An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of aldosterone in human serum and plasma.

OBJECTIVES: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) for aldosterone quantification in human serum and plasma is presented. METHODS: The material used in this RMP was characterized by quantitative nuclear magnetic resonance (qNMR) to assure traceability to SI Units. For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis a two-dimensional heart cut LC approach, in combination with an optimal supported liquid extraction protocol, was established for the accurate analysis of aldosterone in human serum and plasma in order to minimize matrix effects and avoid the co-elution of interferences. Assay validation was performed according to current guidelines. Selectivity and specificity were assessed using spiked serum; potential matrix effects were examined by a post column infusion experiment and the comparison of standard line slopes. An extensive protocol over 5 days was applied to determine precision, accuracy and trueness. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM), for which three individual sample preparations were performed on at least two different days. RESULTS: The RMP allowed aldosterone quantification within the range of 20-1,200 pg/mL without interference from structurally-related compounds and no evidence of matrix effects. Intermediate precision was ≤4.7% and repeatability was 2.8-3.7% for all analyte concentrations. The bias ranged between -2.2 and 0.5% for all levels and matrices. Total measurement uncertainties for target value assignment (n=6) were found to be ≤2.3%; expanded uncertainties were ≤4.6% (k=2) for all levels. CONCLUSIONS: The RMP showed high analytical performance for aldosterone quantification in human serum and plasma. The traceability to SI units was established by qNMR content determination of aldosterone, which was utilized for direct calibration of the RMP. Thus, this candidate RMP is suitable for routine assay standardization and evaluation of clinical samples.

An isotope dilution LC-MS/MS-based candidate reference method for the quantification of androstenedione in human serum and plasma.

The accurate measurement of androstenedione in human serum and plasma is required for steroid profiling to assure the appropriate diagnosis and differential diagnosis of hyperandrogenism. In this work, we introduce an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) candidate reference measurement procedure for the quantification of androstenedione in human serum and plasma. The performance of the procedure enables its use in the evaluation and standardization of routine assays and for the evaluation of patient samples to ensure the traceability of individual patient results. As the primary standard, a certified reference material from NMIA (National Measurement Institute, Australia) was used. Additionally, a quantitative nuclear magnetic resonance (qNMR) method was developed for the value assignment of the primary reference material, which ensures the direct traceability to SI units, as well as the independence from the availability of reference materials. 13C3-labeled androstenedione was used as the internal standard. The introduced method allows the measurement of androstenedione in the range of 0.05-12 ng/mL, and the assay imprecision was found to be <2% between 5 and 12 ng/mL, 3.5% at 1.5 ng/mL, and 5.2% at 0.05 ng/mL, with an accuracy of 95-105% for the serum and 91-103% for the plasma matrix. The transferability to a second laboratory was validated by method comparison based on 112 patient samples. The comparison of the results obtained from the presented method and an LC-MS/MS routine assay, using 150 native patient samples, showed a good correlation with a bias of the routine method of ≤4.0%.