Improved reference measurement method for hemoglobin A1c by use of liquid chromatography-isotope dilution-tandem mass spectrometry.

We have developed an improved reference measurement method for hemoglobin A1c (Hb A1c) 1 based on liquid chromatography–isotope dilution–tandem mass spectrometry (LC-ID-MS/MS) with traceability to the SI unit. The method has a small measurement uncertainty (MU) and gives results in good agreement with the accuracy-based IFCC reference method. Kaiser et al. (1 ) demonstrated the possibility of using ID-MS for Hb A1c measurement, which involved the proteolysis of hemoglobins using endoproteinase Glu-C and hydrolysis of hexapeptide calibration standards using HCl. However, a large MU and significant difference in results compared with the IFCC reference method were observed. Using the same approach, we developed an improved procedure based on LC-ID-MS/MS and used the method for participation in an IFCC ring trial for reference laboratories (RELA 2013) for Hb A1c, in which our results for 2 lyophilized samples were compared against those from the IFCC reference method, including 6 approved IFCC network laboratories for Hb A1c. Key steps in our ID-MS procedure to preserve the unbroken traceability to the SI unit are the hydrolysis of the hexapeptide calibration standards [ -chain N-terminal Val-HisLeu-Thr-Pro-Glu (VE) and 1deoxyfructoxyl-Val-His-Leu-ThrPro-Glu (GE)] and the proteolysis of Hb A0 and Hb A1c. The use of hexapeptide calibration standards allows the traceability to amino acid certified reference materials, which is more reliable than the traceability to reference protein standards in the IFCC reference procedure (1 ). To ensure accurate measurement, complete hydrolysis of the hexapeptides must be achieved. We found that the use of 1% phenol in 6 mol/L HCl markedly reduced the hydrolysis time from the previously reported 65 h (1 ) to 24 h. In addition, amino acids that are stable in acidic environment, such as leucine and proline, were suitable for the quantification of the hexapeptides. This was demonstrated by the consistency between the results of leucine and proline (deviation 0.6%) for both VE and GE. Valine was found to be unsuitable, possibly owing to its linkage to a deoxyfructoxyl group in GE. Because amino acid analysis is affected by both peptide and amino acid impurities, these impurities would need to be quantified (by HPLC and LC-MS/MS, respectively) to ensure the accuracy of the analysis and subsequently the Hb A1c measurement. In our case, VE had satisfactory purity, but GE contained VE as an impurity. The concentration of GE calibration solution was therefore corrected by the quantification of VE in GE to remove the positive bias in the Hb A1c measurement. In proteolysis, incomplete reaction will result in significant deviations between the amounts of hexapeptides obtained and the true amounts of Hb A1c and Hb A0. We found that adding additional endoproteinase Glu-C was crucial to ensure complete proteolysis [125 g/ mg hemoglobin vs the reported amount of 10 g (2 )]. We believe that the complete proteolysis of hemoglobins markedly improves the imprecision and accuracy and further enhances the robustness of our ID-MS method. © 2014 American Association for Clinical Chemistry 1 Hb A1c, hemoglobin A1c; LC-ID-MS/MS, liquid chromatography–isotope dilution–tandem mass spectrometry; MU, measurement uncertainty; VE, -chain N-terminal Val-His-Leu-Thr-Pro-Glu; GE, 1-deoxyfructoxyl-Val-His-Leu-Thr-Pro-Glu; NGSP, National Glycohemoglobin Standardization Program. Table 1. LC-ID-MS/MS measurements of the samples in RELA 2013.