Analysis of Hemoglobin Glycation Using Microfluidic CE-MS: A Rapid, Mass Spectrometry Compatible Method for Assessing Diabetes Management

Diabetes has become a significant health problem worldwide with the rate of diagnosis increasing rapidly in recent years. Measurement of glycated blood proteins, particularly glycated hemoglobin (HbA1c), is an important diagnostic tool used to detect and manage the condition in patients. Described here is a method using microfluidic capillary electrophoresis with mass spectrometry detection (CE-MS) to assess hemoglobin glycation in whole blood lysate. Using denaturing conditions, the hemoglobin (Hb) tetramer dissociates into the alpha and beta subunits (α- and β-Hb), which are then separated via CE directly coupled to MS detection. Nearly baseline resolution is achieved between α-Hb, β-Hb, and glycated β-Hb. A second glycated β-Hb isomer that is partially resolved from β-Hb is detected in extracted ion electropherograms for glycated β-Hb. Glycation on α-Hb is also detected in the α-Hb mass spectrum. Additional modifications to the β-Hb are detected, including acetylation and a +57 Da species that could be the addition of a glyoxal moiety. Patient blood samples were analyzed using the microfluidic CE-MS method and a clinically used immunoassay to measure HbA1c. The percentage of glycated α-Hb and β-Hb was calculated from the microfluidic CE-MS data using peak areas generated from extracted ion electropherograms. The values for glycated β-Hb were found to correlate well with the HbA1c levels derived in the clinic, giving a slope of 1.20 and an R(2) value of 0.99 on a correlation plot. Glycation of human serum albumin (HSA) can also be measured using this technique. It was observed that patients with elevated glycated Hb levels also had higher levels of HSA glycation. Interestingly, the sample with the highest HbA1c levels did not have the highest levels of glycated HSA. Because the lifetime of HSA is shorter than Hb, this could indicate a recent lapse in glycemic control for that patient. The ability to assess both Hb and HSA glycation has the potential to provide a more complete picture of a patient's glycemic control in the months leading up to blood collection. The results presented here demonstrate that the microfluidic CE-MS method is capable of rapidly assessing Hb and HSA glycation from low volumes of whole blood with minimal sample preparation and has the potential to provide more information in a single analysis step than current technologies.