Qualitative and quantitative method of stilbene glucoside and application of stilbene glucoside in pharmacokinetics

Abstract

The invention discloses a qualitative and quantitative method of stilbene glucoside and application of the stilbene glucoside in pharmacokinetics. According to the qualitative and quantitative method, a to-be-detected sample is detected by adopting a UPLC-Q-TOF-MS (Ultra Performance Liquid Chromatography-Quantitative Time of Flight Mass Spectrometry) technology, through SCIEX and Analyst analysis software, an m / z 405.12 ([M-H] <->) most abundant fragmentation ion peak of TSG at 6.050 min is selected for quantification, and the TSG is qualitatively determined by matching characteristic ion fragments of TSG through MS / MS (Mass Spectrometry / Mass Spectrometry). The method provides a rapid determination method which is relatively good in precision and accuracy, relatively low in quantification lower limit, good in specificity, negligible in matrix effect and standard in extraction recovery rate for TSG in-vivo detection.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis and traditional Chinese medicine preparation detection, and in particular to a qualitative and quantitative method for stilbene glycosides and an application thereof in pharmacokinetics. Background Art

[0002] Tetrahydroxy Stilbene Glycoside (TSG), as the main neuroprotective component in Polygonum multiflorum, has multiple pharmacological activities, including cardiovascular protection, neuroprotection, anti-aging, anti-diabetes, anti-inflammatory, etc. However, due to its structural instability, biological matrix background and the setting of detection parameters, the qualitative and quantitative analysis of TSG is challenging.

[0003] In my country, stroke has become the leading cause of death and the main cause of disability among residents. About 80% of strokes are ischemic, usually caused by thromboembolism of the cerebral aorta or its branches. Vascular occlusion leads to hypoxia and interruption of energy supply to brain tissue, resulting in irreversible tissue damage (infarction). In the ischemic penumbra surrounding the infarct core, if timely intervention can be performed within a specific treatment time window, there is still a chance to save damaged brain tissue. At present, there is an urgent need for more extensive and effective treatment options, especially highly effective neuroprotectants. However, the delivery of all central nervous system drugs and the exertion of their efficacy are hindered by the blood-brain barrier (BBB).

[0004] The BBB is a lipid barrier that isolates brain tissue from red blood cells, platelets, and exogenous compounds in the blood. TSG has a strong polarity and is easily soluble in highly polar solvents such as water, methanol, and ethanol, so it can hardly pass through the blood-brain barrier. These properties are not conducive to the development of TSG as a neuroprotectant. However, the physiological and pathological states of the body often have an impact on drug metabolism. Therefore, it will be of great significance to establish an in vivo method for detecting TSG and to conduct a comparative study of its pharmacokinetics in normal rats and stroke rats. Summary of the invention

[0005] In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide a qualitative and quantitative method for stilbene glycosides and its application in pharmacokinetics to evaluate the dynamic changes and tissue distribution of TSG in the body. The present invention uses ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technology to establish a pharmacokinetic analysis method for TSG, and combines ion chromatograms (XIC) and MS / MS spectra to quantitatively and qualitatively analyze TSG.

[0006] The specific technical solutions of the present invention are as follows:

[0007] The present invention provides a qualitative and quantitative method for stilbene glycosides, adopts UPLC-Q-TOF-MS technology to detect the sample to be tested, selects TSG at m / z 405.12 ([MH] 6.050min) through SCIEX and Analyst analysis software, and obtains the sample with the concentration of stilbene glycosides. - ) were quantified by the most abundant fragmentation ion peak, and TSG was identified by matching the characteristic ion fragments of TSG by MS / MS.

[0008] Furthermore, the characteristic ion fragments of the TSG include the parent ion [MH] - m / z = 405.12 and fragment ion [M-Glucose-H] - m / z = 243.06.

[0009] Furthermore, when UPLC-Q-TOF-MS technology was used to detect the samples to be tested: the column was PhenomixLUNA C18 (2.1×100mm, 2.6μm), the sample chamber temperature was 4°C, and the column oven temperature was 40°C; the IDA mode was adopted for qualitative analysis, and the MRM mode was adopted for quantitative analysis; mobile phase A: methanol + 0.1% formic acid; mobile phase B: 0.05% formic acid water, flow rate 0.4mL / min; Negative MS: CUR 35psi; GS150 psi; GS250 psi; ISVF 4500V, TEM 500℃; CE, -10V; DP-80V; Negative MS^2: CE-30V; DP-100V.

[0010] Further, the HPLC gradient elution program is shown in the following table.

[0011]

[0012] The present invention also provides application of the qualitative and quantitative method of diphenylethylene glycosides in pharmacokinetics.

[0013] The present invention also provides a method for analyzing the pharmacokinetics of diphenylethylene glycosides, wherein TSG is injected into the tail vein, plasma and various organ tissue samples are collected at different time points, and the method is used to analyze the dynamic changes and tissue distribution of TSG in vivo.

[0014] Furthermore, the pharmacokinetic parameters and tissue distribution levels of TSG in ischemic stroke rats and normal rats were compared, and DAS software was used to perform statistical moment fitting on the blood drug concentration-time curve and calculate the pharmacokinetic parameters.

[0015] Furthermore, the pharmacokinetic parameters include apparent distribution volume, half-life and total clearance rate.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention uses UPLC-Q-TOF-MS technology to establish a method for the qualitative and quantitative determination of TSG, and applies it to the study of the pharmacokinetics of TSG. This method provides a rapid determination method for the in vivo detection of TSG with good precision and accuracy, low limit of quantification, specificity, negligible matrix effect, and satisfactory extraction recovery rate. Pharmacokinetic parameters show that intravenous injection of TSG conforms to the open two-compartment model, and the apparent distribution volume Vd is 0.445±0.047L; t 1 / 2 It is about 3min; the total clearance CL is 0.065±0.0028. Compared with normal rats, the concentration of TSG in the brain tissue of MCAO rats was significantly increased. The results of pharmacokinetic studies showed that trace accumulation of TSG in the brain tissue of stroke rats was captured, suggesting that the pharmacokinetics of central nervous system drugs, especially, is related to the disease state of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :LC-MS spectrum of rat serum containing TSG under IDA mode and XIC spectrum of 405.12, showing the TSG peak situation.

[0019] Figure 2 : The MS / MS spectra of TSG at 6.05 and 5.13 min indicate that TSG has two isomers in vivo.

[0020] Figure 3 : MS / MS spectrum of TSG 405.12 with a peak at 6.015min, showing the characteristic ion fragment peaks of TSG.

[0021] Figure 4 : TSG standard curve, showing the linear relationship between TSG concentration and the average peak area of ​​primary MS.

[0022] Figure 5 :(A) The average plasma concentration-time distribution of TSG (n=3) and (B) the concentration of TSG in various organs 30 minutes after intravenous injection of TSG (n=5), showing the pharmacokinetic pattern of TSG in normal rats.

[0023] Figure 6 : In stroke rats, (A) the average plasma concentration-time distribution of TSG (n=3) and (B) the concentration of TSG in serum at 10, 30, and 60 min after TSG injection (n=5), showing the difference in the pharmacokinetic patterns of TSG in stroke rats and ischemic stroke rats.

[0024] Figure 7: (A) The concentration of TSG in the heart, liver, spleen, lung, kidney, and testis 30 minutes after TSG injection (n≥3) and (B) the concentration of TSG in the left and right brain regions of brain tissue of normal rats and MCAO rats 30 minutes after TSG injection (n≥3), *p<0.05, MCAO vs. Sham group, showing the difference in tissue distribution of TSG in stroke rats and ischemic stroke rats. DETAILED DESCRIPTION

[0025] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0026] Example 1

[0027] This embodiment provides a qualitative and quantitative method for diphenylethylene glycosides: a qualitative and quantitative method for TSG is established using ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technology. Detection was performed on an ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer Triple TOF 5600+ (SCIEX) instrument, the column was Phenomix LUNA C18 (2.1×100mm, 2.6μm), and the column temperature was set at 40°C. IDA mode was used for qualitative analysis, and MRM mode was used for quantitative analysis. Mobile phase A: methanol + 0.1% formic acid; mobile phase B: 0.05% formic acid water, flow rate 0.4mL / min, elution program see Table 1. Negative MS: CUR35psi; GS150 psi; GS250 psi; ISVF 4500V, TEM 500°C. CE, -10V; DP-80V. Negative MS^2: CE-30V; DP-100V. Equipped with automatic injection, injection volume is 1-2μL, sample chamber temperature is 4℃, column oven temperature is 40℃.

[0028] Table 1. HPLC gradient elution program

[0029]

[0030] The above-mentioned qualitative and quantitative methods of diphenylethylene glycosides were used to detect TSG-containing rat serum. The response and sensitivity of TSG in negative ion mode were good, especially in the background of rat plasma, without endogenous peak interference, as analyzed by SCIEX and Analyst software. The addition of formic acid to the methanol mobile phase improved the peak stability and ionization efficiency of TSG. The most abundant fragmentation ion peak of TSG at m / z 405.12 ([MH]-) at 6.050 min was selected for quantification in extracted ion chromatography (XIC) mode, see for details. Figure 1 At 5.146 min, an isomer of TSG was eluted, which was presumed to be Z-TSG. Figure 2 .

[0031] The MS / MS spectrum data of TSG is qualitative, and the characteristic ion fragments are consistent with the TSG structure: 109.0303, 119.0511, 137.0259, 149.0253, 173.0617, 201.0571, 215.0726, 225.0578, 241.0495, 242.0610, 243.0676, 243.0837, 405.1198. Under the mass spectrometry conditions, the pathways for the generation of fragment ions are speculated, see for details. Figure 3 .

[0032] Example 2

[0033] This embodiment provides the application of the method established in the embodiment in pharmacokinetics: using the method established in Example 1, the pharmacokinetics of TSG were studied in stroke rat models and normal rat models. TSG was injected through the tail vein, plasma and organ tissue samples were collected at different time points, and the dynamic changes and tissue distribution of TSG in the body were analyzed.

[0034] (1) The brief procedure of the middle cerebral artery occlusion (MCAO) surgery is as follows: SD rats were fasted for 12 hours in advance, weighed, and placed in an anesthesia chamber (anesthesia was induced by about 5% isoflurane). The rats underwent MCAO surgery, the common carotid artery (CCA) was ligated, and then a monofilament was inserted from the external carotid artery (ECA) and gently pushed to occlude the ipsilateral middle cerebral artery. After 1 hour of occlusion, reperfusion was achieved by removing the monofilament, and then reperfusion was ensured for 24 hours.

[0035] (2) Pharmacokinetic experimental design: TSG was dissolved in physiological saline to prepare a 3 mg / mL solution. After passing through a 0.22 μm filter membrane, it was administered to rats via tail vein injection at a dose of 10 mg / kg. The sham operation group underwent sham operation after anesthesia, and the drug was administered via tail vein injection 24 hours later. MCAO model rats were injected into the tail vein 24 hours after modeling. 0.5 mL of blood was collected from the eyeball at 0, 3, 6, 9, 12 (2 mL of physiological saline was supplemented by intraperitoneal injection), 20, 30, 40, 50, 60 (2 mL of physiological saline was supplemented by intraperitoneal injection), 120, and 180 minutes after tail vein injection. The heart, liver, spleen, lung, kidney, and testicles of the rats were collected 30 minutes after the blood was collected, and the brain tissue was collected at 10, 30, and 60 minutes after the blood was collected. All serum and tissues were processed within 6 hours, and the test was completed within 48 hours.

[0036] (3) Preparation of drug-containing serum: Collect the collected plasma in an anticoagulant tube containing sodium heparin, immediately invert it upside down to mix, and then centrifuge it at 4000r / min for 5min at 4°C. Take 50μL of supernatant for later use. Add 3 times the volume of methanol solution (150μL) for liquid chromatography to 50μL of serum, vortex for 30s, and mix thoroughly. Place in a refrigerator at -20°C for 1 to 3h (to improve protein precipitation efficiency) to precipitate protein. Centrifuge at 12000rpm at 4°C for 15min. Carefully take 100μL of supernatant and place it in a special liquid chromatography-mass spectrometry insert tube, and store it at -20°C for testing.

[0037] (4) Prepare quality control samples of low, medium and high concentrations of TSG at 20, 200 and 2000 ng / mL (5 μL 0.2, 2, 20 μg / mL TSG quality control standard working solution + 45 μL blank plasma), with 6 replicates for each concentration, and treat according to the serum sample pretreatment method (3) and the test conditions in Example 1. The daytime is a continuous inspection for 3 days on a daily basis. Calculate the peak area As of TSG, and substitute it into the standard curve of the day to obtain the measured concentration and the accuracy of the measured concentration, and calculate the relative standard deviation (RSD%) and deviation (RE%). The low, medium and high concentration samples for intra-day and inter-day inspections require RSD%≤15% and RE% within ±15%.

[0038] (5) The lower limit of quantification (LLOQ) is the lowest drug concentration that can meet the accuracy and precision requirements when measuring samples, that is, the lowest point in the standard curve (S / N ≥ 3), which reflects the sensitivity of the detection method. The LLOQ needs to be tested for intra-day and inter-day accuracy and precision, with 6 replicates, and the inter-day test is conducted for 3 consecutive days on the same basis. The intra-day and inter-day accuracy and precision requirements are RSD% ≤ 20%, and RE% must be within ± 20%. Prepare 6 standard drug-containing plasmas with a TSG concentration of 20 ng / mL as the lower limit of quantification (5 μL 0.2 μg / mL TSG quality control standard working solution + 45 μL blank plasma), and treat them according to the serum sample pretreatment method (3).

[0039] (6) Accurately take 50 μL of blank plasma from 6 different sources of rats, treat according to the serum sample pretreatment method (3) and test under the same test conditions to obtain chromatogram A; take 5 μL of 2000 ng / mL TSG standard solution and add 45 μL of blank rat plasma to prepare 200 ng / mL drug-containing serum sample, and perform the same operation as above to obtain chromatogram B. Take 50 μL of plasma sample 10 minutes after administration of rats, and perform the same operation as above to obtain chromatogram C. It is required that each sample has no interfering peak at the retention time of the reference substance or the peak area of ​​the interfering peak is not greater than 20% of the peak area of ​​the quantitative lower limit sample.

[0040] (7) Matrix effects are caused by other endogenous substances (e.g., salts, amines, fatty acids, glycerides, etc.) that flow out of the spray needle together with the analyte. These substances and the analyte can affect the atomization, volatilization, fragmentation, chemical reaction and charging process of the analyte, resulting in a decrease (ion suppression) or increase (ion enhancement) in the number of ions entering the mass spectrometer, thereby affecting the reliability and accuracy of the quantitative results. When using liquid mass spectrometry, it is necessary to investigate the matrix effect. Take 45 μL of blank rat plasma from 6 sources, add 150 μL of methanol, and perform the remaining operations according to the serum sample pretreatment method (3). Centrifuge to obtain the supernatant. Gently add 5 μL of low, medium, and high concentration (200, 2000, 10000 ng / mL) quality control standard working solution (consistent with the concentration of the pre-dosing method) to the supernatant of the sample in the original tube, respectively. Gently blow and mix, centrifuge again, and remove the supernatant into the liquid mass spectrometry dedicated inner tube. Six samples were prepared in parallel at each concentration level to calculate the peak area in the presence of matrix (QC-B); pure water was used to replace blank rat plasma to calculate the corresponding peak area without matrix (QC-C). The matrix effect of TSG was calculated as (QC-B) / (QC-C), where the RSD of the matrix effect calculated from the six plasmas should not be greater than 15%.

[0041] (8) Same as matrix effect, take 45 μL of blank rat plasma from 6 sources, add 5 μL of low, medium and high concentration (200, 2000, 10000 ng / mL) quality control standard working solution respectively. Then perform protein precipitation operation, the steps are as shown in (3). Prepare 6 samples in parallel at each concentration level, and calculate the peak area in the presence of matrix (QC-A). Calculate the extraction recovery rate of TSG = (QC-A) / (QC-B). The RSD of the extraction recovery calculated from 6 plasma samples shall not be greater than 15%.

[0042] (9) Take 5 μL of TSG standard working solution with concentrations of 50, 100, 200, 500, 1000, 2000, 4000, 6000, 8000, and 10000 ng / mL, add 45 μL of rat blank plasma, and prepare standard plasma samples with concentrations of 5, 10, 20, 50, 100, 200, 400, 600, 800, and 1000 ng / mL. Perform the serum sample pretreatment method (3) and take the supernatant for analysis. Perform linear regression with the MS peak area of ​​the analyte as the y-axis and the concentration of the analyte as the x-axis. The linear equation obtained is the standard curve. The standard curve needs to be re-measured for each test, and the concentration is adjusted accordingly as needed.

[0043] Validation of the pharmacokinetic method: The precision, accuracy, lower limit of quantification, specificity, matrix effect and extraction recovery of the method were investigated. The intra-day RSD% of TSG was <15%, and the RE% was <15%, while the RSD% between 3 days was >15%, and the RE% was >15%, indicating that the established detection method has good intra-day precision and high accuracy, and can accurately determine TSG in plasma. However, due to the poor stability of TSG, TSG samples are easily degraded in the plasma environment, so it is recommended that the samples must be processed and tested within 24 hours. TSG has a quantitative lower limit of 20ng / mL, with high sensitivity and specificity. A standard curve of TSG quality control samples can be established in the matrix background for calibration to eliminate the influence of matrix effects. For details, see Figure 4 When the TSG concentration was high (200, 1000 ng / mL), the matrix effect was almost negligible, and the loss was occasionally large but still within a controllable range.

[0044] Pharmacokinetic data analysis: DAS software was used to perform statistical moment fitting on the blood drug concentration-time curve and calculate pharmacokinetic parameters, including apparent distribution volume (Vd), half-life (t 1 / 2 ) and total clearance (CL). The maximum plasma concentration Cmax of TSG 10mg / kg intravenous injection is 22.70±2.92μg / mL; AUC(0-t) (the area under the concentration-time curve from zero to a certain time t=10) is 125.42±18.17; AUC(0-∞) (the area under the concentration-time curve from zero to a certain time ∞) is 153.8±6.678; the pharmacokinetics of TSG conforms to the one-compartment model, and the apparent distribution volume Vd is 0.445±0.047L; t 1 / 2 About 3 minutes; the total clearance rate CL is 0.065±0.0028. 30 minutes after administration, the concentration of TSG in lung tissue is very high 36.52±7.561μg / mL, which is about 200 times the drug concentration in blood at the same time (0.1837±0.04317μg / mL), which has not been found in previous related literature; the concentration of TSG in heart tissue is 0.1280±0.04697μg / mL, in liver 0.06730±0.03095μg / mL, in spleen 0.3300±0.1450μg / mL, in kidney 0.2162±0.09874μg / mL, in brain tissue 0.03304±0.01237μg / mL, and in testis 0.03076±0.01011μg / mL. See for details. Figure 5 .

[0045] Table 2. Pharmacokinetic method validation precision and accuracy.

[0046]

[0047] Table 3. Determination of the lower limit of quantification for TSG.

[0048]

[0049] Comparative analysis: TSG metabolism in MCAO stroke rats and control rats showed that there was no significant difference in blood drug concentration between the two. There was no significant difference in TSG in serum between normal rats and MCAO rats at three time points of 10, 30, and 60 minutes. When TSG was intravenously injected for 30 minutes, there was no significant difference in the distribution concentration of TSG in various organs; but in the brain tissue, it was observed that the concentration of TSG in the right brain (ischemic area) of MCAO rats was significantly increased, which was not only higher than the concentration in the brain area of ​​sham rats, but also higher than the concentration in the left brain area of ​​the rats themselves, indicating that TSG accumulated in the brain ischemic injury area of ​​stroke rats, and the drug concentration was at a peak around 30 minutes, providing evidence for its ability to cross the blood-brain barrier to alleviate brain I / R injury. This situation is considered to be attributed to the change in the permeability of the blood-brain barrier during the I / R process, which causes TSG to be distributed from the blood (30 minutes, average blood concentration 205.21 ng / mL) to the brain tissue (30 minutes, average tissue concentration 123.025 ng / mL). See for details. Figure 6 and 7 .

[0050] Table 4. Matrix effects of various TSG concentrations.

[0051]

[0052] Table 5. Extraction recovery of TSG at various concentrations.

[0053]

[0054] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A qualitative and quantitative method for stilbene glycosides, characterized in that: UPLC-Q-TOF-MS technology was used to detect the samples. Through SCIEX and Analyst analysis software, TSG was selected at 6.050min m / z 405.12 ([MH] - ) were quantified by the most abundant fragmentation ion peak, and TSG was identified by matching the characteristic ion fragments of TSG by MS / MS.

2. The method according to claim 1, characterized in that The characteristic ion fragments of TSG include parent ion [MH] - m / z = 405.12 and fragment ion [M-Glucose-H] - m / z = 243.

06.

3. The method according to claim 1, characterized in that When UPLC-Q-TOF-MS technology was used to detect the samples to be tested: the column was Phenomix LUNAC18 (2.1×100mm, 2.6μm), the sample chamber temperature was 4℃, and the column oven temperature was 40℃; the IDA mode was adopted for qualitative analysis, and the MRM mode was adopted for quantitative analysis; mobile phase A: methanol + 0.1% formic acid; mobile phase B: 0.05% formic acid water, flow rate 0.4mL / min; Negative MS: CUR 35psi; GS150 psi; GS250 psi; ISVF 4500V, TEM 500℃; CE, -10V; DP-80V; Negative MS^2: CE-30V; DP-100V.

4. The method according to claim 1, characterized in that: The HPLC gradient elution program is shown in the table below.

5. Application of the qualitative and quantitative method of diphenylethylene glycosides according to claim 1 in pharmacokinetics.

6. A method for analyzing the pharmacokinetics of diphenylethylene glycosides, characterized in that: TSG is injected through the tail vein, and plasma and organ tissue samples are collected at different time points. The dynamic changes and tissue distribution of TSG in the body are analyzed using the method described in claim 1.

7. The method according to claim 6, characterized in that The pharmacokinetic parameters and tissue distribution levels of TSG in ischemic stroke rats and normal rats were compared. The DAS software was used to perform statistical moment fitting on the blood concentration-time curve and calculate the pharmacokinetic parameters.

8. The method according to claim 7, characterized in that The pharmacokinetic parameters include apparent volume of distribution, half-life and total clearance.

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