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

Progesterone regulates reproductive processes and is used clinically to monitor ovarian function in people experiencing fertility problems. Measuring serum progesterone is challenging as it is highly protein-bound and exists at very low physiological levels. An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate RMP to quantify progesterone in human serum/plasma has been developed.

To ensure traceability to the SI Units, this RMP utilized primary reference material from the NMIJ. For the determination of progesterone, two-dimensional heart-cut chromatography, in combination with a straightforward protein precipitation protocol, was employed to minimize matrix effects and the coelution of isobaric interferences. Accuracy and precision of the candidate RMP was assessed in a multi-day validation experiment using certified secondary reference materials, spiked serum and plasma samples; measurement uncertainty was evaluated according to the GUM. Equivalence to JCTLM-listed RMPs was determined using leftover samples from the RELA scheme.

The candidate RMP was highly selective for progesterone within a measurement range of 0.0400-72.5 ng/mL (0.127-231 nmol/L) and matrix independent. Intermediate precision was ≤3.3 %, and repeatability ranged from 1.4 to 2.7 % across all concentration levels. The mean bias ranged from 0.1 to 0.7 % for secondary certified reference materials, from -1.6 % to -0.2 % for serum samples, and from -2.3 to 4.0 % for plasma samples. Expanded measurement uncertainty (k=2) for target value assignment (n=6) was found to be ≤3.7 %. Equivalence to JCTLM-listed RMPs demonstrated a relative bias of -2.4 to 2.2 %, all within the measurement uncertainty of the respective RMP.

A candidate RMP based on ID-LC-MS/MS for progesterone quantification is presented, providing metrological traceability, target value assignment, routine test standardization, and the analysis of clinical samples comprising human serum and plasma to ensure the accuracy and traceability of individual patient results.