Method for determining natural product composition in animal plasma

Abstract

The invention relates to the technical field of medicines, in particular to a sensitive and rapid method for measuring the concentration of a natural product composition in plasma, which is suitable for pharmacokinetic research. Specifically, the method comprises a natural product composition plasma sample pretreatment method, a chromatographic separation method and a mass spectrometry detection method. The detection method is high in sensitivity, simple in pretreatment operation, small in sample dosage, high in instrument analysis flux and suitable for preclinical pharmacokinetic research of the natural product composition in prevention and / or treatment of acute respiratory distress, viral pneumonia, cerebral ischemia reperfusion, migraine, depression, spleen and stomach diseases, lipid peroxidation and other diseases.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly relates to a method for determining the concentration of a natural product composition in plasma that is rapidly suitable for pharmacokinetic studies; the natural product composition is used for preventing and / or treating diseases such as acute respiratory distress syndrome, viral pneumonia, cerebral ischemia reperfusion, migraine, depression, spleen and stomach diseases, and lipid peroxidation. Background Art

[0002] Acute lung injury / acute respiratory distress syndrome (ALI / ARDS) refers to acute, progressive, and hypoxic respiratory failure caused by various pathogenic factors inside and outside the lungs other than cardiogenic factors such as severe infection and trauma. Both have the same pathological and physiological changes, namely reduced lung compliance, increased intrapulmonary shunt, and ventilation / perfusion ratio imbalance, etc. Most experts and scholars define severe ALI or the final severe stage of ALI as ARDS. Clinically, the main manifestations are cough, wheezing, chest tightness, and increased respiratory rate, and even cyanosis, etc. In the later stage, it often induces or combines with multiple organ dysfunction syndrome (MODS). Its basic pathological feature lies in extensive damage to pulmonary capillary endothelial cells and alveolar epithelial cells, enhanced vascular permeability causing pulmonary edema, reduced open blood vessels in the lungs forming microthrombi, leading to microcirculation disorders in the lungs. In recent years, although domestic and foreign have made phased progress in understanding the pathogenesis and clinical treatment of ALI / ARDS, and the mortality rate shows a downward trend, it is still as high as 50%. The pathogenesis of ALI / ARDS is intricate, involving multiple links such as inflammatory response, abnormal coagulation function, and oxidative stress. These links do not exist in isolation, but rather influence and connect with each other, and there is currently no specific treatment method. Therefore, actively seeking auxiliary drugs targeting the pathological mechanism of lung injury has positive significance.

[0003] Traditional Chinese medicine has the characteristics of treating diseases with multiple targets, multiple components, and multiple mechanisms, and can provide a new direction for the treatment of ALI / ARDS. It is reported that arecoline can cross the blood-brain barrier due to its unique chemical skeleton, act on M cholinergic receptors, promote body excitement, and thus repair lesions [1] , and it can regulate the activity of adrenaline in the body, thereby enhancing the body's immunity [2,3] , and there are also research reports that arecoline also has unique pharmacological effects such as vasodilation, antithrombosis, inhibition of adipocyte differentiation, antibacterial, anti-inflammatory, and antitumor effects [4-6] ; baicalein and magnolol have pharmacological effects such as anti-inflammatory, antioxidant, antithrombotic, and tissue protection [7,8], Isoquercetin has anti-inflammatory, antioxidant, and neuroprotective effects. [9] ; The combination of these four compounds can comprehensively treat ALI / ARDS from multiple aspects including inflammation, antioxidant, immune enhancement, and the nervous system, providing a new direction for treating diseases. Therefore, we provide a simple, accurate, and rapid bioassay method to accelerate its clinical application.

[0004] Combined with the high separation efficiency of liquid chromatography and the high sensitivity of mass spectrometry, liquid chromatography-mass spectrometry (LC-MS) technology has now become the most effective technical means for quantitative analysis in the field of biomedicine. Currently, there is relatively little research on the pharmacokinetics of each component in this natural product composition. Among them, Li Bing [10,11] et al. established a method for detecting arecoline in beagle dog plasma using liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology. This method uses n-hexane as an internal standard and treats plasma by protein precipitation method; Pan Hong

[12] et al. established a method for simultaneously detecting arecoline and its two metabolites in rat plasma using LC-MS / MS technology; Adrian A Franke

[13] et al. determined the content of arecoline in human saliva, urine, and hair using liquid chromatography-mass spectrometry technology. Xu Caibing

[14] et al. established a method for detecting magnolol in rat plasma using reversed-phase high-performance liquid chromatography. This method uses evodiamine as an internal standard and performs pretreatment by ethyl acetate liquid-liquid extraction, with a lower limit of quantification of 35 ng / mL; Liu Yuan

[15] et al. established a method for detecting magnolol in rat plasma using high-performance liquid chromatography with fluorescence detector (HPLC-FLD) and compared the pharmacokinetic parameters with equal-dose Zhizi Houpu Decoction; Liang Yi

[16] et al. intravenously administered magnolol to beagle dogs and established a method for detecting magnolol in beagle dog plasma using LC-MS / MS technology; Li Hui

[17] et al. established a method for detecting magnolol in rat plasma using LC-MS / MS technology; In Chinese Patent CN110243965B

[18] West China Hospital of Sichuan University established a method for simultaneously determining emodin, vanillic acid, hesperidin, polydatin, and magnolol in Hupi Dachengqi Decoction, providing an effective detection method for the pharmacokinetic detection of this formula. Zhao Zhenyu

[19] et al. intravenously injected baicalein into mice and established a method for detecting baicalein in mouse plasma using high-performance liquid chromatography (HPLC). Its lower limit of quantification is 50 ng / mL; Guo Xiaoyu

[20] et al. simultaneously determined the pharmacokinetic parameters of baicalein and baicalin in rats using high-performance liquid chromatography-electrochemical chromatography detection method and found that the time-concentration curve of the baicalin metabolite baicalein has a double-peak phenomenon; Yumeng Wei

[21] Et al. determined the plasma drug concentration of baicalein in rabbit plasma using LC-UV method and conducted a study on tissue distribution. Yi Hongli

[22] Et al. determined the plasma drug concentration of isoquercetin in rat plasma using HPLC-MS and found that there might be an interconversion among quercetin, isoquercetin and quercetin-3-O-β-D glucuronide in rats; in Chinese Patent CN1190200C

[23] , Merck & Co., Inc. used combinations of two or three bioflavonols including isoquercetin, quercetin-4'-glycoside, rutin and quercetin and found that there were differences in their pharmacokinetics; in Chinese Patent CN112955151B

[24] , Alpine Pharmaceutical Industry Co., Ltd. combined isoquercetin, L-arginine and ascorbic acid or alkali metal salts of ascorbic acid and conducted a study on pharmacokinetics.

[0005] The existing technologies for detecting the concentrations of arecoline, magnolol, baicalein and isoquercetin in plasma cannot balance sensitivity and analysis speed at the same time, and there is currently no method that can simultaneously determine the plasma drug concentration of this natural product composition. There is a need for a highly efficient, accurate, simple and low-cost technical method for accurately analyzing and detecting the concentrations of the components of this natural product composition in a large number of clinical plasma samples. In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] To overcome the defects of the prior art, the purpose of the present invention is to provide a method for determining a natural product composition in animal plasma that is sensitive, rapid and suitable for pharmacokinetic studies;

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] On the one hand, the present invention provides a method for determining a natural product composition in animal plasma that is sensitive, rapid and suitable for pharmacokinetic studies, and the method includes the following steps:

[0009] Add an internal standard and methanol to the plasma sample for vortexing, collect the supernatant after centrifugation, concentrate by nitrogen blowing, add 100 μL of 50% methanol aqueous solution, and obtain the pretreated sample to be measured after vortex mixing;

[0010] Perform liquid chromatography-mass spectrometry detection, separate the sample to be measured using liquid chromatography, then perform mass spectrometry detection, draw a standard curve based on the detected peak area ratio to obtain a regression equation, and finally calculate the concentrations of the components of the natural product composition in the plasma sample to be measured;

[0011] The stable internal standard solution is chrysin and matrine; chrysin is used as the internal standard for detecting baicalin, isoquercetin and magnolol; matrine is used as the internal standard for detecting arecoline and arecaidine;

[0012] The specific methods for the pretreatment include:

[0013] Take 100 μL of plasma, add 10 μL of 120 mg / mL vitamin C solution, 10 μL of 5% formic acid solution and 10 μL of internal standard, shake for 3 min, add 400 μL of precooled methanol, shake for 3 min, centrifuge at 13400 rpm for 15 min, take the supernatant and blow it dry with nitrogen, add 100 μL of 50% methanol-water for reconstitution, shake evenly and load the sample;

[0014] In the above method, preferably, the centrifugation time is 15 min, the centrifugation temperature is 4°C, and the centrifugation speed is 13400 rpm;

[0015] In the above method, preferably, the concentration of the chrysin internal standard is 500 ng / mL, and the concentration of the matrine internal standard is 1000 ng / mL;

[0016] The chromatographic column 1 used for liquid chromatography separation is Agilent Zorbax XDB-C18, and the particle size, inner diameter and column length of this chromatographic column are 5 μm, 2.1 mm and 50 mm respectively. The chromatographic column 2 is: Welch SCX, and the particle size, inner diameter and column length of this chromatographic column are 5 μm, 4.6 mm and 250 mm respectively; The mobile phase 1 used is: Phase A: aqueous solution containing 0.1% formic acid, Phase B: methanol; The mobile phase 2 is: Phase A: aqueous solution containing 0.2% formic acid and the pH is adjusted to 3.8 with ammonia water, Phase B: acetonitrile; Among them, the chromatographic column 1 is used to detect isoquercetin, baicalin and magnolol, and the chromatographic column 2 is used to detect arecoline and arecaidine;

[0017] The elution for liquid chromatography separation is as follows. Chromatographic condition 1 is used to detect isoquercetin, baicalin and magnolol, and chromatographic condition 2 is used to detect arecoline and arecaidine; Chromatographic condition 1 is:

[0018] Gradient elution:

[0019] 0 min 35% B, 8 min 70% B, 13 min 70% B, 13.1 min 35% B;

[0020] Elution time: 17.1 min;

[0021] Injection volume: 10 μL;

[0022] Column temperature: 40°C;

[0023] The elution conditions for liquid chromatography separation 2 are:

[0024] Isocratic elution: 0 - 17 min: 45% B;

[0025] Elution time: 17 min;

[0026] Sample injection volume: 10 μL;

[0027] Column temperature: 30 °C;

[0028] Among them, the mass spectrometry conditions for mass spectrometry detection are as follows. Mass spectrometry condition 1 is used to detect isoquercetin, baicalin, and magnolol, and mass spectrometry condition 2 is used to detect arecoline and arecaidine; Mass spectrometry condition 1 is:

[0029] Electrospray ionization source ESI;

[0030] Dry gas flow rate is 10.0 L / min;

[0031] Dry gas temperature is 350 °C;

[0032] Sprayer pressure is 35.0 psig;

[0033] Capillary voltage is 3000 V;

[0034] Negative ion detection mode;

[0035] Mass spectrometry detection condition 2 is:

[0036] Electrospray ionization source ESI;

[0037] Dry gas flow rate is 10.0 L / min;

[0038] Dry gas temperature is 350 °C;

[0039] Sprayer pressure is 35.0 psig;

[0040] Capillary voltage is 3000 V;

[0041] Positive ion detection mode;

[0042] The ion pairs for quantitative analysis are used for mass spectrometry detection. The quantitative analysis ion pairs are:

[0043] The mass-to-charge ratios m / z generated by magnolol, baicalin, isoquercetin, and chrysin are 265, 445, 463, and 253 respectively; The mass-to-charge ratios m / z generated by arecoline, arecaidine, and sophocarpine are 156, 142, and 247 respectively;

[0044] The specific method for making the standard curve is:

[0045] Using the theoretical concentration of the sample to be measured as the abscissa and the peak area ratio of the sample to be measured to the internal standard as the ordinate, a linear regression equation is obtained through regression analysis calculation;

[0046] Application of the method described in this study in determining natural product compositions in plasma samples; the natural product compositions are applied to the prevention and / or treatment of diseases such as acute respiratory distress syndrome, viral pneumonia, cerebral ischemia-reperfusion, migraine, depression, spleen and stomach diseases, and lipid peroxidation;

[0047] Advantages of the present invention

[0048] (1) The present invention has the characteristic of simple pretreatment operation. After the sample precipitates proteins, it can be loaded for analysis and determination after nitrogen blowing and reconstitution. The extracted sample is easy to preserve and can simultaneously determine multiple compounds. Therefore, the present invention is suitable for the determination of a large number of clinical research samples;

[0049] (2) The present invention has high sensitivity. The lower limit of quantification of arecoline is 10 ng / mL, the lower limit of quantification of magnolol and isoquercitrin is 20 ng / mL, the lower limit of quantification of baicalin is 100 ng / mL, and the lower limit of quantification of arecaidine is 50000 ng / mL; the present invention has good accuracy, high precision, and good reproducibility, and can more accurately determine the concentration of drugs;

[0050] The above description is only an overview of the technical solution of the present invention. In order to be able to more clearly understand the technical means of the present invention and implement it in accordance with the content of the specification, the following takes the examples of the present invention and combines with the attached drawings for detailed description. Description of the drawings

[0051] Figure 1 Product ion scanning mass spectra of chrysin, magnolol, baicalin, and isoquercitrin;

[0052] Figure 2 Product ion scanning mass spectra of arecaidine, arecoline, and sophocarpine;

[0053] Figure 3 SIM chromatograms of arecoline, magnolol, isoquercitrin, baicalin, arecaidine, chrysin, and sophocarpine in blank plasma samples;

[0054] Figure 4 SIM chromatograms of arecoline, magnolol, isoquercitrin, baicalin, arecaidine, chrysin, and sophocarpine in plasma samples at the lower limit of quantification;

[0055] Figure 5 Standard curves of arecoline, magnolol, isoquercitrin, baicalin, and arecaidine in plasma samples. Detailed implementation manners

[0056] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention; obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention; the processes, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and well-known general knowledge in the art, and the present invention has no special restrictive content.

[0057] The development of a method for detecting drug concentration in plasma by liquid chromatography-mass spectrometry can generally be divided into three parts, namely, a pretreatment method, a liquid chromatography method, and a mass spectrometry method; in view of the disadvantages of the prior art, the present invention starts from these three aspects to establish a determination method.

[0058] Embodiment

[0059] I. Pretreatment:

[0060] In the present invention, the plasma dosage used is only 100 μL, which is suitable for the determination of biological samples in clinical research. The extraction method in the present invention selects the protein precipitation method.

[0061] The specific steps of the pretreatment method are as follows:

[0062] Take 100 μL of plasma, add 10 μL of 120 mg / mL vitamin C solution, 10 μL of 5% formic acid solution, and 10 μL of internal standard;

[0063] Vibrate for 3 min, and add 400 μL of pre-cooled methanol;

[0064] Vibrate for 3 min and centrifuge at 13400 rpm for 15 min;

[0065] Take the supernatant and blow it dry with nitrogen, add 100 μL of 50% methanol-water for reconstitution, vibrate evenly and load the sample;

[0066] The injection volume is 10 μL.

[0067] II. Chromatographic analysis

[0068] Chromatographic condition 1: Agilent Zorbax XDB-C18, the particle size, inner diameter, and column length of this chromatographic column are 5 μm, 2.1 mm, and 50 mm respectively; column temperature: 40 °C; mobile phase: phase A: aqueous solution containing 0.1% formic acid, phase B: methanol, 35% B at 0 min, 70% B at 8 min, 70% B at 13 min, 35% B at 13.1 min;

[0069] Chromatographic conditions 2: Welch SCX, with a particle size, inner diameter, and column length of 5 μm, 4.6 mm, and 250 mm respectively; column temperature: 30 °C; mobile phase: aqueous solution containing 0.2% formic acid and adjusted to pH 3.8 with ammonia - acetonitrile, 45:55, v / v;

[0070] Arecoline and arecaidine have high polarity and small molecular weights, making them difficult to separate. The chromatographic column with SCX packing used in this invention has good retention for both the analyte and the internal standard, and the peak shape is symmetric; the Agilent Zorbax XDB - C18 chromatographic column is used to separate baicalin, isoquercetin, magnolol, and chrysin, and it also has good determination and retention for them. It is suitable for the determination of a large number of samples in clinical research.

[0071] III. Mass spectrometry analysis:

[0072] Mass spectrometry conditions 1: ESI source, positive ion monitoring mode, drying gas flow rate of 10.0 L / min, drying gas temperature of 350 °C, nebulizer pressure of 35.0 psig, capillary voltage of 3000 V; mass spectrometry detector: selective ion monitoring mode SIM, the mass - to - charge ratios m / z generated by arecoline, arecaidine, and sophocarpine are 156, 142, and 247 respectively;

[0073] Mass spectrometry conditions 2: ESI source, negative ion monitoring mode, drying gas flow rate of 10.0 L / min, drying gas temperature of 350 °C, nebulizer pressure of 35.0 psig, capillary voltage of 3000 V; mass spectrometry detector: selective ion monitoring mode SIM, the mass - to - charge ratios m / z generated by magnolol, baicalin, isoquercetin, and chrysin are 265, 445, 463, and 253 respectively.

[0074] The present invention will be described in detail below through specific examples:

[0075] Example 1:

[0076] Abbreviation description:

[0077] Abbreviation Full text Chinese description LQC Low quality control Low quality control MQC Medium quality control Medium quality control HQC High quality control High quality control RSD Relative standard deviation Relative standard deviation RE Relative error Relative error

[0078] 1 Materials

[0079] 1.1 Instruments

[0080] Chromatograph: Agilent 1200 high - performance liquid chromatograph, Agilent Technologies Co., Ltd.;

[0081] Mass spectrometer: Agilent 6110 single quadrupole mass spectrometer, Agilent Technologies Co., Ltd.;

[0082] Software for data processing: Drug Analysis System 2.0, MaS Studio Co., Ltd., China

[0083] Centrifuge: Allegra TM X-22R Centrifuge, Beckman Coulter, USA

[0084] Analytical balance: XS105 Analytical Balance, Mettler Toledo, Switzerland

[0085] 1.2 Reference substances and reagents

[0086] Baicalein was purchased from Chengdu Mansite Technology Co., Ltd. with a content of 98%; Baicalin was purchased from Baoji Renshou Pharmaceutical Co., Ltd. with a content of 98%; Isoquercetin was purchased from Chengdu Youli Xinsheng Biotechnology Co., Ltd. with a content of 98%; Magnolol was purchased from Xi'an Guanyu Biotechnology Co., Ltd. with a content of 98%; Sophorcarpine was purchased from Hubei Wande Chemical Industry Co., Ltd. with a content of 98%; Chrysin was purchased from Shaanxi Huike Botanical Development Co., Ltd. with a content of 98%; Arecoline hydrobromide was purchased from ROE Scientific with a content of 98% and Arecaidine was purchased from Tianmo Quality Inspection Co., Ltd. with a content of 99.9%; Water was purified water; Methanol and acetonitrile were LC-MS grade; Formic acid was HPLC grade, and other reagents were all of analytical grade.

[0087] 2 Methods

[0088] 2.1 Preparation of solutions and samples

[0089] Standard series samples: The individual stock solutions of the 7 analytes, namely arecoline, arecaidine, baicalin, isoquercetin, magnolol, sophorcarpine and chrysin, were dissolved in 50% methanol aqueous solution and diluted into a series of concentrations; The final plasma concentrations of arecoline were 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000 ng / mL respectively; The final plasma concentrations of magnolol were 20, 25, 50, 100, 200, 500, 1000, 2000, 5000, 10000 ng / mL respectively; The final plasma concentrations of isoquercetin were 20, 25, 50, 100, 200, 500, 1000, 2000, 5000, 10000 ng / mL respectively; The final plasma concentrations of baicalin were 100, 200, 500, 1000, 2000, 5000, 10000, 20000 ng / mL respectively; The final plasma concentrations of arecaidine were 50, 100, 250, 500, 1000, 2500, 5000, 10000 μg / mL respectively;

[0090] The concentrations of the quality control samples QC are as follows: the low, medium, and high concentrations of the arecoline quality control samples are 50, 500, and 5000 ng / mL respectively; the low, medium, and high concentrations of the isoquercetin and magnolol quality control samples are 50, 250, and 10000 ng / mL respectively; the low, medium, and high concentrations of the baicalin quality control samples are 250, 1000, and 5000 ng / mL respectively; the low, medium, and high concentrations of the arecaidine quality control samples are 50, 500, and 1000 μg / mL respectively;

[0091] Internal standard solution: Weigh accurately the reference standards of chrysin and matrine and make the volume up to the mark with 50% methanol-water. Prepare a solution containing 500 ng / mL of chrysin and 1000 ng / mL of matrine.

[0092] 2.2 Plasma sample treatment

[0093] Take 100 μL of plasma and add 10 μL of 120 mg / mL vitamin C solution, 10 μL of 5% formic acid solution and 10 μL of internal standard;

[0094] Vortex for 3 min, then add 400 μL of pre-cooled methanol;

[0095] Vortex for 3 min and centrifuge at 13400 rpm for 15 min;

[0096] Take the supernatant and dry it by nitrogen blowing, then add 100 μL of 50% methanol-water for reconstitution, vortex evenly and load the sample;

[0097] The injection volume is 10 μL.

[0098] Chromatography-mass spectrometry conditions 1: Agilent Zorbax XDB-C18, the particle size, inner diameter and column length of this chromatographic column are 5 μm, 2.1 mm and 50 mm respectively; column temperature: 40 °C; mobile phase: phase A: aqueous solution containing 0.1% formic acid, phase B: methanol, 35% B at 0 min, 70% B at 8 min, 70% B at 13 min, 35% B at 13.1 min; ESI source, negative ion monitoring mode, drying gas flow rate is 10.0 L / min, drying gas temperature is 350 °C, nebulizer pressure is 35.0 psig, capillary voltage is 3000 V; mass spectrometry detector: selective ion monitoring mode SIM, the mass-to-charge ratios m / z generated by magnolol, baicalin, isoquercetin and chrysin are 265, 445, 463 and 253 respectively;

[0099] Chromatography - Mass Spectrometry Conditions 2: Welch SCX, with the particle size, inner diameter, and column length of the chromatographic column being 5 μm, 4.6 mm, and 250 mm respectively; Mobile phase 1 used is; Column temperature: 30 °C; Mobile phase: Aqueous solution containing 0.2% formic acid, adjusted to pH 3.8 with ammonia - acetonitrile, 45:55, v / v; ESI source, positive ion monitoring mode, drying gas flow rate is 10.0 L / min, drying gas temperature is 350 °C, nebulizer pressure is 35.0 psig, capillary voltage is 3000 V; Mass spectrometry detector: Selective ion monitoring mode SIM, the mass - to - charge ratios m / z generated by arecoline, arecaidine, and sophocarpine are 156, 142, and 247 respectively.

[0100] 2.3 Standard Curve and Quality Control

[0101] Standard Curve

[0102] Using the theoretical concentration to be measured as the abscissa x and the ratio of the peak area of the analyte to the internal standard as the ordinate y, perform regression analysis to calculate the linear regression equation.

[0103] Accuracy and Precision

[0104] For each analytical batch, 5 samples of 3 concentration - level quality control samples are determined. The relative standard deviation RSD (i.e., CV%) of the QC samples at each concentration level needs to be less than 15% to be acceptable, and the accuracy needs to be between 85% and 115% to be acceptable.

[0105] 3 Results and Discussion

[0106] 3.1 Method

[0107] Standard Curve

[0108] The standard curve graphs of arecoline, magnolol, isoquercetin, baicalin, and arecaidine are as Figure 5 shown; The results show that arecoline has a good linear relationship in the range of 10 - 10000 ng / mL, and the average regression equation is R1 = 0.078c - 0.1896. Magnolol has a good linear relationship in the range of 20 - 10000 ng / mL, and the average regression equation is R2 = 0.0079c + 0.8041. Isoquercetin has a good linear relationship in the range of 20 - 10000 ng / mL, and the average regression equation is R3 = 0.0012c + 0.1735. Baicalin has a good linear relationship in the range of 100 - 20000 ng / mL, and the average regression equation is R4 = 0.0012c + 0.241. Arecaidine has a good linear relationship in the range of 50 - 10000 μg / mL, and the average regression equation is R5 = 0.12c + 15.98.

[0109] Lower Limit of Quantification

[0110] The lower limits of quantification of arecoline, magnolol, isoquercitrin, baicalin, and arecaidine were 10, 20, 20, 100, and 50000 ng / mL, respectively.

[0111] Quality control samples

[0112] The results of precision and accuracy both met the standards, and the results are shown in Table 1.

[0113] Table 1 Accuracy and precision of arecoline, arecaidine, magnolol, isoquercitrin, and baicalin in plasma

[0114]

[0115] In the present invention, specific embodiments are used to elaborate on the principles and implementation manners of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention; at the same time, for those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

[0116] References

[0117] [1] SHIH Y T, CHEN P S, WU C H, et al. Arecoline, a major alkaloid of the areca nut, causes neurotoxicity through enhancement of oxidative stress and suppression of the antioxidant protective system[J]. Free Radical Biology and Medicine, 2010, 49(10): 1471-9.

[0118] [2] DASGUPTA R, CHATTERJI U, NAG T C, et al. Ultrastructural and hormonal modulations of the thyroid gland following arecoline treatment in albino mice[J]. Molecular & Cellular Endocrinology, 2010, 319(1-2): 1-7.

[0119] [3]HSU H F, TSOU T C, CHAO H R, et al. Effects of arecoline on adipogenesis, lipolysis, and glucose uptake of adipocytes - A possible role of betel - quid chewing in metabolic syndrome[J]. Toxicol Appl Pharmacol, 2010, 245(3): 370 - 7.

[0120] [4]ZHUQING Q I, QIXIN Y, GUANG W, et al. Effect of arecoline on PDX - 1 mRNA expression in rats with Type 2 diabetes meilitus[J]. International Journal of Pathology and Clinical Medicine, 2010.

[0121] [5]HUANG L W, HSIEH B S, CHENG H L, et al. Arecoline decreases interleukin - 6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells[J]. Toxicology & Applied Pharmacology, 2012, 258(2): 199 - 207.

[0122] [6]CHEN Y J, CHANG L S. Arecoline - induced death of human leukemia K562 cells is associated with surface up - modulation of TNFR2[J]. Journal of Cellular Physiology, 2012, 227(5): 2240 - 51.

[0123] [7] Fang Cheng, Yu Xingbo, Zheng Xiuqian, et al. Research progress on chemical constituents and pharmacological effects of Scutellaria baicalensis Georgi [J]. Chemical Engineer, 2021, 35(3): 3. [8] Jia Yin, Ren Chenxi, Xia Younan, et al. Current research status of pharmacological effects of magnolol [J]. Biochemical Engineering Journal, 2023, 9(3): 170-4.

[0124] [9] NIEMAN D C, HENSON D A, MAXWELL K R, et al. Effects of quercetin and EGCG on mitochondrial biogenesis and immunity [J]. Medicine & Science in Sports & Exercise, 2009, 41(7): 1467.

[0125]

[10] ZHANG B L X Z J L Y Y J N X W X L J L J. Determination and pharmacokinetic studies of arecoline in dog plasma by liquid chromatography-tandem mass spectrometry [J]. Journal of chromatography, B Analytical technologies in the biomedical and life sciences, 2014.

[0126]

[11] Chinese Patent CN103940935A

[0127]

[12] PAN, HONG, LI, et al. Development and validation of a rapid LC-MS / MS method for simultaneous quantification of arecoline and its two active metabolites in rat plasma and its application to a pharmacokinetic study[J]. Journal of Pharmaceutical and Biomedical Analysis: An International Journal on All Drug-Related Topics in Pharmaceutical, Biomedical and Clinical Analysis, 2018.

[0128]

[13] ADRIAN, A., FRANKE, et al. Pilot study of the pharmacokinetics of betel nut and betel quid biomarkers in saliva, urine, and hair of betel consumers[J]. Drug Testing & Analysis, 2016.

[0129]

[14] XU Caibing, TIAN Rui, GAN Lu, et al. Determination of plasma concentration of honokiol by reversed-phase high performance liquid chromatography[J]. Acta Academiae Medicinae Militaris Tertiae, 2014, 36(3): 4.

[0130]

[15] LIU Y, WANG D, YANG G, et al. Comparative pharmacokinetics and brain distribution of magnolol and honokiol after oral administration of Magnolia officinalis cortex extract and its compatibility with other herbal medicines in Zhi-Zi-Hou-Po Decoction to rats[J]. Biomedical Chromatography, 2015.

[0131]

[16] Liang Y, Cui G, Wang X, et al. Pharmacokinetics of honokiol after intravenous guttae in beagle dogs assessed using ultra-performance liquid chromatography-tandem mass spectrometry[J]. Biomedical Chromatography Bmc, 2015, 28(10): 1378-83.

[0132]

[17] Li H, Wen X S, Di W. A simple LC-MS / MS method for determination of magnolol in rat blood and its application in a pharmacokinetic study[J]. Arzneimittelforschung, 2012, 62(02): 83-7.

[0133]

[18] Chinese Patent CN110243965B

[0134]

[19] Zhao Z Y, Dong W J, Liu Y L. Pharmacokinetics and tissue distribution of baicalein in mice[J]. Chinese Journal of Pharmaceuticals, 2009, (9): 4.

[0135]

[20] Guo X Y, Yang L, Chen Y, et al. Comparative study on pharmacokinetics of baicalein and baicalin in rats[J]. Chinese Pharmaceutical Journal, 2008, 43(7): 3.

[0136]

[21] Yu Meng W, Chao P, Gang Y, et al. LC-UV Determination of Baicalin in Rabbit Plasma and Tissues for Application in Pharmacokinetics and Tissue Distribution Studies of Baicalin after Intravenous Administration of Liposomal and Injectable Formulations[J]. Molecules, 2016, 21(4): 444.

[0137]

[22] HONGLI,YIN,JI,et al.Pharmacokinetic comparison of quercetin,isoquercitrin,andquercetin-3-O-β-D-glucuronide in rats by HPLC-MS[J].Peerj,2019.

[0138]

[23] Chinese Patent CN1190200C

[0139]

[24] Chinese Patent CN112955151B

Claims

1. A method for determining a natural product composition in animal plasma, characterized in that the composition consists of compounds isoquercetin, arecoline, magnolol and baicalein, the chemical structure of the composition is shown as follows, the method is used for the pharmacokinetic study of the natural product composition, and the drugs detected by the method are isoquercetin, magnolol, baicalin, arecoline and arecolidine, wherein, Baicalin is the in vivo metabolite of baicalein, and arecoline N-oxide is the in vivo metabolite of arecoline. The method includes the following steps: Add internal standard and methanol to the plasma sample for vortexing. After centrifugation, collect the supernatant, concentrate it by nitrogen blowing, add 100 μL of 50% methanol aqueous solution, and obtain the pretreated sample to be measured after vortex mixing. Perform liquid chromatography-mass spectrometry detection. Separate the sample to be measured using liquid chromatography. Among them, isoquercetin, baicalin, and magnolol are separated using an Agilent Zorbax XDB-C18 chromatographic column with a particle size of 5 μm, an inner diameter of 2.1 mm, and a column length of 50 mm. The mobile phase is methanol and an aqueous solution containing 0.1% formic acid, with gradient elution; Arecoline N-oxide and arecoline are separated using a Welch SCX chromatographic column with a particle size of 5 μm, an inner diameter of 4.6 mm, and a column length of 250 mm. The mobile phase is acetonitrile and ammonium formate solution, with isocratic elution; Then perform mass spectrometry detection. Among them, isoquercetin, baicalin, and magnolol are detected using the negative ion mode, and arecoline N-oxide and arecoline are detected using the positive ion mode; Draw a standard curve based on the detected peak area ratio to obtain a regression equation, and finally calculate the concentration of each component of the natural product composition in the plasma sample to be measured.

2. The method according to claim 1, wherein The stable internal standard solution is chrysin and matrine; Chrysin is used as the internal standard for detecting baicalin, isoquercetin, and magnolol; Matrine is used as the internal standard for detecting arecoline and arecoline N-oxide.

3. The method according to claim 1, wherein The specific method of the pretreatment includes: Take 100 μL of plasma, add 10 μL of 120 mg / mL vitamin C solution, 10 μL of 5% formic acid solution, and 10 μL of internal standard, shake for 3 min, add 400 μL of pre-cooled methanol, shake for 3 min, centrifuge at 13400 rpm for 15 min, take the supernatant and dry it by nitrogen blowing, add 100 μL of 50% methanol water for reconstitution, and shake evenly for sample injection.

4. The method according to claim 1, wherein, The concentration of the internal standard chrysin is 500 ng / mL, and the concentration of the internal standard matrine is 1000 ng / mL.

5. The method according to claim 1, wherein, The chromatographic column 1 used for liquid chromatography separation is Agilent Zorbax XDB-C18 with a particle size of 5 μm, an inner diameter of 2.1 mm, and a column length of 50 mm. The chromatographic column 2 is Welch SCX with a particle size of 5 μm, an inner diameter of 4.6 mm, and a column length of 250 mm; The mobile phase 1 used is: Phase A: aqueous solution containing 0.1% formic acid, Phase B: methanol; The mobile phase 2 is: Phase A: aqueous solution containing 0.2% formic acid and adjust the pH to 3.8 with ammonia water, Phase B: acetonitrile; Among them, chromatographic column 1 is used to detect isoquercetin, baicalin, and magnolol, and chromatographic column 2 is used to detect arecoline and arecoline N-oxide.

6. The method according to claim 5, wherein, The elution conditions for liquid chromatography separation are as follows. Chromatographic condition 1 is used to detect isoquercetin, baicalin, and magnolol, and chromatographic condition 2 is used to detect arecoline and arecoline N-oxide; Chromatographic condition 1 is: Gradient elution: 0 min 35% B, 8 min 70% B, 13 min 70% B, 13.1 min 35% B; Elution time: 17.1 min; Sample volume: 10 μL; Column temperature: 40 °C; The liquid chromatography separation and elution conditions 2 are as follows: Isocratic elution: 0 - 17 min: 45% B; Elution time: 17 min; Sample volume: 10 μL; Column temperature: 30 °C.

7. The method according to claim 1, wherein, The mass spectrometry conditions for mass spectrometry detection are as follows. Mass spectrometry condition 1 is used to detect isoquercetin, baicalin, and magnolol, and mass spectrometry condition 2 is used to detect arecoline and arecaidine; Mass spectrometry condition 1 is: Electrospray ionization source ESI; Dry gas flow rate is 10.0 L / min; Dry gas temperature is 350 °C; Sprayer pressure is 35.0 psig; Capillary voltage is 3000 V; Negative ion detection mode; Mass spectrometry detection condition 2 is: Electrospray ionization source ESI; Dry gas flow rate is 10.0 L / min; Dry gas temperature is 350 °C; Sprayer pressure is 35.0 psig; Capillary voltage is 3000 V; Positive ion detection mode.

8. The method according to claim 7, wherein, The ion pairs for quantitative analysis are used for mass spectrometry detection. The said ion pairs for quantitative analysis are: The mass-to-charge ratios m / z generated by magnolol, baicalin, isoquercetin, and chrysin are 265, 445, 463, and 253 respectively; the mass-to-charge ratios m / z generated by arecoline, arecaidine, and sophocarpine are 156, 142, and 247 respectively.

9. According to the method described in claim 1, wherein the standard curve is specifically prepared as follows: Using the theoretical concentration of the sample to be measured as the abscissa and the peak area ratio of the sample to be measured to the internal standard as the ordinate, a linear regression equation is obtained through regression analysis calculation.

10. The application of the method described in any one of claims 1 to 9 in analyzing the natural product composition in plasma samples; The said natural product composition is used for preventing and / or treating diseases such as acute respiratory distress syndrome, viral pneumonia, cerebral ischemia reperfusion, migraine, depression, spleen and stomach diseases, and lipid peroxidation.

Images