Method for determining glycated hemoglobin A (HbA1C) by mass spectrometry

A method for determining glycated hemoglobin A (HbA1c) in a sample, comprising:

• (a) providing a sample comprising hemoglobin A (HbA) molecules,
• (b) subjecting the sample from step (a) to a partial proteolytic digestion step wherein N-terminal fragments of hemoglobin A β-chain molecules are generated, wherein the partial proteolytic digestion is carried out with the endopeptidase Glu-C in a concentration of about 0.6 mg/mL for a time period of less than about 60 min at a temperature of about 37° C., in a buffer comprising ammonium ions, and stopping the partial proteolytic digestion by adding formic acid in water,
• (c) enriching the N-terminal fragments of hemoglobin A β-chain molecules generated in step (b), and
• (d) subjecting the sample comprising N-terminal fragments of hemoglobin A β-chain molecules to an analysis by mass-spectrometry (MS) wherein the amount or concentration of HbA1c in the sample is determined,
• wherein in the partial proteolytic digestion step (b) about 1% to about 20% of the HbA β-chain molecules present in the sample are digested based on the total amount of HbA β-chain molecules originally present in the sample.