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Hemoglobin A0 is prepared from lysed human blood cells by sequential ion-exchange. It is a mixture of non-glycated and glycated hemoglobin A0 fractions, but is essentially free of HbA1a, HbA1b, HbA1c and HbF. The protein is validated by HPIEC methods, and is suitable as a primary standard in chromatographic or immunologic analyses. The reported concentration is determined by Drabkins assay.