Accuracy, precision, and stability in measurement of hemoglobin A1C by "high-performance" cation-exchange chromatography.

Abstract

We present a "high-performance" liquid-chromatographic method for determination of HbA1c with use of a carboxylate cation-exchange column, ethanolic mobile phases, and a hemolysate-storage reagent that stabilizes a sample for up to five weeks at 4 degrees C. Temperature control and the addition of ethanol to the phosphate step-gradient increase precision by stabilizing peak shape and column activity. Lengthy equilibration of the column between samples is not necessary, because the pKa of the cation-exchange resin is decreased from 6.10 in aqueous media to 5.59 in ethanol/water (5/95 by vol) at 25 degrees C. We identified interfering heme compounds, distinct from HbA1d or HbA1e, that elute with the glycosylated hemoglobins and developed a method of correcting for their presence. The day-to-day coefficient of variation was 3.5%, and the reference interval for a mixed group of 20 healthy adults between the ages of 20 and 56 was 3.2--5.2% HbA1c.