Kandler Kerstin, Singh Neeraj, Bauland Friederike, Fux Elie, Geistanger Andrea, Geletneky Christian, Taibon Judith
Roche Diagnostics GmbH, Penzberg, Germany.
Chrestos Concept GmbH & Co. KG, Essen, Germany.
Clin Chem Lab Med. 2025 Jun 20. doi: 10.1515/cclm-2024-1138.
An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in human serum and plasma is presented.
Quantitative Nuclear Magnetic Resonance (qNMR) spectroscopic methodology has been utilized to assign absolute content (g/g) and International System of Units (SI)-traceability to the reference materials used as primary calibrators. This RMP was developed for the simultaneous quantification of 25OHD2 and 25OHD3 in human samples, utilizing supported liquid extraction (SLE) clean-up and a two-dimensional heart-cut ID-LC-MS/MS method to minimize matrix effects and prevent the co-elution of 3-Epi-25OHD3 and 3-Epi-25OHD2. The method underwent validation in accordance with current guidelines. Selectivity was assessed using spiked samples. To evaluate potential matrix effects, a post-column infusion experiment and a comparison of standard line slopes were performed. A 5-day validation study was conducted to determine precision, accuracy and trueness of the method. Measurement uncertainty for reference value assignment was evaluated in line with the Guide to the Expression of Uncertainty in Measurement (GUM). Equivalence to Joint Committee on Traceability in Laboratory Medicine (JCTLM) listed RMPs was demonstrated through the participation in the CDC Vitamin D Standardization-Certification Program (VDSCP) as well as the RELA scheme.
The RMP enabled the quantification of 25OHD2 and 25OHD3 within the range of 1.50 ng/mL-180 ng/mL (3.64-436 nmol/L for 25OHD2 and 3.74-449 nmol/L for 25OHD3), without interference from their respective epimer and no evidence of matrix effects. Intermediate precision was determined to be ≤4.0 % for 25OHD2 and ≤3.6 % for 25OHD3, while repeatability was ≤3.3 % for 25OHD2 and ≤2.9 % for 25OHD3 across all concentration levels. The relative mean bias for the secondary reference materials varied from -1.0 to 1.1 %, regardless of the analyte. For the spiked samples, the relative mean bias ranged from -4.2 to 1.0 % for 25OHD2 and from -3.9 to 0.9 % for 25OHD3, irrespective of all levels and matrices. Expanded measurement uncertainties (k=2) for target value assignment (n=6) were ≤3.9 % for 25OHD2 and ≤3.2 % for 25OHD3. Participation in the VDSCP and the RELA scheme showed a good agreement with results from the JCTLM listed RMPs and laboratories.
The RMP enables the accurate, precise and consistent determination of 25OHD3 and 25OHD2. The robust performance of this method supports standardization of routine assays and guarantees traceability in the measurement of individual patient samples.
提出一种基于同位素稀释-液相色谱-串联质谱法（ID-LC-MS/MS）的候选参考测量程序（RMP），用于定量测定人血清和血浆中的25-羟基维生素D2（25OHD2）和25-羟基维生素D3（25OHD3）。
已采用定量核磁共振（qNMR）光谱方法来确定用作一级校准物的参考物质的绝对含量（g/g）和国际单位制（SI）可溯源性。该RMP用于同时定量测定人样品中的25OHD2和25OHD3，采用支持液液萃取（SLE）净化和二维中心切割ID-LC-MS/MS方法，以尽量减少基质效应并防止3-表-25OHD3和3-表-25OHD2的共洗脱。该方法按照现行指南进行了验证。使用加标样品评估选择性。为评估潜在的基质效应，进行了柱后注入实验和标准曲线斜率比较。进行了为期5天的验证研究，以确定该方法的精密度、准确度和真实性。根据《测量不确定度表示指南》（GUM）评估参考值赋值的测量不确定度。通过参与美国疾病控制与预防中心维生素D标准化认证计划（VDSCP）以及RELA计划，证明了与实验室医学溯源联合委员会（JCTLM）列出的RMP等效。
该RMP能够在1.50 ng/mL至180 ng/mL范围内定量测定25OHD2和25OHD3（25OHD2为3.64 - 436 nmol/L，25OHD3为3.74 - 449 nmol/L），不受各自差向异构体的干扰，且无基质效应证据。25OHD2的中间精密度测定为≤4.0%，25OHD3为≤3.6%，而在所有浓度水平下，25OHD2的重复性为≤3.3%，25OHD3为≤2.9%。二级参考物质的相对平均偏差在-1.0%至1.1%之间，与分析物无关。对于加标样品，25OHD2的相对平均偏差在-4.2%至1.0%之间，25OHD3在-3.9%至0.9%之间，与所有水平和基质无关。目标值赋值（n = 6）的扩展测量不确定度（k = 2），25OHD2≤3.9%，25OHD3≤3.2%。参与VDSCP和RELA计划表明与JCTLM列出的RMP和实验室的结果具有良好的一致性。
该RMP能够准确、精确且一致地测定25OHD3和25OHD2。该方法的稳健性能支持常规检测的标准化，并保证个体患者样品测量的可溯源性。
