Modified HPLC-electrospray ionization/mass spectrometry method for HbA1c based on IFCC reference measurement...

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Mass Spectrometry

Background:

• Accurate hemoglobin A1c (HbA1c) monitoring is crucial for diabetes mellitus management.
• The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference procedure involves enzymatic digestion and peptide analysis.
• Existing methods require enhancement for improved performance and reliability.

Purpose of the Study:

• To develop and validate a modified reference measurement procedure for HbA1c determination.
• To improve upon the existing IFCC reference method using advanced analytical techniques.

Main Methods:

• Modification of the IFCC reference method incorporating High-Performance Liquid Chromatography (HPLC) coupled with Electrospray Ionization/Mass Spectrometry (ESI/MS).
• Chromatographic separation of hemoglobin beta-chain N-terminal hexapeptides using a C12 reversed-phase column and a binary gradient system (H2O/acetonitrile/formic acid).

Main Results:

• The modified HPLC-ESI/MS method demonstrated significantly higher signal intensities and enhanced system stability compared to the original reference procedure.
• Intralaboratory coefficients of variation (CVs) ranged from 0.71% to 1.86% for HbA1c levels between 3% and 14%.
• Deviations from IFCC target values were within -0.87% to 1.00%, meeting the stringent acceptability criteria for HbA1c standardization.

Conclusions:

• The developed HPLC-ESI/MS procedure represents a significant improvement over the existing IFCC reference measurement procedure for HbA1c.
• This enhanced method provides a more robust and accurate platform for clinical HbA1c measurements, aiding in diabetes patient management.