Temperature dependence of heparin inactivation in acid.

Although the lability of heparin in mild acid has been studied (l-4), the conditions for thermoinactivation have not been clearly defined. Thus, experiments were designed to evaluate the loss in anticoagulant activity of heparin solutions in varying hydrogen ion concentrations over a wide range of temperatures. Samples were adjusted to pH l-4 and 7, then held 1 hr at 25, 37, 48, 58, 68, 78, 88, and 98”. In these experiments, the heparin (sodium salt, 157 USP units/mg) from the Nutritional Biochemical Corporation, Cleveland, Ohio, was dissolved in distilled water to yield a final concentration of 40 USP units/ml when adjusted to the pH values indicated with HCl and then later neutralized with NaOH. (The pH measurements were made on a Zeromatic II pH meter, Beckman Instruments, Palo Alto, California. All reagents used were Reagent Grade from J. T. Baker Chemical Company, Phillipsburg, N. J.). Water bath temperatures for heparin inactivation were maintained at f 0.1” from that indicated and heparin solutions attained these temperatures within 4 min after immersion in the bath. The USP XVII method of anticoagulant activity assay (5) was used in which sheep plasma was recalcified whereby a minimum heparin concentration yielding fluidity of the test sample for 1 hr was considered one standard unit. Close agreement was obtained when duplicate analyses were performed by a commercial laboratory (South Mountain Laboratories, Maplewood, N. J.). Where low heparin concentrations were used (under 40 USP units), agreement between duplicate analyses was f 2 USP units. FIG. 1. This graph depicts the activity (USP units) of samples of heparin held at pH 1-4 for 1 hr.