A method for determining the contents of multiple active ingredients in Qirong tablets

Abstract

The present invention relates to a method for determining the contents of multiple active ingredients in Qirong tablets, wherein liquid chromatography is used to simultaneously determine the contents of 6 active ingredients in Qirong tablets, and a method is used to simultaneously determine the active ingredients of Cynomorium songaricum, protocatechuic acid, catechin, phloridzin, the active ingredient of Ligustrum lucidum, and the active ingredient of Epimedium, icariin. On the one hand, the content determination method can make up for the shortcomings of the existing method, provide a more comprehensive and effective drug control quality standard, further effectively guarantee product quality, and associate drug quality with clinical treatment active ingredients, thereby achieving stable, controllable, efficient and safe standards; on the other hand, it can realize the simultaneous detection of 6 active ingredients, which not only saves the detection cost, but also reduces the amount of reagents used and meets the requirements of green environmental protection.

Description

Technical Field

[0001] The invention belongs to the technical field of drug quality detection and analysis, and specifically relates to a method for determining the contents of multiple effective ingredients in Qirong tablets. Background Art

[0002] At present, the method for treating insomnia and forgetfulness includes taking western medicine, Chinese medicine and Chinese acupuncture treatment. Commonly used western medicines such as receptor agonists and antidepressant drugs with hypnotic effect, although these drugs can alleviate some symptoms, it is difficult to achieve the purpose of complete cure, and the drug side effects are large, and the regulating effect on the whole body is poor. The treatment of Chinese medicine is mainly based on the principle of syndrome differentiation and treatment, mainly based on tonifying the kidney, calming the nerves and nourishing the heart. Commonly used clinical medicines include Qirong tablets, Baizi Yangxin capsules, Huolisu oral liquid, Anshen Yangxin pills, etc., among which Qirong tablets are produced by Xi'an Qianhe Pharmaceutical Co., Ltd., with the functions of tonifying the kidney and consolidating essence, benefiting intelligence and calming the nerves, and are used for spermatorrhea caused by kidney deficiency, impotence and premature ejaculation, insomnia and forgetfulness. Qirong tablets are processed into film-coated tablets by nine medicines of wolfberry, Cistanche, Cynomorium songaricum, Cnidium monnieri, Ligustrum lucidum, Schisandra chinensis, Rosa laevigatae, Epimedium, and Cuscuta australis. This variety is widely recognized by patients and medical workers during clinical use because of its definite efficacy in treating insomnia and forgetfulness caused by kidney deficiency and kidney yang deficiency. However, the current national drug standard WS3-B-2523-97-1 only conducts thin layer identification of single ingredients in the prescription, including epimedium, schisandra, wolfberry, cnidium monnieri and cistanche. Although the content of icariin in epimedium was determined, quality control was not carried out on other key medicinal ingredients in the prescription, such as the main drug Cynomorium songaricum and the auxiliary drug Ligustrum lucidum.

[0003] Cynomorium songaricum is a traditional precious Chinese medicinal material, which has the effects of tonifying kidney yang, benefiting essence and blood, moistening intestines and relieving constipation. It is used for kidney yang deficiency, essence and blood deficiency, waist and knee weakness, impotence and spermatorrhea, dry intestines and constipation, etc. Cynomorium songaricum contains flavonoids, terpenes, steroids, organic acids, sugars and glycosides, volatile components and tannins, etc., which have a good enhancing and promoting effect on human immune function, sexual function, intestinal function, and adrenal cortex secretion function. Modern pharmacological studies have shown that Cynomorium songaricum has a wide range of pharmacological effects such as anti-fatigue, liver protection, enhancing tissue tolerance to hypoxia, improving body immunity, scavenging free radicals, inhibiting human immunodeficiency virus (HIV), inhibiting platelet aggregation and estrogen-like. Epimedium has the effects of tonifying kidney yang, strengthening tendons and bones, and removing rheumatism. It is used for impotence and spermatorrhea, tendon and bone weakness, rheumatic pain, numbness and cramps caused by kidney yang deficiency, and is a commonly used Chinese medicine in clinical practice. Modern pharmacological studies have shown that Epimedium has multiple pharmacological effects, such as promoting osteoblast growth, calming, anti-depression, enhancing immune function, anti-oxidation, and anti-aging. Ligustrum lucidum has the effects of nourishing the liver and kidney, improving eyesight and darkening hair. It is mainly used to treat symptoms such as liver and kidney yin deficiency, dizziness and tinnitus, bone steaming and hot flashes. It is a commonly used yin-tonifying medicine for strengthening the body; chemical composition research shows that the main active ingredients are terpenes, phenylethanol, polysaccharides, etc. Modern pharmacological studies have shown that Ligustrum lucidum has multiple pharmacological effects, such as anti-oxidation, anti-aging, anti-tumor, lowering blood lipids, immunomodulation, and scavenging free radicals.

[0004] Cynomorium songaricum is the main drug in the prescription of Qirong Tablets. It is a traditional precious Chinese medicinal material and is used in the largest amount in the prescription; Ligustrum lucidum is the secondary drug and is used in a large amount. However, there are widespread adulteration, mixing, and abuse of medicinal materials in the market. Especially in the process of drug procurement, acceptance, and identification, it was found that there were many confusing products of Cynomorium songaricum, Ligustrum lucidum, and Epimedium, which was very confusing, and there was also the phenomenon of adding counterfeit products to genuine products. In addition, the Cynomorium songaricum medicinal material included in the 2020 edition of the "Chinese Pharmacopoeia" has no content determination items, which makes it difficult to fully reflect its intrinsic quality. Moreover, the original quality standards cannot fully reflect the quality of Qirong Tablets. Therefore, it is necessary to continue to improve the detection methods of this product to ensure the quality and clinical efficacy of this product. In the original quality standard WS3-B-2523-97-1, only the content of icariin in the epimedium in the prescription was determined, and thin layer identification of single ingredients of epimedium, schisandra, wolfberry, cnidium monnieri and cistanche was performed. No quality control was performed on other key medicinal ingredients in the prescription. The current quality control standards have poor specificity and cannot effectively guarantee the quality of the product. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a method for determining the content of multiple active ingredients in Qirong Tablets, which simultaneously determines the effective ingredients of Cynomorium songaricum, protocatechuic acid, catechin, phloridzin, the effective ingredient of Ligustrum lucidum, and the effective ingredient of Epimedium, icariin, a total of 6 active ingredients. On the one hand, this detection method can make up for the deficiencies of the existing methods, provide a more comprehensive and effective drug control quality standard, and further effectively guarantee product quality; on the other hand, it can realize the simultaneous detection of 6 active ingredients, which not only saves the detection cost, but also reduces the amount of reagents used and meets the requirements of green environmental protection.

[0006] To achieve the above-mentioned object, the present invention is implemented by adopting the following technical solutions:

[0007] A method for determining the content of Qirong tablets, wherein the Qirong tablets are prepared from the following nine medicinal materials: wolfberry fruit, Cistanche deserticola, Cynomorium songaricum, Fructus Cnidium monnieri, Fructus Ligustri Lucidi, Fructus Schisandrae Chinensis, Fructus Rosae Laevigatae, Herba Epimedii, and Fructus Cuscutae; an HPLC method is used to simultaneously determine gallic acid, protocatechuic acid, catechin, and phloridzin in Cynomorium songaricum, salidroside in Fructus Ligustri Lucidi, and icariin in Herba Epimedii, and the method comprises the following steps:

[0008] 1) Chromatographic conditions and system suitability test

[0009] Octadecylsilane bonded silica gel is used as filler; an organic solvent is used as phase A, a certain concentration of dilute acid aqueous solution is used as phase B, gradient elution is performed, the organic solvent is selected from methanol, acetonitrile or acetonitrile-methanol mixed solution, the dilute acid aqueous solution is selected from phosphoric acid, formic acid, glacial acetic acid or a mixture of the above three with a weight or volume percentage concentration of 0.01%-1.0%; the column temperature is 10-40°C; a diode array detector (PDA) is used, the detection wavelength for determining gallic acid, protocatechuic acid, catechin and phlorizin is 275-285nm; the detection wavelength for determining salidroside is 265-275nm; the detection wavelength for determining icariin is 270-280nm; the flow rate is 0.8ml / min-1.2ml / min; the injection volume is 10-20μl.

[0010] 2) Preparation of reference solution

[0011] Accurately weigh an appropriate amount of each reference substance, and add methanol to prepare stock solutions of 0.1 mg / ml gallic acid, 0.06 mg / ml protocatechuic acid, 0.14 mg / ml catechin, 0.12 mg / ml phlorizin, 0.06 mg / ml salidroside, and 0.2 mg / ml icariin; accurately pipette 1 ml of each of the above 6 stock solutions into the same 10 ml brown volumetric flask, add methanol to dissolve and make up to volume, shake well, and use them as mixed reference substance solutions with mass concentrations of 10, 6, 14, 12, 6, and 20 μg / ml, respectively.

[0012] 3) Preparation of test solution

[0013] Take 20 pieces of Qirong tablets, remove the film coating, accurately weigh, grind, mix, take about 0.2-1.0g, accurately weigh, put in a stoppered conical flask, accurately add 20-50ml of methanol, weigh the weight, ultrasonically treat for 30-50min (250W, 50kHz), cool, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

[0014] 4) Determination

[0015] Accurately pipette 10-20 μl of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph, and measure to obtain the measurement result.

[0016] Preferably, the phase A is acetonitrile, the phase B is a 0.05% by volume phosphoric acid aqueous solution; the flow rate is 1.0 ml / min; the column temperature is 30°C; the detection wavelength for determining gallic acid, protocatechuic acid, catechin, and phlorizin is 280 nm; the detection wavelength for determining salidroside and icariin is 273 nm.

[0017] Preferably, during gradient elution, the gradient elution program is: 0-10 min, 8% A; 10-20 min, 8-20% A; 20-30 min, 20%-35% A; 35-35 min, 35-45% A; 35-45 min, 8% A;

[0018] The specific gradient elution program is:

[0019]

[0020]

[0021] Preferably, take 20 pieces of Qirong tablets, remove the film coating, accurately weigh, grind, mix, weigh 0.4 g, accurately weigh, put in a stoppered conical flask, accurately add 25 ml of methanol, weigh the weight, ultrasonically treat for 40 minutes (250W, 50kHz), cool, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

[0022] The specific prescription of Qirong tablets in the present invention is: 70g of wolfberry, 140g of Cistanche deserticola, 210g of Cynomorium songaricum, 42g of Cnidium monnieri, 56g of Ligustrum lucidum, 42g of Schisandra chinensis, 56g of Rosa laevigata, 98g of Epimedium and 84g of Cuscuta australis.

[0023] Compared with the prior art, the present invention has the following beneficial technical effects:

[0024] The content determination method of Qirong tablets of the present invention is based on the content determination of icariin in the prescription of epimedium in the original quality standard WS3-B-2523-97-1, and adds gallic acid, protocatechuic acid, catechin, phloridzin, and salidroside, which are effective components of Cynomorium songaricum in the prescription, and simultaneously determines icariin, an effective component in epimedium, a total of 6 effective components are simultaneously determined, and wavelength switching technology is used for detection, and wavelength switching detection is performed by using a diode array detector to comprehensively consider the wavelengths with the strongest absorption of different effective components and less interference from impurities. The measured components can be better separated in a chromatogram; the interference impurities are removed, which is conducive to the accurate calculation of the component content. The detection method has high sensitivity, strong specificity, good accuracy and reproducibility, and can make up for the shortcomings of the existing method; at the same time, multiple effective components can be determined at one time, the inspection time is saved, the detection efficiency is improved, and the quality of the product is effectively guaranteed; and the reduction in the amount of reagents used meets the requirements of green environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below in conjunction with the accompanying drawings:

[0026] Figure 1 It is the blank reagent chromatogram of the specificity test of the present invention.

[0027] Figure 2 This is the chromatogram of the negative sample of Cynomorium songaricum in the specificity test of the present invention.

[0028] Figure 3 This is the chromatogram of the negative sample lacking Ligustrum lucidum in the specificity test of the present invention.

[0029] Figure 4 This is the chromatogram of the negative sample lacking Epimedium in the specificity test of the present invention.

[0030] Figure 5 It is the chromatogram of the reference substance under chromatographic conditions of the present invention.

[0031] Figure 6 The chromatogram of the test sample under the chromatographic conditions of the present invention; wherein, 1 is the gallic acid peak, 2 is the protocatechuic acid peak, 3 is the catechin peak, 4 is the phloridzin peak, 5 is the salidroside peak, and 6 is the icariin peak.

[0032] Figure 7 It is a linear relationship diagram of the six components in the present invention. DETAILED DESCRIPTION

[0033] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and do not limit the scope of the present invention in any way. The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are commercially available products unless otherwise specified.

[0034] Example 1: Prescription and preparation method of Qirong tablets

[0035] Prescription: Lycium barbarum 70g, Cistanche deserticola 140g, Cynomorium songaricum 210g, Cnidium monnieri 42g, Ligustrum lucidum 56g, Schisandra chinensis 42g, Rosa laevigata 56g, Epimedium 98g, Cuscuta australis 84g

[0036] Preparation method: For the above nine ingredients, crush Cynomorium songaricum into fine powder and sieve; extract Schisandra chinensis and Cnidium monnieri with 60% ethanol by reflux, the first time for 2 hours and the second time for 1 hour, combine the extracts, filter, recover ethanol from the filtrate and concentrate to an appropriate amount; decoct the remaining six ingredients including Lycium barbarum with water twice, the first time for 3 hours and the second time for 2 hours; combine the decoctions, let stand, take the supernatant and concentrate to an appropriate amount, add the above Schisandra chinensis paste and stir well, concentrate to a thick paste, add Cynomorium songaricum powder and mix well, dry, crush, add starch, hydroxypropyl cellulose and silicon dioxide in appropriate amounts, granulate, press into 1000 tablets, and coat with a film.

[0037] Example 2: Selection of detection wavelength

[0038] Through the ultraviolet absorption spectra of six reference solutions of gallic acid, protocatechuic acid, catechin, phlorizin, salidroside, and icariin, and referring to the 2020 edition of the "Chinese Pharmacopoeia" and related literature, it can be seen that gallic acid and protocatechuic acid have maximum absorption at 280nm, catechin has maximum absorption at 210nm and 280nm, phlorizin has maximum absorption at 225nm and 285nm, salidroside has maximum absorption at 224nm and 275nm, icariin has maximum absorption at 240nm and 280nm. The maximum absorption is at 270nm; although catechin has maximum absorption at 210nm, phlorizin at 225nm, and salidroside at 224nm low band, the interference of chromatographic background is also large. Since the maximum absorption wavelengths of gallic acid, protocatechuic acid, catechin and phlorizin in the high band are similar, after comprehensive consideration and analysis, the detection wavelength of gallic acid, protocatechuic acid, catechin and phlorizin is finally determined to be 280nm; similarly, the detection wavelength of salidroside and icariin is determined to be 273nm.

[0039] The experiment shows that the wavelength switching technology of diode array detector (PDA) is used for detection. The wavelength switching detection is performed by using diode array detector to comprehensively consider the wavelengths with the strongest absorption of different effective ingredients and the least interference from impurities. The components to be measured can be better separated in one chromatogram; the removal of interfering impurities is conducive to the accurate calculation of component content.

[0040] Example 3: Selection of mobile phase

[0041] In order to effectively separate the six active ingredients in the test sample and meet the requirements of peak shape, theoretical plate number, etc., the preliminary test used different ratios of fluidity such as acetonitrile-0.5% glacial acetic acid solution, methanol-0.5% phosphoric acid solution, acetonitrile-0.1% phosphoric acid solution, methanol-acetonitrile-0.2% phosphoric acid solution, and acetonitrile-0.05% phosphoric acid solution to test. For example, when the mobile phase acetonitrile-0.5% glacial acetic acid solution was used for isocratic elution at a ratio of 20:60 or 60:40, the retention time of the former was too long, and the latter's main peak and impurities could not be effectively separated and the peak shape was not good. When the mobile phase methanol-0.5% phosphoric acid solution was used for isocratic elution at a ratio of 20:80 or 60:40, the former did not produce a peak within 30 minutes and the baseline drifted, and the latter's main peak and impurities could not be effectively separated and the peak shape was not good. When the ratio of isocratic elution of acetonitrile-0.1% phosphoric acid solution was 20:80 or 60:40, the former had no peak within 30 minutes and the baseline drifted, and the latter had some main peaks and impurities that could not be effectively separated and the peak shape was not good. When the ratio of isocratic elution of methanol-acetonitrile-0.2% phosphoric acid solution was 10:10:80 or 30:30:40, the peak of the effective component had tailing phenomenon, but the retention time of the former was too long, and the main peak of the latter could not be effectively separated from the impurities. When the ratio of isocratic elution of acetonitrile-0.05% phosphoric acid solution was 25:75 or 75:25, the baseline was stable and the retention time was long, but the peaks of salidroside overlapped. The results showed that the linear gradient separation effect of acetonitrile-0.05% phosphoric acid aqueous solution was the best and the baseline was the most stable, but the retention time was longer. Therefore, the mobile phase A was acetonitrile and the mobile phase B was 0.05% phosphoric acid aqueous solution by volume for gradient elution test. After many experiments, none of them were ideal. For example, the mobile phase acetonitrile-0.05% phosphoric acid solution was used for gradient elution: 0-8min, A: 8; 8-20min, A: 8→20; 20-35min, A: 20→35; 35-45min, A: 35→45; 45-50min, A: 8; the results showed that the gallic acid peak and protocatechuic acid peak were not completely separated. The mobile phase was acetonitrile-0.05% phosphoric acid solution, gradient elution: 0-10 min, A: 8; 10-30 min, A: 8→20; 30-35 min, A: 20→35; 35-45 min, A: 35→45; 45-50 min, A: 8; the results showed that the catechin peak and salidroside peak were not completely separated, while the separation degree, peak shape, theoretical plate number, etc. of the other four active ingredients met the requirements.The final test found that the mobile phase acetonitrile-0.05% phosphoric acid solution was used for gradient elution, and the gradient elution program was: 0-10min, 8%A; 10-20min, 8-20%A; 20-30min, 20%-35%A; 35-35min, 35-45%A; 35-45min, 8%A; the results showed that the separation effect was best under the conditions of gradient elution, the baseline was stable, the chromatographic peaks of the six active ingredients could be completely separated, and the peak time, peak shape and separation degree of each component met the requirements, so the conditions can meet the relevant requirements of analytical determination.

[0042] Example 4: Specificity study

[0043] 4.1 Preparation of each negative sample:

[0044] According to the sample prescription process and formula ratio (Example 1), negative samples not containing Cynomorium songaricum, Ligustrum lucidum and Epimedium were prepared respectively; for example, preparation of negative sample not containing Cynomorium songaricum: Schisandra chinensis and Cnidium monnieri were extracted with 60% ethanol by reflux, the first time for 2 hours, the second time for 1 hour, the extracts were combined, filtered, ethanol was recovered from the filtrate, and concentrated to an appropriate amount; Lycium barbarum, Cistanche deserticola, Ligustrum lucidum, Rosacea laevigata, Epimedium and Cuscuta australis were decocted with water twice, the first time for 3 hours, the second time for 2 hours; the decoctions were combined, allowed to stand, the supernatant was concentrated to an appropriate amount, the above-mentioned Schisandra chinensis paste was added and stirred thoroughly, concentrated into a thick paste, starch, hydroxypropyl cellulose and silicon dioxide were added in appropriate amounts, stirred well, vacuum dried, and crushed into fine powder to obtain.

[0045] The negative sample without Ligustrum lucidum and Epimedium was prepared in the same way.

[0046] 4.2 Preparation of negative sample solution:

[0047] Weigh 0.4 g of each negative sample accurately, place in a stoppered conical flask, accurately add 25 ml of methanol, weigh the weight, ultrasonically treat for 50 min (250 W, 50 kHz), cool, make up the lost weight with methanol, shake well, filter, and obtain the filtrate.

[0048] 4.3 Preparation of test solution:

[0049] Test method: Take 20 pieces of Qirong tablets of the same batch, remove the film coating, accurately weigh, grind, mix, take about 0.2-1.0g, accurately weigh, put in a stoppered conical bottle, accurately add 20-50ml of methanol, weigh the weight, ultrasonically treat for 30-90min (250W, 50kHz), cool, make up the lost weight with methanol, shake well, filter, and take the filtrate. Accurately take 10μl of the reference solution and the test solution, inject into the high performance liquid chromatograph, and measure to get the test results.

[0050] Method 1: Accurately add 25 ml of methanol, weigh accurately, extract by ultrasonic for 30 min, cool to make up the weight, and filter;

[0051] Method 2: Accurately add 25 ml of 80% methanol, weigh accurately, extract by ultrasonic for 30 min, cool to make up the weight, and filter;

[0052] Method 3: Accurately add 25 ml of 60% methanol, weigh accurately, extract by ultrasonic for 30 min, cool to make up the weight, and filter;

[0053] Method 4: Accurately add 25 ml of methanol, weigh accurately, heat under reflux for 60 min, cool to make up the weight, and filter;

[0054] Method 5: Accurately add 25 ml of 80% methanol, weigh accurately, heat under reflux for 60 min, cool to make up the weight, and filter;

[0055] Method 6: Accurately add 25 ml of 60% methanol, weigh accurately, heat under reflux for 90 min, cool to make up the weight, and filter;

[0056] Method 7: Accurately add 25 ml of methanol, weigh accurately, extract by ultrasonic for 35 min, cool to make up the weight, and filter;

[0057] Method 8: Accurately add 25 ml of methanol, weigh accurately, extract by ultrasonic for 40 min, cool to make up the weight, and filter;

[0058] Method 9: Accurately add 25 ml of methanol, weigh accurately, extract by ultrasonic for 50 min, cool to make up the weight, and filter;

[0059] Table 1 Test results for selection of test sample preparation method

[0060]

[0061] The results in Table 1 show that: when using method 1, the effective components can be effectively extracted, but the peak area is slightly lower than that of method 8, indicating that the effective components cannot be 100% transferred after 30 minutes of ultrasonic extraction; when using methods 2 and 3, the peak areas of the effective components are significantly lower due to the lower methanol concentration, indicating that the transfer rate of the effective components is low; when using methods 4, 5 and 6, the peak areas of protocatechuic acid and salidroside are smaller, which may be due to the loss of flavonoid components during the heating process; when using method 7, salidroside is not fully extracted, and the peak area is significantly lower When using method 8 and method 9; there is no significant difference in peak area when using method 8 and method 9, but the ultrasonic extraction time of method 9 is longer; therefore, the best method for preparing the test solution is determined to be method 8: 20 pieces of Qirong tablets, remove the film coating, accurately weigh, grind, mix, weigh 0.4g, accurately weigh, place in a stoppered conical flask, accurately add 25ml of methanol, weigh the weight, ultrasonically treat for 40min (250W, 50kHz), cool, make up the lost weight with methanol, shake well, filter, and take the filtrate.

[0062] 4.4 Chromatographic conditions and system suitability test

[0063] A chromatographic column with octadecylsilane bonded silica gel as filler (250mm×4.6mm, 5μm); mobile phase A as determined in Table 1 is acetonitrile, and phase B is a 0.05% by volume phosphoric acid aqueous solution; the flow rate is 1.0ml / min; the column temperature is 30°C; the detection wavelength for determining gallic acid, protocatechuic acid, catechin, and phlorizin is 280nm; the detection wavelength for determining salidroside and icariin is 273nm; the injection volume is 10μl; and the number of theoretical plates should not be less than 3000.

[0064] 4.5 Preparation of reference solution:

[0065] Accurately weigh an appropriate amount of each reference substance, and add methanol to prepare stock solutions of 0.1056 mg / ml gallic acid, 0.0631 mg / ml protocatechuic acid, 0.1452 mg / ml catechin, 0.1265 mg / ml phlorizin, 0.0586 mg / ml salidroside, and 0.2104 mg / ml icariin; accurately pipette 1 ml of each of the above 6 stock solutions in turn and place them in the same 10 ml brown volumetric flask, add methanol to dissolve and make up to volume, shake well, and use them as mixed reference substance solutions with mass concentrations of 10.56, 6.31, 14.52, 12.65, 5.86, and 21.04 μg / ml, respectively.

[0066] 4.6 Determination:

[0067] Accurately pipette 10 μl of each negative sample solution, mixed reference solution, and test solution, respectively, and inject into LC-2030CPlus high performance liquid chromatograph (PDA detector, Shimadzu) for determination.

[0068] The results showed that: in the chromatogram of the test solution, there were identical chromatographic peaks at the corresponding positions of the chromatographic peaks of the six reference substances; there was no interference in the negative, and the separation degree and theoretical plate number met the requirements; indicating that the chromatographic conditions were highly specific. Figure 4 .

[0069] Example 5: Repeatability test

[0070] 5.1 Chromatographic conditions and system suitability test are the same as in Example 4

[0071] 5.2 Accurately pipette 10 μl of the same test solution and inject it into the liquid chromatograph. Repeat the injection 6 times and calculate the peak area RSD.

[0072] 5.3 The results show that the RSD values ​​of the peak areas of the six active ingredients are 0.71%, 1.15%, 1.11%, 0.86%, 1.07%, and 0.93%, respectively, all less than 2.0%, indicating good precision. The specific results are shown in Table 2 below.

[0073] Table 2: Peak area data of 6 active ingredients in repeatability experiment

[0074] frequency gallic acid Protocatechuic acid Catechins Phlorizin Salidroside Icariin 1 870.518 654.426 3556.789 1064.582 294.637 4362.364 2 868.615 658.727 3507.291 1076.758 301.289 4357.849 3 860.361 665.208 3569.289 1058.178 297.246 4397.645 4 859.293 649.375 3509.854 1062.159 301.338 4305.261 5 855.302 669.158 3468.251 1049.353 303.564 4316.257 6 867.492 653.295 3489.386 1057.456 299.689 4403.654 average 863.597 658.365 3516.81 1061.414 299.627 4357.172 RSD% 0.71％ 1.15％ 1.11％ 0.86％ 1.07％ 0.93％

[0075] Example 6: Linear range

[0076] 6.1 Chromatographic conditions and system suitability test are the same as in Example 4

[0077] 6.2 Accurately pipette 1.5 ml of each reference substance stock solution into a 10 ml brown volumetric flask and dilute to volume with methanol to obtain a mixed reference substance solution. Accurately pipette 2 μl, 5 μl, 8 μl, 10 μl, 15 μl, and 20 μl of the mixed reference substance solution, respectively, and perform detection according to the chromatographic conditions described in Example 3. Record the chromatogram, perform linear regression analysis on the peak area (Y) versus the concentration (X), draw a linear relationship diagram, and report the linear equation. The results are shown in Tables 3 and Figure 7 .

[0078] Table 3: Standard curve equations, correlation coefficients and linear ranges of the six components

[0079] Ingredient name Regression equation R Linear range (μg / ml) gallic acid Y=82.379X-1.0894 0.9986 2.1-21.0 Protocatechuic acid Y=109.03X+4.9796 0.9991 1.26-12.60 Catechins Y=239.33X-3.9592 0.9995 2.9-29.0 Phlorizin Y=82.816X+10.58 0.9986 2.52-25.20 Salidroside Y=54.157X+1.9557 0.9974 1.16-11.60 Icariin Y=207.56X-2.4083 0.9992 4.2-42.0

[0080] Example 7: Accuracy (Recovery)

[0081] 7.1 Chromatographic conditions and system suitability test are the same as in Example 4

[0082] 7.2 Preparation of test solution:

[0083] Take 20 pieces of Qirong tablets, remove the film coating, accurately weigh, grind, mix, weigh 6 portions of 0.4g sample, accurately weigh, and place in a 10ml brown volumetric bottle, then accurately pipette 1ml of each stock solution of 0.1056mg / ml of gallic acid, 0.0631mg / ml of protocatechuic acid, 0.1452mg / ml of catechin, 0.1265mg / ml of phloridzin, 0.0586mg / ml of salidroside, and 0.2104mg / ml of icariin into the above brown volumetric bottle, add appropriate amount of methanol, ultrasonically treat (250W, 40kHz) for 40min, cool, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain. Determination: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph, and measure the recovery rate and RSD% of gallic acid, protocatechuic acid, catechin, phloridzin, salidroside and icariin.

[0084] Table 4: Recovery test data table

[0085]

[0086] Recovery rate % = (CA) / B×100%, where A is the amount of the measured component contained in the test sample; B is the amount of reference substance added; and C is the actual measured value.

[0087] The results are shown in Table 4. The standard requirements are: the recovery rate is required to be 85% to 110%, and the recovery rate RSD is less than 4.0%. The experimental results show that the method has good accuracy.

[0088] Example 8: Intermediate Precision

[0089] 8.1 Chromatographic conditions and system suitability test are the same as in Example 4

[0090] 8.2 In the same laboratory, different experimenters A and B conducted experiments on different dates using different chromatographs to calculate the content of each active ingredient. The results are shown in Table 5, which shows that the invention patent method has good precision.

[0091] Table 5: Intermediate precision test data table

[0092]

[0093] Example 9: Durability

[0094] 9.1 Chromatographic conditions and system suitability test are the same as in Example 4

[0095] 9.2 Take the same test solution, place it at room temperature, and accurately pipette 10 μl into the HPLC at 0, 4, 8, 12, and 24 hours respectively; the results are shown in Table 6. The results show that the maximum RSD of the peak area of ​​the test solution within 24 hours is only 0.19%, which is much smaller than the standard 2.0%, indicating that the test solution is stable within 24 hours at room temperature.

[0096] Table 6: Stability test data table

[0097] Injection time point 0h 2h 4h 8h 12h 24h average RSD% gallic acid 870.518 880.513 865.521 879.365 859.987 864.539 870.074 0.96 Protocatechuic acid 654.426 657.892 666.351 659.854 659.301 658.108 659.322 0.60 Catechins 3556.789 3551.615 3569.217 3497.856 3542.572 3501.235 3536.547 0.85 Phlorizin 1064.582 1069.348 1076.324 1059.629 1052.369 1048.968 1061.870 0.97 Salidroside 299.637 300.258 301.598 306.359 302.900 298.364 301.519 0.94 Icariin 4362.364 4359.357 4405.366 4358.654 4358.896 4409.276 4375.652 0.56

[0098] In summary: the method for determining the content of multiple active ingredients in Qirong Tablets of the present invention verifies the scientific nature of the method from the aspects of specificity, repeatability, accuracy (recovery rate), intermediate precision, durability, etc. By determining the content of 6 active ingredients in 3 medicinal materials in the prescription, the product quality standard is further improved, the safety and effectiveness of Qirong Tablets are guaranteed, and the deficiencies of the prior art are made up.

[0099] Example 10: Stability Test

[0100] 10.1 Chromatographic conditions and system suitability test are the same as in Example 4

[0101] 10.2 Commercially available samples of Qirong tablets (batch numbers: 20200301, 20200302, 20200303, produced by Xi'an Qianhe Pharmaceutical Co., Ltd.) were taken for accelerated stability tests and room temperature long-term stability tests. The test results are shown in Table 7.

[0102] Table 7: Accelerated and long-term stability test data of Qirong tablets

[0103]

[0104] Accelerated experimental conditions: Place the samples in a stability test chamber with a relative humidity of 75%±5% and a temperature of 40℃±2℃ for a 6-month accelerated experimental investigation, and take samples for testing at the 0th, 1st, 2nd, 3rd and 6th month respectively; Room temperature experimental conditions: Place the samples in a stability test chamber with a relative humidity of 60%±5% and a temperature of 25℃±2℃ for a long-term experimental investigation for 24 months, and inspect them at the 0th, 3rd, 6th, 12th, 18th and 24th month respectively.

[0105] The experimental results show that the contents of the active ingredients gallic acid, protocatechuic acid, catechins, phloridzin, salidroside and icariin tested did not change significantly in the accelerated test for 6 months and the long-term experiment for 24 months, which met the requirements. This shows that this detection method can effectively control the quality of the drug and can be considered for inclusion in the quality standard. In addition, according to the test results of three consecutive batches of products (batch numbers: 20200301, 20200302, and 20200303) at the end of 24 months, the average content of gallic acid was 0.199 mg / tablet, the average content of protocatechuic acid was 0.114 mg / tablet, the average content of catechins was 0.286 mg / tablet, the average content of phlorizin was 0.112 mg / tablet, the average content of salidroside was 0.100 mg / tablet, and the average content of icariin was 0.415 mg / tablet; taking into account the differences between different raw materials and the errors in the production and inspection processes, it is recommended that the content standard can be set as: gallic acid ≥ 0.18 mg / tablet, protocatechuic acid ≥ 0.10 mg / tablet, catechins ≥ 0.27 mg / tablet, phlorizin ≥ 0.10 mg / tablet, salidroside ≥ 0.09 mg / tablet, and icariin ≥ 0.40 mg / tablet.

[0106] The above embodiments are preferred examples for implementing the present invention, and the present invention is not limited to the above embodiments. Any non-essential additions and substitutions made by those skilled in the art based on the technical features of the technical solution of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for determining the contents of multiple active ingredients in Qirong tablets, characterized in that The liquid chromatography method is used to simultaneously determine the contents of naringin in Rhizoma Drynariae and curculigoside in Rhizoma Curculigoe, which specifically comprises the following steps: 1) Chromatographic conditions and system suitability test Octadecylsilane bonded silica gel is used as filler; organic solvent is used as phase A, and a certain concentration of dilute acid aqueous solution is used as phase B, and gradient elution is performed, wherein the organic solvent is acetonitrile, and the dilute acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage concentration of 0.05%; the column temperature is 10-40°C; a diode array detector PDA is used, and the detection wavelength for determining gallic acid, protocatechuic acid, catechin, and phlorizin is 275-285nm; the detection wavelength for determining salidroside is 265-275nm; the detection wavelength for determining icariin is 270-280nm; the flow rate is 0.8ml / min-1.2ml / min; and the injection volume is 10-20μl; The gradient elution program is: 0-10 min, 8% A; 10-20 min, 8-20% A; 20-30 min, 20%-35% A; 35-35 min, 35-45% A; 35-45 min, 8% A; 2) Preparation of reference solution Accurately weigh appropriate amount of each reference substance, and add methanol to prepare stock solutions of 0.1 mg / ml gallic acid, 0.06 mg / ml protocatechuic acid, 0.14 mg / ml catechin, 0.12 mg / ml phlorizin, 0.06 mg / ml salidroside, and 0.2 mg / ml icariin; accurately pipette 1 ml of each of the above 6 stock solutions into the same 10 ml brown volumetric flask, add methanol to dissolve and make up to volume, shake well, and prepare mixed reference substance solutions with mass concentrations of 10, 6, 14, 12, 6, and 20 μg / ml, respectively; 3) Preparation of test solution Take 20 pieces of Qirong tablets, remove the film coating, accurately weigh, grind, mix, take 0.2-1.0g, accurately weigh, put in a stoppered conical flask, accurately add 20-50ml of methanol, weigh the weight, ultrasonically treat for 30-50min, the ultrasonic treatment conditions are power 250W, frequency 50 kHz, let cool, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain; 4) Determination Accurately pipette 10-20 μl of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph, and measure to obtain the measurement result.

2. The method for determining the contents of multiple active ingredients in Qirong tablets according to claim 1, characterized in that: The phase A is acetonitrile, and the phase B is a 0.05% by volume phosphoric acid aqueous solution; the flow rate is 1.0 ml / min; the column temperature is 30° C.; the detection wavelength for determining gallic acid, protocatechuic acid, catechin, and phlorizin is 280 nm; the detection wavelength for determining salidroside and icariin is 273 nm.

3. The method for determining the contents of multiple active ingredients in Qirong tablets according to claim 1, characterized in that: In step 3), preparation of the test solution: take 20 Qirong tablets, remove the film coating, accurately weigh, grind, mix, weigh 0.4g, accurately weigh, place in a stoppered conical flask, accurately add 25ml of methanol, weigh the weight, ultrasonically treat for 40 min, the ultrasonic treatment conditions are power 250W, frequency 50 kHz, cool, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

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