Long-term evaluation of electrospray ionization mass spectrometric analysis of glycated hemoglobin

Electrospray ionization mass spectrometry (ESIMS) has been successfully applied to the identification of hemoglobin (Hb) variants and the presence of glucose adducts (mass difference of 162 Da) on the separate Hb alpha and beta chains. To establish the potential of ESIMS as a routine and/or a reference method for the quantification of glycohemoglobin (HbA1c), we carried out a detailed evaluation over a 4-month period in a routine laboratory environment.

We optimized a procedure using ESIMS suitable for the routine quantitative analysis of HbA1c. We determined reliability and reproducibility over 4 months and assessed the potential for automated sample injection. We then compared values of 1022 blood samples from diabetic patients with a routine HPLC-based ion-exchange procedure (HA-8140; Menarini).

Results of HbA1c measurement by ESIMS were available within 3 min. The analytical imprecision (CV) was 1.6-5.0% for both manual and automated injections. Data collection over the m/z 980-1400 range confirmed lower glycation of the alpha chain relative to the beta chain (0.66:1). Only one glycation was observed per globin chain. The overall glycohemoglobin (i.e., the average of alpha- and beta-chain glycations) measured by ESIMS (x) on 1022 blood samples was lower than by HPLC (y): y = 1.0432x + 0.4815. However, the ss-chain glycation measured by ESIMS was up to 20% higher than the value measured by ion-exchange HPLC and showed a close conformity, particularly at 5-10% HbA1c, with the ion-exchange Diabetes Control and Complications Trial (DCCT)-corrected and the United Kingdom National External Quality Assessment Scheme DCCT mean return values.

ESIMS provides a precise measurement of HbA1c and, in particular, glycation of the beta chain. The method is robust and could be proposed as a procedure to substantiate HbA1c measurement and/or calibration.