Ultrahigh-performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight tandem mass spectrometry (Q/TOF-MS) in the MS/MS mode and UPLC coupled with triple quadrupole mass spectrometry (QqQ-MS) using the multiple reaction monitoring (MRM) mode were used to make a qualitative and quantitative analysis of twelve bile acids in Bile Arisaema. The fragmentation pathway of twelve bile acids was proposed. The quantification method showed a good linearity over a wide concentration range (
Fermentation is one of the traditional processing technologies commonly used in Traditional Chinese Medicine (TCM) for enhancing efficacy, producing new bioactivities, and alleviating toxicity [
At present, bile acids derived from the pig bile are considered as the main active components in the BA [
In addition, standard processing technology and explicit processing principle are the key to guarantee the clinical efficacy of TCM. However, there are rare researches on the processing technology and mechanism in the BA [
In this work, UPLC-Q/TOF-MS/MS was employed to confirm the bile acids in the methanolic extract of Bile Arisaema. The fragmentation behavior of bile acids was also explored in the negative mode. Then, an UPLC-QqQ-MS/MS method in the MRM mode was established to determine the content of twelve active components in different origins and fermentation times (0 day to 30 days) of BA. It could be used to evaluate the quality and explore the processing mechanism of BA. This study will serve as the first example of comprehensive quality assessment and processing mechanism analysis in Bile Arisaema.
LC-MS grade formic acid was supplied from Merck KGaA (Darmstadt, DE). Methanol and acetonitrile were supplied from Fisher Scientific (Watham, MA, USA). Deionized water was obtained by a Mill-Q system (Billerica, MA, USA). Other chemicals were of analytical purity.
Reference standards including hyodeoxycholic acid (HDCA), cholic acid (CA), chenodeoxycholic acid (CDCA), hyocholic acid (HCA), glycochenodeoxycholic acid (GCDCA), glycocholic acid (GCA), glycohyodeoxycholic acid (GHDCA), taurochenodeoxycholic acid (TCDCA), taurohyodeoxycholic acid (THDCA), taurocholic acid (TCA), glycohyocholic acid (GHCA), and taurohyocholic acid (THCA) were isolated by our library. Their structures were elucidated on the basis of the results of NMR, MS, and IR spectroscopic analysis and compared with the precious references [
The chemical structures of twelve bile acids and IS (internal standard).
The roots and rhizomes of
The bile of pigs was purchased from Dalian Chu-Ming meat federation Co., Ltd., Dalian, Liaoning, China. The twenty batches of Bile Arisaema were collected in December of 2017 from Sichuan, Hebei, Beijing, Anhui provinces of China. The samples were also authenticated by Professor Yan-Jun Zhai. The voucher specimens (no. 20171201–201711220) were deposited in the Key Laboratory of Processing, School of pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, China. Details of the samples are listed in Table
Quantitative analytical results of Bile Arisaema in commercial (
Number | Origins | HDCA | CA | CDCA | HCA | GCDCA | GCA | GHDCA | TCDCA | THDCA | TCA | GHCA | THCA |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
20171201 | Sichuan C&Y Traditional Chinese medicine Co., Ltd. (LOT.160109) | 1950.70 | 27.61 | 2833.93 | 870.70 | 62.74 | 6.12 | 47.23 | 13.98 | 9.12 | 5.85 | 22.34 | 11.25 |
20171202 | Sichuan C&Y Traditional Chinese Medicine Co., Ltd. (LOT.160101) | 2121.04 | 29.19 | 3002.56 | 935.81 | 26.42 | 8.15 | 21.72 | 15.32 | 11.23 | 6.55 | 31.18 | 24.29 |
20171203 | Sichuan C&Y Traditional Chinese Medicine Co., Ltd. (LOT.160601) | 2101.06 | 33.45 | 3136.33 | 969.50 | 23.10 | 4.36 | 17.01 | 12.36 | 10.37 | 4.96 | 24.03 | 15.49 |
20171204 | Sichuan Fuzheng Pharmaceutical Co., Ltd. (LOT.160509) | 104.19 | 4.41 | 212.17 | 68.38 | 2.14 | 0.19 | 3.73 | 5.45 | 0.57 | 2.54 | 2.06 | 1.29 |
20171205 | Sichuan Fuzheng Pharmaceutical Co., Ltd. (LOT.161206) | 78.62 | 2.06 | 169.29 | 61.11 | 5.12 | 1.26 | 5.64 | 4.88 | 3.59 | 0.59 | 1.21 | 3.44 |
20171206 | Sichuan Fuzheng Pharmaceutical Co., Ltd. (LOT.170108) | 122.40 | 5.24 | 235.92 | 76.29 | 0.16 | 2.16 | 3.21 | 0.57 | 1.26 | 0.22 | 1.09 | 3.64 |
20171207 | Neijiang Lianghui Pharmaceutical Co., Ltd. (LOT.170302) | ND | ND | ND | ND | ND | ND | 2.68 | ND | ND | ND | ND | ND |
20171208 | Beijing Tongrentang (Bozhou) Slice Co., Ltd. (LOT. 601002551) | 326.73 | 2.75 | 155.33 | 178.06 | 2.58 | 1.26 | 0.59 | 4.22 | 3.87 | 0.34 | 4.25 | 3.51 |
20171209 | Anhui Jiayu Traditional Chinese Medicine Co., Ltd. (LOT. 170206) | 28.62 | 2.86 | 30.45 | 35.89 | 79.49 | 3.26 | 61.49 | 71.60 | 4.89 | 0.79 | 3.16 | 2.53 |
20171210 | Beijing Huamiao Pharmaceutical Co., Ltd. (LOT. SBB2661) | 834.94 | 6.53 | 1928.04 | 637.88 | 14.86 | 6.39 | 63.18 | 13.95 | 36.25 | 51.28 | 30.24 | 28.56 |
20171211 | Beijing Huamiao Pharmaceutical Co., Ltd. (LOT. SB6191) | 313.17 | 15.31 | 1048.05 | 295.01 | 5032.17 | 154.92 | 3436.47 | 750.92 | 258.18 | 197.53 | 146.55 | 164.23 |
20171212 | Anguo Juyaotang Pharmaceutical Co., Ltd. (LOT. 1701002) | 354.21 | 7700.96 | 1013.32 | 8327.74 | 43.26 | 328.29 | 132.1 | 562.37 | 78.16 | 1121.39 | 129.5 | 821.28 |
20171213 | Sichuan Baisheng Pharmaceutical Co., Ltd. (LOT.170902) | 4216.25 | 47.62 | 6386.69 | 2287.49 | 16.35 | 156.87 | 253.19 | 113.44 | 102.51 | 15.26 | 27.34 | 11.25 |
20171214 | Sichuan Baisheng Pharmaceutical Co., Ltd. (LOT.171204) | 839.32 | 21.16 | 1361.54 | 511.98 | 700.56 | 46.57 | 662.99 | 176.89 | 58.12 | 39.05 | 24.35 | 19.72 |
20171215 | Sichuan Qianfang Traditional Chinese Medicine Co., Ltd. (LOT. 20170601) | 4165.87 | 32.53 | 4444.88 | 1724.25 | 172.47 | 126.34 | 367.21 | 184.37 | 85.25 | 88.52 | 118.23 | 59.69 |
20171216 | Sichuan Jianqu Pharmaceutical Co., Ltd. (LOT.170920) | 2274.11 | 24.35 | 2133.02 | 1117.43 | 102.39 | 52.16 | 18.82 | 110.29 | 49.38 | 89.15 | 52.67 | 47.26 |
20171217 | Yibin Traditional Chinese Medicine Co., Ltd. (LOT. 170501) | ND | 5.45 | ND | 31.98 | 8.20 | ND | 9.05 | ND | ND | ND | ND | ND |
20171218 | Sichuan Hongsheng Pharmaceutical Co., Ltd. (LOT.170903) | 494.08 | 19.66 | 282.36 | 252.93 | 5.32 | 2.23 | 8.69 | 7.56 | 9.16 | 4.61 | 6.56 | 5.31 |
20171219 | Sichuan Guanghan Traditional Chinese Medicine Co., Ltd. (LOT. 170411) | 412.64 | 3607.23 | 918.88 | 3882.89 | 146.58 | 23.59 | 253.46 | 108.16 | 62.04 | 136.77 | 153.4 | 103.72 |
20171220 | Beijing Qiancao Traditional Chinese Medicine Co., Ltd. (LOT. 170806) | 660.79 | 536.16 | 1552.12 | 3818.08 | 100.23 | 55.61 | 165.24 | 253.97 | 176.24 | 248.56 | 236.12 | 118.55 |
ND: not detected.
Appropriate amounts of HDCA, CA, CDCA, HCA, GCDCA, GCA, GHDCA, TCDCA, THDCA, TCA, GHCA, and THCA were separately weighed and dissolved in methanol to get the stock solutions. Then, the twelve stock solutions were mixed and diluted with methanol to prepare a final mixed standard solution containing 8.8
The samples were grinded into powder less than 100 meshes by a pulverizing machine. About 2.0 g of sample powder was weighed accurately into a 100 mL conical flask with cover, and 50 mL methanol was added. After accurate weighing, the mixture was sonicated (power, 250 W; frequency, 50 kHz) for 30 min (Kunshan ultrasonic equipment Co., Ltd, Jiangsu, China). The extracted solution was cooled to room temperature and made up to the original weight with methanol. The supernatants were filtered through a 0.22
The UPLC-Q/TOF-MS/MS analysis was carried out on an Acquity I-Class UPLC system (Waters Corp., Milford, MA, USA) coupled with a Xevo G2-XS mass spectrometer (Waters Corp., Milford, MA, USA). An Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7
The mass spectrometer was performed in the negative MSE mode with a mass range from 50 to 1200 Da. The detection parameters of the ESI source were used as follows: capillary voltage, 2.5 kv; sample cone, 40 V; source offset, 80 V; source temperature, 100°C; flow rate of cone gas, 50 L/h; temperatures and flow rate of desolvation gas (N2), 400°C and 800 L/h; and collision energy, 2 eV in the low energy function and 10 to 30 eV in the high energy function. The software of MassLynx4.1 was used to control the instrument and acquire data.
The UPLC-QqQ-MS/MS analysis was carried out on an Acquity H-Class UPLC system (Waters Corp., Milford, MA, USA) coupled with a Xevo TQ-D mass spectrometer (Waters Corp., Milford, MA, USA). The UPLC conditions were similar to conditions of UPLC-Q/TOF-MS/MS. The mass spectrometer with ESI source was also used in the negative mode. Quantitation was carried out in the multiple reaction monitoring (MRM) mode. The detection parameters of the ESI source were used as follows: capillary voltage, 3.0 kv; cone voltage, 50 V; flow rate of cone gas (N2), 50 L/h; and temperatures and flow rate of desolvation gas (N2), 450°C and 900 L/h. The cone voltage and collision energy of twelve bile acids and IS were optimized by direct infusion into the MS system, respectively. The detailed parameters are listed in Table
The optimized MRM parameters and transitions for each analyst in UPLC-QqQ-MS/MS.
Analyte |
|
[M-H]−( |
MRM transitions (precursor ⟶ product) | Cone voltage (V) | Collision energy (eV) |
---|---|---|---|---|---|
(1) THCA | 1.25 | 514.39 | 514.39 ⟶ 80.14 | 100.0 | 66.0 |
(2) THDCA | 1.60 | 498.39 | 498.39 ⟶ 80.14 | 100.0 | 65.0 |
(3) TCA | 1.74 | 514.39 | 514.39 ⟶ 80.14 | 100.0 | 66.0 |
(4) GHCA | 2.20 | 464.40 | 464.40 ⟶ 74.10 | 76.0 | 38.0 |
(5) GCA | 2.74 | 464.40 | 464.40 ⟶ 74.10 | 76.0 | 38.0 |
(6) GHDCA | 2.77 | 448.41 | 448.41 ⟶ 74.10 | 74.0 | 36.0 |
(7) TCDCA | 2.83 | 498.39 | 498.39 ⟶ 80.14 | 100.0 | 65.0 |
(8) HCA | 3.55 | 407.31 | 407.31 ⟶ 343.42 | 78.0 | 30.0 |
(9) CA | 4.12 | 407.31 | 407.31 ⟶ 343.42 | 78.0 | 30.0 |
(10) GCDCA | 4.41 | 448.41 | 448.41 ⟶ 74.10 | 74.0 | 36.0 |
(11) HDCA | 4.47 | 391.35 | 391.35 ⟶ 345.49 | 80.0 | 34.0 |
(12) CDCA | 8.04 | 391.35 | 391.35 ⟶ 345.49 | 80.0 | 34.0 |
(13) Ginsenoside Rh1 | 1.05 | 637.43 | 637.43 ⟶ 475.26 | 100.0 | 40.0 |
The linearity of the method was constructed by plotting the peak area ratio of the twelve compounds to IS versus their concentration. Each calibration curve was performed with six appropriate concentrations in duplicate. At the same time, the reference standard solution was gradually diluted and detected. The limits of quantitation (LOQs) were determined as the concentration whose S/N was 10, and limits of detection (LODs) were determined as the concentration whose S/N was 3.
The intra- and interday variations were chosen to evaluate the precision of the method. The mixed standard solutions were determined by six replicates within a day for the intraday variability test, while the mixed standard solutions were examined in consecutive three days for the interday variability test. Six copies of sample (20171211) were used to prepare the solution and investigate the repeatability of the method. And one of the solutions was also periodically analyzed at 0, 2, 4, 8, 12, and 24 h to evaluate the stability of the method.
To evaluate the accuracy of this method, a recovery test was performed. Three known amounts (low, middle, and high) of the twelve standards were added to the sample of no. 20171211. Then, the samples were extracted and analyzed using the aforementioned method, and triplicate experiments were performed at each level. Recovery of each analyte was calculated according to the following formula: recovery (%) = (found amount − original amount)/added amount × 100.
The 20 batches of samples were collected from the main producing area of BA in China. Sample preparation and determination were the same as the aforementioned procedure. All the experiments were performed at least in triplicate with constant results.
The powder of
The twelve analytes were firstly detected by UPLC-Q/TOF-MS/MS in both positive and negative ionization modes. It showed that the sensitivity and intensity of analyte signals obtained from the negative ion mode were higher than those from the positive ion mode. Thus, the ESI- mode was selected for qualitative analysis of twelve compounds. To obtain the suitable fragment and product ions, the collision energy was optimized to 10–30 eV.
To obtain satisfactory chromatographic separations, several UPLC analytical parameters were optimized. An Acquity UPLC BEH C18 column was selected, and the optimal mobile phase consisting of acetonitrile (0.1% formic acid) and water (0.1% formic acid) was finally employed. The gradient elution procedure was optimized, and it was also suggested that the separation was operated at the flow rate of 0.4 mL/min and the column temperature at 35°C. The typical chromatogram of standards and samples is shown in Figure
The typical UPLC-Q/TOF-MS/MS chromatogram of (a) mixed standards and (b) samples in Bile Arisaema (1, THCA; 2, THDCA; 3, TCA; 4, GHCA; 5, GCA; 6, GHDCA; 7, TCDCA; 8, HCA; 9, CA; 10, GCDCA; 11, HDCA; 12, CDCA).
As a result of UPLC-Q/TOF-MS/MS, the target compositions were quantitated by UPLC-QqQ-MS/MS in the negative ionization mode. The twelve analytes were detected by the direct full scan mass spectrometry method, and the deprotonated molecules [M-H]− were selected as precursor ions. To obtain the maximum response of precursor and product ions, the parameters of fragment voltage and collision energy were further optimized. All the MRM transitions and parameters applied in the study are shown in Table
UPLC-QqQ-MS/MS multiple reaction mode (MRM) chromatograms of (a) mixed standards and (b) representative sample of BA (1, THCA; 2, THDCA; 3, TCA; 4, GHCA; 5, GCA; 6, GHDCA; 7, TCDCA; 8, HCA; 9, CA; 10, GCDCA; 11, HDCA; 12, CDCA) and IS, Ginsenoside Rh1.
According to the type of structure, the twelve bile acids could be divided into free bile acids and the conjugated bile acids. In addition, it included two pairs of isomers (CA and HCA; HDCA and CDCA) in free bile acids and four pairs of isomers (GCA and GHCA; TCA and THCA; THDCA and TCDCA; GHDCA and GCDCA) in conjugate bile acids. In this study, the deprotonated molecule [M-H]− was detected in the MS/MS spectra of all the analytes within 5.0 ppm (Tables
Mass data of the four free bile acids from Bile Arisaema by UPLC-Q/TOF-MS/MS.
Compound | Formula | Predicted mass ( |
Measured mass ( |
Error (ppm) | MS2 ( |
Side chain eliminated fragments | |
---|---|---|---|---|---|---|---|
Lose H2O fragments | Lose CO, CO2, and H2CO2 fragments | ||||||
CA | C24H40O5 | 407.2797 | 407.2800 [M-H]− | +1.2 | [407]: 389.2697, 371.2588, 353.2483 | 369.2433, 345.2800, 343.2635, 341.243, 325.2537, 323.2379, 309.2588 |
|
HCA | C24H40O5 | 407.2797 | 407.2800 [M-H]− | +1.2 | [407]: 389.2697, 371.2588, 353.2483 | 345.2800, 343.2635, 341.2500, 327.2693, 323.2379, 309.2588 |
|
CDCA | C24H40O4 | 391.2848 | 391.2853 [M-H]− | +1.3 | [ |
343.2638, 329.2839, 327.2685, 325.2533, 299.2366 | 273.2219, 271.2065, 255.2115 |
HDCA | C24H40O4 | 391.2848 | 391.2853 [M-H]− | +1.3 | [ |
343.2638, 329,2839, 327.2685, 325.2533, 299.2366 | 287.2379, 273.2219, 171.2065, 255.2115 |
∗Base peaks are represented in italics.
Mass data of the eight conjugate bile acids from Bile Arisaema by UPLC-Q/TOF-MS/MS.
Compounds | Formula | Predicted mass ( |
Measured mass ( |
Error (ppm) | MS2 ( | ||
---|---|---|---|---|---|---|---|
Lose H2O fragments | Side chain eliminated fragments | Taurine/glycine fragments | |||||
TCA | C26H45NO7S | 514.2838 | 514.2841 [M-H]− | +0.6 | [ |
448.3066, 430.2963, 412.2855, 402.3011, 400.2856, 382.275, 371.2590 | 79.9573, 106.9806, 124.0073 |
THCA | C26H45NO7S | 514.2838 | 514.2841 [M-H]− | +0.6 | [514]: 496.2737, 478.2631 | 448.3066, 430.2963, 412.2855, 402.3011, 382.2751, 371.2590 | 79.9573, 106.9806, |
TCDCA | C26H45NO6S | 498.2889 | 498.2891 [M-H]− | +0.6 | [ |
432.3118, 414.3010, 386.3061, 373.2748, 368.259, 355.2640 | 79.9573, 106.9808, 124.0073 |
THDCA | C26H45NO6S | 498.2889 | 498.2891 [M-H]− | +0.6 | [ |
432.3118, 414.3010, 386.3061, 373.2748, 368.259, 355.2640 | 79.9573, 106.9809, 124.0072 |
GCA | C26H43NO6 | 464.3012 | 464.3016 [M-H]− | +0.9 | [464]: 446.2929 | 418.2961, 402.3012, 371.2593, 369.2430, 354.2612, 323.2377 |
|
GHCA | C26H43NO6 | 464.3012 | 464.3016 [M-H]− | +0.9 | [ |
418.2961, 402.3012, 371.2593, 369.2430, 354.2612, 323.2377 | 74.0245 |
GCDCA | C26H43NO5 | 448.3063 | 448.3068 [M-H]− | +1.1 | [448]: 430.2962 | 404.3168, 402.3007, 386.3065, 368.2958 |
|
GHDCA | C26H43NO5 | 448.3063 | 448.3068 [M-H]− | +1.1 | [448]: 430.2962 | 404.3168, 386.3065, 368.2958 |
|
∗Base peaks are presented in italics.
The typical MS/MS spectra and fragmentation pathway of HCA are shown in Figure
MS/MS spectra (a) and the proposed fragmentation pathway (b) of HCA.
The conjugate bile acids could fall into taurine and glycine type according to the kind of binding amino acid also. Except the deprotonated molecule [M-H]− detected in the MS/MS spectra, the bile acids of the taurine type could produce high abundance fragment ions of [SO3]−, [NH2-CH2-CH2-SO3]−, and [CH2=CH2-SO3]−. Moreover, it could lose the fragment of 66 Da, 82 Da, 94 Da, 96 Da, 108 Da, and 125 Da and product the typical ions of [M-H-H2SO2]−, [M-H-H2SO3]−, [M-H-CH2SO3]−, [M-H-CH4SO3]−, [M-H-C2H4SO3]−, and [M-H-C2H7NSO3]−. The bile acids of the glycine type could produce high abundance fragment ions of [NH2-CH2-COO]−. It could lose the fragment of 44 Da, 46 Da, 58 Da, and 75 Da and product the typical ions of [M-H-CO2]−, [M-H-H2CO2]−, [M-H-C2H2O2]−, and [M-NH2CH2COO]−. The MS/MS spectra and fragmentation pathway of THCA and GHCA are shown in Figures
MS/MS spectra (a) and the proposed fragmentation pathway (b) of THCA.
MS/MS spectra (a) and the proposed fragmentation pathway (b) of GHCA.
Quantitative method was validated by evaluating the linearity, precision, limit of detection (LOD), limit of quantification (LOQ), repeatability, and stability. All results are listed in Table
Calibration curves, linearity ranges, limit of detection (LOD), limit of quantification (LOQ), precision, repeatability, and stability of the twelve analytes.
Analytes | Calibration curves |
|
Linear range ( |
Precision (RSD, %) | LOQ (ng/mL) | LOD (ng/mL) | Repeatability (RSD, %, |
Stability (RSD, %, | |
---|---|---|---|---|---|---|---|---|---|
Intraday | Interday | ||||||||
(1) THCA |
|
0.9998 | 0.22–8.90 | 1.78 | 3.13 | 2.22 | 0.56 | 4.05 | 2.64 |
(2) THDCA |
|
0.9996 | 0.68–27.20 | 0.62 | 2.52 | 2.72 | 0.68 | 4.12 | 2.72 |
(3) TCA |
|
0.9997 | 0.27–10.60 | 2.33 | 3.40 | 5.30 | 1.33 | 3.29 | 4.25 |
(4) GHCA |
|
0.9989 | 0.14–2.80 | 2.86 | 3.11 | 5.60 | 1.40 | 3.57 | 4.19 |
(5) GCA |
|
0.9982 | 0.15–3.00 | 0.82 | 1.42 | 0.75 | 0.19 | 4.04 | 2.35 |
(6) GHDCA |
|
0.9990 | 0.16–3.15 | 0.79 | 1.96 | 3.15 | 0.79 | 3.54 | 1.71 |
(7) TCDCA |
|
0.9997 | 0.55–21.90 | 1.93 | 3.34 | 6.84 | 2.19 | 3.43 | 2.99 |
(8) HCA |
|
0.9994 | 0.20–7.80 | 2.18 | 4.25 | 2.17 | 0.78 | 3.54 | 3.90 |
(9) CA |
|
0.9991 | 0.16–3.25 | 2.99 | 4.01 | 8.13 | 1.63 | 3.27 | 3.97 |
(10) GCDCA |
|
0.9987 | 0.49–9.85 | 1.13 | 1.53 | 4.93 | 1.23 | 4.11 | 2.19 |
(11) HDCA |
|
0.9989 | 0.22–4.40 | 0.85 | 2.43 | 8.80 | 2.20 | 3.65 | 3.82 |
(12) CDCA |
|
0.9985 | 0.42–8.45 | 1.49 | 1.98 | 84.50 | 28.17 | 3.81 | 1.78 |
Recoveries of the twelve compounds (
Analytes | Samples (g) | Origin ( |
Spiked ( |
Found ( |
Mean recovery (%) (RSD, %) |
---|---|---|---|---|---|
(1) THCA | 1.0 | 52.13 | 42.72 | 94.41 | 99.23 (1.11) |
53.40 | 104.52 | 98.27 (1.79) | |||
64.08 | 115.56 | 97.10 (2.96) | |||
|
|||||
(2) THDCA | 1.0 | 103.35 | 82.69 | 185.95 | 99.90 (1.95) |
103.36 | 205.63 | 98.93 (2.43) | |||
124.03 | 227.24 | 95.36 (2.10) | |||
|
|||||
(3) TCA | 1.0 | 70.48 | 56.82 | 126.41 | 98.73 (2.30) |
71.02 | 143.35 | 97.87 (1.46) | |||
85.22 | 154.87 | 99.07 (2.13) | |||
|
|||||
(4) GHCA | 1.0 | 28.20 | 22.40 | 50.42 | 99.13 (0.96) |
28.00 | 56.05 | 96.30 (1.97) | |||
33.60 | 61.37 | 98.63 (2.49) | |||
|
|||||
(5) GCA | 1.0 | 28.35 | 23.04 | 51.42 | 100.63 (0.66) |
28.80 | 56.53 | 97.90 (1.77) | |||
34.56 | 62.50 | 96.17 (0.96) | |||
|
|||||
(6) GHDCA | 1.0 | 264.26 | 211.68 | 476.52 | 100.33 (0.90) |
264.60 | 523.14 | 97.87 (1.76) | |||
317.52 | 580.71 | 98.67 (2.30) | |||
|
|||||
(7) TCDCA | 1.0 | 270.41 | 219.00 | 488.12 | 96.43 (1.99) |
273.75 | 544.43 | 102.15 (0.65) | |||
328.50 | 593.45 | 98.30 (1.99) | |||
|
|||||
(8) HCA | 1.0 | 31.11 | 24.96 | 55.63 | 98.43 (1.12) |
31.20 | 62.12 | 97.10 (1.68) | |||
37.44 | 68.41 | 99.73 (2.21) | |||
|
|||||
(9) CA | 1.0 | 28.71 | 22.88 | 51.34 | 97.10 (1.36) |
28.60 | 57.18 | 98.73 (1.57) | |||
34.32 | 62.56 | 100.97 (1.53) | |||
|
|||||
(10) GCDCA | 1.0 | 651.34 | 520.08 | 1161.43 | 98.13 (1.48) |
650.10 | 1301.54 | 100.15 (1.45) | |||
780.12 | 1434.82 | 101.40 (1.08) | |||
|
|||||
(11) HDCA | 1.0 | 21.62 | 16.72 | 38.43 | 96.33 (0.85) |
21.12 | 42.71 | 98.73 (1.42) | |||
24.64 | 46.08 | 99.17 (0.44) | |||
|
|||||
(12) CDCA | 1.0 | 127.84 | 101.40 | 228.02 | 99.17 (0.78) |
126.75 | 253.71 | 97.60 (1.06) | |||
152.10 | 279.73 | 101.07 (1.50) |
The validated UPLC-QqQ-MS/MS method was applied to simultaneously quantify the twelve compounds in twenty batches of BA samples collected from different regions in China. Table
In order to optimize the fermentation technology of BA, the same sample of BA which had different fermentation times was determined. HCA (hierarchical clustering analysis) and PCA (principal components analysis) were performed on the basis of the content of twelve bile acids compounds from UPLC-QqQ-MS/MS profiles by employing MetaboAnalyst 4.0 software (
(a) HCA of different fermentation times of Bile Arisaema; (b) PCA of different fermentation times of Bile Arisaema.
Compare of the content of 12 bile acids between Bile Arisaema and pig bile in different fermentation times (
Samples | HDCA | CA | CDCA | HCA | GCDCA | GCA | GHDCA | TCDCA | THDCA | TCA | GHCA | THCA |
---|---|---|---|---|---|---|---|---|---|---|---|---|
0 days | ND (ND) | ND (ND) | 0.82 (1.35) | 0.47 (0.58) | 253.30 (297.43) | 10.90 (12.36) | 97.25 (123.46) | 97.97 (134.19) | 36.50 (45.36) | 24.26 (34.26) | 10.11 (15.26) | 20.19 (29.38) |
1 day | ND (ND) | ND (ND) | 1.23 (1.42) | 0.61 (0.69) | 251.87 (295.67) | 10.76 (12.22) | 97.26 (123.23) | 97.88 (133.99) | 36.45 (45.13) | 24.21 (34.27) | 10.09 (15.29) | 20.11 (29.41) |
3 days | 10.25 (ND) | ND (ND) | 2.46 (1.54) | 0.96 (0.72) | 230.34 (289.37) | 9.55 (12.17) | 96.87 (120.39) | 96.26 (132.45) | 35.68 (45.09) | 23.89 (33.75) | 9.86 (15.11) | 20.03 (27.99) |
5 days | 50.21 (5.21) | ND (ND) | 40.55 (2.57) | 4.28 (0.93) | 216.42 (285.63) | 8.86 (12.06) | 87.25 (118.54) | 90.03 (130.58) | 30.42 (44.18) | 20.15 (33.45) | 9.26 (15.03) | 18.85 (26.45) |
7 days | 80.54 (11.49) | 1.24 (ND) | 88.75 (3.47) | 8.21 (1.23) | 197.57 (277.54) | 8.13 (11.96) | 77.52 (114.86) | 77.25 (125.29) | 21.73 (43.09) | 18.52 (32.98) | 8.55 (14.56) | 15.56 (25.13) |
10 days | 110.37 (20.18) | 2.37 (ND) | 187.89 (8.24) | 20.34 (1.46) | 186.25 (265.12) | 7.46 (11.68) | 68.39 (110.43) | 54.29 (121.36) | 11.51 (42.58) | 14.75 (31.52) | 7.23 (13.87) | 10.23 (24.02) |
15 days | 142.48 (31.11) | 3.65 (ND) | 376.88 (12.31) | 84.00 (1.57) | 153.39 (254.31) | 6.78 (11.27) | 55.84 (105.16) | 45.83 (118.33) | 5.40 (41.16) | 12.54 (30.37) | 6.27 (12.45) | 6.70 (23.45) |
20 days | 146.21 (42.15) | 4.17 (ND) | 388.03 (20.15) | 88.14 (1.89) | 151.76 (246.77) | 6.62 (10.79) | 54.46 (100.29) | 45.26 (112.20) | 5.26 (38.73) | 12.26 (28.32) | 6.12 (11.01) | 6.43 (21.36) |
25 days | 147.06 (53.32) | 4.37 (ND) | 391.16 (30.49) | 90.91 (2.16) | 149.11 (220.10) | 6.43 (9.42) | 53.92 (95.01) | 45.09 (103.15) | 5.12 (35.22) | 12.14 (27.16) | 6.03 (10.28) | 6.29 (18.19) |
30 days | 149.35 (60.43) | 4.49 (ND) | 399.24 (50.49) | 91.16 (3.26) | 147.21 (201.23) | 6.24 (8.79) | 53.02 (90.12) | 44.88 (95.37) | 5.03 (30.11) | 11.94 (25.46) | 5.98 (8.99) | 6.11 (16.33) |
ND, not detected; (), content of bile acids in the pig bile.
In Table
In this study, the proposed fragmentation behaviors of the bile acids were illuminated. It could provide a reference for screening bile acids in Bile Arisaema due to similarity in their skeleton and fragment groups. A simple, sensitive, and feasible UPLC-QqQ-MS/MS method was developed and validated for the simultaneous determination of twelve bile acids in Bile Arisaema. The developed method offered the advantages of simple sample preparation and high sensitivity. It was successfully applied to simultaneously quantify the twelve bioactive components in twenty batches of Bile Arisaema samples collected from different regions of China. In addition, comparative analysis of twelve bioactive components in different fermentation times which confirmed the time of 15 days was suitable for BA. Furthermore, one of the possible processing mechanisms of BA was promoting the conjugated bile acids to transform into free bile acids.
Bile Arisaema
Ultrahigh-performance liquid chromatography
Quadrupole time-of-flight tandem mass spectrometry
Triple quadrupole mass spectrometry
Multiple reaction monitoring
Hyodeoxycholic acid
Cholic acid
Chenodeoxycholic acid
Hyocholic acid
Glycochenodeoxycholic acid
Glycocholic acid
Glycohyodeoxycholic acid
Taurochenodeoxycholic acid
Taurohyodeoxycholic acid
Taurocholic acid
Glycohyocholic acid
Taurohyocholic acid
Hierarchical clustering analysis
Principal components analysis.
The data used to support the findings of this study are included within the article.
The authors declare there are no conflicts of interest.
This research was financially supported by Industry Special Funds of National Development and Reform Commission (201507004-03) and Standardization of Traditional Chinese Medicine of Ministry of Science and Technology (ZYBZH-Y-ZY-45).