Detection method for characteristic chromatogram of wolfberry and chrysanthemum granules and application of detection method in quality detection

Abstract

The method for detecting the characteristic chromatogram of the wolfberry and chrysanthemum granules is established for the first time, a wolfberry and chrysanthemum granule sample is precisely weighed, a solvent is added for dissolution and extraction, filtration is performed, a subsequent filtrate is taken, a test solution is obtained, high performance liquid chromatography detection is performed on the test solution, and the wolfberry and chrysanthemum granule control characteristic chromatogram generated by common characteristic peaks is obtained. When the method is used for determining the high performance liquid characteristic chromatogram of the wolfberry and chrysanthemum granules, the following chemical components in the wolfberry and chrysanthemum granules can be quickly and effectively identified: puerarin-4 '-O-glucoside, chlorogenic acid, puerarin, 3'-methoxy puerarin, puerarin apigenin, typhaenoside, isochlorogenic acid B, 3, 5-O-dicaffeoylquinic acid and isochlorogenic acid C; the characteristic peaks of the chrysanthemum, the radix puerariae and the pollen typhae are reflected, the method has the advantages of good integrity, good stability, high precision, good repeatability and good durability, and a scientific basis is provided for better establishing a quality control evaluation system of the wolfberry and chrysanthemum granules.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine component detection, and particularly relates to a method for detecting a characteristic spectrum of Qiju granules and an application thereof in quality inspection. Background Art

[0002] Qiju Granules are made from seven Chinese medicinal ingredients: wolfberry, kudzu root, chrysanthemum, astragalus, dendrobium, Panax notoginseng, and pollen. They nourish the liver and kidneys, nourish yin, and improve eyesight. They are used for early- to mid-stage and dry-stage age-related macular degeneration with liver and kidney yin deficiency, characterized by blurred vision and distorted vision. Drusen, macular pigmentation disorders, and atrophic changes can be seen in the fundus; accompanied by dry eyes, dizziness, tinnitus, soreness in the waist and knees, insomnia, frequent dreams, a red tongue with little coating, and a thin pulse. The treatment is highly effective.

[0003] Qiju Granules' inspection items primarily include thin-layer chromatography (TLC) identification of wolfberry, chrysanthemum, kudzu root, and Panax notoginseng, as well as content determination of kudzu root and chrysanthemum (HPLC determination of puerarin, chlorogenic acid, and 3,5-O-dicaffeoylquinic acid). Qiju Granules are made from seven diverse and complex Chinese medicinal materials. Using only TLC for qualitative identification and HPLC for puerarin, chlorogenic acid, and 3,5-O-dicaffeoylquinic acid determination is insufficient to fully reflect the quality of Qiju Granules.

[0004] As a quality control technology, the characteristic profile of traditional Chinese medicine (TCM) can comprehensively control the quality of drugs, with characteristics of systematicity, integrity, and stability. High-performance liquid chromatography (HPLC), drawing on the theories and principles of HPLC, incorporates new technologies such as small particle size, very low system volume, and rapid detection methods, increasing analytical throughput and chromatographic peak capacity. It is simple, accurate, highly sensitive, reproducible, and uses a wide variety of detectors, making it a key method for constructing characteristic profiles.

[0005] Currently, there are no patents or literature reports on the use of HPLC to study the characteristic spectrum of Qiju Granules. The present invention discloses an HPLC characteristic spectrum of Qiju Granules. Using this method, a reference characteristic spectrum is established, and specified values ​​for the relative retention times of each characteristic peak are established. This reference characteristic spectrum can be used to comprehensively evaluate and control the quality of Qiju Granules, thereby ensuring the stability of product quality and the safety and effectiveness of clinical use. Summary of the Invention

[0006] The present invention provides a method for establishing a characteristic spectrum of Qiju granules, which is used for more comprehensive control and evaluation of the quality of Qiju granules.

[0007] To achieve the above object, the present invention provides a method for detecting the characteristic spectrum of Qiju granules, comprising the following steps:

[0008] a. Preparation of test solution: Accurately weigh the Qiju granule sample, add solvent to dissolve and extract, filter, and take the filtrate to obtain the test solution;

[0009] b. The test solution obtained in step a was subjected to HPLC to obtain a control characteristic spectrum of Qiju particles generated by a common characteristic peak;

[0010] In the high performance liquid chromatography method, the chromatographic column is a C18 chromatographic column; acetonitrile is used as mobile phase A, and 0.1% by volume phosphoric acid aqueous solution is used as mobile phase B, and gradient elution is performed;

[0011] The gradient elution procedure is:

[0012] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～30 9→14 91→86 30～35 14→18 86→82 35～80 18 82

[0013] In one or more preferred embodiments, the preparation method of the test solution described in step a and the determination method described in step b are used to determine the characteristic spectra of 10 batches of samples, and the obtained characteristic spectra are analyzed, and the chromatographic peaks with good stability and appropriate response values ​​in the sample characteristic spectra are selected as characteristic peaks. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System 2012 Edition A" promulgated by the State Pharmacopoeia Commission is used to automatically match the chromatographic peaks of the HPLC spectra of 10 batches of samples to form a common pattern diagram, and establish a control spectrum.

[0014] In one or more preferred embodiments, the flow rate of the mobile phase in the high performance liquid chromatography method in step b is 0.9-1.1 mL / min; more preferably 1.0 ml / min

[0015] In one or more preferred embodiments, the detection wavelength in the high performance liquid chromatography method in step b is 265-345 nm; more preferably 305-325 nm.

[0016] In one or more preferred embodiments, in the high performance liquid chromatography method of step b, the C18 chromatographic column is 250 mm×4.6 mm, 5 μm; the injection volume is 8 μL-12 μL, more preferably 5-10 μL; and the column temperature is 30°C.

[0017] In one or more preferred embodiments, the mass volume ratio of the Qiju granule sample to the solvent in step a is 1 g: (20-50) mL; more preferably 1 g: 25 mL.

[0018] In one or more preferred embodiments, in step a, the solvent is a methanol aqueous solution, wherein the volume percentage concentration of the methanol aqueous solution is 50%-70%.

[0019] In one or more preferred embodiments, in step a, the extraction method is ultrasound, shaking, or reflux; more preferably, the extraction method is ultrasonic dissolution, and the ultrasonic time is 15-45 minutes.

[0020] On the other hand, the present invention provides the application of the detection method of the characteristic spectrum of Qiju granules in the quality detection of the components of Qiju granules.

[0021] The present invention also provides a quality detection method for Qiju granules, comprising obtaining a characteristic spectrum of Qiju granules using the above-mentioned detection method of the characteristic spectrum of Qiju granules; and comparing the relative retention time of the obtained characteristic spectrum of Qiju granules with that of the control characteristic spectrum.

[0022] In one or more preferred embodiments, the quality detection method of the Qiju Granules adopts the same conditions as the detection method of the above-mentioned Qiju Granule characteristic spectrum to obtain the characteristic spectrum of the Qiju Granules. Preferably, the Qiju Granule characteristic spectrum contains 10 characteristic peaks, wherein peak 2 is the reference peak of the chlorogenic acid reference substance, peak 3 is the reference peak of the puerarin reference substance, and peak 8 is the reference peak of the 3,5-O-dicaffeoylquinic acid reference substance. The relative retention time of each characteristic peak is calculated based on the puerarin reference substance reference peak as the S peak: peak 1 is 0.63±10%, peak 4 is 1.13±10%, peak 5 is 1.19±10%, peak 6 is 1.93±10%, peak 7 is 2.36±10%, peak 9 is 2.58±10%, and peak 10 is 2.93±10%.

[0023] In one or more preferred embodiments, the characteristic peaks of the characteristic spectrum of the Qiju granules are attributed as follows: Peak 1 is the characteristic peak of puerarin-4'-O-glucoside, and the medicinal flavor is Pueraria lobata; Peak 2 is the characteristic peak of chlorogenic acid, and the medicinal flavor is chrysanthemum; Peak 3 is the characteristic peak of puerarin, and the medicinal flavor is Pueraria lobata; Peak 4 is the characteristic peak of 3'-methoxypuerarin, and the medicinal flavor is Pueraria lobata; Peak 5 is the characteristic peak of puerarin apiglycoside, and the medicinal flavor is Pueraria lobata; Peak 6 is the characteristic peak of typhaneoglucoside, and the medicinal flavor is Typha pollen; Peak 7 is the characteristic peak of isochlorogenic acid B, and the medicinal flavor is chrysanthemum; Peak 8 is the characteristic peak of 3,5-O-dicaffeoylquinic acid (isochlorogenic acid A), and the medicinal flavor is chrysanthemum; Peak 9 is the characteristic peak of 1,3-dicaffeoylquinic acid, and the medicinal flavor is chrysanthemum; Peak 10 is the characteristic peak of isochlorogenic acid C, and the medicinal flavor is chrysanthemum.

[0024] In one or more preferred embodiments, the Qiju granules are prepared from the following raw materials in parts by weight: 10-15 parts of wolfberry, 6-10 parts of chrysanthemum, 7-12 parts of complanatus, 3-8 parts of dendrobium, 1-5 parts of Panax notoginseng, 3-8 parts of raw pollen, and 6-10 parts of kudzu root.

[0025] The preparation method of the Qiju granules comprises the following steps:

[0026] Step 1: Weigh wolfberry, chrysanthemum, complanatus, dendrobium, Panax notoginseng, raw pollen, and kudzu root according to weight;

[0027] Step 2: Crush the Panax notoginseng from step 1, sieve it, collect the Panax notoginseng powder and set aside;

[0028] Step 3: decoct the Panax notoginseng powder prepared in step 2 with the other six herbs including Astragalus complanatus and Astragalus complanatus twice with water, each time for 1 hour, filter, concentrate the filtrate under reduced pressure, dry, and grind into a paste powder;

[0029] Step 4: Add appropriate amount of auxiliary materials to the paste powder obtained in step 3, granulate, dry, and granulate to obtain granules.

[0030] Beneficial effects of the present invention:

[0031] The present invention establishes for the first time a method for detecting the characteristic spectrum of Qiju Granules. The high-performance liquid chromatography characteristic spectrum of Qiju Granules is determined by the method of the present invention, and the following chemical components in Qiju Granules can be quickly and effectively identified: puerarin-4'-O-glucoside, chlorogenic acid, puerarin, 3'-methoxypuerarin, puerarin apiglycoside, typhaneuroside, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, and isochlorogenic acid C. It embodies the characteristic peaks of the three medicinal flavors of chrysanthemum, kudzu root, and pollen cattail, and has the advantages of good integrity, good stability, high precision, good repeatability, and good durability, providing a scientific basis for better establishing a quality control and evaluation system for Qiju Granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the HPLC characteristic spectrum of 10 batches of Qiju granule samples;

[0033] Figure 2 This is the HPLC reference spectrum of Qiju Granules;

[0034] Figure 3 It is the HPLC characteristic spectrum of Qiju Granules and the HPLC characteristic spectrum of each single herbal medicine reference substance;

[0035] Figure 4 This is the HPLC profile obtained according to the mobile phase gradient in Table 5;

[0036] Figure 5 This is the HPLC profile obtained according to the mobile phase gradient in Table 6;

[0037] Figure 6 This is the HPLC profile obtained according to the mobile phase gradient in Table 7;

[0038] Figure 7 It is the HPLC spectrum of the test solution in the wavelength range of 265-365nm;

[0039] Figure 8It is the HPLC spectrum of the test solution obtained with different extraction solvents;

[0040] Figure 9 It is the HPLC spectrum of the test solution obtained by different extraction methods;

[0041] Figure 10 is the HPLC spectrum of the test solution obtained at different extraction times;

[0042] Figure 11 It is an HPLC spectrum obtained by investigating specificity and integrity;

[0043] Figure 12 It is the HPLC spectrum obtained by precision test;

[0044] Figure 13 This is the HPLC spectrum obtained by conducting stability experiments;

[0045] Figure 14 This is the HPLC spectrum obtained by repeatable experimental investigation;

[0046] Figure 15 It is the HPLC spectrum at different flow rates;

[0047] Figure 16 It is the HPLC spectrum at different column temperatures;

[0048] Figure 17 It is the HPLC spectrum of different chromatographic columns;

[0049] Figure 18 It is the HPLC spectrum measured by different instruments; DETAILED DESCRIPTION

[0050] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0051] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0052] The instruments, reagents and their sources used in the following examples are as follows:

[0053] Instrument: Shimadzu LC-20AT high performance liquid chromatograph; PDA detector;

[0054] Reagents: Qiju Granules were prepared by Beijing Yingke Rui Innovative Pharmaceutical Research Co., Ltd., and the sample batch numbers are shown in Table 1; acetonitrile and phosphoric acid were chromatographically pure, water was purified water, and the remaining reagents were of analytical grade.

[0055] Table 1 Batch numbers of Qiju granules tested samples

[0056] sample batch number S2 C-QJ200302-KL1-3 S3 C-QJ200303-KL1-3 S4 C-QJ200304-KL1-3 S5 221119-01 S6 221119-02 S7 221119-03 S8 C-QJ200401 S9 C-QJ-200601 S10 C-QJ-200602 S11 C-QJ-200603

[0057] Example 1 Establishment of HPLC Characteristic Spectrum of Qiju Granules

[0058] Preparation of reference solution:

[0059] Accurately weigh an appropriate amount of chlorogenic acid reference substance and 3,5-O-dicaffeoylquinic acid reference substance, place in a brown volumetric flask, and add 70% methanol to make a mixed solution containing 20 μg of chlorogenic acid and 50 μg of 3,5-O-dicaffeoylquinic acid per 1 ml. (Store below 10°C). Accurately weigh an appropriate amount of puerarin reference substance, add 70% methanol to make a solution containing 0.2 mg per 1 ml.

[0060] a. Preparation of test solution:

[0061] Take an appropriate amount of this product, grind it into powder, take about 1.0g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, stopper it tightly, weigh it accurately, and treat it ultrasonically (power 500W, frequency 40kHz) for 30 minutes. Remove it, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain;

[0062] b. Chromatographic conditions and determination:

[0063] Use octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, using gradient elution as specified in Table 2; maintain a column temperature of 30°C, and a detection wavelength of 325 nm. The theoretical plate number calculated based on the puerarin peak should be no less than 4000. Accurately pipette 10 μl of each reference solution and test solution into a liquid chromatograph for determination.

[0064] Table 2

[0065] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～30 9→14 91→86 30～35 14→18 86→82 35～80 18 82

[0066] Calibration of characteristic peaks:

[0067] According to the preparation and determination of the test solution, the characteristic spectra of 10 batches of samples were measured and analyzed. The chromatographic peaks with good stability and appropriate response values ​​in the characteristic spectra of the samples were selected as characteristic peaks. A total of 10 characteristic peaks were calibrated. Figure 1 .

[0068] Generation of control feature maps:

[0069] The Chinese Pharmacopoeia Committee issued the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation Software System 2012 Edition A", and 10 batches of sample HPLC chromatogram peaks were automatically matched to form a common pattern map and establish a reference map. The results are shown in Figure 2 .

[0070] Relative retention time result analysis:

[0071] Taking puerarin as peak S, the relative retention times of 10 batches of samples were calculated, and the results are shown in Table 3. By measuring the characteristic spectra and calculating the relative retention times of 10 batches of test samples, 10 characteristic peaks were presented in the characteristic spectra of the test samples, of which 3 peaks should correspond to the retention times of the reference peaks, peak 2 is the chlorogenic acid reference peak, peak 8 is the 3,5-O-dicaffeoylquinic acid reference peak, and the peak corresponding to the puerarin reference peak (peak 3) is the S peak. The relative retention times of peaks 1, 4 to 7, 9, and 10 to the S peak were calculated and should be within the specified value ±10%. The specified values ​​of relative retention times are: 0.63 (peak 1), 1.13 (peak 4), 1.19 (peak 5), 1.93 (peak 6), 2.36 (peak 7), 2.58 (peak 9), and 2.93 (peak 10).

[0072] Table 3 Relative retention time results of 10 batches of samples

[0073] sample 1 2 3(S) 4 5 6 7 8 9 10 S2 0.636 0.693 1.000 1.127 1.188 1.907 2.344 2.455 2.560 2.919 S3 0.636 0.693 1.000 1.127 1.188 1.906 2.342 2.452 2.556 2.916 S4 0.635 0.692 1.000 1.126 1.188 1.903 2.340 2.450 2.555 2.915 S5 0.630 0.691 1.000 1.133 1.194 1.979 2.411 2.516 2.619 2.965 S6 0.629 0.690 1.000 1.134 1.195 1.977 2.408 2.512 2.616 2.962 S7 0.629 0.690 1.000 1.134 1.196 1.981 2.413 2.517 2.621 2.966 S8 0.629 0.693 1.000 1.132 1.191 1.960 2.398 2.507 2.611 2.962 S9 0.637 0.692 1.000 1.127 1.189 1.893 2.329 2.440 2.543 2.904 S10 0.637 0.692 1.000 1.127 1.189 1.889 2.327 2.439 2.542 2.907 S11 0.638 0.692 1.000 1.126 1.189 1.886 2.323 2.434 2.538 2.902

[0074] The characteristic peaks of Qiju Granules’ characteristic spectrum belong to:

[0075] Weigh 0.12g of wolfberry powder, 0.08g of chrysanthemum powder, 0.08g of kudzu root powder, 0.1g of astragalus powder, 0.06g of dendrobium powder, 0.06g of pollen cattail powder, and 0.03g of notoginseng powder, add 50ml of water respectively, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of 70% methanol to the residue, ultrasonically treat (power 500w, frequency 40kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain the solution of each medicinal piece for single-flavor drug identification. At the same time, prepare the reference solution contained in each medicinal piece for single peak identification, see Table 4 and Figure 3 .

[0076] Table 4 Chromatographic peaks of medicinal flavors

[0077]

[0078] Example 2 Selection of mobile phase elution gradient in the assay method

[0079] Octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as the mobile phase A, 0.1% phosphoric acid solution was used as the mobile phase B, and the gradient elution was performed according to Table 5. Other chromatographic conditions were the same as those in Example 1. Under these chromatographic conditions, the chromatographic peak separation was poor, and the chromatographic peaks were too concentrated at 35 to 45 minutes. Figure 4 ; The elution gradient was adjusted to Table 6, and it was found that the chromatographic peak separation was poor at 0 to 10 minutes, and the chromatographic peaks were more concentrated at 35 to 50 minutes. Figure 5 Continue to adjust the elution gradient, gradient elution was performed according to Table 7, the detection wavelength was 325nm, the chromatographic peak information was detected to be comprehensive, and the chromatographic peaks were evenly distributed, the separation was good, the detection time was 80 minutes, and the results were shown Figure 6 .

[0080] Table 5

[0081] Time (min) Acetonitrile (%) 0.1% phosphoric acid (%) 0～20 9→14 91→86 20～30 14→18 86→82 30～35 18→22 82→78 35～40 22→25 78→75 40～50 25→30 75→70

[0082] Table 6

[0083] Time (min) Acetonitrile (%) 0.1% phosphoric acid (%) 0～20 9→14 91→86 20～30 14→18 86→82 30～35 18→22 82→78 35～65 22 78

[0084] Table 7

[0085] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～30 9→14 91→86 30～35 14→18 86→82 35～80 18 82

[0086] Example 3 Selection of detection wavelength in the determination method

[0087] The test solution was scanned at a wavelength of 190-400 nm using a diode array detector. The results showed that within the wavelength range of 210-250 nm, there were few chromatographic peaks and an unstable baseline. The spectrum within the wavelength range of 265-365 nm was selected for comparison. Figure 7 At wavelengths of 265nm and 285nm, the chromatographic peak response values ​​between 40 and 65 minutes were low, indicating incomplete information. At wavelengths of 345nm and 365nm, the number of chromatographic peaks between 0 and 35 minutes was relatively small, and the response values ​​were also low. Comparison of different wavelengths revealed that at detection wavelengths of 305nm or 325nm, each chromatographic peak provided more information, with moderate response values, good resolution, and a relatively stable baseline.

[0088] Example 4 Investigation of the extraction solvent in the preparation of the test solution

[0089] Take Qiju granules, grind them into powder, take about 1.0g (a total of 4 groups), accurately weigh, and place them in stoppered conical flasks, accurately add 25ml of methanol, 70% methanol, 50% methanol, and 30% ethanol respectively, plug them tightly, weigh the weight, and ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, take them out, let them cool, weigh them again, and make up the lost weight with methanol, 70% methanol, 50% methanol, and 30% ethanol respectively, shake them well, filter, and take the filtrate to obtain. Accurately draw 10μl of the test solution respectively, inject it into the liquid chromatograph, measure it according to the chromatographic conditions in Example 1, record the chromatogram, and the results are shown in the figure. Figure 8 Results: When the solvent was 30% ethanol, the baseline was unstable from 40 to 46 minutes. When the solvent was methanol, the response values ​​of individual peaks decreased. When the solvent was 70% methanol or 50% methanol, there was little effect on the peak shape and number of characteristic spectra.

[0090] Example 5 Investigation of the extraction method in the preparation of the test solution

[0091] Take Qiju granules, grind them into powder, take about 1.0g (3 groups in total), weigh them accurately, place them in stoppered conical flasks, add 25ml of 70% methanol accurately, stopper them tightly, weigh them, ultrasonically treat them (power 500W, frequency 40kHz) for 30 minutes, reflux extract them for 30 minutes, and shake them for 30 minutes, take them out, let them cool, weigh them again, make up the lost weight with 70% methanol, shake them well, filter them, and take the filtrate. Accurately take 10μl of the filtrate separately, inject them into the liquid chromatograph, measure them, and record the chromatogram. The results are shown in the figure. Figure 9 Results: Using ultrasound, shaking and reflux, it was found that different extraction methods had no significant effect on the characteristic spectrum.

[0092] Example 6 Investigation of extraction time in preparation of test solution

[0093] Take Qiju granules, grind them into powder, take about 1.0g (3 groups in total), weigh accurately, and place them in stoppered conical flasks. Accurately add 25ml of 70% methanol, seal tightly, weigh the weight, and ultrasonically treat (power 500W, frequency 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. Take out, let cool, weigh the weight again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate. Accurately take 10μl of the filtrate separately, inject it into the liquid chromatograph, measure, record the chromatogram, and the results are shown in the figure. Figure 10 The results showed that different extraction times had no significant effect on the characteristic spectra.

[0094] Example 7 Specificity and integrity inspection

[0095] In order to investigate whether the blank solvent interferes with the determination, the test solution, reference solution and blank solvent were injected into the chromatograph respectively and the chromatogram was recorded. Figure 11As shown, the results show that the blank solvent has no interference; at the same time, the integrity of the test sample was also investigated, and the investigation was extended for 30 minutes. The results showed that there was no chromatographic peak within the 30-minute extended detection period. The results basically met the principle of maximum information content and had good integrity.

[0096] Example 8 Precision Test

[0097] Take Qiju granules, grind them into powder, take about 1.0g, weigh accurately, and operate according to the preparation and determination method of the test solution. Inject the sample 6 times continuously and calculate the relative retention time of each chromatographic peak and the S peak. The determination results are shown in Table 8 and Figure 12 .

[0098] Table 8 Relative retention time results of instrument precision test

[0099]

[0100]

[0101] The results showed that the relative retention times were basically consistent, RSD<2.0, and the instrument precision was good.

[0102] Example 9 Stability Test

[0103] Take Qiju granules, grind them into powder, take about 1.0g, weigh accurately, and operate according to the preparation and determination method of the test solution. After preparation, place the sample for determination at 0, 2, 4, 8, 12, and 24 hours, calculate the relative retention time of each chromatographic peak and the S peak. The determination results are shown in Table 9 and Figure 13 .

[0104] Table 9 Relative retention time results of stability test

[0105]

[0106] The results showed that the relative retention times were basically consistent, RSD<2.0%, and the test solution had good stability within 24 hours.

[0107] Example 10 Repeatability test

[0108] Take Qiju granules, grind them into powder, take about 1.0g (a total of 6 parts), accurately weigh, and operate according to the preparation and determination method of the test solution in Example 1 to calculate the relative retention time of each chromatographic peak and the S peak. The determination results are shown in Table 10 and Figure 14 .

[0109] Table 10 Relative retention time results of repeatability test

[0110]

[0111]

[0112] The results showed that the relative retention times were basically consistent with RSD<2.0%, indicating that the method had good repeatability.

[0113] Example 11 Durability Investigation

[0114] The durability of the chromatographic conditions under three different flow rates, three different column temperatures, three different brands of chromatographic columns, and two different manufacturers' instruments was investigated. The characteristic spectrum of the test sample should show a chromatographic peak with the same retention time as the reference chromatographic peak. The relative retention times of the remaining peaks were calculated with the S peak as the reference. The specific measurement results are shown in Tables 11 to 14. Figures 15 to 18 .

[0115] Table 11 Relative retention time results at different flow rates

[0116]

[0117] The results showed that the relative retention times at different flow rates were basically consistent, with RSD < 10.0%, indicating that the method had good durability.

[0118] Table 12 Relative retention time results at different column temperatures

[0119]

[0120] The results showed that the relative retention times at different column temperatures were basically consistent, with RSD<10.0%. However, when the column temperature was 32°C, the separation of peak 10 in the chromatogram was poor, so the column temperature was fixed at 30°C.

[0121] Table 13 Relative retention time results of different chromatographic columns

[0122]

[0123] The results showed that the relative retention times of different chromatographic columns were basically consistent, with RSD less than 10.0%, indicating that the method was robust.

[0124] Table 14 Relative retention time results of different instruments

[0125]

[0126] The results showed that the relative retention times of different instruments were basically consistent, with RSD less than 10.0%, indicating that the method had good durability.

[0127] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for detecting the characteristic spectrum of Qiju granules, characterized in that: The following steps are involved: a. Preparation of test solution: accurately weigh the Qiju granule sample, add solvent to dissolve and extract, filter, take the filtrate to obtain the test solution; wherein the Qiju granule is composed of wolfberry, chrysanthemum, Astragalus, Dendrobium, Panax notoginseng, raw pollen, and Pueraria; b. The test solution obtained in step a is subjected to high performance liquid chromatography to obtain a control characteristic spectrum of Qiju particles generated by common characteristic peaks; wherein, In the high performance liquid chromatography method, the chromatographic column is a C18 chromatographic column; acetonitrile is used as mobile phase A, and a 0.1% by volume phosphoric acid aqueous solution is used as mobile phase B, and gradient elution is performed; the gradient elution program is: 。 2. The detection method according to claim 1, characterized in that: The chromatographic conditions described in step b were used for analysis and determination, and the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012" software recommended by the State Pharmacopoeia Committee was used for analysis to obtain the HPLC control characteristic spectrum of Qiju Granules.

3. The detection method according to claim 1, characterized in that: The detection wavelength in the high performance liquid chromatography of step b is 265-345 nm, preferably 305-325 nm.

4. The detection method according to claim 1, characterized in that: In the high performance liquid chromatography method of step b, the C18 chromatographic column is 250 mm×4.6 mm, 5 μm; the injection volume is 8 μL-12 μL; and the flow rate of the mobile phase is 0.9-1.1 mL / min.

5. The detection method according to claim 1, characterized in that: In step b, the high performance liquid chromatography method, the column temperature of the C18 chromatographic column is 30°C.

6. The detection method according to claim 1, characterized in that: In step a, the solvent is a methanol aqueous solution, wherein the volume percentage concentration of the methanol aqueous solution is 50%-70%.

7. The detection method according to claim 1, characterized in that: In step a, the extraction method is ultrasound, shaking or reflux.

8. Application of the detection method of the characteristic spectrum of Qiju granules according to any one of claims 1-7 in the quality detection of the ingredients of Qiju granules.

9. A method for detecting the quality of Qiju granules, comprising: The characteristic spectrum of Qiju granules is obtained under the same conditions as the detection method of the characteristic spectrum of Qiju granules described in any one of claims 1 to 7, characterized in that the characteristic spectrum of Qiju granules contains 10 characteristic peaks, wherein peak 2 is a reference peak of chlorogenic acid reference substance, peak 3 is a reference peak of puerarin reference substance, peak 8 is a reference peak of 3,5-O-dicaffeoylquinic acid reference substance, and the relative retention time of each characteristic peak is calculated based on the puerarin reference substance reference peak as the S peak: peak 1 is 0.63±10%, peak 4 is 1.13±10%, peak 5 is 1.19±10%, peak 6 is 1.93±10%, peak 7 is 2.36±10%, peak 9 is 2.58±10%, and peak 10 is 2.93±10%.

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