Determination of scutellarin plasma concentration and its clinical pharmacokinetics by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS)
2019-09-15 07:04:39
Abstract Objective: To study the pharmacokinetics of breviscapine tablets in Chinese human body. METHODS: Twenty healthy volunteers received a single oral dose of 120 mg breviscapine tablets in a high-performance liquid phase. The total aglycone of scutellarin in plasma was determined by mass spectrometry . RESULTS: In the blood concentration determination method established in this experiment, the impurities in the plasma did not interfere with the determination of the sample, and the linear range was 0.0126~3.24 mg/L; the intra-day and inter-day precision were less than 12.0%. The main pharmacokinetic parameters of 20 healthy subjects after single-dose oral breviscapine tablets (120 mg): Tmax=7.0±2.3h, Cmax=0.9±0.5 mg/L, AUC0-∞= 5.6±1.6 mg/ h/L, AUC0-∞=5.8±1.6 mg/h/L, MRT0-∞=8.0±1.1 h, MRT0-∞=8.6±1.4 h. Conclusion: The established LC-MS method is suitable for the pharmacokinetic study of scutellarin. The oral pharmacokinetics of breviscapine tablets is characterized by a long peak time, and the drug-time curve of about 45% of the total number of subjects has a bimodal phenomenon.
Key words: breviscapine tablets; scutellarin; high performance liquid chromatography-mass spectrometry; blood concentration; pharmacokinetics breviscapine is a whole grass from the genus Erigeron breviscapus extraction) known as the a prime erigeron fleabane, acetic pigment, the main active ingredient scutellarin (scularin), also known as scutellarin, i.e. 4 ', 5,6 trihydroxyflavone -7- Glucuronide, which has dilated blood vessels, improves cerebral blood circulation, enhances cerebral blood flow, reduces vascular resistance, and counteracts platelet aggregation caused by adenosine diphosphate. It is mainly used for the treatment of cardiovascular diseases such as hypertension and cerebral thrombosis. In this study, LC-MS was used to determine the total aglycones in scutellarin in plasma, and the pharmacokinetics of breviscapine tablets in healthy volunteers in China were studied.
1 Materials and Methods 1.1 Drugs and reagents scutellarin tablets: Yunnan Pharmaceutical Institute pharmaceutical factory production, batch number, specifications: 20mg / tablet; scutellaria ethyl sulphate reference product (China National Institute for the Control of Pharmaceutical and Biological Products, 842-200102); Quercetin reference substance (internal standard, China National Institute for the Control of Pharmaceutical and Biological Products, 081-9003); scutellarin aglycone reference substance, provided by Kunming Institute of Botany, Chinese Academy of Sciences; acetonitrile (chromatographically pure); other reagents are AR reagents.
1.2 Instrument and chromatographic conditions Waters liquid chromatography. Mass spectrometer (Waters 2695 high performance liquid chromatography , Micromass ZQ2OOO mass spectrometer, Masslynx 4.0 software); Drict-Q5 ultrapure water machine (Millipore), Sartoris BP211D electronic balance. Liquid phase conditions: Zorb-ax SBC18 column (2.1 mm × 50 mm, 3.5 μm); mobile phase: acetonitrile: 2 mmol / L ammonium acetate: formic acid (25: 75: 0.1); flow rate: 0.2 ml / min; column temperature: 30 ° C. Mass spectrometry conditions: selective ion detection (SIM); scutellarin aglycone, [M+H]+ ion, m·z=287.2; quercetin, [M+H]+ ion, m·z: 303.2; capillary Voltage: 3.5 KV; cone voltage: 50 V; ion source temperature: 120 ° C; nitrogen flow: 380 L / h.
1.3 Treatment of plasma samples Accurately add 0.6 ml of plasma sample in a 10 ml centrifuge tube, vortex for 10 s, add 0.6 ml of 1 mol/L hydrochloric acid, vortex for 30 s, heat in a water bath at 80 ° C for 2 h, and cool in an ice bath. Accurately add 200μl of internal standard solution (0.99mg/L), vortex for 30s, add 4.5ml of ether, vortex for 3min, centrifuge at 3000r/min for 10min, absorb 3ml of supernatant and transfer to another centrifuge tube, blow dry at 40°C N2. Add 250 μl of mobile phase, vortex for 60 s, centrifuge at 12000 r/min for 5 min, and aspirate the supernatant for analysis.
1.4 Subjects selected 20 male healthy subjects with a body weight of 61.4±5.1 kg and an age of 23.1±1.7 years. After passing the physical examination, the informed consent was signed and approved by the ethics committee. The subjects did not take any other medication within 2 weeks and during the trial period.
1.5 The trial designed 20 male healthy subjects with a single dose of oral breviscapine tablets at a dose of 120 mg. Subjects should not drink alcoholic beverages and coffee beverages for 1 week before and during the trial period; fasting for at least 10 hours before the test, taking the medicine on an empty stomach the next morning, drinking water for 2 hours, and entering the standard meal after 4 hours. Subjects took 4 ml of blood from the elbow vein in the heparinized test tube before and after taking the drug at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 24 h, and centrifuged. The plasma was taken and stored in a refrigerator at -75 ° C until the measurement.
1.6 Data Processing The blood concentration-time data of the subjects were processed using DAS (ver1.0) software to calculate the pharmacokinetic parameters.
2 Results 2.1 Chromatographic behavior (specificity) Under the chromatographic conditions used in this experiment, the retention time of scutellarin was about 3.4min, and the retention time of internal standard quercetin was about 5.6min. The scutellarin and the internal standard quercetin have good peak shape, no peak interference measurement, and the baseline is stable.
2.2 In the plasma, the standard curve of scutellarin was taken from a 10 ml centrifuge tube, and the appropriate amount of scutellarin standard solution was precisely added. After drying with N2, add 0.6 ml of blank plasma, vortex for 30 s, and prepare a mixture containing scutellarin. Drug-containing plasma at concentrations of 3.24, 1.62, 0.81, 0.405, 0.202, 0.101, 0.0506, 0.0253, and 0.0126 mg/L, each of which was 5 parts, and was processed according to the "Processing of Plasma Samples" to record the chromatogram. Calculate the ratio f (f = As · Ai) of the area of ​​the lamp A peak and the area of ​​the internal standard peak Ai. The peak area ratio factory was used to calculate the blood concentration C, and the regression equation was obtained: f=0.00299+3.49×C, r=0.9992, and the weight coefficient W=1/C squared.
2.3 Precision Take 10ml centrifuge tube, prepare the drug-containing plasma containing scutellarin concentration of 1.62, 0.202, 0.0506mg/L according to the standard curve preparation method, and operate according to the “treatment of plasma sampleâ€. In the daytime and during the day (5d), 5 samples were prepared for each concentration, and the chromatogram was recorded. The ratio f of the area of ​​the lamp A peak and the area of ​​the internal standard peak Ai was calculated, and the concentration of scutellarin in each sample was calculated. The intraday RSD was 3.51%, 6.40%, and 10.55%, respectively; the daytime RSD was 6.59%, 7.93%, and 11.03%.
2.4 Recovery rate Take a few tubes of clean centrifuge tube, prepare the operation with the concentration of scutellarin 1.62 and 0.202 according to the standard curve preparation method, record the chromatogram, and calculate the ratio of the area of ​​the fluoranoid peak area As and the internal standard peak area Ai. f, the ratio is calculated on the same day as the standard curve equation, the ratio of the calculated concentration to the added concentration is the relative recovery rate. The relative recoveries (%) of the high, medium and low concentrations are 109±8, 118 respectively. ±9, 117±12.
2.5 Stability According to the standard curve preparation method, the drug-containing plasma containing scutellarin concentration of 1.62, 0.202, and 0.0506 mg/L was prepared, and each concentration was made up to 12 parts, and 3 parts were prepared according to the "plasma sample". The treatment item was immediately extracted and analyzed. Three parts were placed at room temperature for 3 hours, then extracted and analyzed. Three parts were frozen and thawed for 3 times, and then extracted and analyzed. After the preparation, 3 parts were prepared and placed in a refrigerator at -75 ° C for 15 days, and then frozen and then extracted and analyzed. The results showed that the plasma samples of scutellarin were stable at room temperature for 3 h, freeze-thaw 3 times and frozen for 15 days.
2.6 Pharmacokinetics of breviscapine tablets in human body Blood concentration determination results: average concentration-time curve of plasma scutellarin in 20 healthy subjects after fasting single-dose oral breviscapine tablets (120 mg) . After breviscapine tablets were taken orally, the drug-time curve of about 45% of the total number of subjects had a bimodal phenomenon, so the pharmacokinetic parameters were calculated using a nonparametric method. The main pharmacokinetic parameters of 20 healthy subjects after fasting single-dose oral breviscapine tablets (120 mg).
3 Discussion Dog oral scutellarin, its absolute bioavailability is only 0.4%, almost no absorption, and breviscapine tablets clinically effective. Since it is effective, there must be a material basis. In the pre-test, healthy volunteers received a single-dose H dose of scutellarin 360 mg from the elbow vein before and 1 , 3, 5, and 8 h after taking the drug. The plasma was treated by solid phase extraction using LC-MS. The scutellarin was measured, and as a result, the plasma concentration of the prototype drug was measured at about 5 h at a time of about 20 μg/L. At the same time, we found a large number of scutellarin aglycones in plasma and urine. According to the literature method, after the hydrolysis conditions are optimized and determined, the peak area of ​​the aglycon is larger after the plasma sample is hydrolyzed. The presence of a large number of aglycones in plasma can explain its efficacy and "material balance" and is consistent with the oral absorption of flavonoid glycosides.
For the bimodal phenomenon of the drug CT curve, due to its various causes, until now there is no general ideal method for statistical analysis. At present, the internationally more conventional treatment methods mostly use the non-compartmental model method, which analyzes the in vivo process of drugs, mainly based on the area under the curve of the drug, is not limited by the mathematical model, and is generally applicable to any compartment state. In this experiment, DAS (ver1.0) software was used for data processing, and the obtained statistical distance parameters were in line with the trend of pharmacokinetics.
references
1 Jiang Xuehua, Li Suhua, Lan Lan, Yang Junyi, Zhou Jing. Pharmacokinetics of breviscapine in dogs [J]. Journal of Pharmaceutical Sciences, 2003; 38:371-3
2 Ge Qinghua, Zhou Wei, Zhi Xiaotong, Ma Lili, Chen Xiuhua. Study on pharmacokinetics and absolute bioavailability of breviscapine in dogs [J]. China Pharmaceutical Industry Journal, 2003; 34: 618-20
3 Wang Fengmei, Wu Meizhen, Zhou Li, Yao Wei, Yan Heyong. Methodological study on the determination of ginkgo flavonoids in plasma by high performance liquid chromatography [J]. Chinese Journal of Traditional Chinese Medicine, 2003:28:279-81
4 Wang Hongxi, Yi Wei, Ping Qi Neng. Application of effect surface optimization method in the hydrolysis of flavonol glycosides of Ginkgo biloba [J]. Chinese Natural Medicine, 2004; 2: 28-32
5 Chen Shujuan, Zhang Bin, Yang Yimei, Dai Zongshun, Zeng Fandian. Pharmacokinetics of batsine in dogs [J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2000; 5:213-7
Key words: breviscapine tablets; scutellarin; high performance liquid chromatography-mass spectrometry; blood concentration; pharmacokinetics breviscapine is a whole grass from the genus Erigeron breviscapus extraction) known as the a prime erigeron fleabane, acetic pigment, the main active ingredient scutellarin (scularin), also known as scutellarin, i.e. 4 ', 5,6 trihydroxyflavone -7- Glucuronide, which has dilated blood vessels, improves cerebral blood circulation, enhances cerebral blood flow, reduces vascular resistance, and counteracts platelet aggregation caused by adenosine diphosphate. It is mainly used for the treatment of cardiovascular diseases such as hypertension and cerebral thrombosis. In this study, LC-MS was used to determine the total aglycones in scutellarin in plasma, and the pharmacokinetics of breviscapine tablets in healthy volunteers in China were studied.
1 Materials and Methods 1.1 Drugs and reagents scutellarin tablets: Yunnan Pharmaceutical Institute pharmaceutical factory production, batch number, specifications: 20mg / tablet; scutellaria ethyl sulphate reference product (China National Institute for the Control of Pharmaceutical and Biological Products, 842-200102); Quercetin reference substance (internal standard, China National Institute for the Control of Pharmaceutical and Biological Products, 081-9003); scutellarin aglycone reference substance, provided by Kunming Institute of Botany, Chinese Academy of Sciences; acetonitrile (chromatographically pure); other reagents are AR reagents.
1.2 Instrument and chromatographic conditions Waters liquid chromatography. Mass spectrometer (Waters 2695 high performance liquid chromatography , Micromass ZQ2OOO mass spectrometer, Masslynx 4.0 software); Drict-Q5 ultrapure water machine (Millipore), Sartoris BP211D electronic balance. Liquid phase conditions: Zorb-ax SBC18 column (2.1 mm × 50 mm, 3.5 μm); mobile phase: acetonitrile: 2 mmol / L ammonium acetate: formic acid (25: 75: 0.1); flow rate: 0.2 ml / min; column temperature: 30 ° C. Mass spectrometry conditions: selective ion detection (SIM); scutellarin aglycone, [M+H]+ ion, m·z=287.2; quercetin, [M+H]+ ion, m·z: 303.2; capillary Voltage: 3.5 KV; cone voltage: 50 V; ion source temperature: 120 ° C; nitrogen flow: 380 L / h.
1.3 Treatment of plasma samples Accurately add 0.6 ml of plasma sample in a 10 ml centrifuge tube, vortex for 10 s, add 0.6 ml of 1 mol/L hydrochloric acid, vortex for 30 s, heat in a water bath at 80 ° C for 2 h, and cool in an ice bath. Accurately add 200μl of internal standard solution (0.99mg/L), vortex for 30s, add 4.5ml of ether, vortex for 3min, centrifuge at 3000r/min for 10min, absorb 3ml of supernatant and transfer to another centrifuge tube, blow dry at 40°C N2. Add 250 μl of mobile phase, vortex for 60 s, centrifuge at 12000 r/min for 5 min, and aspirate the supernatant for analysis.
1.4 Subjects selected 20 male healthy subjects with a body weight of 61.4±5.1 kg and an age of 23.1±1.7 years. After passing the physical examination, the informed consent was signed and approved by the ethics committee. The subjects did not take any other medication within 2 weeks and during the trial period.
1.5 The trial designed 20 male healthy subjects with a single dose of oral breviscapine tablets at a dose of 120 mg. Subjects should not drink alcoholic beverages and coffee beverages for 1 week before and during the trial period; fasting for at least 10 hours before the test, taking the medicine on an empty stomach the next morning, drinking water for 2 hours, and entering the standard meal after 4 hours. Subjects took 4 ml of blood from the elbow vein in the heparinized test tube before and after taking the drug at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 24 h, and centrifuged. The plasma was taken and stored in a refrigerator at -75 ° C until the measurement.
1.6 Data Processing The blood concentration-time data of the subjects were processed using DAS (ver1.0) software to calculate the pharmacokinetic parameters.
2 Results 2.1 Chromatographic behavior (specificity) Under the chromatographic conditions used in this experiment, the retention time of scutellarin was about 3.4min, and the retention time of internal standard quercetin was about 5.6min. The scutellarin and the internal standard quercetin have good peak shape, no peak interference measurement, and the baseline is stable.
2.2 In the plasma, the standard curve of scutellarin was taken from a 10 ml centrifuge tube, and the appropriate amount of scutellarin standard solution was precisely added. After drying with N2, add 0.6 ml of blank plasma, vortex for 30 s, and prepare a mixture containing scutellarin. Drug-containing plasma at concentrations of 3.24, 1.62, 0.81, 0.405, 0.202, 0.101, 0.0506, 0.0253, and 0.0126 mg/L, each of which was 5 parts, and was processed according to the "Processing of Plasma Samples" to record the chromatogram. Calculate the ratio f (f = As · Ai) of the area of ​​the lamp A peak and the area of ​​the internal standard peak Ai. The peak area ratio factory was used to calculate the blood concentration C, and the regression equation was obtained: f=0.00299+3.49×C, r=0.9992, and the weight coefficient W=1/C squared.
2.3 Precision Take 10ml centrifuge tube, prepare the drug-containing plasma containing scutellarin concentration of 1.62, 0.202, 0.0506mg/L according to the standard curve preparation method, and operate according to the “treatment of plasma sampleâ€. In the daytime and during the day (5d), 5 samples were prepared for each concentration, and the chromatogram was recorded. The ratio f of the area of ​​the lamp A peak and the area of ​​the internal standard peak Ai was calculated, and the concentration of scutellarin in each sample was calculated. The intraday RSD was 3.51%, 6.40%, and 10.55%, respectively; the daytime RSD was 6.59%, 7.93%, and 11.03%.
2.4 Recovery rate Take a few tubes of clean centrifuge tube, prepare the operation with the concentration of scutellarin 1.62 and 0.202 according to the standard curve preparation method, record the chromatogram, and calculate the ratio of the area of ​​the fluoranoid peak area As and the internal standard peak area Ai. f, the ratio is calculated on the same day as the standard curve equation, the ratio of the calculated concentration to the added concentration is the relative recovery rate. The relative recoveries (%) of the high, medium and low concentrations are 109±8, 118 respectively. ±9, 117±12.
2.5 Stability According to the standard curve preparation method, the drug-containing plasma containing scutellarin concentration of 1.62, 0.202, and 0.0506 mg/L was prepared, and each concentration was made up to 12 parts, and 3 parts were prepared according to the "plasma sample". The treatment item was immediately extracted and analyzed. Three parts were placed at room temperature for 3 hours, then extracted and analyzed. Three parts were frozen and thawed for 3 times, and then extracted and analyzed. After the preparation, 3 parts were prepared and placed in a refrigerator at -75 ° C for 15 days, and then frozen and then extracted and analyzed. The results showed that the plasma samples of scutellarin were stable at room temperature for 3 h, freeze-thaw 3 times and frozen for 15 days.
2.6 Pharmacokinetics of breviscapine tablets in human body Blood concentration determination results: average concentration-time curve of plasma scutellarin in 20 healthy subjects after fasting single-dose oral breviscapine tablets (120 mg) . After breviscapine tablets were taken orally, the drug-time curve of about 45% of the total number of subjects had a bimodal phenomenon, so the pharmacokinetic parameters were calculated using a nonparametric method. The main pharmacokinetic parameters of 20 healthy subjects after fasting single-dose oral breviscapine tablets (120 mg).
3 Discussion Dog oral scutellarin, its absolute bioavailability is only 0.4%, almost no absorption, and breviscapine tablets clinically effective. Since it is effective, there must be a material basis. In the pre-test, healthy volunteers received a single-dose H dose of scutellarin 360 mg from the elbow vein before and 1 , 3, 5, and 8 h after taking the drug. The plasma was treated by solid phase extraction using LC-MS. The scutellarin was measured, and as a result, the plasma concentration of the prototype drug was measured at about 5 h at a time of about 20 μg/L. At the same time, we found a large number of scutellarin aglycones in plasma and urine. According to the literature method, after the hydrolysis conditions are optimized and determined, the peak area of ​​the aglycon is larger after the plasma sample is hydrolyzed. The presence of a large number of aglycones in plasma can explain its efficacy and "material balance" and is consistent with the oral absorption of flavonoid glycosides.
For the bimodal phenomenon of the drug CT curve, due to its various causes, until now there is no general ideal method for statistical analysis. At present, the internationally more conventional treatment methods mostly use the non-compartmental model method, which analyzes the in vivo process of drugs, mainly based on the area under the curve of the drug, is not limited by the mathematical model, and is generally applicable to any compartment state. In this experiment, DAS (ver1.0) software was used for data processing, and the obtained statistical distance parameters were in line with the trend of pharmacokinetics.
references
1 Jiang Xuehua, Li Suhua, Lan Lan, Yang Junyi, Zhou Jing. Pharmacokinetics of breviscapine in dogs [J]. Journal of Pharmaceutical Sciences, 2003; 38:371-3
2 Ge Qinghua, Zhou Wei, Zhi Xiaotong, Ma Lili, Chen Xiuhua. Study on pharmacokinetics and absolute bioavailability of breviscapine in dogs [J]. China Pharmaceutical Industry Journal, 2003; 34: 618-20
3 Wang Fengmei, Wu Meizhen, Zhou Li, Yao Wei, Yan Heyong. Methodological study on the determination of ginkgo flavonoids in plasma by high performance liquid chromatography [J]. Chinese Journal of Traditional Chinese Medicine, 2003:28:279-81
4 Wang Hongxi, Yi Wei, Ping Qi Neng. Application of effect surface optimization method in the hydrolysis of flavonol glycosides of Ginkgo biloba [J]. Chinese Natural Medicine, 2004; 2: 28-32
5 Chen Shujuan, Zhang Bin, Yang Yimei, Dai Zongshun, Zeng Fandian. Pharmacokinetics of batsine in dogs [J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2000; 5:213-7
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