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Background: Standardization of hemoglobin (Hb)A1c measurements is a process of considerable interest for quality assurance in diabetes management. To contribute to continuous quality improvement and fulfillment of the requirements for reference measurement procedures according to the standards of the International Organization for Standardization, we developed a calibration system of highest metrological order using isotope dilution–mass spectrometry with a reference material. Method: Samples were prepared by enzymatic cleavage based on the IFCC reference measurement procedure for LC-MS analysis. After digestion the samples were spiked with [D7]-labeled glycated and nonglycated hexapeptides as internal standards for quantification. LC-MS analysis was performed by using a C12 reversed-phase column and a gradient of acetonitrile/H2O containing 0.1% formic acid. Results: Calibration systems for HbA1c determination based on liquid chromatography–isotope dilution–mass spectrometry (LC-ID-MS) and on the IFCC reference measurement procedure were compared. A linear regression analysis demonstrated a correlation of r2 = 1.00 between the 2 different calibration systems. Mean deviation was 5.5% for the calibration and 3.3% for hemolysate samples, with a mean expanded uncertainty of 4.9%. Conclusions: This LC-ID-MS procedure allows the current IFCC reference measurement procedure for HbA1c to be raised to a higher order of accuracy.

Liquid Chromatography–Isotope Dilution–Mass Spectrometry as a New Basis for the Reference Measurement Procedure for Hemoglobin A1c Determination