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Simultaneous quantification of the major bile acids in Artificial Calculus bovis by high‐performance liquid chromatography with precolumn derivatization and its application in quality control
Author(s) -
Shi Yan,
Xiong Jing,
Sun Dongmei,
Liu Wei,
Wei Feng,
Ma Shuangcheng,
Lin Ruichao
Publication year - 2015
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201500139
Subject(s) - chromatography , derivatization , chemistry , chenodeoxycholic acid , cholic acid , extraction (chemistry) , deoxycholic acid , high performance liquid chromatography , bile acid , biochemistry
An accurate and sensitive high‐performance liquid chromatography method coupled with ultralviolet detection and precolumn derivatization was developed for the simultaneous quantification of the major bile acids in Artificial Calculus bovis , including cholic acid, hyodeoxycholic acid, chenodeoxycholic acid, and deoxycholic acid. The extraction, derivatization, chromatographic separation, and detection parameters were fully optimized. The samples were extracted with methanol by ultrasonic extraction. Then, 2‐bromine‐4’‐nitroacetophenone and 18‐crown ether‐6 were used for derivatization. The chromatographic separation was performed on an Agilent SB‐C18 column (250 × 4.6 mm id, 5 μm) at a column temperature of 30°C and liquid flow rate of 1.0 mL/min using water and methanol as the mobile phase with a gradient elution. The detection wavelength was 263 nm. The method was extensively validated by evaluating the linearity ( r 2 ≥ 0.9980), recovery (94.24–98.91%), limits of detection (0.25–0.31 ng) and limits of quantification (0.83–1.02 ng). Seventeen samples were analyzed using the developed and validated method. Then, the amounts of bile acids were analyzed by hierarchical agglomerative clustering analysis and principal component analysis. The results of the chemometric analysis showed that the contents of these compounds reflect the intrinsic quality of artificial Calculus bovis , and two compounds (hyodeoxycholic acid and chenodeoxycholic acid) were the most important markers for quality evaluating.