Abstract:

To promote the standardization and harmonization of glycated hemoglobin A1c （HbA1c） testing results in medical laboratories across Beijing， we established a reference method for HbA1c. This method participated in the International Comparison Program for HbA1c Reference Laboratories organized by the European Reference Laboratory Network for HbA1c to validate its accuracy. The experimental samples consisted of HbA1c network reference laboratory proficiency testing samples imported annually from the Netherlands， including six calibrators （A-F）， ten intercomparison samples， several quality control materials， and additional auxiliary samples with International Federation of Clinical Chemistry and Laboratory Medicine （IFCC）-assigned values. The pre-treatment process involved digesting samples with protein endopeptidase Glu-C. The experimental samples were removed from the -80 ℃ freezer and allowed to reach room temperature. Subsequently， 50 μL of Glu-C （mass concentration： 200 μg/mL） was added to each sample vial at a ratio of total hemoglobin to enzyme of 1 mg∶0.01 mg. The volume was then adjusted to a final volume of 500 μL with ammonium acetate solution （50 mmol/L， pH 4.3）. After thorough mixing， the samples were incubated at 37 ℃ for 18-20 h. Using mobile phases of methanol and 0.1% formic acid aqueous solution under gradient elution conditions， the detection of HbA1c in international samples was performed via high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS）. A gradient elution mode was employed for liquid chromatography separation using a Shimadzu C18 column （50 mm×3 mm， 2.2 μm）. The injector temperature was maintained at 4-8 ℃， with a column temperature of 30 ℃. The flow rate was kept constant at 0.6 mL/min， with an injection volume of 5 μL and a total run time of 8 min. The MS detection was performed using electrospray ionization （ESI） in positive ion mode with multiple reaction monitoring （MRM）. The monitored ion pairs for the precursor and product ions of non-glycated and glycated N-terminal hexapeptides are m/z 348.2/237.2 and m/z 429.2/245.2， respectively. The linearity was evaluated by performing regression analysis. The HbA1c （mmol/mol） quantification in unknown samples was achieved by performing linear regression using Analyst 1.6.2 software. Peak areas were integrated， with the abscissa （X-axis） representing the known concentration ratio of HbA1c to hemoglobin A0 （HbA0）， and the ordinate （Y-axis） representing the average peak area ratio of glycated to non-glycated hexapeptides. Both glycated and non-glycated hexapeptides eluted within 3 min， with linear correlation coefficients ranging from 0.999 6 to 0.999 8. The quality control （QC） materials （≥3 types） annually provided by the IFCC were analyzed. Since each QC material in every shipment typically consists of two replicated vials， enzymatic digestion was performed in two separate batches. For each batch， five parallel samples were processed， followed by triplicate injections of each sample. Intra-assay and total coefficients of variation （CV） were calculated. The intra-assay coefficients of variation （CVs） were 0.35%-2.20%， and the total CVs were 0.83%-2.39%. From 2018 to 2024， IFCC compared individual laboratory results with the overall median， calculating proportional bias （slope） and systematic bias （intercept） based on linear regression. Our laboratory’s combined statistical test value （computed from slope and intercept） ranged from 0.1 to 3.0. The systematic bias ranged from -0.69 to 1.27， and the proportional bias ranged from -0.009 to 0.021. The residual values from all testing points in the 2024 proficiency testing （PT） program were evenly distributed around zero. Furthermore， the magnitude of residual deviations from zero was relatively small compared to those observed across the 15 participating laboratories， demonstrating satisfactory consistency in our laboratory’s analytical results. The established HbA1c reference method demonstrated robust performance， achieving satisfactory results in IFCC international comparisons from 2018 to 2024.

Key words: glycated hemoglobin, liquid chromatography-tandem mass spectrometry （LC-MS/MS）, international comparison