Determination of Defibrase in Pharmaceutical Formulation by LC with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate Derivatization and Fluorescence Detection

A sensitive LC method with pre-column fluorescence derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate has been developed for the determination of defibrase from the venom of Agkistrodon acutus (also named defibrase in the Chinese Pharmacopoeia) in pharmaceutical formulation. By a denaturing procedure prior to derivatization, full and homogeneously derivatization of defibrase was obtained and demonstrated using MALDI-TOF-MS. The specificity of the method was validated by assessing interference from the placebo and by forced degradation. The method has demonstrated good linearity (r = 0.9998), accuracy (mean recovery 98.53%) and precision (RSD < 2.5%). Moreover, the method was found to be sensitive with a low limit of detection (3.35 ng mL^?1) and limit of quantitation (10.16 ng mL^?1). The method was successfully applied to the quantitative determination of defibrase in pharmaceutical formulation.