[Qualitative and quantitative analysis of major cholic acids in Suis Fellis Pulvis].

This study is to establish a qualitative method for rapid identification of bile acids in Suis Fellis Pulvis based on UHPLC-LTQ-Orbitrap-MS technology,and an HPLC-ELSD internal standard method for the quantitative determination of two glycine-conjugated BAs in Suis Fellis Pulvis.The chromatographic separation of the UHPLC-LTQ-Orbitrap-MS qualitative analysis was achieved on a Waters Acquity UPLC HSS T_3column(2.1 mm×100 mm,1.8µm),with 0.2%formic acid aqueous solution(A)-acetonitrile(B)as mobile phase ingradient elution.Electrospray ionization(ESI)source was applied and operated in negative ion mode.Quantitative analysis was performed at 30℃on a Diamonsil-C_(18)column(4.6 mm×250 mm,5µm).The mobile phase consisted of 0.2%formic acid solution and acetonitrile with gradient elution and the flow rate was 1.0 m L·min~(-1).An ELSD was used with a nitrogen flow-rate of1.4 L·min~(-1)at a drift tube temperature of 60℃and the gain was 1.A total of 14 bile acids in Suis Fellis Pulvis were characterized based on the accurate mass measurements,fragmentation patterns,chromatographic retention times,and reference materials.For the quantitative analysis method,the glycohyodeoxycholic acid and glycochenodeoxycholic acid had good linear relationship in the range of26.52-265.20 mg·L~(-1)(r=0.999 8)and 19.84-198.40 mg·L~(-1)(r=0.999 1),respectively.The average recoveries(n=6)were104.1%and 103.1%,and the RSD were 2.0%and 2.4%.The UHPLC-LTQ-Orbitrap-MS technology provides a fast and efficient qualitative analysis method for identification of bile acids in Suis Fellis Pulvis.The HPLC-ELSD internal standard method is accurate and reliable,which has reference value for the quality control of Suis Fellis Pulvis.

Development and validation of a sensitive LC-MS-MS method for the simultaneous determination of multicomponent contents in artificial Calculus Bovis.

Artificial Calculus Bovis is a major substitute in clinical treatment for Niuhuang, a widely used, efficacious but rare traditional Chinese medicine. However, its chemical structures and the physicochemical properties of its components are complicated, which causes difficulty in establishing a set of effective and comprehensive methods for its identification and quality control. In this study, a simple, sensitive and reliable liquid chromatography-tandem mass spectrometry method was successfully developed and validated for the simultaneous determination of bilirubin, taurine and major bile acids (including six unconjugated bile acids, two glycine-conjugated bile acids and three taurine-conjugated bile acids) in artificial Calculus Bovis using a Zorbax SB-C18 column with a gradient elution of methanol and 10 mmol/L ammonium acetate in aqueous solution (adjusted to pH 3.0 with formic acid). The mass spectra were obtained in the negative ion mode using dehydrocholic acid as the internal standard. The content of each analyte in artificial Calculus Bovis was determined by monitoring specific ion pairs in the selected reaction monitoring mode. All analytes demonstrated perfect linearity (r(2) > 0.994) in a wide dynamic range, and 10 batches of samples from different sources were further analyzed. This study provided a comprehensive method for the quality control of artificial Calculus Bovis.

Metabolism and effects on cholestasis of isoursodeoxycholic and ursodeoxycholic acids in bile duct ligated rats.

Isoursodeoxycholic acid (isoUDCA), the 3 beta-epimer of ursodeoxycholic acid (UDCA), may have pharmaceutical potential because of its similar hydrophilicity and in vitro cytoprotection as compared with UDCA. We compared metabolism and effects on cholestasis of UDCA and isoUDCA in experimental cholestasis in rats. Cholestasis was induced by bile duct ligation. For bile flow and biliary bile acid analysis, UDCA or isoUDCA were infused intraduodenally. For the study of chronic effects, chow was supplemented with 2.5 g/kg UDCA or isoUDCA for 3 weeks. Sham-operated animals served as controls. IsoUDCA became completely converted to UDCA in the liver. Choleresis and biliary bile acids were the same after the intraduodenal administration of either compound. Oral administration of UDCA or isoUDCA significantly improved liver biochemistry but not clinical and histological parameters in chronic cholestasis. The decrease of serum cholic acid in control animals was more pronounced after isoUDCA (-93%) than after UDCA (-76%). Only after UDCA, this decrease was compensated by increases of UDCA, beta-muricholic acid (MCA), and Delta(22)-beta-MCA. Our results show that isoUDCA has the same effect on choleresis and liver biochemistry as UDCA. IsoUDCA features pro-drug characteristics of UDCA and causes compared to the latter lower serum bile acid concentrations in non-cholestatic animals.

Reversed-phase LC assay method for deoxycholate in influenza vaccine.

Sodium deoxycholate is used for the disruption of particles in the manufacturing of some influenza vaccines. Residual deoxycholate in inactivated vaccines is currently determined using a labour-intensive colorimetric method which lacks complete specificity. An alternative assay method for residual deoxycholate in vaccine preparations was developed using reversed-phase LC. Cholic acid was used as internal standard and the ratio of internal standard to test solute was used for all calculations. Prior to LC analysis, deoxycholic acid was concentrated by solid-phase extraction, a procedure that also removed proteinaceous material in vaccine samples. The clean-up/concentration procedure recovery was examined using untreated samples and was found to be quantitative. The linearity range of the LC method was between 3 and 200 micrograms ml-1, with a limit of detection of approximately 0.4 micrograms on column, and a lower limit of quantitation of 1.6 micrograms on column. Replicate assays during intra-and inter-day experiments gave acceptable levels of variability. The DCA content of samples from three lots of influenza vaccine varied between 10 and 16 micrograms ml-1. These values were appreciably lower than those measured spectrophotometrically, indicating the higher specificity of the LC method.

[Determination of cholic acid in man-made bezoar by derivative spectrometry].

Cholic acid in man-made bezoar can be determined by the suggested first order derivative spectrometric method. The peak amplitude within 310-290 nm is independent of the other components and only varies linearly with cholic acid concentration. The method is simple and accurate with good reproducibility.

[HPTLC densitometric determination of free bile acids in bezoar].

Cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) which are extracted with CH3OH from Bezoar can be separated on HPTLC silica gel plate (made in China) with isooctane-n-butyl acetate-acetic acid (4:2:1), and the three bile acids were determined by TLC densitometry.

Effect of ursodeoxycholic acid administration on bile acid composition in hamster bile.

The modification in the composition of bile acids in hamster by the administration of high dose of ursodeoxycholic acid (UDCA) was investigated. Male Golden Syrian hamsters were divided into five groups: a control group, two groups that received 0.5 g of UDCA per 100 g of standard diet during 30 and 60 days and another two groups that received 1 g of UDCA per 100 g of standard diet during 30 and 60 days. After ether anaesthesia the gallbladder was removed and bile was immediately aspirated. Bile acids were determined by high performance liquid chromatography (HPLC). Taurolithocholic (TLCA) and glycolithocholic acids (GLCA) increased significantly in all treated groups. The glyco/tauro ratio of 0.69 in controls became more than 1 in treated animals except in the case of lithocholic acid (LCA) conjugates which remained less than 1. UDCA derivatives increased proportionally to the administered dose and the cholic/cheno ratio diminished significantly. A moderate increase of 3- and 7-keto derivatives of chenodeoxycholic acid (CDCA) was observed in all treated groups but the above mentioned increment was especially evident in 3-keto derivatives. A high percentage of UDCA administered in the hamster was likely transformed to CDCA and the glyco conjugates of the bile acids were the predominant species except for the LCA derivatives.

[Application of colorimetric-equal value derivative spectrometry to the determination of cholic acid in niuhuang jiedu pian (tablet)].

A colorimetric-equal value derivative spectrometry for the quantitative analysis of cholic acid in Niuhuang Jiedu Pian(tablet)containing 8 crude drugs is reported. The method is free from the interferuce of borneol rhubarb and other components without separation. The average recovery of cholic acid is 99.75 +/- 0.79% (X +/- CV).

[Studies on calculus bovis by FTIR-PAS].

This article reports the characteristics of different colour layers of Calculus Bovis by FTIR-PAS. It is unnecessary to treat the sample before analysis, and the sample can be reused after analysis. The method is fast accurate and easy to operate.

Bile salts in breast milk.

Breast milk was studied for the presence of bile salts. Cholate and chenodeoxycholate were found in all samples examined. In both colostrum and milk there was a predominance of cholate. Their biological activity remains to be determined.

[Reactivity of the liver to administration of cholic acid to rats subjected to polychloropinene expsoure for a long time]. / Reaktivnost' pecheni na vvedenie kholevoi kisloty u krys, podvergavshikhsia dlitel'nomu vozdeistviiu polikhlorpinena.

It was found that in healthy rats cholic acid caused a moderately increased intensity of bile secretion and raised the content of bile acids in it. In animals with toxic hepatitis produced by a long-term action of the chemical poison polychlorpinene in a dose of 1/10 DL50 and following introduction to such rats of cholic acid in a dose of 30 mg/100 g an intensified elinination of cholates and cholesterol excretion were evident. Combined action of polychlorpinene and cholic acid was conductive to a reduced conjugation of cholic acid with taurine and to a greater conjugation with glycine, this being attended by an increased content of free bile acids in the bile.

[Role of incomplete dietary protein in experimental formation of gallstones]. / Rol' nepolnotsennogo belka pishchi v obrazovanii zhelchnykh kammei v éksperimente

The influence exerted by the quality of alimentary protein on the composition of the secreted bile, morphology of the liver and gallbladder, as well as on the formation of gallstones was studied in experiments with rats and hamsters. It is shown that with inclusion in an experimental fat-free ration of qualitatively incomplete protein gelatin (instead of caseine) the frequency of the gallstones formation increases by almost 6 times. In animals this is attended by a reduced concentration of a number of major bile components. Upon feeding for 2 weeks on lithogenic ration with gelatin morphological investigations disclose inflammatory changes in the gallbladder wall, and after a lapse of 3 weeks--multiple exulcerations in the mucosa and a dense gross sediment in the gallbladder lumen.