B-129 Uncertainty Calculations for Reference Measurement Procedures
Authors: Albrecht E , Geistanger A , Hilger A , Bauland F , Taibon J 
Published: September 2023

Abstract

Background
Quantitative results based on reference measurement procedures (RMP) require the calculation of the uncertainty of the result. In order to take the uncertainty of the full traceability chain into account a modularisation of the different execution steps is needed, starting from the primary reference material until the final concentration value. Different strategies have to be applied and combined to obtain the final uncertainty.We combine different ways for calculating the uncertainty of an isotope dilution -liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure for Cortisol, and explain the differences between Type A and Type B uncertainties and best strategies to choose from them. In addition, combinations of results from subsequent steps to a final overall uncertainty are presented.

Methods
An ID-LC-MS/MS based reference measurement procedure requires as a starting point a primary reference material with known purity of the analyte under consideration. Nowadays this purity assessment is often done with quantitative nuclear magnetic resonance (qNMR) spectroscopy. The qNMR uncertainty calculation is a Type A approach, based on replicate sample preparations and measurements. With this primary material a first set of primary calibrators is produced, often through multiple steps of weighings, preparation of stock solutions and dilutions. For these steps balances, volumetric flasks, or pipettes are used, all of them coming with an error budget which has to enter the uncertainty budget of the primary calibrator preparation. We will show how this can be done through Type B uncertainty calculation. Based on the primary calibrators the LC/MS instrument is calibrated and measurements of individual samples are carried out. We will introduce a hierarchical variability experiment, which takes into account the most important error sources and results in a Type A uncertainty for the LC/MS measurements.Finally, the errors of the LC/MS measurements have to be combined with those of the primary calibrator preparation to obtain the overall uncertainty of an LC/MS reference measurement procedure.

Results
We show the approaches and calculation strategies based on the Cortisol candidate reference measurement procedure, developed within the Roche LC/MS Reference Measurement Procedure project. The purity assessment by qNMR carries an uncertainty of 0.18%. This is incorporated into the uncertainty calculations of the primary calibrators, where in addition uncertainties from weighing and dilutions are incorporated resulting in uncertainties of the primary calibrator values between 0.6% and 1.1%. Combining these uncertainties with the variabilities of the LC/MS measurement procedure results in final uncertainties between 1.6% and 2.8%.

Conclusion
We have developed a sound framework for uncertainty calculation of reference measurement procedures, taking into account the different procedure steps, and included this into a process for the management of the different input variables up to comprehensive reports of the calculations.