New Validated Method for Quantification of Glycated Hemoglobin by LC-QToF-MS: Is HRMS Able to Quantify Clinical Samples?

High-resolution mass spectrometry is a powerful tool in clinical analysis but remains less explored due to its lower dynamic range and sensitivity compared to triple quadrupoles. Glycated hemoglobin (HbA1c) is the current gold standard biomarker to monitor the control of diabetes, representing long-term plasma glycemic levels. Due to its clinical importance, several methods have been developed for HbA1c quantification, using different principles; however, the results obtained with these techniques may differ according to the method adopted. Hence, there is a great need to standardize the current methods to quantify glycated hemoglobin. A new UPLC-QToF-MS method was fully validated and tested to quantify HbA1c in human samples. The peptides VHLTPE m/z 695.373 and gly-VHLTPE m/z 857.426, obtained via Glu-C digestion, were the selected peptides for quantification of HbA1c (mmol/mol). Chromatographic separation was obtained in a C18 column, maintained at 40 °C. The mobile phase was composed of water and acetonitrile, both containing 0.02% TFA and 0.1% acetic acid, and eluted in gradient mode. The method was fully validated, being considered linear in the range of 25-107 mmol/mol of HbA1c, and was sensitive, selective, precise, accurate, and free of matrix and carryover effects. The method was successfully applied to real samples, reaching about 90% agreement with reference method results, providing accurate and precise information on peptide mass, without laborious sample preparation. These results support the use of HRMS to improve the quality of quantitative results of HbA1c in health services and demonstrate a possible application of peptide investigation for clinical analysis in the near future.