CN107643341B - Method for determining active ingredients in cistanche deserticola total glycoside capsules - Google Patents
Method for determining active ingredients in cistanche deserticola total glycoside capsules Download PDFInfo
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Abstract
The invention provides a method for measuring active ingredients in a desert cistanche total glycoside capsule, which adopts ultra-high performance liquid chromatography to detect a control sample to obtain a correction factor f of verbascoside and tubular anthocyanin AActeoside/Tuliposide ACorrection factors f for acteoside and isoacteosideActeoside/IsoverbascosideCorrection factors f for acteoside and echinacosideActeoside/EchinacosideAnd then obtaining the contents of other three components through the obtained correction factor and the chromatogram of the sample to be detected, wherein the mobile phase is acetonitrile and 0.1vol% formic acid aqueous solution during the detection of the ultra-high performance liquid chromatography; the acteoside is used as an internal reference, so that the content of the angionoside A, the isoacteoside and the echinacoside obtained by calculating the obtained correction factors is accurate, and the simultaneous determination of four samples can be realized by only using one reference substance, thereby greatly saving the cost.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a method for measuring active ingredients in a cistanche total glycoside capsule.
Background
The cistanche total glycosides capsule is a single preparation prepared by taking total cistanche glycosides extracted from cistanche tubulosa as raw materials, has the effects of tonifying kidney and benefiting marrow, and strengthening brain and improving intelligence, and is clinically used for treating mild and moderate vascular dementia with the syndrome of insufficient marrow-sea. According to the research result of the chemical components of the desert cistanche total glycoside capsule, the known organic phenolic acids and the phenethyl alcohol glycosides are respectively chlorogenic acid, caffeic acid, echinacoside, verbascoside, angioside A and isoverbascoside, and a quantitative method for simultaneously measuring the phenylethanoid glycosides is established. However, the reference substance is not easy to obtain and expensive, so that the aim of realizing multi-index quality control by using a conventional external standard method is directly limited, and the method cannot be applied to production practice.
Therefore, the technical problem to be solved at present is to provide a method for reducing the cost of testing the active ingredients in the cistanche total glycoside capsules.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method for determining active ingredients in a desert cistanche total glycosides capsule, and the detection method provided by the present invention has low detection cost.
The invention provides a method for measuring active ingredients in a desert cistanche total glycoside capsule, which comprises the following steps:
1) detecting the control sample by ultra-high performance liquid chromatography to obtain correction factor f of acteoside and tubular flower glycoside AActeoside/angionoside ACorrection factors f for acteoside and isoacteosideacteoside/IsoacteosideCorrection factors f for acteoside and echinacosideActeoside/echinacoside，
The mobile phase for the ultra-high performance liquid chromatography detection is acetonitrile and 0.1vol% formic acid aqueous solution;
2) detecting the content of verbascoside in a sample to be detected of the cistanche total-glycoside capsule by adopting the same ultra-performance liquid chromatography detection conditions as the step 1), and obtaining the content of verbascoside in the sample to be detected and peak areas of tubular acteoside A, isoverbascoside and echinacoside;
3) calculating the contents of the tubular glycoside A, the isoverbascoside and the echinacoside by the correction factor obtained in the step 1), the contents of the verbascoside obtained in the step 2 and the peak areas of the tubular glycoside A, the isoverbascoside and the echinacoside.
Preferably, in the step 1) detection, the elution of the mobile phase is gradient elution.
Preferably, in the gradient elution, acetonitrile is used as a phase A, 0.1vol% formic acid water solution is used as a phase B, and the elution procedure is as follows: 0-5 min: 9-13% of A, 5-15 min: 13-15% of A, 15-22 min: 15-17% of A, 22-27 min: 17-23% of A, 27-35 min: 23-35% of A.
Preferably, the flow rate of the mobile phase in the step 1) is 0.15-0.25 mL/min.
Preferably, the column temperature detected in the step 1) is 28-32 ℃.
Preferably, the wavelength detected in the step 1) is 325-335 nm.
Preferably, the desert cistanche total glycoside capsule to-be-tested sample is prepared according to the following method:
mixing the content of the total glucosides of the desertliving cistanche capsules with methanol according to the filling quantity difference, performing ultrasonic treatment, and taking the supernatant to obtain the total glucosides of the desertliving cistanche capsules to be tested.
Preferably, the dosage ratio of the content of the desert cistanche total glycosides capsules to the methanol is 0.1 g: (300-400) mL.
Preferably, said obtained fActeoside/angionoside AThe value of (A) is preferably 1.250 to 1.350, more preferably 1.280 to 1.346, most preferably 1.346; said obtained facteoside/IsoacteosideThe value of (A) is preferably 1.250 to 1.330, more preferably 1.266 to 1.270, and most preferably 1.266; said obtained fActeoside/echinacosideThe value of (A) is preferably 1.050 to 1.200, more preferably 1.135 to 1.145, and most preferably 1.135;
the ultrasonic time is 20-40 min.
Compared with the prior art, the invention provides a method for measuring active ingredients in cistanche total glycoside capsules, which adopts ultra-high performance liquid chromatography to detect a control sample to obtain a correction factor f of verbascoside and tubular anthocyanin AActeoside/angionoside ACorrection factors f for acteoside and isoacteosideacteoside/IsoacteosideCorrection factors f for acteoside and echinacosideActeoside/echinacosideDetecting the content of verbascoside in a sample to be detected of the cistanche total-glycoside capsule by adopting the same ultra-high performance liquid chromatography detection condition as a detection control sample to obtain the content of verbascoside in the sample to be detected and peak areas of tubular acteoside A, isoverbascoside and echinacoside; then obtaining the contents of other three components through the obtained correction factor and a chromatogram of a sample to be detected, wherein the mobile phase is acetonitrile and 0.1vol% formic acid aqueous solution during the detection of the ultra-high performance liquid chromatography; the verbascoside is used as an internal reference, so that the content of the tubular acteoside A, the isoverbascoside and the echinacoside which are calculated by the obtained correction factors is accurate, and the simultaneous determination of four samples can be realized by only using one reference substance, thereby greatly saving the cost; the experimental result shows that the content of each component in the sample to be detected obtained by the detection method provided by the invention has no significant difference with the content of each component obtained by a standard curve method; in addition, the detection condition provided by the invention can be used for mixing chlorogenic acid and coffeeCaffeic acid was well isolated.
Drawings
FIG. 1 is a UPLC plot at 330nm for a mixed control solution and a test solution.
Detailed Description
The invention provides a method for measuring active ingredients in a desert cistanche total glycoside capsule, which comprises the following steps:
1) detecting the control sample by ultra-high performance liquid chromatography to obtain correction factor f of acteoside and tubular flower glycoside AActeoside/angionoside ACorrection factors f for acteoside and isoacteosideacteoside/IsoacteosideCorrection factors f for acteoside and echinacosideActeoside/echinacoside，
The mobile phase for the ultra-high performance liquid chromatography detection is acetonitrile and 0.1vol% formic acid aqueous solution;
2) detecting the content of verbascoside in a sample to be detected of the cistanche total-glycoside capsule by adopting the same ultra-performance liquid chromatography detection conditions as the step 1), and obtaining the content of verbascoside in the sample to be detected and peak areas of tubular acteoside A, isoverbascoside and echinacoside;
3) calculating the contents of the tubular glycoside A, the isoverbascoside and the echinacoside by the correction factor obtained in the step 1), the contents of the verbascoside obtained in the step 2 and the peak areas of the tubular glycoside A, the isoverbascoside and the echinacoside.
According to the invention, the correction factor f of verbascoside and tubular anthocyanin A is obtained by detecting a control sample by adopting ultra-high performance liquid chromatographyActeoside/angionoside ACorrection factors f for acteoside and isoacteosideacteoside/IsoacteosideCorrection factors f for acteoside and echinacosideActeoside/echinacoside(ii) a Preferably, the chromatographic column for detection is Agilent SB-C18RRHD; the mobile phase for detection is acetonitrile and 0.1vol% formic acid aqueous solution; the elution of the mobile phase is preferably a gradient elution in which acetonitrile is used as phase A and 0.1vol% formic acid aqueous solution is used as phase B, and the elution procedure is carried outComprises the following steps: 0-5 min: 9-13% of A, 5-15 min: 13-15%, 15-22 min: 15-17% of A, 22-27 min: 17-23% of A, 27-35 min: 23-35% of A; the flow rate of the mobile phase is preferably 0.15-0.25 mL/min, and more preferably 0.19-0.21 mL/min; the detected column temperature is preferably 28-32 ℃, and more preferably 30-31 ℃; the detection wavelength is preferably 325-335 nm, and more preferably 330 nm; the sample size is preferably 2. mu.L.
Said obtained fActeoside/angionoside AThe value of (A) is preferably 1.250 to 1.350, more preferably 1.280 to 1.346, most preferably 1.346; said obtained facteoside/IsoacteosideThe value of (A) is preferably 1.250 to 1.330, more preferably 1.266 to 1.270, and most preferably 1.266; said obtained fActeoside/echinacosideThe value of (A) is preferably 1.050 to 1.200, more preferably 1.135 to 1.145, and most preferably 1.135;
in addition, in the gradient elution, the% A refers to the volume ratio, for example, 9-13% A refers to that each 100mL of eluent contains 9-13 mL of A; vol% refers to volume percent.
According to the invention, the content of verbascoside in the to-be-detected sample of the desert cistanche total glycoside capsule is detected by adopting the same ultra-performance liquid chromatography detection condition as the step 1), so that the content of verbascoside in the to-be-detected sample, the peak area of tubular acteoside A, the peak area of isoverbascoside and the peak area of echinacoside are obtained; in the step, the detection conditions the same as those in the step 1) are the same except that the sample to be detected is different, and the other detection conditions are the same, such as the detected mobile phase, the elution mode, the detected column temperature, the flow rate of the mobile phase and the like.
The sample to be tested of the cistanche total glycosides capsule is preferably prepared according to the following method: mixing the content of the total glucosides of the desertliving cistanche capsules with methanol according to the filling quantity difference, performing ultrasonic treatment, and taking the supernatant to obtain the total glucosides of the desertliving cistanche capsules to be tested. The dosage ratio of the content to the methanol under the condition of the filling quantity difference of the cistanche total glycosides capsules is preferably 0.1 g: (300-400) mL, more preferably 0.1 g: (330-350) mL; the ultrasonic time is 20-40 min, more preferably 30-35 min, and the ultrasonic power is preferably 200-300W, more preferably 250-280W.
According to the invention, the contents of the tubular glycoside A, the isoverbascoside and the echinacoside are calculated by the correction factor obtained in the step 1) and the contents of the verbascoside obtained in the step 2 and the peak areas of the tubular glycoside A, the isoverbascoside and the echinacoside.
The invention provides a method for measuring active ingredients in a desert cistanche total glycoside capsule, which adopts ultra-high performance liquid chromatography to detect a control sample to obtain a correction factor f of verbascoside and tubular anthocyanin AActeoside/angionoside ACorrection factors f for acteoside and isoacteosideacteoside/IsoacteosideCorrection factors f for acteoside and echinacosideActeoside/echinacosideDetecting the content of verbascoside in a sample to be detected of the cistanche total-glycoside capsule by adopting the same ultra-high performance liquid chromatography detection condition as a detection control sample to obtain the content of verbascoside in the sample to be detected and peak areas of tubular acteoside A, isoverbascoside and echinacoside; then obtaining the contents of other three components through the obtained correction factor and a chromatogram of a sample to be detected, wherein the mobile phase is acetonitrile and 0.1vol% formic acid aqueous solution during the detection of the ultra-high performance liquid chromatography; the verbascoside is used as an internal reference, so that the content of the tubular acteoside A, the isoverbascoside and the echinacoside which are calculated by the obtained correction factors is accurate, and the simultaneous determination of four samples can be realized by only using one reference substance, thereby greatly saving the cost; the content of each component in the sample to be detected obtained by the detection method has no significant difference with the content of each component obtained by a standard curve method, and in addition, chlorogenic acid and caffeic acid can be well separated by the detection condition provided by the invention.
The following will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Apparatus and materials
Agilent 1290 ultra performance liquid chromatograph (DAD uv detector); a chromatographic column: agilent SB-C18RRHD(3.0×100mm，1.8μm)，Agilent SB-C18RRHT(3.0×100mm，1.8μm)，Agilent XDB-C18RRHT (3.0X 100mm, 1.8 μm). Acetonitrile, formic acid as chromatographic pure (separately packed in the inlet), water as ultrapure water, and the rest of the reagents as analytical pure.
Chlorogenic acid reference (batch: 110753-201415), caffeic acid reference (batch: 110885-200102), echinacoside reference (batch: 111670-201505) and verbascoside reference (batch: 111530-201310) are all provided by the China food and drug testing institute; the angioside A control (batch: 150629) and the isoverbascoside control (batch: 15111802) were provided by Wodofield Biotechnology GmbH. Cistanchis herba Total glycosides capsules (batch No. 1310011,1310021,1310031) are provided by Jiangsu Kangyuan pharmaceutical industry Co., Ltd.
2. Method and results
2.1 selection of chromatographic conditions
2.1.1 selection of detection wavelength
According to the experiment, the components to be detected in the test solution are scanned at the full wavelength of 190-400 nm, and the result shows that chlorogenic acid, caffeic acid, echinacoside, verbascoside, angioside A and isoverbascoside have maximum absorption peaks at the wavelength of 325-335 nm, so that 330nm is selected as the detection wavelength, and the result shows that the separation degree of each component to be detected is good under the condition of the detection wavelength.
2.1.2 selection of column temperature
For f durability investigation of different column temperatures, an Aglient1290 ultra-efficient liquid-phase chromatographic system and Agilent C are adopted18The chromatographic column respectively examines the influence of different column temperatures (25, 28, 30, 32 and 35 ℃) on each relative correction factor, and the results show that verbascoside and tubular anthocyanin A can not be completely separated under the condition of 25 ℃ and caffeic acid can not be completely separated from adjacent chromatographic peaks under the condition of 35 ℃, which indicates that the method has the effect on the column temperatureThe certain durability range is limited, and the durability is good within the range of 28-32 ℃.
2.1.3 specific detection method
The chromatographic column is Agilent SB-C18RRHD (3.0X 100mm, 1.8 μm); acetonitrile (A) -0.1% formic acid water solution (B) is used as a mobile phase; gradient elution: 0-5 min: 9-13% of A, 5-15 min: 13-15%, 15-22 min: 15-17% of A, 22-27 min: 17-23% of A, 27-35 min: 23-35%; volume flow rate: 0.2mL min-1; column temperature: 30 ℃; detection wavelength: 330 nm; sample introduction amount: 2 μ L. Under the condition, the separation degree of each component to be detected is good, the chromatogram is shown in figure 1, the UPLC diagram of the mixed reference substance solution and the test solution at 330nm in figure 1 is shown, wherein, the mixed reference substance A, the cistanche total glycoside capsule test sample, 1, chlorogenic acid 2, caffeic acid 3, echinacoside 4, verbascoside 5, tubular glycoside A6, isoverbascoside
2.2 preparation of control solutions and test solutions
2.2.1 selection of preparation method of test solution (desert cistanche Total glycosides Capsule to be tested)
The experiment examines the preparation methods of the test solution with different extraction solvents, different extraction modes, different extraction time and different solvent dosage. By examining 30% methanol, 50% methanol, 70% methanol, 100% methanol, 30% ethanol, 50% ethanol, 70% ethanol, and 100% ethanol, it was found that the peak shape obtained when 100% methanol was used as the extraction solvent was good and the content of the component to be measured was high; examining different extraction times (5min, 10min, 15min, 30min, 45min and 60min) of different extraction modes (ultrasonic and reflux) shows that the ultrasonic and reflux modes have little influence on the content, the ultrasonic is selected as the extraction mode because the ultrasonic is convenient, the content of the component to be detected increases along with the ultrasonic time, but the content changes little after 30min, so the ultrasonic is finally selected for 30 min.
2.2.2 preparation of control solutions (control)
Precisely weighing appropriate amount of chlorogenic acid, caffeic acid, verbascoside, angioside A, isoverbascoside and echinacoside, respectively adding methanol to dissolve and dilute into mixed control solution containing 7.43 μ g, 10.20 μ g, 109.46 μ g, 123.97 μ g, 210.40 μ g and 1214.24 μ g per 1mL, and shaking to obtain control sample (control).
2.2.3 preparation of test solutions
Taking about 0.15g of the content of the total glycosides of cistanche deserticola capsules under the term of filling quantity difference, precisely weighing, placing in a conical flask, precisely adding 50mL of methanol, performing ultrasonic treatment for 30min (250W, 40Hz), cooling, precisely weighing 1mL to 10mL measuring flask, adding methanol to dilute to scale, shaking up, centrifuging (12000 r.min)-1And 5min), taking the supernatant to obtain the desert cistanche total glycoside capsule sample to be tested.
2.3 Linear relationship inspection
Precisely sucking mixed reference substance solutions 0.25mL, 0.5 mL, 1.0 mL, 2.0 mL, 4.0 mL and 10.0mL respectively, placing in a 10mL measuring flask, adding methanol to dilute to scale, shaking uniformly, precisely sucking the solutions 2 μ L respectively, injecting into a super-high performance liquid chromatograph, measuring, and performing linear regression with reference mass concentration as abscissa (X) and peak area integral value as ordinate (Y) to obtain a regression equation: chlorogenic acid: 26.11X +0.07223, r20.99998, linear range: 0.1858-7.4315 μ g/mL-1(ii) a Caffeic acid: Y46.899X +2.2435, r20.99998, linear range: 0.2550-10.2000 μ g/mL-1(ii) a Verbascoside: Y16.21X +0.99338, r20.99998, linear range: 2.7364-109.4550 μ g/mL-1(ii) a Angionoside A: Y12.104X-0.54241, r2Linear range 0.99995: 3.0993-123.9702 μ g/mL-1(ii) a Isoverbascoside: Y12.825X +1.8102, r2Linear range 0.99999: 5.2602-210.4064 μ g/mL-1(ii) a Echinacoside: Y14.792X-82.167, r2Linear range 0.9997: 30.3559-1214.2355 μ g/mL-1。
2.4 precision test
Accurately sucking mixed reference solution with high, medium and low concentrations, continuously sampling for 6 times, and recording peak areas, wherein the RSD values of the peak areas of chlorogenic acid, caffeic acid, verbascoside, angioside A, isoverbascoside and echinacoside are 0.44%, 0.19%, 0.16%, 0.15%, 0.20% and 0.95% (high), respectively; 1.37%, 1.17%, 1.05%, 1.04%, 1.06%, 1.66% (middle); 0.87%, 0.60%, 0.58%, 0.60%, 0.74%, 0.75% (low), indicating good injection precision under these conditions.
2.5 stability test
And taking a mixed reference substance solution and a test substance solution (batch number: 1310011), injecting samples for 0h, 2h, 4h, 8h, 12h, 16h, 20h and 24h respectively, recording peak areas, and calculating the RSD value of the peak areas. The results show that the RSD values of the peak areas of chlorogenic acid, caffeic acid, verbascoside, angioside A, isoverbascoside and echinacoside in the mixed control solution are respectively 1.01%, 0.70%, 0.71%, 0.56%, 0.49% and 0.49%; the RSD values of the peak areas of chlorogenic acid, caffeic acid, verbascoside, angioside A, isoverbascoside and echinacoside in the test solution are respectively 1.01%, 1.96%, 1.82%, 1.58%, 0.89% and 1.16%, which indicates that the mixed reference solution and the test solution have better stability within 24 hours.
2.6 repeatability test
The sample solution (batch number: 1310011) is prepared into 6 parts in parallel according to the sample solution preparation method, the mass fractions of the components are measured according to the chromatographic conditions, and the RSD values are calculated, so that the RSD values of the contents of chlorogenic acid, caffeic acid, verbascoside, tubular glycoside A, isoverbascoside and echinacoside in the sample solution are respectively 2.24%, 1.44%, 1.82%, 2.04%, 0.60% and 0.44%, which shows that the method has good repeatability.
2.7 sample recovery test
Precisely weighing the content (batch number: 1310011) of the total glucosides of the desertliving cistanche capsules under the term of filling quantity difference, parallelly weighing 9 parts and dividing into 3 groups, respectively and sequentially adding low, medium and high chlorogenic acid, caffeic acid, verbascoside, tubuloside A, isoverbascoside and echinacoside reference substances with 3 different concentrations (the adding quantity of the low, medium and high concentration reference substance solution is 50%, 100% and 150% of the content of the components to be measured of the sample solution), measuring the mass fractions of the components according to the chromatographic conditions, calculating the sample adding recovery rate and the RSD value, and respectively obtaining the average sample adding recovery rates of the chlorogenic acid, the caffeic acid, the verbascoside, the tubular glucoside A, the isoverbascoside and the echinacoside of 97.34%, 100.96%, 99.29%, 103.27%, 102.41% and 98.72%; the RSD values were 1.05%, 1.97%, 1.61%, 1.30%, 1.50%, 1.75%, respectively, indicating good accuracy of the method.
Example 2
Determination of relative correction factors
1 determination of relative correction factor
Taking the mixed control solution in item 2.3 of example 1, carrying out sample injection for 2 times according to the detection method of item 2.1.3 in example 1, measuring the peak area of each component, calculating the average peak area of each component, calculating f of caffeic acid by taking chlorogenic acid as an internal reference according to a calculation formula of a relative correction factor, and calculating f of angioside A, isoverbascoside and echinacoside by taking verbascoside as an internal reference, so that f of chlorogenic acid/caffeic acid, f of verbascoside/tubular glycoside A, f of verbascoside/isoverbascoside and f of verbascoside/echinacoside are respectively 0.538, 1.346, 1.266 and 1.135; the RSD values were 2.11%, 0.28%, 0.37%, 1.65%, respectively, and the results are shown in Table 1.
TABLE 1 relative correction factor for each component
2 influence of different volume flows on f
The different volume flow rates are respectively 0.19 mL/min-1，0.20mL·min-1，0.21mL·min-1When the influence of the flow rate on each component f is examined, the results show that the RSD values are 0.39%, 1.90%, 1.48% and 1.95%, respectively, and the fine-tuning volume flow rate has no significant influence on each component f.
3 Effect of different column temperatures on f
When the column temperatures were 28 ℃, 30 ℃ and 32 ℃, respectively, the influence of the column temperatures on each component f was examined, and the results showed that the RSD values were 0.50%, 2.65%, 1.90% and 2.47%, respectively, indicating that fine adjustment of the column temperature had no significant influence on each component f.
4 Effect of different detection wavelengths on f
When the different detection wavelengths are 328nm, 330nm and 332nm respectively, the influence of the detection wavelengths on each component f is examined, and the results show that the RSD values are 2.06%, 0.71%, 1.77% and 0.00% respectively, which indicates that the fine-tuning detection wavelength has no significant influence on each component f.
5 Effect of different chromatography columns on f
Investigating different chromatographic columns (Agilent SB-C) with the same brand and same specification18RRHD(3.0×100mm，1.8μm)，Agilent SB-C18RRHT(3.0×100mm，1.8μm)，Agilent XDB-C18RRHT (3.0X 100mm, 1.8 μm)) on each component f, resulting in RSD values of 0.48%, 1.01%, 1.48%, 0.40%, respectively, indicating that these 3 columns of the same brand had no significant effect on each component f.
6 Effect of different experimenters on f
The influence of different experimenters (experimenters A, experimenters B and experimenters C) on each component f is examined, and the RSD values are respectively 1.69%, 1.79%, 0.64% and 1.09%, which shows that each component f obtained by the operation of different experimenters has no significant influence.
7 Combined Effect of various durability factors on f
Combining the above factors, the RSD values of f chlorogenic acid/caffeic acid, f verbascoside/tubulin A, f verbascoside/isoverbascoside, and f verbascoside/echinacoside are 1.09%, 1.69%, 1.58%, and 1.88%, respectively, and the results are shown in Table 2.
TABLE 2 Combined Effect of various factors on relative correction factors
Example 3
Location of chromatographic peak of component to be measured
The analyte was detected according to the method of example 1, part 2.1.3
By adopting the relative retention time to position the chromatographic peak of the component to be detected, the average relative retention time of caffeic acid, angionoside A, isoverbascoside and echinacoside is respectively 0.790, 0.987, 0.928 and 1.603, and the RSD value is respectively 0.96%, 0.21%, 0.38% and 0.45%.
Example 4
Comparison of measurement results of QAMS method and standard curve method
The method comprises the steps of detecting an object to be detected according to the detection method of part 2.1.3 in the embodiment 1, simultaneously measuring multiple components of the cistanche total glycoside capsule by adopting a standard method, and quantitatively calculating by using an established QAMS method (one-measurement and multiple-evaluation method) to verify the accuracy of the QAMS method for multi-index quality evaluation in the cistanche total glycoside capsule, wherein the result is shown in a table 3, the quantity of each component of the phenylethanoid glycosides measured by the two methods has no significant difference, and the Relative Deviation (RD) is less than 5%, which indicates that the established method has reliability.
Table 3 shows the results of assay of angioside A, isoverbascoside and echinacoside by the koji method (a) and QAMS method (b)
The results in table 3 show that, when the results obtained by the standard curve method and the QAMS method are compared, the content deviation of each ingredient of the phenylethanoid glycosides, namely tubular glycoside a, isoverbascoside and echinacoside, is less than 5%, and the content difference between the contents of the verbascoside and the echinacoside is large, so that the result deviation obtained by the two methods is slightly larger than that obtained by the other two ingredients, but is within the allowable deviation range, which indicates that the QAMS method established in the experiment can reliably determine the content of the phenylethanoid glycosides in the cistanche total glycoside capsules.
And the comparison of the results of the two methods for measuring the organic phenolic acid components
For organic phenolic acid components caffeic acid and chlorogenic acid, although other feasibility researches of the QAMS method meet the requirements, the content results measured by the two methods have large deviation. The possible reasons for analyzing the content of the caffeic acid and the chlorogenic acid in the cistanche total glycoside capsules are low, and the structure difference is generated, so that the ultraviolet absorption has certain difference due to the difference of the structures of the caffeic acid and the chlorogenic acid in a quinic acid structure, and the content results of the QAMS method and the regression curve method are different. Therefore, from the viewpoint of the accuracy of content measurement, the content measurement of chlorogenic acid and caffeic acid in the cistanche total glycoside capsules is recommended to be carried out by adopting an external standard method or a linear regression equation method.
The research adopts UPLC ultra-performance liquid chromatography to simultaneously determine the multi-component content of the cistanche total glycosides capsule, thereby greatly saving the analysis time and the dosage of the mobile phase solvent; the QAMS method is applied to the multi-index quality evaluation mode of the cistanche total glycoside capsule for the first time, and feasibility research is carried out on the established QAMS method for simultaneously measuring various components, and the result shows that the QAMS method shows great advantages under the conditions of shortage of reference substances and high detection cost of the multi-index components, thereby proving that the QAMS method has good application prospect in the multi-index quality control and evaluation mode of the traditional Chinese medicine.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (7)
1. A method for determining active ingredients in cistanche deserticola total glycoside capsules comprises the following steps:
1) detecting control sample by ultra-high performance liquid chromatography to obtain correction factor of acteoside and tubular flower glycoside AfCalycoside/angionoside A, acteoside and isoacteoside correction factorfCalycoside/Isoverbascoside, acteoside and echinacoside correction factorfThe mobile phase for the ultra-high performance liquid chromatography detection is acetonitrile and 0.1vol% formic acid water solution;
the above-mentionedfThe value of verbascoside/angioside A is 1.250-1.350;
the above-mentionedfThe verbascoside/isoverbascoside value is 1.250-1.330;
the above-mentionedfThe value of verbascoside/echinacoside is 1.050-1.200;
the elution of the mobile phase is gradient elution; in the gradient elution, acetonitrile is used as a phase A, 0.1vol% formic acid water solution is used as a phase B, and the elution procedure is as follows: 0-5 min: 9-13% of A, 5-15 min: 13-15% of A, 15-22 min: 15-17% of A, 22-27 min: 17-23% of A, 27-35 min: 23-35% of A; the column temperature detected in the step 1) is 28-32 ℃; the wavelength detected in the step 1) is 325-335 nm;
the chromatographic column is as follows: c18 ，3.0×100mm，1.8 μm；
2) Detecting the content of verbascoside in a sample to be detected of the cistanche total-glycoside capsule by adopting the same ultra-performance liquid chromatography detection conditions as the step 1), and obtaining the content of verbascoside in the sample to be detected and peak areas of tubular acteoside A, isoverbascoside and echinacoside;
3) calculating the contents of the tubular glycoside A, the isoverbascoside and the echinacoside according to the correction factor obtained in the step 1), the contents of the verbascoside obtained in the step 2 and peak areas of the tubular glycoside A, the isoverbascoside and the echinacoside;
the chlorogenic acid and the caffeic acid in the cistanche deserticola total glycoside capsule are subjected to content determination by adopting an external standard method or a linear regression equation method.
2. The method for determining active ingredients in desert cistanche glycosides capsules as claimed in claim 1, wherein the flow rate of the mobile phase in step 1) is 0.15-0.25 mL/min.
3. The method for determining the active ingredients in the desert cistanche total glycosides capsule as claimed in claim 1, wherein the desert cistanche total glycosides capsule to-be-tested sample is prepared according to the following method:
mixing the content of the capsule with methanol, performing ultrasonic treatment, and collecting the supernatant to obtain the sample of the capsule.
4. The method for determining active ingredients in the desert cistanche total glycosides capsule as claimed in claim 3, wherein the dosage ratio of the content of the desert cistanche total glycosides capsule to the methanol is 0.1 g: (300-400) mL.
5. The method for determining the active ingredients in the desert cistanche total glycosides capsule as claimed in claim 3, wherein the time of the ultrasound is 20-40 min.
6. The method for determining the active ingredients in the desert cistanche total glycosides capsule as claimed in claim 1, wherein the method comprises the step of measuring the active ingredients in the desert cistanche total glycosides capsulefThe verbascoside/angioside A value is 1.280-1.346; the above-mentionedfThe verbascoside/isoverbascoside value is 1.266-1.270; the above-mentionedfThe acteoside/echinacoside value is 1.135-1.145.
7. The method for determining the active ingredients in the desert cistanche total glycosides capsule as claimed in claim 1, wherein the method comprises the step of measuring the active ingredients in the desert cistanche total glycosides capsulefVerbascoside/angioside A value of 1.346; the above-mentionedfVerbascoside/isoverbascoside value of 1.266; the above-mentionedfThe verbascoside/echinacoside value was 1.135.
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