-LC-MS/MS candidate reference measurement procedure, provides a traceable and reliable platform for the standardization of routine tests and for the evaluation of cortisol in clinical samples.