CN111272940B - Limited detection method for bear gall powder extract in phlegm-heat clearing injection and fingerprint spectrum thereof - Google Patents
Limited detection method for bear gall powder extract in phlegm-heat clearing injection and fingerprint spectrum thereof Download PDFInfo
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Abstract
The invention relates to a limit detection method of bear gall powder extract in a phlegm-heat clearing injection, wherein the bear gall powder extract comprises ursodeoxycholic acid, chenodeoxycholic acid, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid; the limit detection method is an HPLC-DAD method, and comprises the following steps: s1, preparing a reference substance solution; s2, preparing a sample solution; s3, limit detection. The fingerprint spectrum obtained by the limit detection method also comprises 4 characteristic peaks in total, wherein each peak takes ursodeoxycholic acid peak as a reference peak. The limit detection method calculates the limit detection of the bear gall powder extract by an external standard method of the peak area of the sample and the peak area of the reference substance; the similarity between the fingerprint of the sample and the reference fingerprint is calculated, so that the product quality is accurately, efficiently and comprehensively monitored, and the authenticity of the product is identified.
Description
Technical Field
The invention relates to the technical field of medicine analysis and detection, in particular to a limit detection method of bear gall powder extract in a phlegm-heat clearing injection and a fingerprint spectrum thereof.
Background
The main components of bear gall powder in the phlegm-heat clearing injection comprise ursodeoxycholic acid, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid, chenodeoxycholic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid. The main components of the raw materials bear gall powder and decoction pieces of the phlegm heat injection are tauro-ursodesoxycholic acid and taurochenoxycholic acid, and in the process of preparing the phlegm heat injection, two tauro-deoxycholic acids are hydrolyzed due to the process, and in the bear gall powder extract, the ursodesoxycholic acid and the chenodeoxycholic acid are taken as main materials, and a small amount of 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid are also produced.
In the current standard, because ursodeoxycholic acid is a main medicinal component and a characteristic component, the ursodeoxycholic acid is controlled as a content measurement item, and only the upper limit of the content of the chenodeoxycholic acid in the bear gall powder is controlled. The clinical effectiveness of two components of 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid is not reported, and the content of the 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid in the injection is increased due to the process, so that the injection and chenodeoxycholic acid are required to be controlled in a limited amount.
Therefore, ursodeoxycholic acid, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid, chenodeoxycholic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid are presented in a fingerprint, and limit detection of the ursodeoxycholic acid, the 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid is a problem to be solved by a person skilled in the art.
Disclosure of Invention
The invention aims at overcoming the defects in the prior art and provides a limit detection method and a fingerprint spectrum of bear gall powder extract in a phlegm-heat clearing injection.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides a limit detection method of bear gall powder extract in a phlegm-heat clearing injection, wherein the bear gall powder extract comprises ursodeoxycholic acid, chenodeoxycholic acid, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid; the limit detection method is an HPLC-DAD method, and comprises the following steps:
s1, preparing a reference substance solution: taking ursodeoxycholic acid reference substance, chenodeoxycholic acid reference substance, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid reference substance and a proper amount of 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid reference substance, precisely weighing, adding methanol to dissolve and dilute into mixed standard solution containing 4.0mg of ursodeoxycholic acid, 0.6mg of chenodeoxycholic acid, 0.02mg of 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid and 0.06mg of 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid in each 1mL, and taking the mixed standard solution as a reference substance solution;
s2, preparing a sample solution: providing different batches of hot phlegm injection as sample solutions;
s3, limit detection: respectively precisely sucking the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph for diode array scanning limit detection, wherein chromatographic determination conditions are as follows: porosill 120EC-C18 (2.7 μm,0.46×5 cm) was used as the column; acetonitrile-0.1% phosphoric acid is adopted as a mobile phase, gradient elution is carried out, and the elution time and the volume of the mobile phase are respectively as follows:
0min, 20% acetonitrile, 80% phosphoric acid, 0.1%;
13min, 38% acetonitrile, 62% phosphoric acid 0.1%;
30min, 50% acetonitrile, 50% 0.1% phosphoric acid;
35min, acetonitrile 50%,0.1% phosphoric acid 50%;
35.1min, 20% acetonitrile, 80% 0.1% phosphoric acid;
40min, 20% acetonitrile, 0.1% phosphoric acid 80%.
Preferably, each 1mL of the phlegm heat clear injection contains 3.5-6.4 mg of ursodeoxycholic acid; contains chenodeoxycholic acid, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid with a total content of not more than 1.4mg.
Preferably, the chromatographic assay conditions further comprise: the column temperature is 25-40 ℃.
Preferably, the chromatographic assay conditions further comprise: the detection wavelength is 200-400 nm.
Further preferably, the detection wavelength is 203nm.
Preferably, the chromatographic assay conditions further comprise: the flow rate is 0.8-1.2 mL/min.
Preferably, the chromatographic assay conditions further comprise: the sample injection amount is 10-30 mu L.
Further preferably, the sample injection amount is 10. Mu.L.
The second aspect of the present invention provides a fingerprint obtained by the limit detection method as described above, wherein the fingerprint has 4 characteristic peaks, and the relative retention time and the relative peak area of each peak using ursodeoxycholic acid peak as a reference peak are as follows:
relative retention time: the peak of 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid is 1.128-1.129; chenodeoxycholic acid peak is 1.377-1.378; peak of 7α -hydroxy-3-oxo-5β -cholanic acid is 1.442-1.444;
relative peak area: the peak of 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid is 0.086-0.087; chenodeoxycholic acid peak is 0.184-0.185; the peak of 7alpha-hydroxy-3-oxo-5beta-cholanic acid is 0.070-0.071.
Compared with the prior art, the invention has the following technical effects:
the limit detection method has the advantages of simple pretreatment method for the test sample, simple operation, stable result, good reproducibility, high precision and the like; the fingerprint spectrum has good separation effect of each characteristic peak, can reflect the quality information of the product more comprehensively, and is beneficial to monitoring the quality of the product comprehensively and identifying the authenticity of the product; the limit detection method calculates the limit detection of the bear gall powder extract by an external standard method of the peak area of the sample and the peak area of the reference substance; the similarity between the fingerprint of the sample and the reference fingerprint is calculated, so that the product quality is accurately, efficiently and comprehensively monitored, and the authenticity of the product is identified.
Drawings
FIG. 1 is a fingerprint of bear gall powder extract in the injection for clearing phlegm heat, obtained in example 1;
wherein, peak 1 is ursodeoxycholic acid; peak 2 is 3α -hydroxy-7-oxo-5β -cholanic acid; peak 3 is chenodeoxycholic acid; peak 4 is 7α -hydroxy-3-oxo-5β -cholanic acid;
FIG. 2 is a superimposed graph of the fingerprint of bear gall powder extract in the phlegm-heat clearing injection obtained in example 1;
FIG. 3 is a DAD spectrum of a bear gall powder extract control in the phlegm-heat clearing injection obtained in example 5;
wherein, FIG. 3a is a DAD spectrum of ursodeoxycholic acid; FIG. 3b is a spectrum of 3 a-hydroxy-7-oxo-5 β -cholanic acid DAD; FIG. 3c is a DAD spectrum of chenodeoxycholic acid; FIG. 3d is a spectrum of 7α -hydroxy-3-oxo-5β -cholanic acid DAD;
FIG. 4 is a fingerprint spectrum of the example 5 sputum heat clearing injection obtained with a sample injection amount of 10. Mu.L of bear gall powder extract.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
The invention is further described below with reference to the drawings and specific examples, which are not intended to be limiting.
Example 1
The limit detection method of the bear gall powder extract in the phlegm-heat clearing injection is characterized by comprising ursodeoxycholic acid, chenodeoxycholic acid, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid; the limit detection method is an HPLC-DAD method, and comprises the following steps:
s1, preparing a reference substance solution: taking ursodeoxycholic acid reference substance, chenodeoxycholic acid reference substance, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid reference substance and a proper amount of 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid reference substance, precisely weighing, adding methanol to dissolve and dilute into mixed standard solution containing 4.0mg of ursodeoxycholic acid, 0.6mg of chenodeoxycholic acid, 0.02mg of 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid and 0.06mg of 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid in each 1mL, and taking the mixed standard solution as a reference substance solution;
s2, preparing a sample solution: providing different batches of hot phlegm injection as sample solutions;
s3, limit detection: respectively precisely sucking the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph for diode array scanning limit detection, wherein chromatographic determination conditions are as follows: porosill 120EC-C18 (2.7 μm,0.46×5 cm) was used as the column; acetonitrile-0.1% phosphoric acid is adopted as a mobile phase, gradient elution is carried out, and the elution time and the volume of the mobile phase are respectively as follows:
0min, 20% acetonitrile, 80% phosphoric acid, 0.1%;
13min, 38% acetonitrile, 62% phosphoric acid 0.1%;
30min, 50% acetonitrile, 50% 0.1% phosphoric acid;
35min, acetonitrile 50%,0.1% phosphoric acid 50%;
35.1min, 20% acetonitrile, 80% 0.1% phosphoric acid;
40min, 20% acetonitrile, 80% phosphoric acid, 0.1%;
preferably, each 1mL of the phlegm heat clear injection contains 3.5-6.4 mg of ursodeoxycholic acid; contains chenodeoxycholic acid, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid with a total content of not more than 1.4mg.
Preferably, the chromatographic assay conditions further comprise: the column temperature was 35 ℃.
Preferably, the chromatographic assay conditions further comprise: the detection wavelength is 203nm.
Preferably, the chromatographic assay conditions further comprise: the flow rate was 1.0mL/min.
Preferably, the chromatographic assay conditions further comprise: the sample injection amount is 10 mu L.
Taking 33 batches of samples for measurement, recording the retention time and the peak area of each common peak, and taking the retention time and the peak area of ursodeoxycholic acid as references, and converting the relative retention time and the relative peak area of each common peak, so that the RSD of each common peak relative retention time is 0-0.2%.
Table 1 sample relative retention time calculation results
TABLE 2 sample concentration calculation results
Example 2
Taking ursodeoxycholic acid reference substance, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid reference substance, chenodeoxycholic acid reference substance and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid reference substance, precisely weighing, adding methanol to dissolve and dilute into a mixed solution containing 8.0mg of ursodeoxycholic acid, 0.04mg of 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid, 1.2mg of chenodeoxycholic acid and 0.12mg of 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid in each 1mL, and shaking uniformly to obtain the mixed reference substance solution. And precisely sucking the mixed reference substance solutions 1, 2, 5, 10 and 15mL to 20mL of measuring flask respectively to obtain a series of standard solutions, drawing a standard curve by taking the reference substance concentration (mg/mL) as an abscissa X and the peak area as an ordinate Y, and calculating a regression equation. The result shows that the peak area of ursodeoxycholic acid in the range of 0.365792-7.31584 mg/mL has good linear relation with the concentration of the reference substance; the peak area of the 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid in the range of 0.003595-0.0719 mg/mL has good linear relation with the concentration of the reference substance; the peak area of chenodeoxycholic acid in the range of 0.101787-2.03574 mg/mL has good linear relation with the concentration of the reference substance; the peak area of 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid in the range of 0.00574-0.1148 mg/mL has good linear relation with the concentration of the reference substance.
TABLE 3 examination of the linear relationship between ursodeoxycholic acid content measurement
Table 4 3 linear relationship examination of alpha-hydroxy-7-oxo-5 beta-cholanic acid content measurement
Table 5 linear relationship investigation of chenodeoxycholic acid content determination
Table 6 7 linear relationship examination of alpha-hydroxy-3-oxo-5 beta-cholanic acid content measurement
Precisely sucking 10 mu L of the sample solution, continuously sampling and measuring for 6 times, recording the retention time and the peak area of each common peak, and converting the relative retention time and the relative peak area of each common peak by taking the retention time and the peak area of ursodeoxycholic acid as references, wherein the RSD of the relative retention time of each common peak is 0.01-0.02%; the relative peak area RSD is 0.23 to 0.34%. The result shows that the method has high precision.
TABLE 7 calculation of relative retention time for precision experiments
TABLE 8 calculation of relative peak area for precision test
Precisely sucking 10 mu L of each sample solution, measuring in six parts according to a law, recording the retention time and the peak area of each common peak, taking the retention time and the peak area of ursodeoxycholic acid as references, and converting the relative retention time and the relative peak area of each common peak, wherein the RSD of each common peak relative retention time is 0.0%; the relative peak area RSD is 0.0 to 2.5%. The result shows that the method has good reproducibility.
TABLE 9 relative retention time calculation results of repeatability tests
TABLE 10 relative peak area calculation results for repeatability test
Sample solutions were taken and analyzed at 0, 4, 8, 12, 16, 20, 24 hours, peak areas were recorded, and RSD values for the peak areas were calculated, showing that ursodeoxycholic acid, 3 a-hydroxy-7-oxo-5 β -cholanic acid, chenodeoxycholic acid, and 7a-hydroxy-3-oxo-5 β -cholanic acid control solutions and sample solutions were substantially stable within 0 to 24 hours.
TABLE 11 stability test peak area results for control solutions
TABLE 12 test sample solution stability test peak area results
Example 3
Investigation of column temperature: the sample solutions were taken and measured at a column temperature of 25℃and 30℃and 35℃and 40℃respectively, and as a result, the separation of the common peaks and the peak shape were not adversely affected by the change in the column temperature.
Inspection of detection wavelength: the diode array scanning is carried out on ursodeoxycholic acid, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid, chenodeoxycholic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid reference chromatograms and sample chromatograms, and the peaks of ursodeoxycholic acid, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid, chenodeoxycholic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid in the chromatograms of the samples, and the results show that, as the ursodeoxycholic acid, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid, chenodeoxycholic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid have no characteristic maximum absorption wavelength between 200 nm and 400nm, 203nm with larger absorption and less interference peak in the ultraviolet absorption spectrogram is adopted as detection wavelength, as shown in fig. 3.
Investigation of flow rate: the sample solutions were taken and measured at flow rates of 0.8mL/min, 1.0mL/min and 1.2mL/min, respectively, and as a result, the change in flow rate did not adversely affect the separation condition and peak shape of each common peak.
Investigation of sample injection amount: sample solutions were taken and measured at sample amounts of 10. Mu.L, 20. Mu.L and 30. Mu.L, respectively, and as a result, the peak shape and the separation degree of each peak of the fingerprint were optimal at the sample amounts of 10. Mu.L, as shown in FIG. 4.
In conclusion, the limit detection method has the advantages of simple pretreatment method for the test sample, simple operation, stable result, good reproducibility, high precision and the like; the fingerprint spectrum has good separation effect of each characteristic peak, can comprehensively reflect the quality information of the product, and is beneficial to comprehensively monitoring the quality of the product and identifying the authenticity of the product; the limit detection method of the invention calculates the limit detection of bear gall powder extract by using an external standard method of the peak area of the fingerprint of the sample to be detected and the peak area of the linear equation reference substance; the similarity between the fingerprint of the sample and the reference fingerprint is calculated, so that the product quality is accurately, efficiently and comprehensively monitored, and the authenticity of the product is identified.
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, and it will be appreciated by those skilled in the art that equivalent substitutions and obvious variations may be made using the description and illustrations of the present invention, and are intended to be included within the scope of the present invention.
Claims (7)
1. A limit detection method of bear gall powder extract in a phlegm-heat clearing injection is characterized in that the bear gall powder extract comprises ursodeoxycholic acid, chenodeoxycholic acid, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid; the limit detection method is an HPLC-DAD method, and comprises the following steps:
s1, preparing a reference substance solution: taking ursodeoxycholic acid reference substance, chenodeoxycholic acid reference substance, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid reference substance and a proper amount of 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid reference substance, precisely weighing, adding methanol to dissolve and dilute into mixed standard solution containing 4.0mg of ursodeoxycholic acid, 0.6mg of chenodeoxycholic acid, 0.02mg of 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid and 0.06mg of 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid in each 1mL, and taking the mixed standard solution as a reference substance solution;
s2, preparing a sample solution: providing different batches of hot phlegm injection as sample solutions;
s3, limit detection: respectively precisely sucking the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph for diode array scanning limit detection, wherein chromatographic determination conditions are as follows: porosill 120EC-C18,2.7 μm, 0.46X 5cm was used as column; acetonitrile-0.1% phosphoric acid is adopted as a mobile phase, gradient elution is carried out, and the elution time and the volume of the mobile phase are respectively as follows:
0min, 20% acetonitrile, 80% phosphoric acid, 0.1%;
13min, 38% acetonitrile, 62% phosphoric acid 0.1%;
30min, 50% acetonitrile, 50% 0.1% phosphoric acid;
35min, acetonitrile 50%,0.1% phosphoric acid 50%;
35.1min, 20% acetonitrile, 80% 0.1% phosphoric acid;
40min, 20% acetonitrile, 80% phosphoric acid, 0.1%;
each 1mL of the phlegm heat clearing injection contains 3.5-6.4 mg of ursodeoxycholic acid; contains chenodeoxycholic acid, 3 alpha-hydroxy-7-oxo-5 beta-cholanic acid and 7 alpha-hydroxy-3-oxo-5 beta-cholanic acid with a total content of not more than 1.4mg.
2. The limit amount detection method according to claim 1, wherein the chromatographic assay conditions further comprise: the column temperature is 25-40 ℃.
3. The limit amount detection method according to claim 1, wherein the chromatographic assay conditions further comprise: the detection wavelength is 200-400 nm.
4. A limit amount detection method according to claim 3, characterized in that the detection wavelength is 203nm.
5. The limit amount detection method according to claim 1, wherein the chromatographic assay conditions further comprise: the flow rate is 0.8-1.2 mL/min.
6. The limit amount detection method according to claim 1, wherein the chromatographic assay conditions further comprise: the sample injection amount is 10-30 mu L.
7. The limit amount detection method according to claim 6, wherein the sample injection amount is 10 μl.
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