Method for determining contents of multiple active ingredients in Zhikang Capsules by HPLC

Abstract

The present invention relates to a method for determining the contents of multiple active ingredients in Zhikang Capsules by HPLC. This method simultaneously determines three active ingredients, namely imperatorin in Angelica dahurica in the preparation, 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalate in Bletilla striata, and dracorhodin B in Dracaena cochinchinensis, and adopts a dual-wavelength mode for detection. This detection method has high sensitivity, strong specificity, good accuracy and reproducibility. At the same time, it can determine multiple active ingredients at one time, saving the inspection time and improving the detection efficiency. This content determination method is sensitive, specific, accurate and reproducible, can make up for the deficiencies of existing methods, effectively control the quality of drugs, associate the drug quality with the active ingredients of clinical treatment, so as to meet the clinical needs better by reaching the standards of stability, controllability, high efficiency and safety.

Description

Technical Field

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

[0002] Zhikang Capsule is a traditional Chinese medicine produced by Xi'an Qianhe Pharmaceutical Co., Ltd. Its prescription consists of 14 ingredients: rhubarb, coptis root, notoginseng, angelica root, donkey-hide gelatin, dragon bone (calcined), white schizonepeta, vinegar myrrh, sea cucumber, madder, dragon's blood, licorice, pearl, and borneol. It can clear away heat, cool blood, stop bleeding, dissipate blood stasis, promote tissue regeneration and relieve pain. It is used for traumatic bleeding, metrorrhagia, hematemesis and blood in the stool. Since it was launched on the market for more than 20 years, its significant effects of stopping bleeding, removing blood stasis, reducing swelling and relieving pain, and astringing and promoting tissue regeneration have been highly recognized by patients and clinicians. Zhikang Capsule is included in the 2020 edition of the Chinese Pharmacopoeia. The drug standard only performs thin layer identification on rhubarb, coptis root, notoginseng and dragon's blood in the prescription, and determines the content of rhubarb and borneol. The quality control needs to be improved, which cannot fully reflect the quality of the drug. It is difficult to control the clinical efficacy indicators and ensure the consistency of clinical efficacy. Therefore, it is necessary to further improve the content detection method of the product and associate the control indicators with the clinical efficacy, so as to further ensure the quality and efficacy of the product. Imperatorin is a major active ingredient of Angelica dahurica, which has anti-inflammatory, antiviral, anti-tumor and anti-proliferative effects; 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate is the main active ingredient of Bletilla striata, which has the effects of hemostasis and wound healing; and lordreya B is the active ingredient of Dragon's Blood, which has the effects of promoting blood circulation and dispersing blood stasis, relieving pain and hemostasis, astringing sores and promoting tissue regeneration, and has anti-inflammatory and antioxidant activities. Introducing the content determination of the above three active ingredients into the quality control method of Zhikang Capsules is of great significance to ensure the quality of the product. In this study, the three active ingredients of imperatorin in Angelica dahurica, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate in Bletilla striata, and lordreya B in Dragon's Blood were simultaneously determined as quality control indicators to further improve and improve the quality standard research. The contents of the above three ingredients were simultaneously determined by high performance liquid chromatography, and the multi-index ingredient quality control model of Zhikang Capsules was established, in order to provide a reference for the control and improvement of the quality standard of Zhikang Capsules. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for determining the contents of multiple active ingredients in Zhikang Capsules by HPLC. The method simultaneously determines three active ingredients in the prescription of Zhikang Capsules, namely, imperatorin in Angelica dahurica, 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate in Bletilla striata, and dragon's blood dregs B. The detection method can make up for the deficiencies of the existing quality control methods of Zhikang Capsules, provide an easy-to-operate, sensitive and efficient drug quality control method, and further effectively guarantee the product quality of Zhikang Capsules.

[0004] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme:

[0005] A high performance liquid chromatography method for detecting the contents of multiple components in Zhikang capsules, wherein the medicine is prepared from the following 14 medicinal materials: rhubarb, coptis root, notoginseng, angelica root, donkey-hide gelatin, dragon bone (calcined), bletilla striata, myrrh, cuttlebone, madder, dragon's blood, liquorice, pearl, and borneol; the high performance liquid chromatography method is used to simultaneously determine the contents of imperatorin, an effective component of angelica root, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, an effective component of bletilla striata, and dragon's blood B, an effective component of dragon's blood, in the Zhikang capsule preparation, and the method comprises the following steps:

[0006] (1) Chromatographic conditions and system suitability test

[0007] 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 selected from methanol, acetonitrile or acetonitrile-methanol mixture, and 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%; column temperature is 20-40°C; detection wavelength is 200-350nm; flow rate is 0.5ml / min-1.2ml / min; injection volume is 10-20μl; and the number of theoretical plates should not be less than 5000;

[0008] Preferably, the phase A is acetonitrile, the phase B is a 0.2% by volume phosphoric acid aqueous solution; the flow rate is 0.8 ml / min; the column temperature is 30°C; the detection wavelength for determining 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate and luerurein B is 275 nm; the detection wavelength for determining imperatorin is 245 nm; the theoretical plate number calculated based on the luerurein B peak should be no less than 10,000.

[0009] Preferably, during gradient elution, the gradient elution program is 0-15 min, 20-25% A; 15-20 min, 25-35% A; 20-30 min, 35-45% A; 30-40 min, 45-70% A; 40-50 min, 70-20% A; the specific gradient elution program is:

[0010] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～15 20→25 80→75 15～20 25→35 75→65 20～30 35→45 65→55 30～40 45→70 55→30 40～50 70→20 30→80

[0011] (2) Preparation of reference solution

[0012] Accurately weigh appropriate amounts of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, luerurein B, and imperatorin reference substances, respectively, and add 80% methanol to prepare stock solutions containing 1.6 mg / ml of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, 0.1 mg / ml of luerurein B, and 0.1 mg / ml of imperatorin; accurately pipette 5 ml, 3 ml, and 1 ml of the above three stock solutions into the same 50 ml brown volumetric flask, add 80% methanol to dissolve and make up to volume, shake well, and prepare a mixed reference solution containing 160 μg 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, 6 μg luerurein B, and 2 μg imperatorin per 1 ml.

[0013] (3) Preparation of test solution

[0014] Take the contents of Zhikang Capsules, grind them into powder, weigh 0.3-1.2g, accurately weigh, place in a stoppered conical flask, accurately add 20-50ml of 80% methanol, weigh the weight, ultrasonically treat (250W, 50kHz) for 20-60min, cool, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain.

[0015] Preferably, the contents of Zhikang capsules are taken, ground into powder, 0.6 g is weighed, accurately weighed, placed in a stoppered conical flask, 25 ml of 80% methanol is accurately added, the weight is weighed, ultrasonic treatment (250 W, 50 kHz) is performed for 30 min, cooled, 80% methanol is used to make up the lost weight, shaken well, filtered, and the filtrate is obtained.

[0016] (4) Determination

[0017] 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.

[0018] The prescription of 1000 Zhikang capsules in the present invention is: 65g of rhubarb, 50g of coptis root, 50g of panax notoginseng, 31g of angelica dahurica, 50g of donkey-hide gelatin, 44g of dragon bone (calcined), 44g of bletilla striata, 31g of vinegar myrrh, 44g of cuttlebone, 50g of madder, 12g of dragon's blood, 11g of liquorice, 4g of pearl, and 4g of borneol.

[0019] Compared with the prior art, the present invention has the following beneficial technical effects:

[0020] The present invention is a detection method further improved on the basis of the original quality standard, and adds a method for simultaneously determining the content of three effective components in the prescription, namely, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, dragon's blood effective component dragon's blood B, and imperatorin, an effective component of Angelica dahurica. The dual-wavelength mode is used for detection, and the wavelength with the strongest absorption of different effective components and less interference from impurities is selected for detection. The measured components can be better separated in a chromatogram; the removal of interfering impurities is conducive to the accurate calculation of the component content. The detection method has high sensitivity, strong specificity, good accuracy and reproducibility, can make up for the shortcomings of the existing methods, and can simultaneously achieve the determination of multiple effective components at one time, save inspection time, improve detection efficiency, and effectively guarantee the quality of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a chromatogram of the mixed reference substance of the present invention under the chromatographic conditions of the present invention at a detection wavelength of 275 nm;

[0022] Figure 2 The chromatogram of the mixed reference substance of the present invention under the chromatographic conditions of the present invention at a detection wavelength of 245 nm;

[0023] Figure 3 This is the chromatogram of the test product of the present invention at a detection wavelength of 275nm;

[0024] Figure 4 This is the chromatogram of the test product of the present invention at a detection wavelength of 245nm;

[0025] Figure 5 The chromatogram of the negative sample lacking Bletilla striata at the detection wavelength of 275 nm;

[0026] Figure 6 The chromatogram of the negative sample lacking dragon's blood at the detection wavelength of 275nm;

[0027] Figure 7 The chromatogram of the negative sample lacking Angelica dahurica at the detection wavelength of 245nm;

[0028] in Figure 1-7 Among them, 1 is the peak of 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate, 2 is the peak of louretin B, and 3 is the peak of imperatorin.

[0029] Figure 8 This is a linear relationship diagram of the three active ingredients. DETAILED DESCRIPTION

[0030] The present invention is described below with reference to specific examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, auxiliary materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0031] Example 1: Prescription and preparation method of Zhikang Capsules

[0032] Prescription: 65g of rhubarb, 50g of coptis root, 50g of panax notoginseng, 31g of angelica dahurica, 50g of donkey-hide gelatin, 44g of dragon bone (calcined), 44g of bletilla striata, 31g of myrrh, 44g of cuttlebone, 50g of madder, 12g of dragon's blood, 11g of licorice, 4g of pearl, and 4g of borneol.

[0033] Preparation method: Take the above 14 ingredients, add 80% ethanol, heat and reflux twice, each time for 2 hours, combine the alcohol extract, filter, recover the ethanol, and make a clear paste for later use. Mix the residue with madder and licorice, add water and boil twice, each time for 2 hours, combine the decoction, and filter. Mix the filtrate with the above alcohol extract, concentrate under reduced pressure to form a thick paste with a relative density of 1.35-1.38 (60°C), dry, grind into fine powder, and set aside. Take Panax notoginseng, sea cucumber, and white ginseng, dry at 60°C, mix and grind with dragon bone (calcined), vinegar myrrh, and donkey-hide gelatin, and set aside as fine powder. Grind dragon's blood and pearl into fine powder separately, mix with the above two fine powders, add appropriate amount of starch, make granules with 80% ethanol, add borneol fine powder, mix well, put into capsules, and make 1000 capsules.

[0034] Example 2: Selection of detection wavelength

[0035] The wavelength of imperatorin, 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, and lordreyacin B reference solution and sample solution was scanned in the range of 200-400nm. The results showed that 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate and lordreyacin B had large absorption at 275nm, and the separation effect of components in the sample was good, with less interference. Imperatorin had large absorption at 245nm, and the separation effect of components in the sample was good, with less interference. Considering the wavelengths with the strongest absorption of different active ingredients and less interference from impurities, the dual wavelength mode was finally selected. The content of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate and lordreyacin B was determined at 275nm, and the content of imperatorin was determined at 245nm. Two chromatograms can be obtained simultaneously in one injection analysis. Under the premise of good separation of the target active ingredients, interfering impurities are eliminated, which is conducive to the accurate calculation of component content.

[0036] Example 3: Selection of mobile phase

[0037] In order to effectively separate the three active ingredients in the test sample, and to meet the requirements of peak shape, theoretical plate number, etc., the preliminary test was conducted using different proportions of mobile phases such as acetonitrile-0.5% glacial acetic acid solution, methanol-0.1% phosphoric acid solution, acetonitrile-0.1% formic acid solution, methanol-acetonitrile-0.2% phosphoric acid solution, and acetonitrile-0.2% phosphoric acid solution. The results showed that when eluting with acetonitrile-0.5% glacial acetic acid solution, methanol-0.1% phosphoric acid solution, acetonitrile-0.1% formic acid solution, and methanol-acetonitrile-0.2% phosphoric acid solution, the peak shape of individual active ingredients was not good, or the baseline drift was not stable; the separation effect of acetonitrile-0.2% phosphoric acid aqueous solution was the best, the baseline was the most stable, and the peak shape of the three active ingredients was good, but some peaks could not be effectively separated. Therefore, the mobile phase A was acetonitrile, and the mobile phase B was a 0.2% volume percentage phosphoric acid aqueous solution for gradient elution. The detailed results are shown in Table 1.

[0038] Table 1: Mobile phase selection test results

[0039]

[0040] The experimental results show that the gradient elution mobile phase system composed of acetonitrile-0.2% phosphoric acid aqueous solution in Experiment No. 9 (0-15min, 20-25% A; 15-20min, 25-35% A; 20-30min, 35-45% A; 30-40min, 45-70% A; 40-50min, 70-20% A) has the best separation effect under the conditions of gradient elution, a stable baseline, and the chromatographic peaks of the three active ingredients can be completely separated. In addition, dragon's blood A and dragon's blood B can be completely separated with a separation degree greater than 2.0. The peak time, peak shape and separation degree of each component meet the requirements. Therefore, the conditions can meet the relevant requirements of analytical determination.

[0041] Example 4: Specificity study

[0042] 4.1 Preparation of each negative sample:

[0043] According to the sample's prescription process and formula ratio (Example 1), negative samples without Bletilla striata, Angelica dahurica and Dragon's Blood were prepared respectively; for example, the preparation of the negative sample of Bletilla striata: Take rhubarb, coptis root and Angelica dahurica, add 80% ethanol, heat and reflux twice, each time for 2 hours, combine the alcohol extract, filter, recover the ethanol, and prepare the clear paste for use. The residue is mixed with madder and licorice and decocted with water twice, each time for 2 hours, the decoction is combined and filtered. The filtrate is mixed with the above alcohol extract, concentrated under reduced pressure to a thick paste with a relative density of 1.35 to 1.38 (60°C), dried, crushed into fine powder, and prepared for use. Take Panax notoginseng and sea cucumber, dry at 60°C, mix and crush with dragon bone (calcined), vinegar myrrh and donkey-hide gelatin, and prepare fine powder for use. Dragon's Blood and pearl are crushed into fine powder separately, mixed with the above two fine powders, added with appropriate amount of starch, granulated with 80% ethanol, added with borneol fine powder, mixed, and obtained.

[0044] 4.2 Preparation of negative sample solution:

[0045] Weigh 0.6 g of each negative sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 80% methanol, weigh it, ultrasonically treat it (250W, 50kHz) for 30 minutes, cool it, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0046] 4.3 Preparation of test solution:

[0047] Test method: Take the contents of Zhikang capsules of the same batch, grind them into powder, weigh 0.6g, accurately weigh, put them in a stoppered conical bottle, accurately add 25ml of extraction solvent methanol or 80% methanol or 70% methanol, weigh the weight, ultrasonically extract, cool, make up the weight, filter, take the filtrate, and get; accurately take 10μl of the test solution, inject it into the high performance liquid chromatograph, and measure, and get the test results. The results are shown in Table 2.

[0048] Method 1: Accurately add 25 ml of methanol, weigh accurately, extract by ultrasonic for 20 min, cool to make up the weight, and filter;

[0049] Method 2: Accurately add 25 ml of methanol, weigh accurately, extract by ultrasonic for 30 min, cool to make up the weight, and filter;

[0050] Method 3: Accurately add 25 ml of 80% methanol, weigh accurately, extract by ultrasonic for 20 min, cool to make up the weight, and filter;

[0051] Method 4: Accurately add 25 ml of 70% methanol, weigh accurately, extract by ultrasonic for 20 min, cool to make up the weight, and filter;

[0052] Method 5: Accurately add 25 ml of 80% methanol, weigh accurately, extract by ultrasonic for 30 min, cool to make up the weight, and filter;

[0053] Method 6: Accurately add 25 ml of 80% methanol, weigh accurately, extract by ultrasonic for 40 min, cool to make up the weight, and filter;

[0054] Table 2: Results of the test for the selection of test sample preparation methods

[0055]

[0056] The results showed that: when using methods 1 and 2, the extraction of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate was not sufficient, and the peak area was significantly lower than that of other methods; when using methods 3 and 4, the effective components could be effectively extracted, but the peak area of ​​1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate and imperatorin 4 was significantly lower than that of method 3, indicating that the transfer rate of effective components extracted by 80% methanol was better than that by 70% methanol; when using method 3, the peak area was significantly lower than that of method 5, indicating that the effective components could not be 100% transferred after 20 minutes of ultrasonic extraction; when using method 6, there was no significant difference in peak area between that of method 5, but its ultrasonic extraction was longer. Therefore, the best method for preparing the test solution is determined as follows: take the contents of Zhikang Capsules, grind them into powder, weigh 0.6 g, accurately weigh, place in a stoppered conical flask, accurately add 25 ml of 80% methanol, weigh the weight, ultrasonically treat (250 W, 50 kHz) for 30 min, cool, make up the lost weight with 80% methanol, shake well, filter, and obtain the filtrate.

[0057] 4.4 Chromatographic conditions and system suitability test

[0058] Chromatographic column: 250mm×4.6mm, 5μm, with octadecylsilane bonded silica gel as filler; gradient elution, mobile phase, phase A is acetonitrile, phase B is 0.2% by volume phosphoric acid aqueous solution; gradient elution program is 0-15min, 20-25%A; 15-20min, 25-35%A; 20-30min, 35-45%A; 30-40min, 45-70%A; 40-50min, 70-20%A), flow rate is 0.8ml / min; column temperature is 30℃; detection wavelength for determination of 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate and lordreya grandis B is 275nm; detection wavelength for determination of imperatorin is 245nm; injection volume is 10μl; theoretical plate number calculated based on lordreya grandis B peak should not be less than 10000.

[0059] Preparation of reference solution: accurately weigh appropriate amount of each reference substance, add 80% methanol to prepare stock solutions containing 1.6022 mg / ml of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, 0.1044 mg / ml of luerurein B, and 0.1060 mg / ml of imperatorin; accurately pipette 5 ml, 3 ml, and 1 ml of the above three stock solutions into the same 50 ml brown volumetric flask, add 80% methanol to dissolve and make up to volume, shake well, and prepare mixed reference solutions with mass concentrations of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, luerurein B, and imperatorin of 160.22 μg / ml, 6.26 μg / ml, and 2.12 μg / ml, respectively.

[0060] Determination: Accurately pipette 10 μl of each negative sample solution, mixed reference solution, and test solution, and inject into LC-2030C Plus high performance liquid chromatograph for determination.

[0061] 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 three 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 1-7 .

[0062] Example 5: Repeatability test

[0063] 5.1 Chromatographic conditions and system suitability test

[0064] Same as Example 4

[0065] 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.

[0066] 5.3 The results show that the RSD values ​​of the peak areas of the three active ingredients are 0.22%, 0.17%, and 0.11%, respectively, indicating that the instrument has good precision. The specific results are shown in Table 3 below.

[0067] Table 3: Peak area data of three active ingredients in repeatability experiment

[0068]

[0069] Example 6: Linear range

[0070] 6.1 Chromatographic conditions and system suitability test

[0071] Same as Example 4

[0072] 6.2 Accurately pipette 5ml, 3ml and 1ml of the reference substance stock solutions of 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate, lordreerain B and imperatorin into a 50ml brown volumetric flask and dilute to volume with 80% methanol to obtain mixed reference substance solution C. 5 10ml, 6ml and 2ml of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutylmalate, loueruerin B and imperatorin reference substance stock solutions were accurately pipetted into a 50ml brown volumetric flask and fixed to volume with 80% methanol to obtain mixed reference substance solution C. 6 ; Accurately pipette mixed reference solution C 5 Place 5 ml in a 10 ml brown volumetric flask and dilute to volume with 80% methanol to obtain mixed reference solution C. 3 ; Accurately pipette the mixed reference solution C 3 and mixed reference solution C 5 1 ml each, mix well to obtain mixed reference solution C 4 ; Accurately pipette mixed reference solution C 3 Place 5 ml in a 10 ml brown volumetric flask and dilute to volume with 80% methanol to obtain mixed reference solution C. 2 ; Accurately pipette mixed reference solution C 2 Place 2 ml in a 10 ml brown volumetric flask and dilute to volume with 80% methanol to obtain mixed reference solution C. 1 ; Mixed reference solution C 1 , C 2 , C 3 , C 4 , C 5 , C 6 Representing 5%, 25%, 50%, 75%, 100%, 200% linear levels respectively. The detection was carried out according to the chromatographic conditions described in Example 4, and the chromatogram was recorded. The linear regression analysis was performed on the peak area (Y) versus the concentration (X), and a linear relationship diagram was drawn. The results are shown in Table 4 (linear regression equations and correlation coefficients and linear ranges of the three components). Figure 8 This is a linear relationship diagram of the three components.

[0073] Table 4: Linear regression equations, correlation coefficients and linear ranges of the three components

[0074]

[0075] Example 7: Accuracy (Recovery)

[0076] 7.1 Chromatographic conditions and system suitability test

[0077] Same as Example 4

[0078] 7.2 Preparation of test solution:

[0079] Accurately measure 1 ml of 1.6022 mg / ml stock solution of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutylmalate, 0.5 ml of 0.1044 mg / ml stock solution of lureerain B, and 0.1 ml of 0.1060 mg / ml stock solution of imperatorin, place in a stoppered conical flask, evaporate the solvent, and perform the operation three times in parallel; accurately measure 2 ml of 1.6022 mg / ml stock solution of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutylmalate, and .... 1 ml of the stock solution of 0.1044 mg / ml and 0.2 ml of the stock solution of 0.1060 mg / ml of imperatorin were placed in a stoppered conical flask, the solvent was evaporated, and the operation was repeated for 3 times; 3 ml of the stock solution of 1.6022 mg / ml of 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate, 1.5 ml of the stock solution of 0.1044 mg / ml of dragon's blood B, and 0.3 ml of the stock solution of 0.1060 mg / ml of imperatorin were accurately measured and placed in a stoppered conical flask, the solvent was evaporated, and the operation was repeated for 3 times; 0.6 g of the ground content of the Zhikang capsule prepared in Example 1 with a known content was respectively taken and added to the above 9 stoppered conical flasks, 25 ml of 80% methanol was accurately added, and the test solution was prepared according to the above test solution preparation method.

[0080] Determination: Accurately pipette 10 μl of each test solution and inject it into the high performance liquid chromatograph for determination. Calculate the content and recovery rate by external standard method.

[0081] Recovery rate (%) = (total amount measured - content in sample) / amount of reference substance added × 100%

[0082] The results are shown in Table 5. For 9 samples, the recovery of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate was between 96.3% and 101.1%, with an RSD value of 1.7%; the recovery of lureerain B was between 96.9% and 102.4%, with an RSD value of 2.2%; the recovery of imperatorin was between 95.6% and 102.1%, with an RSD value of 2.3%. The experimental results show that the method has good accuracy.

[0083] Table 5: Recovery test data table

[0084]

[0085] Example 8: Intermediate Precision

[0086] 8.1 Chromatographic conditions and system suitability test

[0087] Same as Example 4

[0088] 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 6, which show that the invention patent method has good precision.

[0089] Table 6: Intermediate precision test data table

[0090]

[0091] Example 9: Durability

[0092] 9.1 Chromatographic conditions and system suitability test

[0093] Same as Example 4

[0094] 9.2 Take the same test solution, place it at room temperature, and accurately pipette 10 μl into the HPLC at 0, 2, 4, 8, 12, and 24 hours respectively; the results show that the maximum RSD of the peak area of ​​the test solution within 24 hours is only 0.61%, which is much smaller than the standard 2.0%; the results are shown in Table 7, indicating that the test solution is stable within 24 hours at room temperature.

[0095] Table 7: Stability test data table

[0096]

[0097] In summary: the method for determining the content of multiple active ingredients in Zhikang Capsules 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 three active ingredients in the prescription Chinese medicinal materials, the product quality assurance is further improved, the safety and effectiveness of Zhikang Capsules are guaranteed, and the deficiencies of the prior art are made up.

[0098] Example 10: Stability Test

[0099] 10.1 Chromatographic conditions and system suitability test

[0100] Same as Example 4

[0101] 10.2 Take commercial samples of Zhikang Capsules (batch numbers: 20220401, 20220402, 20220403, produced by Xi'an Qianhe Pharmaceutical Co., Ltd.) for accelerated stability tests and room temperature long-term stability tests. Accelerated test conditions: Place the samples in a stability test chamber with a relative humidity of 75% ± 5% and a temperature of 40°C ± 2°C for 6 months of accelerated experimental investigation, and take samples for testing in the 1st, 2nd, 3rd and 6th months respectively; Room temperature test conditions: Place the samples in a stability test chamber with a relative humidity of 60% ± 5% and a temperature of 25°C ± 2°C for a long-term 6-month experimental investigation, and check them in the 0th, 3rd and 6th months respectively. The test results are shown in Table 8.

[0102] Table 8: Accelerated and long-term stability test data of Zhikang capsules

[0103]

[0104] The experimental results show that after 6 months of accelerated test and 6 months of long-term test, the contents of the active ingredients 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate, dragon's blood B and imperatorin did not change significantly, which met the requirements. This shows that this detection method can effectively control the quality of drugs and can be considered for inclusion in the quality standards.

[0105] The above embodiments are preferred examples for implementing the present invention and are not limited to the above embodiments. Any non-essential additions and substitutions made by those skilled in the art based on the present technical solution shall fall within the protection scope of the present invention.

Claims

1. A method for determining the contents of multiple active ingredients in Zhikang Capsules by HPLC. Features The content of imperatorin, an effective component of Angelica dahurica, 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate, an effective component of Bletilla striata, and dragon's blood active component dragon's blood B in Zhikang capsule preparation was simultaneously determined by high performance liquid chromatography, which specifically comprises the following steps: (1) Chromatographic conditions and system suitability test Octadecylsilane bonded silica gel is used as filler; gradient elution, mobile phase, phase A is acetonitrile, phase B is 0.2% by volume phosphoric acid aqueous solution; gradient elution program is: 0-15min, 20-25% A; 15-20min, 25-35% A; 20-30min, 35-45% A; 30-40min, 45-70% A; 40-50min, 70-20% A; flow rate is 0.8ml / min; column temperature is 30℃; detection wavelength for determination of 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate and lureerain B is 275nm; detection wavelength for determination of imperatorin is 245nm; injection volume is 10-20μl; theoretical plate number calculated based on lureerain B peak should be not less than 10000; (2) Preparation of reference solution Accurately weigh appropriate amounts of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, luerurein B, and imperatorin reference substances, respectively, and add 80% methanol to prepare stock solutions containing 1.6 mg / ml of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, 0.1 mg / ml of luerurein B, and 0.1 mg / ml of imperatorin; accurately pipette 5 ml, 3 ml, and 1 ml of the above three stock solutions into the same 50 ml brown volumetric flask, add 80% methanol to dissolve and make up to volume, shake well, and prepare a mixed reference substance solution containing 160 μg 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, 6 μg luerurein B, and 2 μg imperatorin per 1 ml. (3) Preparation of test solution Take the contents of Zhikang Capsules, grind them into powder, weigh 0.3-1.2g, accurately weigh, put into a stoppered conical flask, accurately add 20-50ml of 80% methanol, weigh the weight, ultrasonically treat for 20-60 min, cool, make up the lost weight with 80% 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 Zhikang Capsules by HPLC according to claim 1, Features: In step (3), the test solution is prepared as follows: the contents of Zhikang capsules are taken, ground into powder, 0.6 g is weighed, accurately weighed, placed in a stoppered conical flask, 25 ml of 80% methanol is accurately added, the weight is weighed, ultrasonic treatment is performed for 30 min, cooled, and the lost weight is supplemented with 80% methanol, shaken, filtered, and the filtrate is obtained; the ultrasonic treatment power is 250 W and the Hz is 50 kHz.

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