Evaluation of the Suitability of Kinetic Chromogenic LAL Assay for Determination of Endotoxin Levels in Heparin Sodium Injection

摘要

Determination of the levels of endotoxins in injectable products has always been one of the concerns of regulatory authorities and manufacturers. Since a number of pharmaceuticals interfere with the LAL test to some degree, overcoming the inhibition or enhancement properties of a product is required as part of the validation of the LAL test for use in the final release testing of parenteral products. In this study, interference profile of Heparin injection in quantitative chromogenic LAL test was evaluated and the method of overcoming was investigated and validated. The results indicate that dilution as the most widely used technique for overcoming interference could not eliminate LAL interference in the aforementioned medicinal product. The inhibitory nature of heparin occurs due to its anticoagulant properties and can be overcome by using divalent cations such as magnesium. Three concentrations of magnesium chloride were evaluated for elimination of heparin’s inhibitory effect. All three concentrations studied (5, 10 and 25 mM) could effectively eliminate the inhibitory effects of heparin. Hence, one-way analysis of variance was used to determine statistically significant differences between these three concentrations. The results of ANOVA statistical method showed the optimal recovery of spiked endotoxin was at a concentration of 10 mM of magnesium chloride. In consequence, chromogenic LAL test using 10 mM of magnesium chloride as diluent could be a validated method of choice for heparin LAL assay. Highlights Bacterial endotoxins are important contaminants associated with injectable pharmaceuticals. Kinetic chromogenic LAL assay was used as the method to determine endotoxin levels in heparin injections. Selectivity, linearity and repeatability of the endotoxin chromogenic method was validated.