Modified HPLC-ESI-MS Method for Glycated Hemoglobin Quantification Based on the IFCC Reference Measurement Procedure and Its Application for Quantitative Analyses in Clinical Laboratories of China

Abstract

Background: The level of glycated hemoglobin (HbA1c ) has been recognized as an important indicator of long-term glycemic control. However, the HbA1c measurement is not currently included as a diagnostic determinant in China. Current study aims to assess a candidate modified International Federation of Clinical Chemistry reference method for the forthcoming standardization of HbA1c measurements in China.

Methods: The HbA1c concentration was measured using a modified high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method. The modified method replaces the propylcyanide column with a C18 reversed-phase column, which has a lower cost and is more commonly used in China, and uses 0.1% (26.5 mmol/l) formic acid instead of trifluoroacetic acid. Moreover, in order to minimize matrix interference and reduce the running time, a solid-phase extraction was employed. The discrepancies between HbA1c measurements using conventional methods and the HPLC-ESI-MS method were clarified in clinical samples from healthy people and diabetic patients. Corresponding samples were distributed to 89 hospitals in Beijing for external quality assessment.

Results: The linearity, reliability, and accuracy of the modified HPLC-ESI-MS method with a shortened running time of 6 min were successfully validated. Out of 89 hospitals evaluated, the relative biases of HbA1c concentrations were < 8% for 74 hospitals and < 5% for 60 hospitals. Compared with other conventional methods, HbA1c concentrations determined by HPLC methods were similar to the values obtained from the current HPLC-ESI-MS method.

Conclusion: The HPLC-ESI-MS method represents an improvement over existing methods and provides a simple, stable, and rapid HbA1c measurement with strong signal intensities and reduced ion suppression.

Keywords: diabetes; external quality assessment; glycated hemoglobin; high-performance liquid chromatography; mass spectrometry.

Figure 1

Standard curve of the current modified HPLC–ESI–MS method for glycated hemoglobin (HbA_1c). The peak areas of HbA_1c and HbA₀ were measured with the modified HPLC–ESI–MS method. The percentage concentrations of HbA_1c in the standard curve were 0, 36.7, 66, 88.7, and 118 mmol/mol. Each standard was measured three times, and the mean value was used to plot the standard curve. HPLC–ESI–MS, high‐performance liquid chromatography–electrospray ionization–mass spectrometry.

Figure 2

External quality assessments. Hemolysate samples A: 0901 and B: 0903 were tested by clinical laboratories in 89 hospitals. The accepted relative bias was ±8%, labeled by the upper and lower red lines.

Figure 3

Reliability of glycated hemoglobin (HbA_1c) measurements. The reliability of HbA_1c measurements from the external quality assessments was validated by comparing values from two batches of samples (Nos. 0901 and 0903) obtained by the 89 clinical laboratories. The assigned values of HbA_1c% by the modified high‐performance liquid chromatography–electrospray ionization–mass spectrometry method were 32.46 and 74.32 mmol/mol, for Nos. 0901 and 0903, respectively; the National Glycohemoglobin Standardization Program (NGSP) values were calculated to be 5.12% and 8.95%, respectively, according to the following equation NGSP(%) = 0.0915 × IFCC + 2.15 mmol/mol.