Human antithrombin (AT) inhibits several proteases in the coagulation system, including thrombin and factor Xa, and thus, plays an important role in the regulation of blood coagulation. The predominant form of AT in plasma is ATalpha, which contains four glycosylated asparagine residues, and the minor form is ATbeta, which lacks the Asn-135 glycosylation. In this study, hydroxyapatite high-performance liquid chromatography, using a segmented sodium phosphate gradient, was utilized for the high-resolution separation of ATalpha and ATbeta. The detection limit (signal-to-noise ratio of 3) for ATbeta was 30 mug/mL, corresponding to 0.5% of the injected concentration of AT. Two analyzed commercial AT products both contained about 2% ATbeta. This method is suitable for the determination of ATP in pure samples of native AT. (C) 2002 Elsevier Science (USA). All rights reserved.