Liu Huixia, Han Yanlin, Wang Huimin, Zhang Yuefeng, Liu Chunlong, Liang Man
Reference Laboratory, Autobio Diagnostics Co., Ltd, Zhengzhou, 450016, Henan, China.
Department of Laboratory Medicine, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu, China.
Anal Bioanal Chem. 2025 May 12. doi: 10.1007/s00216-025-05900-5.
Accurate measurement of human insulin is critical for proper diagnosis, monitoring, and treatment of diabetes. But the insulin results of clinical immunoassay are inconsistent mainly due to antibody cross-reactivity. To standardize and ensure the accuracy of clinical assays, reference measurement procedures (RMPs) with higher metrological order are required. An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for quantification of human insulin in serum as a candidate reference measurement procedure (cRMP) was developed and validated. Insulin was enriched from human serum by insulin antibodies immobilized on magnetic beads. The eluent was analyzed by ID-LC-MS/MS. The cRMP separated human insulin from potentially interfering compounds and enabled measurement over a range of 0.05-40 ng/g, with no matrix effects and carryover. The limit of detection (LOD) and the limit of quantitation (LOQ) in serum were 24.6 pg/g and 48.8 pg/g, respectively. Imprecision (intra-assay and inter-assay) was <2.77% at 0.436, 2.003, and 11.449 ng/g. Recoveries ranged from 99.5% to 101.7% at three spiked levels. Extraction recovery was measured at 85% or higher. Insulin analogues caused no interference for the determination of endogenous insulin. Expanded measurement uncertainty of target value-assigned samples was ≤3.5%. The cRMP was applied to measure human insulin in serum and was compared with two immunoassays using 46 serum samples. Also, a discrepancy of three candidate reference materials for the calibration of cRMP was discussed.
准确测量人胰岛素对于糖尿病的正确诊断、监测和治疗至关重要。但临床免疫测定的胰岛素结果不一致，主要是由于抗体交叉反应性。为了规范并确保临床检测的准确性，需要具有更高计量等级的参考测量程序（RMP）。开发并验证了一种用于定量血清中人胰岛素的同位素稀释液相色谱-串联质谱法（ID-LC-MS/MS）作为候选参考测量程序（cRMP）。通过固定在磁珠上的胰岛素抗体从人血清中富集胰岛素。用ID-LC-MS/MS分析洗脱液。该cRMP将人胰岛素与潜在干扰化合物分离，并能够在0.05-40 ng/g范围内进行测量，无基质效应和残留。血清中的检测限（LOD）和定量限（LOQ）分别为24.6 pg/g和48.8 pg/g。在0.436、2.003和11.449 ng/g时，不精密度（批内和批间）<2.77%。在三个加标水平下，回收率在99.5%至101.7%之间。萃取回收率测量值为85%或更高。胰岛素类似物对内源性胰岛素的测定无干扰。目标值赋值样品的扩展测量不确定度≤3.5%。将该cRMP应用于测量血清中的人胰岛素，并与使用46份血清样品的两种免疫测定法进行比较。此外，还讨论了用于校准cRMP的三种候选参考物质的差异。
