Ott Myriam, Singh Neeraj, Kubicova Marie, Bauland Friederike, Köppl Daniel, Gaudl Alexander, Geistanger Andrea, Ceglarek Uta, Rauh Manfred, Geletneky Christian, Taibon Judith
Roche Diagnostics GmbH, Penzberg, Germany.
Chrestos Concept GmbH & Co. KG, Essen, Germany.
Clin Chem Lab Med. 2025 Apr 17. doi: 10.1515/cclm-2024-1478.
Cortisone is an inert precursor/metabolite of the potent steroid hormone cortisol. Measurement of serum cortisone levels and the cortisol-cortisone ratio can be useful for the diagnosis of dysfunction in the regulation of cortisol levels (i.e., severe and subtle apparent mineralocorticoid excess, low-renin primary aldosteronism). Therefore, an isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) to quantify cortisone in human serum/plasma was developed and validated.
Quantitative nuclear magnetic resonance (qNMR) was utilized to assign absolute content (g/g) and SI-traceability to reference materials used as primary calibrators. A supported liquid extraction sample preparation protocol as well as a two-dimensional heart-cut LC approach for LC-MS/MS analysis were employed to mitigate matrix effects and prevent co-elution of interferences. Selectivity was determined by analyzing a matrix sample containing the analyte, the internal standard and six potential interferents. A post-column infusion experiment and a comparison of standard line slopes were performed to evaluate matrix effects. An extensive protocol over five days was applied to determine precision, accuracy and trueness. Measurement uncertainty (MU) was evaluated in compliance with current guidelines.
This RMP is suitable for analyzing cortisone within the 0.0800-120 ng/mL (0.222-333 nmol/L) range, demonstrating selectivity, sensitivity and matrix independence. Intermediate precision was ≤3.4 %, repeatability was ≤2.9 % across all concentration levels and relative mean bias ranged from -3.7 to 2.8 % across all tested matrices and concentrations. Expanded MU (k=2) for target value assignment (n=6) ranged from 2.1 to 5.5 %, irrespective of concentration or sample type.
This RMP allows for accurate and reproducible determination of cortisone in human serum and plasma. Implementation of this method supports routine assay standardization and patient sample measurement with confirmed traceability.
可的松是强效甾体激素皮质醇的惰性前体/代谢产物。测定血清可的松水平以及皮质醇-可的松比值，对于诊断皮质醇水平调节功能障碍（即严重和轻微的表观盐皮质激素过多、低肾素原发性醛固酮增多症）可能有用。因此，开发并验证了一种基于同位素稀释-液相色谱-串联质谱（ID-LC MS/MS）的候选参考测量程序（RMP），用于定量人血清/血浆中的可的松。
利用定量核磁共振（qNMR）确定用作一级校准品的参考物质的绝对含量（g/g）和国际单位制可溯源性。采用支持液液萃取样品制备方案以及二维中心切割液相色谱法进行LC-MS/MS分析，以减轻基质效应并防止干扰物共洗脱。通过分析含有分析物、内标和六种潜在干扰物的基质样品来确定选择性。进行柱后注入实验和标准曲线斜率比较，以评估基质效应。应用为期五天的广泛方案来确定精密度、准确度和真实性。按照现行指南评估测量不确定度（MU）。
该RMP适用于分析0.0800 - 120 ng/mL（0.222 - 333 nmol/L）范围内的可的松，具有选择性、灵敏度和基质独立性。中间精密度≤3.4%，在所有浓度水平下重复性≤2.9%，在所有测试基质和浓度下相对平均偏差范围为-3.7%至2.8%。目标值赋值（n = 6）的扩展MU（k = 2）范围为2.1%至5.5%，与浓度或样品类型无关。
该RMP能够准确且可重复地测定人血清和血浆中的可的松。该方法的实施有助于常规检测标准化以及对具有确认可溯源性的患者样品进行测量。
