CN113281439A - Quality control detection method of Shenbao tablets - Google Patents
Quality control detection method of Shenbao tablets Download PDFInfo
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Abstract
The invention relates to a quality control detection method of Shenbao tablets, which comprises the following steps: the method comprises the following steps of content measurement, identification and fingerprint spectrum measurement of active ingredients, wherein the content measurement of the active ingredients comprises the following steps: (1) chromatographic conditions are as follows: acetonitrile is taken as a mobile phase A, and a phosphoric acid solution is taken as a mobile phase B; gradient elution; (2) preparation of control solutions: taking appropriate amount of echinacoside reference substance and icariin reference substance, and adding methanol to obtain reference substance solution; (3) preparation of a test solution: taking a proper amount of Shenbao tablets, precisely weighing, grinding, taking a proper amount of fine powder, precisely weighing, placing in a volumetric flask, adding a proper amount of an extraction solvent, carrying out ultrasonic treatment, cooling to room temperature, adding the extraction solvent to a scale, shaking uniformly, centrifuging, filtering, and taking a subsequent filtrate; (4) the determination method comprises the following steps: and respectively sucking the reference solution and the test solution, injecting into an ultra-high performance liquid chromatograph, and recording the chromatogram.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a quality control detection method of Shenbao tablets.
Background
The syndrome of kidney yang deficiency is a common syndrome and cause of disease in traditional Chinese medicine, and is encoded by SF97 "kidney yang deficiency syndrome (TM 1)" in the eleventh revision of the International Classification of diseases (ICD-11). Shenbao tablets are a Chinese patent medicine (national medicine Standard Z20080627) on the market, are representative medicines for treating kidney-yang deficiency, and are recorded in clinical common prescriptions and Chinese patent medicines (national health publishing house, 2020, first edition, page 73). The SHENBAO tablet is prepared from herba Epimedii, semen Trigonellae, fructus Rosae Laevigatae, radix rehmanniae Preparata, fructus Psoraleae, fructus Cnidii, radix Polygoni Multiflori Preparata, Cistanchis herba, fructus Lycii, semen Cuscutae, fructus Schisandrae chinensis, Rubi fructus, radix astragali, Ginseng radix Rubri, Atractylodis rhizoma, rhizoma Dioscoreae, Poria, radix Angelicae sinensis, rhizoma Ligustici Chuanxiong, fructus Foeniculi, semen plantaginis, radix Glycyrrhizae Preparata, etc., by extracting, and making into preparation; has effects of harmonizing yin and yang, warming yang, invigorating kidney, and strengthening body resistance, and can be used for treating soreness of waist and legs, listlessness, nocturia, aversion to cold, and clear and thin leucorrhea.
At present, the quality control method of Shenbao tablets adopts a quality control mode (method) of Chinese patent medicine in Chinese pharmacopoeia, namely, the quality control is carried out by a prescription, a preparation method, a preparation rule, thin-layer chromatography (TLC) identification of individual raw medicinal materials and chemical components thereof, content determination of individual components such as icariin and the like. This method is not sufficient to reflect the overall quality information of shenbao tablets and their prescriptions; the operation is repeated, for example, the same chemical component is qualitatively identified by a thin layer chromatography mode and quantitatively determined by a content determination mode. Patent application No. 202010734686.2 discloses a method for controlling the quality of volatile components of Shenbao tablets, but only relates to a small part of the components contained in Shenbao tablets.
Therefore, the invention improves the detection method of the Shenbao tablet on the basis of the prior art, and effectively improves the quality of the product.
Disclosure of Invention
The invention aims to provide a more comprehensive and efficient detection method of Shenbao tablets, and provides a quality evaluation basis for the safety and effectiveness of the Shenbao tablets.
The invention provides a detection method of Shenbao tablets, which simultaneously realizes content evaluation of a plurality of components, integral chromatogram-spectrum information and identification of a plurality of prescription raw materials through one-time measurement and analysis.
The detection method adopts Ultra Performance Liquid Chromatography (UPLC) to detect the Shenbao tablets.
The detection method comprises the following steps: the method comprises the following steps of content measurement, identification and fingerprint spectrum measurement of active ingredients, wherein the content measurement of the active ingredients comprises the following steps:
(1) chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, and 0.1% phosphoric acid solution is used as a mobile phase B; gradient elution;
(2) preparation of control solutions: taking appropriate amount of echinacoside reference substance and icariin reference substance, and adding methanol to obtain reference substance solution;
(3) preparation of a test solution: precisely weighing appropriate amount of SHENBAO tablet, grinding, adding appropriate amount of fine powder, precisely weighing, placing in a volumetric flask, adding appropriate amount of extraction solvent, ultrasonic treating, cooling to room temperature, adding extraction solvent to scale, shaking, centrifuging, filtering, and collecting filtrate;
(4) the determination method comprises the following steps: respectively sucking 1 μ l of each of the reference solution and the sample solution, injecting into an ultra high performance liquid chromatograph, and recording chromatogram.
Wherein, in the step (1), the gradient elution is carried out, and in the gradient elution process, the proportion of the mobile phase A is changed as follows: 0-2 minutes, 7% -10% acetonitrile; 2-10 minutes, 10% -14% acetonitrile; 10-18 minutes, 14% -23% acetonitrile; 18-25 minutes, 23% -34% acetonitrile; from 25 to 29 minutes, from 34 to 50 percent of acetonitrile; 29-32 minutes, 50% -80% acetonitrile; 32-36 minutes, 7% acetonitrile.
Wherein, the chromatographic column of the octadecylsilane chemically bonded silica as a filler in the step (1) is Waters CORTECS T3, 2.1 × 150mm, 1.6 μm.
Wherein, in the preparation of the test solution,
the extraction solvent is selected from: purified water, ethanol, acetonitrile or methanol; preferably 50% ethanol;
extraction time: 10-40 minutes; preferably 20 minutes;
ultrasonic conditions are as follows: 150-350W, 20-50 kHz; preferably 250W, 25 kHz;
centrifugation conditions: 3000-; preferably 5000 revolutions per minute for 5 minutes.
Wherein, the preparation of the reference substance solution in the step (2): taking appropriate amount of echinacoside and icariin as reference substances, adding methanol to obtain solutions containing 30 μ g of echinacoside and icariin per 1 ml;
wherein, the preparation of the test solution in the step (3): taking 10 Shenbao tablets, precisely weighing, grinding, taking a proper amount of fine powder (about equivalent to 1 tablet weight), precisely weighing, placing in a 50ml volumetric flask, adding an extraction solvent, carrying out ultrasonic treatment, cooling, adding the extraction solvent to a scale, shaking up, centrifuging, filtering, and taking a subsequent filtrate to obtain the Shenbao tablet.
The detection method further comprises the following steps:
(5) obtaining a comparison fingerprint spectrum: introducing the sample chromatogram of the qualified sample evaluated by the current standard into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system to synthesize a comparison fingerprint, and calibrating a characteristic peak 6, a characteristic peak 10 and a characteristic peak 12 in the chromatogram by using psoralen, icariin and osthole reference substance solutions;
(6) and (3) similarity evaluation: introducing the fingerprint of the sample to be evaluated into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and calculating the similarity, wherein the similarity between the sample and the reference fingerprint is not less than 0.9;
(7) and (3) identification: comparing the sample spectrum with the reference fingerprint spectrum, wherein the sample spectrum has reserved peaks at the corresponding positions of the characteristic peak 6, the characteristic peak 10 and the characteristic peak 12 of the reference fingerprint spectrum;
content determination: calculating the contents of echinacoside and icariin according to an external standard method of Chinese pharmacopoeia; each tablet of the product contains icariin no less than 1.2mg, and contains echinacoside no less than 1.0 mg.
In the step (5), the sample chromatogram qualified by the current standard evaluation of not less than 20 batches is processed by a traditional Chinese medicine chromatogram fingerprint similarity evaluation system to obtain the kidney essence tablet comparison fingerprint.
And (3) similarity evaluation: introducing the test sample fingerprint and the reference fingerprint into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and calculating the similarity.
The fingerprint spectrum of Shenbao tablet at least includes 12 characteristic chromatographic peaks of chlorogenic acid, echinacoside, stilbene glucoside, hyperin, psoralen, epimedin A, epimedin B, epimedin C, icariin (S), baohuoside I, cnidium fruit element, etc. its confirming method is to use corresponding reference substance to obtain chromatographic peaks with same retention time under the same condition, and at the same time to assist multi-wavelength scanning to confirm its spectrum consistency.
Wherein, the fingerprint of Shenbao tablet contains the following characteristic chromatographic peaks, and the relative retention time obtained by comparing the retention time with the retention time of an icariin chromatographic peak (S peak) is respectively: 0.20 (characteristic peak 1), 0.48 (characteristic peak 2), 0.54 (characteristic peak 3), 0.55 (characteristic peak 4), 0.64 (characteristic peak 5), 0.90 (characteristic peak 6), 0.96 (characteristic peak 7), 0.97 (characteristic peak 8), 0.99 (characteristic peak 9), 1.00 (characteristic peak 10), 1.25 (characteristic peak 11), 1.33 (characteristic peak 12), and the respective relative retention times should be within ± 10% of the predetermined values.
Wherein, the fingerprint of Shenbao tablet includes characteristic peak 6 (0.90), characteristic peak 10 (1.00) and characteristic peak 12 (1.33) which represent traditional Chinese medicines such as fructus psoraleae, herba epimedii and fructus cnidii.
The relative retention time calculation method comprises the following steps: retention time of each peak/retention time of icariin peak.
The relative retention times may vary within a range, with a relative variation of no more than 10%.
The identification method of each chromatographic peak is to use corresponding reference substance to obtain chromatographic peaks with the same retention time under the same condition, and simultaneously assist multi-wavelength scanning to identify the spectral consistency.
Preferably, the detection method of the present invention comprises the following steps:
(1) chromatographic conditions and system applicability test:
using a Waters CORTECS T3, 2.1X 150mm, 1.6 μm type chromatographic column, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B; the column temperature is 40 ℃; the detection wavelength is 330 nm; the flow rate of the mobile phase is 0.3ml/min, the theoretical plate number calculated according to icariin peak is not lower than 20000,
gradient elution process:
(2) preparation of control solutions: accurately weighing appropriate amount of icariin and echinacoside reference substances respectively, adding methanol to each 1ml reference substance mother liquor containing icariin or echinacoside 0.2mg, adding icariin and echinacoside mother liquor 3ml each into 20ml measuring flask, adding methanol to scale, and shaking.
(3) Preparation of a test solution: taking 10 Shenbao tablets, precisely weighing, grinding, taking a proper amount of fine powder (about equal to 1 tablet weight), precisely weighing, placing in a 50ml volumetric flask, adding a proper amount of 50% ethanol, carrying out ultrasonic treatment (250W, 25 kHz) for 20 minutes, taking out, cooling, adding an extraction solvent to scale, shaking up, centrifuging (5000 revolutions per minute, 5 minutes), filtering with a 0.22 mu m filter membrane, and taking a subsequent filtrate.
(4) The determination method comprises the following steps: respectively sucking 1 μ l of each of the reference solution and the test solution, injecting into an ultra-high performance liquid chromatograph, recording chromatogram peak area, and calculating echinacoside and icariin content by external standard method;
(5) obtaining a comparison fingerprint spectrum: introducing the sample chromatogram of the qualified sample evaluated by the current standard into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system to synthesize a comparison fingerprint, and calibrating a characteristic peak 6, a characteristic peak 10 and a characteristic peak 12 in the chromatogram by using psoralen, icariin and osthole reference substance solutions;
(6) and (3) similarity evaluation: introducing the fingerprint of the sample to be evaluated into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and calculating the similarity, wherein the similarity between the sample and the reference fingerprint is not less than 0.9;
(7) and (3) identification: comparing the sample spectrum with the reference fingerprint spectrum, wherein the sample spectrum has reserved peaks at the corresponding positions of the characteristic peak 6, the characteristic peak 10 and the characteristic peak 12 of the reference fingerprint spectrum.
The technical scheme provided by the invention also comprises the steps of obtaining the UPLC spectrum of the Shenbao tablet, (1) judging the quantity and relative retention time of the common peaks to judge whether the Shenbao tablet is qualified or not, (2) comparing with a comparison fingerprint spectrum to qualitatively identify (whether the Shenbao tablet exists or not) the medicinal materials such as the fructus psoraleae, the epimedium herb, the fructus cnidii and the like, (3) carrying out one-time quantification on the contents of a plurality of common peak components, and (4) further judging the stability of the overall quality of the product by comparing and determining the similarity between the sample spectrum and the comparison fingerprint spectrum.
The technical scheme of the invention adopts one-time measurement, simultaneously realizes the quality evaluation content, realizes one-time measurement and multiple evaluations, and has more efficient and more comprehensive quality control on the Shenbao tablets.
Drawings
FIG. 1 shows UPLC spectrum of Shenbao tablet obtained by the present invention
FIG. 2 is UPLC spectrum of Shenbao tablet obtained by the present invention under different wavelengths
FIG. 3 is a calibration curve of echinacoside reference substance
FIG. 4 is a standard curve of icariin control
FIG. 5 is the overlay of the fingerprint of 21 batches of Shenbao tablets obtained by the present invention
FIG. 6 shows the comparison fingerprint of Shenbao tablets obtained by the present invention
FIG. 7 is the overlay of the fingerprint of Shenbao tablet and the comparison fingerprint obtained by the present invention
FIG. 8 is a thin-layer chromatogram (photograph) of psoralen, icariin and osthole in SHENBAO tablet, wherein the left part is identification of herba Epimedii, the middle part is identification of fructus Psoraleae, and the right part is identification of fructus Cnidii.
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention.
Example 1 screening Process of the Shenbao tablet one-test-multiple-evaluation quality control method
1.1 instruments and materials
Chromatograph: waters H-class ultra-high performance liquid chromatograph
A chromatographic column: waters CORTECS T3, 2.1X 150mm, 1.6 μm;
reagent: methanol, ethanol, acetonitrile, analytically pure; phosphoric acid, methanol, acetonitrile, and chromatographic purity;
comparison products: echinacoside, China institute for food and drug assay, purity 91.8%, lot No. 111670-;
icariin, China institute for food and drug assay, purity 98.1%, lot No. 110737-;
shenbao tablets for kidney: jiangxi Hui ren pharmaceutical industry, batch number: **279.
1.2 method selection
According to the prescription of Shenbao tablet, echinacoside derived from cistanche and icariin derived from epimedium are selected as index components for content determination. When the UPLC method is adopted to detect the Shenbao tablet sample, the icariin is found to be better separated, the echinacoside is not separated from the front peak and the rear peak, and the quantitative detection needs to optimize the elution gradient of the method.
1.3 examination of preparation method of test solution
1.3.1 examination of extraction solvent
The influence of solvents such as methanol, ethanol, acetonitrile, water and the like on the extraction of the Shenbao tablet sample is examined. The results show that the components can not be completely extracted by using each solvent alone except methanol. In consideration of safety, the extraction capacities of methanol and ethanol-water solution are compared according to the polarity relationship, the ethanol-water solution is found to have better extraction effect than methanol, the extraction capacities are not greatly different when ethanol solutions with different concentrations are compared, and a 50% ethanol solution is selected as an extraction solvent in comprehensive consideration, and the results are shown in tables 1 and 2.
The extraction method comprises the following steps: taking 10 Shenbao tablets, precisely weighing, grinding, taking a proper amount of fine powder (about equivalent to 1 tablet weight), precisely weighing, placing in a 50ml volumetric flask, adding an extraction solvent, treating for 30 minutes by using ultrasound (250W and 25 kHz), cooling, adding the extraction solvent to a scale, shaking up, centrifuging (8000 rpm and 5 minutes), filtering by using a 0.22 mu m filter membrane, and taking a subsequent filtrate to obtain the Shenbao tablet;
TABLE 1 results of different solvent extractions
(Note: peak area corrected by using the amount of sample to eliminate the influence of the difference in the amount of sample)
TABLE 2 ethanol extraction results at different concentrations
1.3.2 examination of extraction time
The influence of different extraction times on the extraction capacity was investigated. The result shows that the ultrasonic treatment is carried out for 10-40 minutes, the extraction effect of the Shenbao tablet is not very different, and no obvious difference exists. Considering the risk of insufficient extraction due to the fact that the Shenbao tablet fine powder is easy to coagulate into blocks when dissolved, 20 minutes is selected as the ultrasonic time, and the results are shown in Table 3.
TABLE 3 different ultrasound time extraction results
1.3.3 ultrasonic conditions
The influence of different ultrasonic powers and frequencies on the extraction effect is investigated. The result shows that the extraction results are obviously different from the extraction results under other extraction conditions except for the extraction result of 150W-30kHz, the extraction results of 250W with different frequency and the extraction results of 350W with different frequency are not obviously different from the extraction results of 450W-50kHz, namely, the extraction capacities under other conditions are not greatly different except for the condition that the power of 150W cannot be completely extracted. Therefore, the conditions of 250W-20kHz and above are selected. The results are shown in Table 4.
TABLE 4 different Power-frequency extraction results
1.3.4 examination of centrifugation conditions
The influence of the centrifugation at different rotation speeds on the test sample was examined. The results show that the elution results are not obviously different under the centrifugation conditions with different rotating speeds, and the conditions of 5000rpm and above are better in filtration from the viewpoint of the difficulty of filtration, so that 5000rpm is selected as the centrifugation conditions, and the results are shown in Table 5.
TABLE 5 measurement results of various rotational speeds
1.3.5 method of preparing Final defined test solutions
Taking 10 Shenbao tablets, precisely weighing, grinding, taking a proper amount of fine powder (about equal to 1 tablet weight), precisely weighing, placing in a 50ml volumetric flask, adding a proper amount of 50% ethanol, carrying out ultrasonic treatment (250W, 25 kHz) for 20 minutes, taking out, cooling, adding an extraction solvent to scale, shaking up, centrifuging (5000 revolutions per minute, 5 minutes), filtering with a 0.22 mu m filter membrane, and taking a subsequent filtrate.
1.4 chromatographic conditions and System suitability test
1.4.1 elution gradient investigation
The acetonitrile is used as a mobile phase A, a 0.1% phosphoric acid solution is used as a mobile phase B, the separation effect of different elution gradients on chromatographic peaks of echinacoside and icariin is examined, and the result shows that the separation effect of the following gradient elution procedures on chromatographic peaks of echinacoside and icariin is the best, and a chromatogram is shown in figure 1.
1.4.2 chromatographic column inspection
3 columns were investigated: waters ACQUITY UPLC HST 3 (2.1X 150mm, 1.8 μm) chromatographic column, Waters ACQUITY UPLC BEH C18 (2.1X 150mm, 1.7 μm) chromatographic column, Waters CORTECS T3 (2.1X 150mm, 1.6 μm) chromatographic column. As a result, the separation effect of the Waters CORTECS T3 (2.1X 150mm, 1.6 μm) column was the best, the separation effect of echinacoside from icariin peak was the best, and the separation effect of other peaks was also the best, so the Waters CORTECS T3 (2.1X 150mm, 1.6 μm) column was selected for detection.
1.4.3 detection wavelength investigation
Carrying out full-wavelength scanning (190-.
1.4.4 chromatographic conditions for Final confirmation
Using a Waters CORTECS T3, 2.1X 150mm, 1.6 μm type chromatographic column, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B; during the gradient elution, the ratio of mobile phase a changes as: 0-2 minutes, 7% -10% acetonitrile; 2-10 minutes, 10% -14% acetonitrile; 10-18 minutes, 14% -23% acetonitrile; 18-25 minutes, 23% -34% acetonitrile; from 25 to 29 minutes, from 34 to 50 percent of acetonitrile; 29-32 minutes, 50% -80% acetonitrile; 32-36 minutes, 7% acetonitrile, and 40 ℃ of column temperature; the detection wavelength is 330 nm; the flow rate of the mobile phase is 0.3ml/min, and the theoretical plate number is not lower than 20000 calculated according to icariin peak.
1.5 methodological considerations
1.5.1 sample preparation
Preparing reference mother liquor: accurately weighing appropriate amount of icariin and echinacoside control, respectively, adding methanol to obtain control mother liquor containing icariin or echinacoside 0.2mg per 1 ml.
Preparing a test solution: taking 10 Shenbao tablets, precisely weighing, grinding, taking a proper amount of fine powder (about equivalent to 1 tablet weight), precisely weighing, adding a proper amount of 50% ethanol into a 50ml measuring flask, carrying out ultrasonic treatment (250W and 25 kHz) for 20 minutes, taking out, cooling, adding 50% ethanol to scale marks, shaking up, centrifuging (5000 revolutions per minute and 5 minutes), taking supernatant, filtering with a 0.22 mu m microporous membrane, and taking a subsequent filtrate.
Negative sample solution preparation: according to the prescription of Shenbao tablet, the medicinal materials except cistanche and epimedium are taken, the Shenbao tablet negative total mixed granules are prepared according to the Shenbao tablet standard (preparation method), and the negative sample solution is prepared according to the preparation method of test solution by taking the total mixed granules.
Preparation of mixed control solution: taking icariin and echinacoside mother liquor 3ml respectively in a 20ml measuring flask, adding methanol to scale, and shaking up to obtain the final product.
Preparation of recovery rate test solution: taking a Shenbao tablet negative sample without cistanche and epimedium medicinal materials, grinding, precisely weighing an appropriate amount of fine powder (about equivalent to 1 tablet) in a 50ml measuring flask, adding about 30ml of 50% ethanol, carrying out ultrasonic treatment (250W and 25 kHz) for 20 minutes, adding 7.0ml of each mother solution of a reference substance, adding 50% ethanol to the scale, shaking up, taking an appropriate amount of centrifugal liquid (5000 revolutions per minute and 5 minutes), taking centrifugal supernatant, filtering with a 0.22 mu m filter membrane, and taking a subsequent filtrate to obtain the Shenbao tablet negative sample.
1.5.2 precision investigation
Accurately weighing appropriate amount of SHENBAO tablet (lot number X279), preparing sample solution according to sample solution preparation method, continuously sampling for 6 times, recording chromatogram, measuring content, and keeping peak area RSD not more than 2% and relative retention time deviation of common peak within 5%; according to the similarity evaluation of the chromatographic fingerprint of the traditional Chinese medicine, the similarity is not lower than 0.95, the precision of the instrument is good, and the results are shown in tables 6 and 7.
TABLE 6
Results of precision measurement
TABLE 7
Precision relative peak retention time of each common peak
1.5.3 repeatability test
Taking a proper amount of Shenbao tablets (batch number x 279), preparing 6 parts of sample solution according to the preparation operation of the test solution, measuring each needle, recording the peak area RSD of the content measurement peak not more than 2%, ensuring that the relative retention time deviation of the common peak is within 5%, and evaluating according to the similarity of the chromatographic fingerprint of the traditional Chinese medicine, wherein the similarity is not less than 0.95. The results show that the method has good repeatability, and are shown in tables 8 and 9.
TABLE 8
Measurement results of repetitive samples
TABLE 9
Relative retention time of each common peak of repetitive samples
1.5.4 durability examination
The stability of the sample solution is good; and small changes of column temperature, flow rate and the like have no influence on detection.
1.5.4.1 solution stability
Taking a proper amount of Shenbao tablets (batch number x 279), preparing a sample solution according to the preparation operation of the sample solution, respectively measuring at 0h, 2h, 4h, 8h, 12h, 16h, 20h and 24h, recording a chromatogram, recording the content measurement peak area RSD (mean shift of the content measurement peak area RSD) which is not more than 2 percent, and keeping the relative retention time deviation of the common peak within 5 percent; according to the evaluation of the similarity of the chromatographic fingerprint of the traditional Chinese medicine, the similarity is not lower than 0.95, the stability of the sample solution is good, and the results are shown in tables 10 and 11.
Results of solution stability measurement
TABLE 11
Solution stability Each common Peak relative Retention time
1.5.4.2 column temperature examination
Taking a proper amount of Shenbao tablets (batch number x 279), preparing a sample solution according to the preparation operation of a test solution, respectively adjusting the column temperature to 38 ℃ and 42 ℃ in the chromatographic condition, measuring, recording a chromatogram, wherein the RSD (content measurement peak area) is not more than 2%, and the relative retention time deviation of the common peak is within 5% of the relative retention time under the condition of 40 ℃; according to the evaluation of the similarity of the chromatographic fingerprint of the traditional Chinese medicine, the similarity is not lower than 0.95, the column temperature has no influence on the detection, and the results are shown in tables 12 and 13.
TABLE 12
Results of different column temperatures
Common peak retention time under different column temperature conditions
1.5.4.3 investigation of flow Rate
Taking a proper amount of Shenbao tablets (batch number x 279), preparing a sample solution according to the preparation operation of the sample solution, respectively adjusting the flow rates in chromatographic conditions to be 0.29ml/min and 0.31ml/min, measuring, recording a chromatogram, wherein the area of a content measurement peak is integrally reduced along with the increase of the flow rate, but the ratio of the relative total peak area is not greatly changed, and the deviation of the relative retention time of the common peak is within 5% of the relative retention time of the condition of 0.30 ml/min; according to the evaluation of the similarity of the chromatographic fingerprint of the traditional Chinese medicine, the similarity is not lower than 0.95, the flow rate has no influence on the detection, and the results are shown in tables 12 and 13.
TABLE 14
Results of different flow rate measurements
Relative retention time of common peaks under different flow rate conditions
1.5.5 line survey
Taking reference mother liquor, diluting according to tables 16 and 17 to prepare reference solutions containing icariin and echinacoside respectively at 20-80 mu g/ml, precisely measuring the reference solutions respectively at 1 mu l, injecting into an ultra-high performance liquid phase, recording a chromatogram, drawing a standard curve (see attached figures 3 and 4) by taking the reference concentration as a horizontal coordinate and the peak area as a vertical coordinate, and solving a linear regression equation, wherein the result is shown in tables 16 and 17.
TABLE 16
Echinacoside linear determination data table
TABLE 17
Icariin linearity determination data table
1.5.6 accuracy examination
The above "1.5.1" mixed control solution and recovery rate test solution were taken, 1. mu.l of each of the control solutions was measured precisely, and injected into an ultra high performance liquid phase, and chromatograms were recorded, and the recovery rates were calculated, and the average recovery rate of icariin was 93.2%, the average recovery rate of RSD was 0.4%, the average recovery rate of echinacoside was 96.0%, and the RSD was 1.1%, and the results are shown in tables 18 and 19.
Echinacoside recovery results
Icariin recovery rate results
Example 2 identification of Shenbao tablets, measurement of finger-print, and measurement of icariin and echinacoside contents
2.1 instruments and materials
Chromatograph: waters H-class ultra-high performance liquid chromatograph
A chromatographic column: waters CORTECS T3, 2.1X 150mm, 1.6 μm;
reagent: methanol, ethanol, acetonitrile, analytically pure; phosphoric acid, methanol, acetonitrile, and chromatographic purity;
comparison products:
chlorogenic acid, 110753-
Echinacoside 111670-
Stilbene glucoside 110844 and 201713, 93.6 percent, China institute for testing food and drug
Hyperin, 111521-
Verbascoside 17051902, 98% of Dopperadine organisms
Psoralen, 110739-
Epimedin A, 19121605, 99.68%, Dolphin organisms
Epimedin B, 19120605, 98.97%, Dolphin organisms
Epimedin C, 111780-contained and 201302, 95.6 percent, China institute for testing food and drug
Icariin, 110737-
Baohuoside I, 111852-
Osthole, 110822-containing 201710, 99.50 percent, China institute for testing and testing food and drug
Shenbao tablets for kidney: jiangxi Hui ren pharmaceutical industry, batch number: 395, 396 and 397.
2.2 sample solution preparation
Reference solution: weighing chlorogenic acid, echinacoside, stilbene glucoside, hyperoside, acteoside, psoralen, epimedin A, epimedin B, epimedin C, icariin, baohuoside I and osthole reference substances respectively at 2mg, placing into a 100ml measuring flask, adding methanol to dissolve and dilute to scale, shaking, filtering with 0.22 μm microporous membrane, and collecting the subsequent filtrate.
Preparation of a reference solution: accurately weighing appropriate amount of icariin and echinacoside reference substances respectively, adding methanol to each 1ml reference substance mother liquor containing icariin or echinacoside 0.2mg, adding icariin and echinacoside mother liquor 3ml each into 20ml measuring flask, adding methanol to scale, and shaking.
Preparing a test solution: taking 10 Shenbao tablets, precisely weighing, grinding, taking a proper amount of fine powder (about equivalent to 1 tablet weight), precisely weighing, adding a proper amount of 50% ethanol into a 50ml measuring flask, carrying out ultrasonic treatment (250W and 25 kHz) for 20 minutes, taking out, cooling, adding 50% ethanol to scale marks, shaking up, centrifuging (5000 revolutions per minute and 5 minutes), taking supernatant, filtering with a 0.22 mu m microporous membrane, and taking a subsequent filtrate.
2.3 chromatographic conditions and system applicability: using a Waters CORTECS T3, 2.1X 150mm, 1.6 μm type chromatographic column, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B; during the gradient elution, the ratio of mobile phase a changes as: 0-2 minutes, 7% -10% acetonitrile; 2-10 minutes, 10% -14% acetonitrile; 10-18 minutes, 14% -23% acetonitrile; 18-25 minutes, 23% -34% acetonitrile; from 25 to 29 minutes, from 34 to 50 percent of acetonitrile; 29-32 minutes, 50% -80% acetonitrile; 32-36 minutes, 7% acetonitrile; the column temperature is 40 ℃; the detection wavelength is 330 nm; the flow rate of the mobile phase is 0.3ml/min, and the theoretical plate number is not lower than 20000 calculated according to icariin peak.
2.4 assay: respectively and precisely sucking 1 mu l of the mixed control solution and the test solution, injecting into an ultra-high performance liquid chromatograph, recording a chromatogram, calculating the contents of echinacoside and icariin by using an external standard method, and processing the chromatogram by using a 2012 edition of the similarity evaluation system of traditional Chinese medicine fingerprints to obtain the fingerprint of the Shenbao tablet. See figure 5 (overlay of 21 batches of shenbao tablets) and figure 6 (generation of control map).
2.5 content evaluation: three batches (batch numbers 395, 396 and 397) of Shenbao tablets were prepared by 2.2 method, test solution was precisely extracted 1 μ l each of the reference solution and the test solution, injected into the ultra high performance liquid chromatograph, chromatogram peak area was recorded, echinacoside and icariin content were calculated by external standard method, and the measurement results are shown in Table 20.
Continuous 3 batches of assay results
Quantification of the product from this and the following 20 batches (mean ± standard deviation SD): icariin 1.96 plus or minus 0.26 mg/tablet, echinacoside 2.03 plus or minus 0.34 mg/tablet, the limit of the product is determined as 'mean-3 SD', and the ten parts are rounded: each tablet of the product contains icariin no less than 1.2mg, and contains echinacoside no less than 1.0 mg.
2.6 fingerprint evaluation: taking the 3 batches of shenbao tablets, preparing a test solution according to a 2.2 method, precisely absorbing 1 mu l of each of a reference solution, a reference solution and the test solution, respectively, injecting an ultra-high performance liquid chromatogram, recording the chromatogram, sequentially introducing the acquired UPLC (ultra performance liquid chromatogram) into a 2012 edition of a traditional Chinese medicine fingerprint similarity evaluation system in an AIA format, and comparing the similarity with a reference fingerprint generated by 2.4, wherein the similarity is respectively 0.979, 0.978 and 0.978, and the common limit is more than 0.9.
The test sample has 12 characteristic chromatographic peaks, wherein each characteristic chromatographic peak and retention time are respectively as follows:
characteristic peak 1: chlorogenic acid (0.20 ± 10%), characteristic peak 2: echinacoside (0.48 ± 10%), characteristic peak 3: stilbene glycoside (0.54 ± 10%), characteristic peak 4: hyperin (0.55 ± 10%), characteristic peak 5: acteoside (0.64 ± 10%) characteristic peak 6: psoralen (0.90 ± 10%), characteristic peak 7: epimedin a (0.96 ± 10%), characteristic peak 8: epimedin B (0.97 ± 10%), characteristic peak 9: epimedin C (0.99 ± 10%), characteristic peak 10: icariin (S ± 10%), characteristic peak 11: baohuoside I (1.25 ± 10%), characteristic peak 12: osthole (1.33 + -10%).
2.7, identification: comparing the sample spectrum with the reference fingerprint spectrum, and taking icariin chromatographic peak as reference peak, wherein the sample spectrum has retention peak at corresponding positions of characteristic peak 6 (psoralen), characteristic peak 10 (icariin) and characteristic peak 12 (osthole) of the reference fingerprint spectrum, as shown in figure 7. The results of thin layer chromatography identification of the above three components by current methods are shown in figure 8.
Example 3 comparison of Performance of quality control method
The embodiment 1 and the embodiment 2 show that the technical scheme of the invention not only can at least determine 2 index components of echinacoside and icariin, but also can compare the detected fingerprint with a reference fingerprint through similarity evaluation software to obtain more comprehensive medicine quality information (similarity), and can also use the fingerprint to replace thin-layer chromatography for identification.
The icariin content of the Shenbao tablet is not lower than 1.20mg per tablet according to the current product standard. As can be seen from table 21, the difference in icariin content between 15 batches was large (RSD = 8.77%), with the quality control method of the present invention, the RSD of icariin content between 15 batches was 11.17%, and the double-sample t test result p > 0.05, there was no significant difference between the two; the similarity results showed little difference between the 15 batches (RSD = 0.83%).
TABLE 21 quality control of SHENBAO tablet
The method of the invention is compared and analyzed with the existing quality control method, and the operation steps, time cost and consumable cost of 2 methods are compared.
TABLE 22 comparison of Current quality control methods with the present invention
TABLE 23 analysis of time cost (same sample treatment part time not calculated)
Cost analysis of single test consumables
Compared with the existing quality control operation steps, the quality control method of the invention is simpler, omits the complicated preparation process of the test sample in the Thin Layer Chromatography (TLC) identification process, consumes less time and is about one half of the existing standard. In the quality control method, the echinacoside reference substance is high in price (about 900/count), which can increase the cost of consumable materials for single content detection, and the correction factor can be used for replacing the echinacoside reference substance to determine the echinacoside content, so that the cost of the consumable materials for single detection is reduced to 313, which is one third of the cost of consumable materials of the current detection standard.
Claims (8)
1. A quality control detection method of Shenbao tablets is characterized by comprising the following steps: the method comprises the following steps of content measurement, identification and fingerprint spectrum measurement of active ingredients, wherein the content measurement of the active ingredients comprises the following steps:
(1) chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, and 0.1% phosphoric acid solution is used as a mobile phase B; gradient elution;
(2) preparation of control solutions: taking appropriate amount of echinacoside reference substance and icariin reference substance, and adding methanol to obtain reference substance solution;
(3) preparation of a test solution: precisely weighing appropriate amount of SHENBAO tablet, grinding, adding appropriate amount of fine powder, precisely weighing, placing in a volumetric flask, adding appropriate amount of extraction solvent, ultrasonic treating, cooling to room temperature, adding extraction solvent to scale, shaking, centrifuging, filtering, and collecting filtrate;
(4) the determination method comprises the following steps: respectively sucking 1 μ l of each of the reference solution and the sample solution, injecting into an ultra high performance liquid chromatograph, and recording chromatogram.
2. The detection method according to claim 1, wherein the gradient elution in step (1) is performed in such a manner that the ratio of mobile phase A changes as follows: 0-2 minutes, 7% -10% acetonitrile; 2-10 minutes, 10% -14% acetonitrile; 10-18 minutes, 14% -23% acetonitrile; 18-25 minutes, 23% -34% acetonitrile; from 25 to 29 minutes, from 34 to 50 percent of acetonitrile; 29-32 minutes, 50% -80% acetonitrile; 32-36 minutes, 7% acetonitrile.
3. The detection method according to claim 1, wherein the octadecylsilane chemically bonded silica in the step (1) is used as a filler of a Waters CORTECS T3, 2.1X 150mm, 1.6 μm.
4. The detection method according to claim 1, wherein in the preparation of the test solution,
the extraction solvent is selected from: purified water, ethanol, acetonitrile or methanol;
extraction time: 10-40 minutes;
ultrasonic conditions are as follows: 150-350W, 20-50 kHz;
centrifugation conditions: 3000 and 9000 revolutions per minute for 5 minutes.
5. The assay of claim 1, wherein the step (2) of preparing the control solution comprises: taking appropriate amount of echinacoside and icariin as reference substances, and adding methanol to obtain solutions containing 30 μ g of echinacoside and icariin per 1 ml.
6. The detection method according to claim 1, wherein the step (3) of preparing the test solution: precisely weighing 10 Shenbao tablets, grinding, taking a proper amount of fine powder, precisely weighing, placing into a 50ml volumetric flask, adding an extraction solvent, carrying out ultrasonic treatment, cooling, adding the extraction solvent to a scale, shaking uniformly, centrifuging, filtering, and taking a subsequent filtrate.
7. The detection method according to claim 1, further comprising the steps of:
(1) obtaining a comparison fingerprint spectrum: importing the sample atlas of the qualified sample of the current standard evaluation into the synthesized comparison fingerprint atlas of the traditional Chinese medicine chromatogram fingerprint atlas similarity evaluation system;
(2) and (3) similarity evaluation: introducing the fingerprint of the sample to be evaluated into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and calculating the similarity, wherein the similarity between the sample and the reference fingerprint is not less than 0.9;
(3) and (3) identification: comparing the test sample spectrum with the comparison fingerprint spectrum.
8. The detection method according to claim 1, further comprising the steps of:
(1) chromatographic conditions and system applicability test: using a Waters CORTECS T3, 2.1X 150mm, 1.6 μm type chromatographic column, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B; the column temperature is 40 ℃; the detection wavelength is 330 nm; the flow rate of the mobile phase is 0.3ml/min, the theoretical plate number is not lower than 20000 calculated according to icariin peak, and gradient elution is carried out;
(2) preparation of control solutions: accurately weighing appropriate amount of icariin and echinacoside reference substances respectively, adding methanol to each 1ml reference substance mother liquor containing icariin or echinacoside 0.2mg, adding icariin and echinacoside mother liquor 3ml each into 20ml measuring flask, adding methanol to scale, and shaking to obtain the final product;
(3) preparation of a test solution: taking 10 Shenbao tablets, precisely weighing, grinding, taking a proper amount of fine powder, precisely weighing, placing into a 50ml volumetric flask, adding a proper amount of 50% ethanol, carrying out ultrasonic treatment for 20 minutes, taking out, cooling, adding an extraction solvent to a scale, shaking up, centrifuging, filtering with a 0.22 mu m filter membrane, and taking a subsequent filtrate;
(4) the determination method comprises the following steps: respectively sucking 1 μ l of each of the reference solution and the test solution, injecting into an ultra-high performance liquid chromatograph, recording chromatogram peak area, and calculating echinacoside and icariin content by external standard method;
(5) obtaining a comparison fingerprint spectrum: introducing the sample chromatogram of the qualified sample evaluated by the current standard into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system to synthesize a comparison fingerprint, and calibrating a characteristic peak 6, a characteristic peak 10 and a characteristic peak 12 in the chromatogram by using psoralen, icariin and osthole reference substance solutions;
(6) and (3) similarity evaluation: introducing a test sample fingerprint of a sample to be evaluated into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and calculating the similarity, wherein the similarity between the test sample fingerprint and a reference fingerprint is not less than 0.9;
(7) and (3) identification: comparing the sample spectrum with the reference fingerprint spectrum, wherein the sample spectrum has reserved peaks at the corresponding positions of the characteristic peak 6, the characteristic peak 10 and the characteristic peak 12 of the reference fingerprint spectrum.
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