An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of 17-hydroxyprogesterone in human serum and plasma.

OBJECTIVES

Accurate measurement of 17α-hydroxyprogesterone (17-OHP) in human serum and plasma is required for steroid profiling, to ensure the appropriate diagnosis of congenital adrenal hyperplasia. Therefore, an isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) to quantify 17-OHP in human serum and plasma has been developed.

METHODS

A two-dimensional heart-cut LC approach was used to minimize matrix effects and prevent co-elution of interferences. A post-column infusion experiment was conducted to assess the potential impact of different matrices on ion suppression or enhancement effects. A multi-day validation experiment assessed precision, trueness and accuracy. Measurement uncertainty was evaluated in compliance with current guidelines.

RESULTS

The working range of this RMP was 0.0200-60.0 ng/mL (0.0605-182 nmol/L) and the method proved to be matrix-independent. The intermediate precision coefficient of variation (CV) ranged from 1.5 % to 2.1 % and repeatability CV from 1.3 % to 1.9 %, across all concentration levels. The relative mean bias ranged from -4.0 % to 2.0 %, regardless of the matrix and concentration level. The measurement process for single measurements showed an expanded uncertainty (k=2) between 3.6 % and 4.5 %. This uncertainty was further reduced performing the target value assignment (n=6), resulting in a range of 2.0-2.5 %. Equivalence between the candidate RMP and the Joint Committee on Traceability in Laboratory Medicine-listed RMPs was established through participation in the External Quality Assessment scheme for reference Laboratories in Laboratory Medicine (RELA) ring trial.

CONCLUSIONS

The performance of this procedure enables the evaluation and standardization of routine assays, and the evaluation of patient samples, to ensure traceability of individual patient results.