A candidate reference measurement procedure for quantification of human insulin in serum based on immunoaffinity...

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.