Abstract
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration. Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by in-house (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials. Using CAL1, intra-laboratory CVs ranged between 2.8-7.4%, 4.4-18.0% and 5.2-22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p<0.001), respectively. Using CAL1, median bias vs. all laboratory-medians ranged from-6.6 to 6.9%,-17.2 to 7.8% and-12.0 to 16.8% for cortisol, 17OH-progesterone and aldosterone, respectively. Regression lines significantly deviated from the best fit for most laboratories. Using CAL3 improved cortisol and 17OH-progesterone between-method bias and correlation. Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements.
Highlights
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for steroid measurement in high-throughput settings thanks to its analytical specificity and large dynamic range [1,2,3,4,5]
The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration
CAL3 significantly reduced bias median, ranging from −8.8 to 3.4%, in three laboratories and variance in one (Figure 3, Supplemental Table 7 and Supplemental Figure 6). This is the first study examining the variability among LC-MS/MS measurements for circulating cortisol, 17OHprogesterone and aldosterone
Summary
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for steroid measurement in high-throughput settings thanks to its analytical specificity and large dynamic range [1,2,3,4,5]. Due to the abundance of isobars within the steroid family, chromatographic separation is critical for LC-MS/ MS specificity. Using stable isotope-labeled analytes as internal standards (IS) minimizes procedural variability and matrix interference. IS with different isotopes and substitutions may influence measurement accuracy [15,16,17]. LC-MS/MS methods may display different susceptibilities to isobaric and matrix-related interferences. Whether performance of LC-MS/MS methods differs according to serum or plasma sample matrix and associated anticoagulants or coagulation supports has received limited examination [18]
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