Early developments toward HbA_1c determination in whole blood by high-speed sample preparation and LC-MS/MS analysis

We report a method to determine HbA_1c (glycated hemoglobin) where whole blood samples are prepared by fast hemolysis (dilution with deionized water and vortex mixing), digestion with 0.6 mg/mL endoproteinase Glu C (Glu C) in 30 mM ammonium acetate buffer (pH 4.3) at 37 °C for 45 min, and termination of the digestion by diluting with 0.1% formic acid in water, and then analysis by a gradient liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with a run time of 36 s. The method is linear between 0 and 200 HbA_1c/mol Hb (IFCC) with a correlation coefficient of 0.999, providing an inter-day reproducibility between 1.3 and 2.3% CV, and comparable with results from analysis of the same samples on the Roche Cobas® c 513 clinical analyzer with a correlation coefficient of 0.998. In two alternative detection workflows that were not characterized in detail, the same digested samples were purified by a magnetic bead-based solid-phase extraction (SPE) method requiring about 10 min and then analyzed using either an isocratic LC-MS/MS method or a flow injection analysis (FIA)-MS/MS method with run times of 12 s and 18 s, respectively. Our work demonstrates the feasibility of LC-MS-based methods for HbA_1c determination that minimize the time required for sample preparation and measurement while preserving analytical performance and are thereby more suitable for routine clinical settings compared to traditional methods which require up to 25 h and 23 min, respectively, to prepare and measure samples.