Huang-Lian Jie-Du decoction (HLJDD) has been used to treat cardiovascular and cerebrovascular disease for many years in China. Currently, the determination of effect components in HLJDD is focusing either on the formula or on the extract, while quantification of that in biological samples is scarce, especially simultaneous determination of multicomponent. In this paper, a rapid, specific, and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was developed and fully validated for the simultaneous determination of seven main active constituents, i.e., baicalin, baicalein, wogonoside, wogonin, berberine, palmatine, jatrorrhizine in rat plasma. The method was also successfully applied to a quantitative study after oral administration of HLJDD at different doses of 1.5, 3, and 6 g/kg body weight to high fat-induced atherosclerosis rats. The analytes were detected by ESI source and multiple reactions monitoring (MRM) using positive scanning mode. The blood was collected from the abdominal aorta of rats at predetermined time and preprepared with icariin and tetrahydropalmatine as internal standards (IS). Sample preparation was achieved by protein precipitation (PPT). The validation parameters (linearity, sensitivity, intra-/interday precision and accuracy, extraction recovery, and matrix effect) were within acceptable ranges, and biological extracts were stable during the entire storing and preparing process. And the result of determination of HLJDD-containing plasma, baicalin, baicalein, wogonoside, and wogonin could be highly detected in a dose-dependent manner while berberine, jatrorrhizine, and palmatine were determined in a very low level and in a dose-independent mode. Thus, the established method was sensitive enough and successfully applied to the determination of seven effective components in plasma taken from 24 high fat-induced atherosclerosis rats after oral administration of three dosages of HLJDD.
Atherosclerosis (AS) is a chronic disease with a typical pathological process of abnormal lipid metabolism, i.e., infiltrating into the arterial intima and depositing on the vessel wall which triggers inflammation and then in turn leads to the abnormal response to injury of the vessel wall [
Hang-Lian Jie-Du Decoction (HLJDD), a classic prescription of TCM, was composed of four common herbs at the ratio of 3:2:2:3:
Chemical structures of baicalin, baicalein, wogonoside, wogonin, berberine, palmatine, jatrorrhizine, icariin (I.S.), and tetrahydropalmatine (I.S.).
The medicinal materials of
An amount of crude drug equivalent to a daily dose of Huang-Lian-Jie-Du decoction was weighed, and the four medicinal herbs (
Accurately weigh a certain amount of baicalin, baicalein, wogonoside, wogonin, berberine, palmatine, jatrorrhizine, icariin, and tetrahydropalmatine in a 25mL volumetric flask, dissolved and diluted to the concentration of 144, 92, 176, 140, 200, 116, 423, 480, and 488
The calibration standard samples were prepared by freshly spiking the appropriate working solution into blank plasma yielding the concentrations of 2.1-2625, 2.0-1750, 2.0-1875, 2.0-136.72, 0.25-78.13, 0.25-56.64, and 0.25-25 ng/mL for baicalin, baicalein, wogonoside, wogonin, berberine, palmatine, and jatrorrhizine, respectively, and processed as described in the sample preparation. Quality control (QC) samples used for the intra- and interday accuracy and precision, extraction recovery, and stability study were prepared in the same way as calibration standard samples at concentrations of 2.1, 25.0, and 2625 ng/ml for baicalin; 2.0, 25.0, and 575 ng/ml for baicalein; 2.0, 25.0, and 1875 ng/ml for wogonoside; 2.0, 25.7, and 136.72 ng/ml for wogonin; and 0.25, 2.5, and 25.0 ng/ml for berberine, palmatine, and jatrorrhizine.
An aliquot of 100
The analysis was performed using the Shimadzu UHPLC system (Shimadzu Corporation, Kyoto, Japan) consisting of an LC-30AD binary pump, a DGU-20A5 degassing unit, a SIL-30AC autosampler, and a CTO-30A5R column oven. Mass spectrometric detection was conducted on an AB Sciex Qtrap 5500 System (Applied Biosystems, Foster City, CA, USA) and equipped with Analyst software (version 1.6.2) for data processing. Chromatographic separation was achieved on a Shimadzu Shim-pack XR-ODS III (1.6
For mass detection, the electrospray ionization source was operated in positive mode. The operating parameters were optimized under the following conditions: 500°C for the interface temperature and 5.5 kV for the ion spray voltage; ion source gas 1 and gas 2 were fixed at 50 psi. Vacuum was obtained by a Turbo molecular pump (Agilent Technologies, USA). Nitrogen generated by the high purity nitrogen generator (99.999%, Peak Scientific Instruments Ltd., UK) was used as the source of curtain gas (30 psi) and collision gas (7 psi). The optimized multiple reaction monitoring (MRM) parameters including collision energy and declustering potential are listed in Table
Optimized precursor/production pairs and multiple reaction monitoring (MRM) parameters for the analytes and IS.
Analyte | m/z | DP/V | CE/eV |
---|---|---|---|
Baicalin | 447.1→271.0 | 95 | 30 |
Baicalein | 271.0→123.0 | 190 | 44 |
Wogonoside | 461.1→285.0 | 90 | 28 |
Wogonin | 285.0→270.0 | 130 | 34 |
Berberine | 337.8→294.0 | 80 | 38 |
Palmatine | 353.9→322.1 | 50 | 54 |
Jatrorrhizine | 339.1→295.0 | 100 | 27 |
Icariin(IS) | 677.3→369.1 | 200 | 70 |
Tetrahydropalmatine(IS) | 357.2→192.1 | 210 | 35 |
The proposed method was validated for specificity, extraction recovery, matrix effect, LLOQ, linearity, and stability. Meanwhile, intra-and interday validation were performed to evaluate the accuracy and precision of the measurements.
To investigate the specificity of this method, chromatogram comparison of blank plasma, blank plasma spiked with IS/analyte, and rat plasma samples were conducted to assay for the exclusion of any endogenous interference existing at or close to the expected retention time of the analytes. Calibration curves were established from peak area ratios (analyte peak area to the internal standard peak area, As/Ai) versus nominal concentrations using a linear least-squares regression model (1/X2 weighting).
Intra- and interday precisions and accuracy were denoted by assessing measured results of QC samples at low, medium, and high concentrations. Each of the five samples was processed in parallel. Continuously measure a batch of samples and calculate intraday precision. Then, five samples of low, medium, and high plasma samples of each component were prepared in parallel on different consecutive days, and the 45 samples were tested in three batches, and the interday precision was calculated by referring to the accompanying standard. Precisions were expressed by the relative standard deviation (RSD, %), while accuracy (%) was presented as the percentage difference between the mean measured concentrations and the spiked concentrations.
Extraction recoveries of the seven analytes from rat blank plasma were determined by comparing the mean peak areas of the QC samples spiked before protein precipitation with those spiked after protein extraction. Matrix effects occurred when endogenous molecules were coeluting with the analytes of interest enhancing or decreasing the ionization efficiency of the electrospray interface. The matrix effect was assessed via comparing the mean peak areas of the QC samples spiked after the pretreatment with those of the pure solution.
Stability of seven active constituents was checked by comparing measured results with those of freshly prepared samples. The short- and long-term stabilities were evaluated by analyzing QC plasma samples kept at room temperature for 4 h and in the freezer (−20°C) for 30 days, respectively; the freeze-thaw stability was carried out by detecting QC samples undergoing three freeze-thaw cycles; the postpreparation stability was assessed by determining the extracted QC samples stored under autosampler conditions (4°C) for 12 h.
Specifically pathogen-free Sprague-Dawley rats (male, weighing 200 ± 20 g) were purchased from Hunan Slac Laboratory Animal Co. LTD (Hunan, China, Certificate No. SCXK-2013-0004) and acclimated in Exhaust Ventilated Closed-System Cage Rack (EVC) for at least a week with environmentally controlled quarters (22±2°C and 12/12-h light/dark cycle) and free access to standard chow and water. Animal welfare and experimental procedures were strictly in accordance with the guide for the care and use of laboratory animal by the Animal Ethics Committee of Jiangxi University of TCM. After one week of acclimatization, the mice were randomly divided into five groups (
The total separation time for all analytes was 16 minutes and there were little interferential substances with the analytes and IS in the blank plasma. Representative chromatogram of analytes and IS in rat plasma was shown in Figure
The MRM spectrum of each component (a) blank plasma; (b) blank plasma spiked with analytes and IS; (c) rat plasma sample collected after i.g. administration of HLJDD.
The calibration curve of each analyte was established with at least six points of standard solution, and each point was repeated five times. The calibration curves of all analytes exhibited good linearity, and the regression equations with correlation coefficients and linear range were listed in Table
Regression data and LLOQs of the multi-components determined in HLJDD.
Analyte | Linear range | Linear regression equations | Correlation coefficient ( | LLOQs |
---|---|---|---|---|
Baicalin | 2.1-2625 | y =35.46x+1561 | 0.9995 | 2.1 |
Baicalein | 2.0-1750 | y=2.522x+233.2 | 0.9989 | 2.0 |
Wogonoside | 2.0-1875 | y=669.6.x+55174 | 0.9949 | 2.0 |
Wogonin | 2.0-136.7 | y=5885x+30206 | 0.9985 | 2.0 |
Berberine | 0.25-78.13 | y=28947x+29485 | 0.9989 | 0.25 |
Palmatine | 0.25-56.64 | y=4786x+1293 | 0.9955 | 0.25 |
Jatrorrhizine | 0.25-25.0 | y =2877x+2720 | 0.9975 | 0.25 |
The intraday and interday precision of all analytes were all less than 15%, whilst the accuracy deviation values were all within 96.4±6.0% of the actual values at each QC level (shown in Table
Intra-/inter-day precision and accuracy for the determination of the components in rat plasma.
Analytes | Spiked Concentration | Precision (%) | Accuracy (%, Mean ± SD) | ||
---|---|---|---|---|---|
Intra-day | Inter-day | Intra-day | Inter-day | ||
Baicalin | 2.1 | 6.54 | 2.96 | 85.12 ± 5.82 | 90.10 ± 9.84 |
25.0 | 5.82 | 10.87 | 90.73 ± 7.80 | 87.45 ± 4.95 | |
2625 | 10.76 | 11.71 | 91.19 ± 8.37 | 94.14 ± 7.30 | |
Baicalein | 2.0 | 6.60 | 5.41 | 95.19 ± 8.95 | 92.61 ± 7.28 |
25.0 | 11.72 | 7.98 | 96.46 ± 8.17 | 98.19 ± 6.89 | |
575 | 6.86 | 4.24 | 100.15 ± 9.36 | 105.85 ± 7.94 | |
Wogonoside | 2.0 | 3.45 | 14.31 | 87.12 ± 5.84 | 102.19 ± 9.78 |
25.0 | 7.85 | 7.54 | 90.73 ± 7.91 | 85.96 ± 5.85 | |
1875 | 7.19 | 0.77 | 101.14 ± 5.38 | 98.89 ± 7.23 | |
Wogonin | 2.0 | 2.91 | 5.97 | 96.45 ± 5.89 | 88.77 ± 4.93 |
25.7 | 4.53 | 14.11 | 92.76 ± 6.89 | 94.75 ± 5.73 | |
136.7 | 2.06 | 9.92 | 95.71 ± 8.78 | 90.50 ± 3.35 | |
Berberine | 0.25 | 3.92 | 13.28 | 88.19 ± 6.59 | 88.14 ± 5.05 |
2.50 | 9.48 | 2.99 | 99.45 ± 8.84 | 85.79 ± 8.78 | |
25.0 | 1.29 | 12.25 | 100.89 ± 8.73 | 91.16 ± 4.38 | |
Palmatine | 0.25 | 3.75 | 10.54 | 85.12 ± 5.81 | 95.19 ± 8.95 |
2.50 | 7.36 | 9.66 | 90.89 ± 7.93 | 106.49 ± 8.19 | |
25.0 | 6.28 | 0.78 | 108.78 ± 4.73 | 110.14 ± 9.24 | |
Jatrorrhizine | 0.25 | 3.55 | 13.06 | 104.12 ± 7.46 | 87.02 ± 6.24 |
2.50 | 7.18 | 6.57 | 92.57 ± 6.65 | 90.64 ± 5.93 | |
25.0 | 4.38 | 0.86 | 98.10 ± 4.35 | 96.18 ± 4.97 |
The extraction recoveries (absolute recoveries) of each component were more than 80% at each QC level, which satisfied the quantitative requirements of biological samples. With respect to matrix effect, no suppressive or enhancing effect was found on the analytes and IS. That is to say, the responses of all components in the matrix were consistent with that in pure solution. The results are shown in Table
Extraction recovery and matrix effect of the components in rat plasma.
Analyte | Spiked Concentration | Extraction recovery | Matrix effect |
---|---|---|---|
Baicalin | 2.1 | 83.78 ± 7.53 | 106.60 ± 4.96 |
25.0 | 93.53 ± 9.84 | 104.82 ± 9.12 | |
2625 | 93.45 ± 7.15 | 95.12 ± 4.89 | |
Baicalein | 2.0 | 82.18 ± 5.85 | 96.88 ± 5.45 |
25.0 | 89.45 ± 4.56 | 91.18 ± 14.56 | |
575 | 103.29 ± 8.59 | 93.13 ± 4.89 | |
Wogonoside | 2.0 | 89.78 ± 6.66 | 102.51 ± 11.29 |
25.0 | 89.40 ± 5.56 | 103.75 ± 13.05 | |
1875 | 96.75 ± 12.82 | 101.02 ± 11.82 | |
Wogonin | 2.0 | 92.51 ± 4.12 | 96.14 ± 6.85 |
25.7 | 80.52 ± 9.20 | 98.16 ± 10.85 | |
136.7 | 89.66 ± 12.13 | 99.02 ± 13.63 | |
Berberine | 0.25 | 95.35 ± 12.51 | 91.97 ± 9.50 |
2.50 | 96.26 ± 14.99 | 105.17 ± 8.57 | |
25.0 | 91.89 ± 12.23 | 107.46 ± 5.36 | |
Palmatine | 0.25 | 91.06 ± 10.29 | 92.25 ± 7.42 |
2.50 | 92.17 ± 6.98 | 107.22 ± 11.41 | |
25.0 | 89.80 ± 8.45 | 87.49 ± 9.29 | |
Jatrorrhizine | 0.25 | 88.91 ± 13.32 | 90.24 ± 9.03 |
2.50 | 93.33 ± 12.98 | 98.79 ± 8.57 | |
25.0 | 87.78 ± 4.89 | 110.67 ± 10.97 |
Results of the stability (shown in Table
Stability of the components in rat plasma under a variety of storage and process conditions.
Analyte | Spiked Concentration | RSD% | |||
---|---|---|---|---|---|
Freeze-thaw cycles | Short-term stability | Long-term stability | Auto-sampler stability | ||
(three freeze-thaw cycles) | (room temperature, 4 h) | (-20°C, 30 d) | (4°C, 12 h) | ||
Baicalin | 2.1 | 13.86 | 10.53 | 5.63 | 6.93 |
25.0 | 5.34 | 4.56 | 5.91 | 8.72 | |
2625 | 10.64 | 8.45 | 1.25 | 2.16 | |
Baicalein | 2.0 | 8.17 | 6.48 | 3.99 | 12.79 |
25.0 | 7.89 | 5.78 | 6.23 | 7.89 | |
575 | 3.73 | 6.78 | 5.69 | 11.36 | |
Wogonoside | 2.0 | 5.54 | 3.91 | 2.46 | 7.88 |
25.0 | 10.35 | 4.99 | 14.16 | 2.52 | |
1875 | 11.95 | 11.56 | 11.99 | 8.38 | |
Wogonin | 2.0 | 4.25 | 8.72 | 1.96 | 4.53 |
25.7 | 7.11 | 12.12 | 3.79 | 3.73 | |
136.7 | 13.62 | 10.56 | 2.88 | 4.79 | |
Berberine | 0.25 | 12.30 | 7.63 | 3.73 | 7.82 |
2.50 | 11.01 | 8.43 | 12.19 | 10.13 | |
25.0 | 14.52 | 4.96 | 13.13 | 7.40 | |
Palmatine | 0.25 | 15.75 | 8.45 | 13.83 | 9.93 |
2.50 | 11.82 | 8.31 | 11.98 | 6.67 | |
25.0 | 10.07 | 4.97 | 2.54 | 7.99 | |
Jatrorrhizine | 0.25 | 11.20 | 7.54 | 2.86 | 7.87 |
2.50 | 11.16 | 6.08 | 2.11 | 11.69 | |
25.0 | 14.82 | 9.05 | 1.31 | 4.09 |
The present method was successfully used for the determination of three dosages of HLJDD in 24 high fat-induced AS rat plasmas. The concentration of the analytes in plasma after i.g. administration was shown in Table
The concentration of seven analytes in rat plasma after oral administration of three dosages of HLJDD.
Analyte | Groups | Concentration |
---|---|---|
Baicalin | L | 262.95 ± 4.75 |
M | 489.06 ± 7.29 | |
H | 1159.32 ± 126.39 | |
| ||
Baicalein | L | 406.48 ± 62.62 |
M | 804.98 ± 104.02 | |
H | 1885.93 ± 143.94 | |
| ||
Wogonoside | L | 690.56 ± 4.33 |
M | 1542.70 ± 165.74 | |
H | 4079.52 ± 21.00 | |
| ||
Wogonin | L | 9.89 ± 9.76 |
M | 14.21 ± 8.17 | |
H | 36.50 ± 15.72 | |
| ||
Berberine | L | 0.77 ± 0.19 |
M | 0.83 ± 0.19 | |
H | 0.79 ± 0.34 | |
| ||
Palmatine | L | 15.75 ± 1.82 |
M | 27.73 ± 3.10 | |
H | 25.64 ± 2.49 | |
| ||
Jatrorrhizine | L | 0.18 ± 0.17 |
M | 0.39 ± 0.27 | |
H | 0.03 ± 0.01 |
The concentration of seven analytes in rat plasma after oral administration of three dosages of HLJDD.
The choice of mobile phase was a crucial factor in achieving fine chromatographic behavior and appropriate ionization. Modifiers such as formic acid and ammonium formate alone or in combination with different concentrations were compared. The best peak shape and ionization were achieved adapting 5 mM ammonium formate buffer. Linear gradient elution was used to elute endogenous substances residue from the column. In addition, all analytes and IS were both fully scanned by positive and negative mode. As alkaloids were detected overwhelmingly in the positive mode while flavonoids with little difference in both modes, the positive mode was used in the MRM acquisition.
According to the previous phytochemical and HPLC–MS studies, iridoids, alkaloids, and flavonoids were the predominant constituents in HLJDD [
Although the determination of HLJDD was reported before [
From the result of determination of HLJDD-containing plasma, baicalin, baicalein, wogonoside, and wogonin could be highly detected in a dose-dependent manner while berberine, jatrorrhizine, and palmatine were determined in a very low level and in a dose-independent mode. For one thing, the former absorptions were relatively better than the latter. For another, flavonoids are easily bound to glucuronic acid or sulfuric acid to form two-phase metabolism so their plasma concentration-time curves showed obvious bimodal phenomena and concentration increased slowly from the 5th day [
As we all know, the absorption of the intestinal tract and metabolism of the liver may affect the bioavailability of the drug [
Quantification of ingredients at a low level was the obstacles in the study of active components of traditional Chinese medicine in biological fluids. Simply using chromatography was usually time-consuming, insensitive, and nonselective enough. In the present study, a highly selective and sensitive UHPLC–ESI-MS method was developed and validated to simultaneously determine the seven active components in rat plasma and successfully applied to 24 high fat-induced AS rats after oral administration of HLJDD. It could apply for further pharmacokinetic study of the analytes and may provide a scientific basis for clinical application of HLJDD.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declared no conflicts of interest.
Li Jiang and Yanling Xiong contributed equally to this work.
This work was supported by grants of National Natural Science Foundation of China (Nos. 81703823 and 81560744), Jiangxi Provincial Medical and Health Science & Technology Plan (No. 2018B131), Jiangxi Provincial Educational Science & Technology Plan (No. GJJ170753), and Jiangxi Provincial Chinese Medicine First Class Discipline Research Fund (JXSYLXK-ZHYAO120).