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

Abstract

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.

Liquid-Chromatographic Methods for Carboxylic Acids in Biological Samples

“…On the other hand, both isomers of 2-HG in human tissue were significantly increased. of enantiomers by TSPC derivatization [78]. It was revealed that levels of both (R)-and (S)-2-HG in human urine were comparable among patients with type 2 diabetes mellitus, lung cancer, colorectal cancer, and nasopharyngeal carcinoma.…”

“…One of the strategies for separating enantiomeric isomers is the chiral derivatization method. (+)-O,O'-Diacetyl-L-tartaric anhydride (DATAN) [77] and N-(ptoluenesulfonyl)-L-phenylalanyl chloride (TSPC) [78] are two reagents developed for this purpose. Both reagents have been used for RPLC-MS/MS analysis.…”

Detection technologies and metabolic profiling of bile acids: a comprehensive review

“… HPLC High sensitivity and specificity, applicable to hardly volatile substances. Complicated sample treatment that requires derivatization to enhance the UV absorption of BAs, with the efficiency affecting detection results; long detection time that is unsuitable for a large number of samples [ 33 ]. GC-MS High degree of separation giving accurate molecular information, applicable to separation and quantification of mixtures of non-conjugated BAs [ 105 ].…”

“…Different derivatization methods give various detection wavelengths. For example, Shi et al [ 33 ] detected hydeoxycholic acid (HDCA), CA, CDCA and DCA in Calculus Bovis Sativus using nitrophenacyl bromide as the derivatization reagent at the wavelength of 263 nm. Kakiyama et al [ 34 ] performed HPLC for BAs in feces after methylation derivatization of C24-COOH using benzoyl compound.…”

Micellar electrokinetic capillary chromatography fingerprints combined with multivariate statistical analyses to evaluate the quality consistency and predict the fingerprint–efficacy relationship of Salviae miltiorrhizae Radix et Rhizoma (Danshen)

“…In addition, principle component analysis (PCA) was also used to assess the fingerprint of the DS samples. PCA is the multivariate statistics method that can simplify the problem and finds main factors through dimensionality reduction .…”