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A new method for identification of natural, artificial and in vitro cultured Calculus bovis using high-performance liquid chromatography-mass spectrometry.

Liu Y, Tan P, Liu S, Shi H, Feng X, Ma Q - Pharmacogn Mag (2015 Apr-Jun)

Bottom Line: Whereas, as bile acids are with poor ultraviolet absorbance and high structural similarity, effective technology for identification and quality control is still lacking.A new compound with protonated molecule at m/z 405 was found, which we called 3α, 12α-dihydroxy-7-oxo-5α-cholanic acid.In addition, the characteristics of bile acids were illustrated.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China.

ABSTRACT

Objective: Calculus bovis have been widely used in Chinese herbology for the treatment of hyperpyrexia, convulsions, and epilepsy. Nowadays, due to the limited source and high market price, the substitutes, artificial and in vitro cultured Calculus bovis, are getting more and more commonly used. The adulteration phenomenon is serious. Therefore, it is crucial to establish a fast and simple method in discriminating the natural, artificial and in vitro cultured Calculus bovis. Bile acids, one of the main active constituents, are taken as an important indicator for evaluating the quality of Calculus bovis and the substitutes. Several techniques have been built to analyze bile acids in Calculus bovis. Whereas, as bile acids are with poor ultraviolet absorbance and high structural similarity, effective technology for identification and quality control is still lacking.

Methods: In this study, high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) was applied in the analysis of bile acids, which effectively identified natural, artificial and in vitro cultured Calculus bovis and provide a new method for their quality control.

Results: Natural, artificial and in vitro cultured Calculus bovis were differentiated by bile acids analysis. A new compound with protonated molecule at m/z 405 was found, which we called 3α, 12α-dihydroxy-7-oxo-5α-cholanic acid. This compound was discovered in in vitro cultured Calculus bovis, but almost not detected in natural and artificial Calculus bovis. A total of 13 constituents was identified. Among them, three bio-markers, including glycocholic acid, glycodeoxycholic acid and taurocholic acid (TCA) were detected in both natural and artificial Calculus bovis, but the density of TCA was different in two kinds of Calculus bovis. In addition, the characteristics of bile acids were illustrated.

Conclusions: The HPLC coupled with tandem MS (LC/MS/MS) method was feasible, easy, rapid and accurate in identifying natural, artificial and in vitro cultured Calculus bovis.

No MeSH data available.


Related in: MedlinePlus

The mass spectrometry1 (MS) and MS2 of 3α, 12α-dihydroxy-7-oxo-5β-cholanic acid
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Figure 2: The mass spectrometry1 (MS) and MS2 of 3α, 12α-dihydroxy-7-oxo-5β-cholanic acid

Mentions: A compound, with its pseudo-molecular ion at m/z 405, was found in Calculus bovis [Figure 2]. The prominent [M-H]− ion of cholic acid was at m/z 407.2798, while this new compound was at m/z 405.2636. Its MS2 was similar to cholic acid, and its MS2 was at m/z 343 and m/z 289. According to above data, we deduced the compound has similar core structure with cholic acid and has lost H2 from cholic acid. However, according to the structure of cholic acid, there are three hydroxyls in C3, C7 and C12, respectively, which could generate carbonyl by losing hydrogen on O and C. There are three kinds of possible structure in this compound. In this paper, we solved this problem using quantum chemistry. The theoretical calculation of Gaussian calculation was done using Gaussian 03w pack. Quantum chemical calculations were also performed for a better analysis of the results, which would help to have a deep insight into the compound.[161718] Density functional theory and 6-31 g basis set were used in the calculation. Geometry optimization analysis was completed at the B3LYP/6-31 g level. Three optimal structure and energy were obtained by Gaussian calculation. As shown in Figure 3, the structure of this compound was the most stable, with the lowest energy. The HF value was −1313.1769. The same method was used to optimize the structure of cholic acid, which was with the HF value at −1313.1769, and the structure is shown in Figure 4. Based on the result of Gaussian calculation, the structure of the new compound was for C7 carbonyl structure, which could be called 3α, 12α-dihydroxy-7-oxo-5β-cholanic acid. It was discovered in in vitro cultured and almost not detected in natural and artificial Calculus bovis.


A new method for identification of natural, artificial and in vitro cultured Calculus bovis using high-performance liquid chromatography-mass spectrometry.

Liu Y, Tan P, Liu S, Shi H, Feng X, Ma Q - Pharmacogn Mag (2015 Apr-Jun)

The mass spectrometry1 (MS) and MS2 of 3α, 12α-dihydroxy-7-oxo-5β-cholanic acid
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4378128&req=5

Figure 2: The mass spectrometry1 (MS) and MS2 of 3α, 12α-dihydroxy-7-oxo-5β-cholanic acid
Mentions: A compound, with its pseudo-molecular ion at m/z 405, was found in Calculus bovis [Figure 2]. The prominent [M-H]− ion of cholic acid was at m/z 407.2798, while this new compound was at m/z 405.2636. Its MS2 was similar to cholic acid, and its MS2 was at m/z 343 and m/z 289. According to above data, we deduced the compound has similar core structure with cholic acid and has lost H2 from cholic acid. However, according to the structure of cholic acid, there are three hydroxyls in C3, C7 and C12, respectively, which could generate carbonyl by losing hydrogen on O and C. There are three kinds of possible structure in this compound. In this paper, we solved this problem using quantum chemistry. The theoretical calculation of Gaussian calculation was done using Gaussian 03w pack. Quantum chemical calculations were also performed for a better analysis of the results, which would help to have a deep insight into the compound.[161718] Density functional theory and 6-31 g basis set were used in the calculation. Geometry optimization analysis was completed at the B3LYP/6-31 g level. Three optimal structure and energy were obtained by Gaussian calculation. As shown in Figure 3, the structure of this compound was the most stable, with the lowest energy. The HF value was −1313.1769. The same method was used to optimize the structure of cholic acid, which was with the HF value at −1313.1769, and the structure is shown in Figure 4. Based on the result of Gaussian calculation, the structure of the new compound was for C7 carbonyl structure, which could be called 3α, 12α-dihydroxy-7-oxo-5β-cholanic acid. It was discovered in in vitro cultured and almost not detected in natural and artificial Calculus bovis.

Bottom Line: Whereas, as bile acids are with poor ultraviolet absorbance and high structural similarity, effective technology for identification and quality control is still lacking.A new compound with protonated molecule at m/z 405 was found, which we called 3α, 12α-dihydroxy-7-oxo-5α-cholanic acid.In addition, the characteristics of bile acids were illustrated.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China.

ABSTRACT

Objective: Calculus bovis have been widely used in Chinese herbology for the treatment of hyperpyrexia, convulsions, and epilepsy. Nowadays, due to the limited source and high market price, the substitutes, artificial and in vitro cultured Calculus bovis, are getting more and more commonly used. The adulteration phenomenon is serious. Therefore, it is crucial to establish a fast and simple method in discriminating the natural, artificial and in vitro cultured Calculus bovis. Bile acids, one of the main active constituents, are taken as an important indicator for evaluating the quality of Calculus bovis and the substitutes. Several techniques have been built to analyze bile acids in Calculus bovis. Whereas, as bile acids are with poor ultraviolet absorbance and high structural similarity, effective technology for identification and quality control is still lacking.

Methods: In this study, high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) was applied in the analysis of bile acids, which effectively identified natural, artificial and in vitro cultured Calculus bovis and provide a new method for their quality control.

Results: Natural, artificial and in vitro cultured Calculus bovis were differentiated by bile acids analysis. A new compound with protonated molecule at m/z 405 was found, which we called 3α, 12α-dihydroxy-7-oxo-5α-cholanic acid. This compound was discovered in in vitro cultured Calculus bovis, but almost not detected in natural and artificial Calculus bovis. A total of 13 constituents was identified. Among them, three bio-markers, including glycocholic acid, glycodeoxycholic acid and taurocholic acid (TCA) were detected in both natural and artificial Calculus bovis, but the density of TCA was different in two kinds of Calculus bovis. In addition, the characteristics of bile acids were illustrated.

Conclusions: The HPLC coupled with tandem MS (LC/MS/MS) method was feasible, easy, rapid and accurate in identifying natural, artificial and in vitro cultured Calculus bovis.

No MeSH data available.


Related in: MedlinePlus