| Part 1 Optimization of the Extraction Process of Qianliean Granules by Orthogonal TestObjective: To develop a HPLC-MS/MS method for simultaneous determination of 4 effective components(Phillyrin, Berberine, Paeoniflorin and TanshinoneⅡA)in Qianliean Granules. And to optimize the Extraction Process of Qianliean Granules by Orthogonal Test.Methods: The main factors which affect the Extraction Process of Qianliean Granules are ethanol concentration, Liquid-solid ratio and extraction time,which are used as effective factors to study the Extraction Process of Qianliean Granules. We use the extraction content of Phillyrin, Berberine, Paeoniflorin and TanshinoneⅡA as the evaluation index, and select the L9(34) Orthogonal Test table as the the experimental scheme.The concentrations of the four effective components were determined by HPLC-MS/MS. A Waters C18 chromatographic column(150 mm×4.6 mm, 3.5 μm) was used; The mobile phase was consisted of the mobile phase A(acetonitrile) and B formic acid-water(2 m M ammonium acetate))with a flow rate of 0.6 m L·min-1, using gradient elution: A 0~7 min: 20 %'60 %; 7~8 min: 60 %'90 %; 8~15 min: 90 %; 15~17 min: 90 %'20 %; 17~20 min: 20 %. at the temperature of 35 ℃, Then 10 μl sample was injected to analysis. Dtetctor: API 4000 type- three weight four grade rod mass spectrometer; The mass spectrometer was operated in the positive and anion electrospray ionization mode with an electrospray ionization interface(ESI); the source voltage was maintained at 5.5 k V and the ion source temperature was set at 550 °C; The curtain gas(CUR) pressure was fixed at 10 psi and the collision gas(CAD) pressure was at 4 psi; Nitrogen was used as GS1(pressure was 55 psi) and GS2(pressure was 55 psi); The DP and CE values of Paeoniflorin were- 69 and- 17, respectively; The DP and CE values of Berberine were 90 and 39, respectively; The DP and CE values of Phillyrin were 65 and 18, respectively; The DP and CE values of TanshinoneⅡA were 137 and 32, respectively. Quantification was performed using multiple reaction monitoring(MRM) of the transitions of m/z 525.2 ' m/z 449.0 for Paeoniflorin, m/z 336.2 ' m/z 320.2 for Berberine, m/z 552.3 ' m/z 355.3 for Phillyrin, m/z 295.3 ' m/z 277.2 for TanshinoneⅡA, respectively.Results: The HPLC-MS/MS of determinating the concentration of Phillyrin, Berberine, Paeoniflorin and TanshinoneⅡA are established. There were good linear relationships for Phillyrin, Berberine, Paeoniflorin and TanshinoneⅡA within the range of 2.868-28.680μg/m L, 0.215-2.148μg/m L, 0.207-20.7 μg/m L and 0.587-5.87μg/m L, respectively(r >0.997). The average recoveries were, 98.28 %(RSD=2.9%) 、 95.81 %(RSD=5.0 %) '99.20%(RSD=2.3%), 99.31%(RSD=5.6%). The precision was evaluated and the relative standard deviation(RSD)values were 5.0%、5.1%、2.4% and 3.1%, respectively. The stability and Repeatability were conformed to the test.The best extraction method of Qianliean Granules is established, namely ethanol concentration is 75%, Liquid-solid ratio is 10 times and extraction time is 6 h.Conclusion: Established a HPLC-MS/MS method for simultaneous determination of 4 effective components(Phillyrin, Berberine, Paeoniflorin and TanshinoneⅡA.)in Qianliean Granules. This method was simple,accurate and reproducible.They can set the basis for reasonable application and quality control for Qianliean Granules. Through using the Orthogonal Test to optimize the Extraction Process of Qianliean Granules, which improved the extraction efficiency and utilization degree, and improved the production efficiency of Qianliean Granules. Part 2 Pharmacokinetics study on the main components of Qianliean Granules in ratsObjective:To establish the Pharmacokinetics method of 2 effective constituent of Qianliean Granules: Phillyrin and Paeoniflorin in rats.Methods: Seven rats was given 2 m L/kg of Qianliean Granules. The blood samples were collected from epicanthal folds at 0 h, 0.25 h, 0.5 h,0.75 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h after treatmen. The blood concentration of drugs was determined by HPLC-MS/MS, the pharmacokinetic parameters of the rats were calculated by DAS 2.1.1.The concentrations were determined with Waters C18 chromatographic column(150 mm×4.6 mm, 3.5 μm); the mobile phase was consisted of acetonitrile-water-formic acid(2 m M ammonium acetate) with a flow rate of 0.6 m L·min-1, The mobile phase A is acetonitrile, B is formic acid-water(2 m M ammonium acetate) and using gradient elution: 0~9 min: 15 %'50 %; 9~11 min: 50 %'90 %; 11~17 min: 90 %; 17~19 min: 90 %'15 %; 19~20 min: 15 %, at the temperature of 35 ℃, 20 μl sample was injected to analysis. Dtetctor: API 4000 type- three weight four grade rod mass spectrometer. The mass spectrometer was operated in the positive and anion electrospray ionization mode with an electrospray ionization interface(ESI); the source voltage was maintained at 5.5 k V and the ion source temperature was set at 550 °C; The curtain gas(CUR) pressure was fixed at 10 psi and the collision gas(CAD) pressure was at 4 psi; Nitrogen was used as GS1(pressure was 55 psi) and GS2(pressure was 55 psi); The DP and CE values of Paeoniflorin were-69 and-17, respectively; The DP and CE values of Berberine were 90 and 39, respectively; The DP and CE values of Phillyrin were 65 and 18, respectively; The DP and CE values of TanshinoneⅡA were 137 and 32, respectively. Quantification was performed using multiple reaction monitoring(MRM) of the transitions of m/z 525.2 ' m/z 449.0 for Paeoniflorin, m/z 336.2 ' m/z 320.2 for Berberine, m/z 552.3 ' m/z 355.3 for Phillyrin, m/z 295.3 ' m/z 277.2 for TanshinoneⅡA, respectively.Results: The main pharmacokinetic parameters of Paeoniflorin and Phillyrin were obtained as follows: AUC0-t were(4897.645±2207.577) μg?h/L and(263.475±54.795)μg?h/L; AUC0-∞ were(4909.699±2219.68)μg?h/L and(270.399±51.943)μg?h/L; t1/2 were(2.206±0.631)h and(1.355±0.317)h; Cmax were(1504.069±620.885)μg/L and(79.043±15.568)μg /L;V were(14.342±4.633)L and(151±54.935)L; CL were(5.025±2.773)L/(h?kg)and(76.253±13.986)L/(h?kg).Conclusion: The LC-MS/MS method has a good performance in terms of accuracy, sensitivity and precision. It is suitable to determine the plasma concentration of Paeoniflori and Phillyrin in rats. |
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