A Quantitative Method for the Clean Analysis of Bilirubin in Bezoar Bovis

Abstract

The origin of Bezoar Bovis (BB) is a stone formed in the gall sac of Bos taurus Linné var. domesticus Gmelin (Bovidae), which is designated in the Japanese Pharmacopoeia Seventeenth Edition (JP17). It is important to determine the quantity of its principal component, bilirubin, for the BB quality evaluation. Although a semi-quantitative method for measuring chloroform-soluble BB ingredients including bilirubin is available, a quantitative assay for bilirubin is not described in JP17. Furthermore, from the viewpoint of clean analysis, it is undesirable that chloroform is used as the solvent for the identification test of BB in JP17. In our previous paper in 2010, we reported a HPLC-based quantification of bilirubin in BB that excluded chloroform and used a column packed with octylsilanized silica gel (C₈). In the present study, we improved the analytical conditions and established the quantitative HPLC method of bilirubin in BB with widely-used octadesylsilanized silica gel (C₁₈) to achieve constancy of extraction efficiency and stability of standard solution.