A method for determining the contents of multiple components in Qufengzhitong capsules and constructing fingerprints

Abstract

The present invention relates to the technical field of traditional Chinese medicine analysis, and in particular to a method for determining the content of multiple components in a Qufeng Zhitong capsule and a method for constructing a fingerprint spectrum. The present invention optimizes the preparation method of a test solution and the chromatographic conditions, and the constructed content determination method and fingerprint spectrum method can be respectively applied to the simultaneous quantitative determination and fingerprint spectrum research of multiple components such as gallic acid, protocatechuic acid, neochlorogenic acid, and loganinic acid in Qufeng Zhitong capsules, and has good accuracy, high sensitivity, good stability, and simple operation, and can be applied to subsequent quality control research of Qufeng Zhitong capsules.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine analysis, and in particular to a method for determining the contents of multiple components in a Qufeng Zhitong capsule and constructing a fingerprint spectrum. Background Art

[0002] Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method. It is based on the systematic study of the chemical components of traditional Chinese medicine and is mainly used to evaluate the authenticity, quality and stability of traditional Chinese medicine and its preparations. Traditional Chinese medicine and its preparations are multi-component complex systems. Therefore, the evaluation of their quality should adopt a detection method that is suitable for them and can provide rich identification information. The establishment of traditional Chinese medicine fingerprint can more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparations, and then provide an overall description and evaluation of the quality of the medicine.

[0003] Qufengzhitong capsule is a traditional Chinese medicine preparation made of 7 medicinal materials: Geranium, Mistletoe, Dipsacus asper, Clematis chinensis, Angelica dahurica, Radix Aconiti Kusnezoffii, and Carthamus tinctorius. The contents of Qufengzhitong capsule are brown to brown powder, bitter and astringent, with the effects of dispelling wind and relieving pain, relaxing muscles and activating blood circulation, and strengthening muscles and bones. It is used to treat arthralgia caused by wind, cold and dampness, liver and kidney deficiency, such as numbness of the limbs, pain in the waist and knees, and arthralgia caused by wind, cold and dampness.

[0004] The current standard of Qufengzhitong Capsules is included in the 2020 edition of the Chinese Pharmacopoeia. The inspection items include thin layer chromatography identification items of Dipsacus asper, Viscum album, Clematis chinensis and Angelica dahurica; limit inspection of aconitine; content determination items of Geranium and Dipsacus asper. However, the above quality control methods are difficult to systematically and comprehensively control the quality of drugs. Therefore, there is an urgent need for a comprehensive, efficient and safe quality inspection method that can simultaneously qualitatively and quantitatively determine the active ingredients of Qufengzhitong Capsules, so as to effectively control the quality of Qufengzhitong Capsules. Summary of the invention

[0005] In view of the problem that the prior art cannot perform multi-component analysis on Qufengzhitong Capsules, the present invention provides a method for determining the contents of multiple components in Qufengzhitong Capsules and constructing a fingerprint spectrum.

[0006] In order to achieve the above-mentioned object of the invention, the embodiment of the present invention adopts the following technical solution:

[0007] A method for determining the contents of multiple components in a Qufengzhitong capsule comprises extracting the contents of the Qufengzhitong capsule with a methanol aqueous solution, and determining the contents of the target components by ultra-high performance liquid chromatography (UPLC), wherein the chromatographic conditions of the ultra-high performance liquid chromatography are:

[0008] Chromatographic column: C18 bonded silica gel column;

[0009] Mobile phase: Mobile phase A is 0.1% formic acid aqueous solution (v / v), mobile phase B is methanol, and mobile phase A and mobile phase B are used for gradient elution. The elution gradient is as follows:

[0010]

[0011] The detection wavelengths are as follows:

[0012]

[0013]

[0014] Dipsaconin VI was detected alone at 211 nm;

[0015] The column temperature is 35-45°C; preferably 40°C;

[0016] The flow rate was 0.3 mL / min.

[0017] The content detection method provided by the present invention can simply, quickly and accurately determine the content of multiple components in Qufeng Zhitong Capsules, and the method has good accuracy, high sensitivity and good stability, and can be used for subsequent related research such as the determination of the content of multiple components in Qufeng Zhitong Capsules.

[0018] The gradient elution specifically means that: from 0 to 5 min, the volume percentage of mobile phase A decreases uniformly from 97% to 91%, from 5 to 9 min, the volume percentage of mobile phase A decreases uniformly from 91% to 85%, from 9 to 11 min, the volume percentage of mobile phase A decreases uniformly from 85% to 83%, from 11 to 15 min, the volume percentage of mobile phase A decreases uniformly from 83% to 73%, from 15 to 18 min, the volume percentage of mobile phase A decreases uniformly from 73% to 72%, from 18 to 26 min, the volume percentage of mobile phase A decreases uniformly from 72% to 50%, from 26 to 31 min, the volume percentage of mobile phase A decreases uniformly from 50% to 34%, and from 31 to 35 min, the volume percentage of mobile phase A decreases uniformly from 34% to 26%.

[0019] Preferably, the chemical components to be tested include gallic acid, protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, loganinic acid, corilagin, loganin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angelicol A, dihydroanthenoyl acetate, dipsaponin VI, osthole, and dihydroanthenoyl angelic acid.

[0020] Preferably, the specific operation of extracting the Qufengzhitong capsule with methanol aqueous solution is: ultrasonically extracting the contents of the Qufengzhitong capsule with 25% to 100% methanol aqueous solution (v / v). More preferably, ultrasonically extracting the contents of the Qufengzhitong capsule with 75% methanol aqueous solution (v / v).

[0021] Preferably, the temperature of the ultrasonic extraction is 25-60° C.; the time of the ultrasonic extraction is 20-40 min.

[0022] Optionally, the temperature of the ultrasonic extraction is 60°C; the time of the ultrasonic extraction is 30 minutes.

[0023] Preferably, the mass volume ratio of the content of the Qufeng Zhitong Capsule to the methanol aqueous solution is 1:25-125 (g:mL), and more preferably 1:50 (g:mL).

[0024] Preferably, the content determination method comprises the following steps:

[0025] Step a, extracting the contents of Qufeng Zhitong Capsules with methanol aqueous solution to obtain a test solution;

[0026] Step b, preparing a reference solution of the index component to be tested;

[0027] Step c, using the ultra-high performance liquid chromatography method to measure the reference solution and the test solution, and using the external standard method to calculate the content of the index component to be measured in the test solution.

[0028] The present invention also provides a method for constructing a fingerprint spectrum of Qufengzhitong Capsule, comprising the following steps:

[0029] S1, preparing a reference solution of the ingredients;

[0030] S2. Take different batches of Qufeng Zhitong Capsules, extract the contents respectively with methanol-water solution to obtain test sample solutions of different batches of Qufeng Zhitong Capsules, determine the reference solution and the test sample solution with the ultra-high performance liquid chromatography in the above-mentioned content determination method, record the fingerprints, import all the fingerprints into the Chinese medicine chromatographic fingerprint similarity evaluation system software, select the common characteristic chromatographic peaks in the fingerprints of different batches of Qufeng Zhitong Capsules as characteristic fingerprint peaks, generate standard control fingerprints, and calculate the relative retention time and relative peak area of ​​each common characteristic fingerprint peak.

[0031] Beneficial effects of the patent application of this invention:

[0032] Based on the UPLC-PDA timed wavelength switching technology, a method for the simultaneous detection of 16 index components in Qufengzhitong Capsules was established and systematically verified. The constructed method was applied to determine 28 batches of Qufengzhitong Capsules, and the 16 index components in Qufengzhitong Capsules were determined quickly and effectively. The constructed method can characterize the content of index components in Qufengzhitong Capsules more comprehensively and effectively, which helps to ensure the safety and effectiveness of the clinical application of Qufengzhitong Capsules. The establishment of UPLC fingerprint provides a reliable basis for the quality inspection of Qufengzhitong Capsules, which can be used to control and evaluate the intrinsic quality of Qufengzhitong Capsules and improve the quality inspection standards of Qufengzhitong Capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0034] Figure 1 is a wavelength-switched ultra-high performance liquid chromatogram of the mixed reference solution in Example 1 of the present invention;

[0035] Figure 2 is a wavelength-switched ultra-high performance liquid chromatogram of the test solution in Example 1 of the present invention;

[0036] Figure 3 is an ultra-high performance liquid chromatogram at 211 nm of the mixed reference substance solution in Example 1 of the present invention;

[0037] Figure 4 It is the ultra-high performance liquid chromatogram of the test solution at 211 nm in Example 1 of the present invention;

[0038] Figure 5 The ultra-high performance liquid chromatography fingerprints of 28 batches of Qufeng Zhitong Capsules medicinal materials in Example 2 of the present invention;

[0039] Figure 6 This is the characteristic fingerprint of the Qufeng Zhitong Capsule in Example 2 of the present invention. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0041] The Qufeng Zhitong Capsules used in the following examples are 28 batches of medicines produced by Shaanxi Buchang Pharmaceutical Co., Ltd. from 2018 to 2020.

[0042] Example 1

[0043] The embodiment of the present invention provides a method for determining the contents of multiple components in Qufeng Zhitong Capsules, comprising the following steps:

[0044] Step a, preparation of test solution

[0045] Accurately weigh 0.5 g of the contents of Qufeng Zhitong Capsules, put it in a 25 mL volumetric flask, add an appropriate amount of 75% methanol aqueous solution (v / v), shake well, extract at 60 ° C for 30 min, let it stand at room temperature, add 75% methanol aqueous solution (v / v) to the scale, shake well, take 2 mL and centrifuge for 10 min, take 1 mL of the supernatant, add 2 mL of water to dilute, and the Qufeng Zhitong Capsules test solution is obtained.

[0046] Step b, preparation of reference solution

[0047] Take appropriate amount of 16 reference substances, weigh accurately, dissolve gallic acid, protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and loganic acid reference substances in 10% methanol aqueous solution (v / v) to prepare reference substance stock solutions with concentrations of 3.620 mg / mL, 0.3228 mg / mL, 0.714 mg / mL, 1.804 mg / mL, 1.012 mg / mL, and 3.618 mg / mL, respectively; dissolve corilagin, loganin, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C in 50% methanol aqueous solution (v / v) The reference substances of angelica alcohol, dihydroanthenoyl acetate, osthole, dihydroanthenoyl angelica ester and dipsaponin VI were dissolved in methanol to prepare reference substance stock solutions with concentrations of 1.612 mg / mL, 1.618 mg / mL, 0.4980 mg / mL, 0.1996 mg / mL and 0.7980 mg / mL, respectively; the reference substances of angelica alcohol, dihydroanthenoyl acetate, osthole, dihydroanthenoyl angelica ester and dipsaponin VI were dissolved in methanol to prepare reference substance stock solutions with concentrations of 1.086 mg / mL, 1.003 mg / mL, 1.974 mg / mL, 0.3204 mg / mL and 13.996 mg / mL, respectively. Take an appropriate amount of the stock solution to prepare a mixed reference solution with concentrations of gallic acid 24.89 μg / mL, protocatechuic acid 1.332 μg / mL, neochlorogenic acid 3.034 μg / mL, chlorogenic acid 7.442 μg / mL, cryptochlorogenic acid 3.036 μg / mL, loganin 33.92 μg / mL, corilagin 10.68 μg / mL, loganin 7.686 μg / mL, isochlorogenic acid B 2.490 μg / mL, isochlorogenic acid A 1.747 μg / mL, isochlorogenic acid C 3.192μg / mL, angelica alcohol A 9.231μg / mL, dihydroanthellol acetate 6.269μg / mL, osthole 10.86μg / mL, dihydroanthellol angelate 1.602μg / mL, and dipsaponin VI 138.2μg / mL.

[0048] Step c: using Waters ACQUITY UPLC H-Class PLUS ultra high performance liquid chromatograph to measure the reference solution and the test solution, the chromatographic conditions are:

[0049] Column: ACQUITY BEH C 18 (2.1×100mm, 1.7μm);

[0050] Column temperature: 40°C; injection volume: 2 μL, flow rate: 0.3 mL / min;

[0051] The detection wavelengths are as follows:

[0052]

[0053] Dipsaconin VI was detected alone at 211 nm;

[0054] Mobile phase: Phase A is 0.1% formic acid aqueous solution, phase B is methanol;

[0055] The gradient elution conditions were:

[0056]

[0057] The ultra-high performance liquid chromatogram of the reference solution is shown in Figure 1 , 3 As shown;

[0058] The ultra-high performance liquid chromatogram of the test solution is as follows Figure 2 , 4 shown.

[0059] The external standard method was used to calculate the contents of 16 components in the sample. The formula is as follows:

[0060]

[0061] Wherein, w is the content of the target component in the Qufengzhitong capsule to be tested, in mg / g;

[0062] C s is the concentration of the component in the reference solution, in mg / mL;

[0063] A n is the peak area of ​​the target component to be measured in the test solution;

[0064] A s is the peak area of ​​the component in the reference solution;

[0065] W is the actual weighed amount of the contents of the Qufeng Zhitong Capsule to be tested.

[0066] Test Example 1

[0067] The test example of the present invention provides a methodological investigation of the method for determining the content of multiple components in the Qufeng Zhitong Capsule in Example 1:

[0068] (1) Study on linear relationship and detection limit and quantification limit

[0069] A mixed reference solution was prepared according to the method of step b in Example 1, and diluted stepwise into a series of mixed reference solutions of different concentrations, and the peak area was determined by ultra-high performance liquid chromatography, and the chromatographic conditions were the same as those of step c in Example 1. The samples were injected twice in parallel, and the concentration of each compound reference substance was taken as the abscissa x (μg / mL), and the corresponding reference substance peak area was taken as the ordinate y, and a regression equation was drawn; the detection limit and quantification limit of gallic acid, protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, loganinic acid, corilagin, loganin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angelic alcohol A, dihydro-eucryl acetate, Dipsacoside VI, osthole, and dihydro-eucryl angelic acid were determined with a signal-to-noise ratio S / N=3 as the detection limit (LOD) and S / N=10 as the quantification limit (LOQ). The results are shown in Table 1.

[0070] Table 1 Linear regression equation, linear range, correlation coefficient, detection limit and quantification limit of indicator components

[0071]

[0072] As shown in Table 1, each component has a good linear relationship within its own concentration range (r 2 ≥0.999), the concentration range of the detection limit was 0.02118-1.728 μg / mL, and the concentration range of the quantification limit was 0.08707-3.455 μg / mL, indicating that the method for determining the content of multiple components in the Qufeng Zhitong Capsule provided by the present invention has high sensitivity.

[0073] (2) Precision test

[0074] (a) Intra-day precision test

[0075] The test solution of Qufeng Zhitong Capsules was prepared according to the method of step a in Example 1, and the same test solution was accurately aspirated and injected 6 times, and the peak area was determined by ultra-high performance liquid chromatography, and the chromatographic conditions were the same as step c in Example 1. The RSD values ​​were calculated based on the peak areas of gallic acid, protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, loganinic acid, corilagin, loganin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angelicol A, dihydroanthenoyl acetate, chuanxiong saponin VI, osthole, and dihydroanthenoyl angelic acid, and the results are shown in Table 2.

[0076] Table 2 Intra-day precision test results of the determination of multiple components in Qufengzhitong Capsules (n=6)

[0077]

[0078]

[0079] As shown in Table 2, the intra-day precision RSD of the method for determining the contents of multiple components in the Qufeng Zhitong Capsule provided by the present invention is less than 2.6%.

[0080] (b) Inter-day precision test

[0081] The test solution of Qufeng Zhitong Capsule was prepared according to the method of step a in Example 1. The same sample solution was accurately drawn every day for 3 consecutive days, and the injection was repeated 6 times. The peak area was determined by ultra-high performance liquid chromatography, and the chromatographic conditions were the same as step c in Example 1. The RSD value was calculated based on the average peak area of ​​gallic acid, protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, loganinic acid, corilagin, loganin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angelic alcohol A, dihydroanthenoyl acetate, chuanxiong saponin VI, osthole, and dihydroanthenoyl angelic acid. The results are shown in Table 3.

[0082] Table 3 Results of the daytime precision test for the determination of multiple components in Qufengzhitong Capsules (n=3)

[0083]

[0084] As shown in Table 3, the inter-day precision RSD of the method for determining the contents of multiple components in the Qufengzhitong capsule provided by the present invention is less than 2.9%.

[0085] It can be seen from Table 2 and Table 3 that the method for determining the contents of multiple components in the Qufeng Zhitong Capsule provided by the present invention has good precision.

[0086] (3) Repeatability test

[0087] According to the method of step a in Example 1, 6 test solutions of Qufeng Zhitong Capsules were prepared in parallel, and the test solutions were determined by ultra-high performance liquid chromatography to calculate the content. The chromatographic conditions were the same as those of step c in Example 1. The contents of gallic acid, protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, loganinic acid, corilagin, loganin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angelicol A, dihydroanthenoyl acetate, radix dipsaponins VI, osthole, and dihydroanthenoyl angelic acid were calculated by external standard method and the RSD values ​​were calculated. The results are shown in Table 4.

[0088] Table 4 Repeatability test results of the determination of multiple components in Qufengzhitong capsules (n=6)

[0089]

[0090] As shown in Table 4, in this embodiment, the gallic acid content is 3.343 mg / g, the protocatechuic acid content is 0.1758 mg / g, the neochlorogenic acid content is 0.4951 mg / g, the chlorogenic acid content is 1.541 mg / g, the cryptochlorogenic acid content is 0.5343 mg / g, the loganin content is 5.402 mg / g, the corilagin content is 1.692 mg / g, the loganin content is 1.034 mg / g, the isochlorogenic acid B content is 0.6951 mg / g, and the isochlorogenic acid A content is 0. The content of chlorogenic acid C was 0.7192 mg / g, the content of angelicol A was 1.105 mg / g, the content of dihydroanthellate acetate was 0.7873 mg / g, the content of osthole was 1.568 mg / g, the content of dihydroanthellate angelate was 0.2064 mg / g, and the content of dipsaponin VI was 18.30 mg / g; the repeatability test RSD of the method for determining the content of multiple components in the Qufeng Zhitong Capsules provided by the present invention was less than 2.9%, indicating that the detection method had good repeatability.

[0091] (4) Stability test

[0092] The test solution of Qufeng Zhitong Capsule was prepared according to the method of step a in Example 1, and stored at room temperature. The same portion of the test solution was accurately drawn at 0, 2, 4, 6, 8, 10, and 12 hours, and the test solution was measured by ultra-high performance liquid chromatography. The chromatographic conditions were the same as those of step c in Example 1. The RSD values ​​were calculated based on the peak areas of gallic acid, protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, loganinic acid, corilagin, loganin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angelicol A, dihydroanthenoyl acetate, radix dipsaponins VI, osthole, and dihydroanthenoyl angelic acid. The results are shown in Table 5.

[0093] Table 5 Stability test results of multiple component content determination of Qufengzhitong capsule (n=7)

[0094]

[0095]

[0096] As shown in Table 5, the stability test RSD of the method for determining the content of multiple components of the Qufeng Zhitong Capsule provided by the present invention is less than 2.4%, indicating that the test solution has good stability within 12 hours at room temperature.

[0097] (5) Sample recovery test

[0098] Take about 0.25g of the contents of Qufeng Zhitong Capsules, accurately weigh, and accurately add 5mL of mixed reference solution with the same mass as the components in the sample (gallic acid 167.6μg / mL, protocatechuic acid 8.651μg / mL, neochlorogenic acid 24.70μg / mL, chlorogenic acid 75.30μg / mL, cryptochlorogenic acid 27.75μg / mL, loganin 260.5μg / mL, corilagin 81.28μg / mL, loganin 50.42μg / mL, isochlorogenic acid B 36.35μg / mL, isochlorogenic acid A 21.43μg / mL, isochlorogenic acid C 36.71μg / mL, angelica alcohol A 54.84μg / mL, dihydroanthellol acetate 39.16μg / mL, osthole 78.79μg / mL, dihydroanthellol angelate 10.09μg / mL, and chuanxiong saponin VI 881.7μg / mL), and 6 portions of Qufengzhitong Capsule test solutions were prepared according to the method of step a in Example 1, and each sample was measured in parallel twice, and the test solutions were measured by ultra performance liquid chromatography, and the chromatographic conditions were the same as those in step c in Example 1. The sample recovery rates and RSD values ​​of gallic acid, protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, loganinic acid, corilagin, loganin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angelicol A, dihydroanthenoyl acetate, dipsaponin VI, osthole, and dihydroanthenoyl angelate were calculated according to the sample recovery formula to investigate the accuracy of the method.

[0099] The recovery rate formula is as follows:

[0100]

[0101] The results are shown in Table 6.

[0102] Table 6 The results of the sample recovery test for the determination of the content of multiple components in Qufeng Zhitong Capsules (n=6)

[0103]

[0104]

[0105]

[0106] As can be seen from Table 6, the average sample recovery rate of the method for determining the content of multiple components in the Qufeng Zhitong Capsules provided by the present invention is between 92.1% and 108.2%, indicating that the sample recovery rate of each compound basically meets the requirements and the method is accurate and feasible.

[0107] It can be seen from Tables 1 to 6 that the method for determining the contents of multiple components in the Qufeng Zhitong Capsule provided by the present invention meets the requirements of content determination methodology research and can be used for quantitative analysis of Qufeng Zhitong Capsule samples.

[0108] The results of the determination of the contents of multiple components in different batches of Qufeng Zhitong Capsules are shown in Table 7.

[0109] Table 7 Determination results of 16 chemical components in 28 batches of Qufengzhitong capsules (n=2, mg / g)

[0110]

[0111] As shown in Table 7, the content of gallic acid, protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, loganinic acid, corilagin, loganin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angelicol A, dihydroanthenoside acetate, chuanxiong saponin VI, osthole, dihydroanthenoside angelic acid in different batches of Qufengzhitong capsule samples were studied by the content determination method of the multiple components of Qufengzhitong capsule provided by the present invention. The content of ingredients, among which, in 28 batches of Qufeng Zhitong Capsule samples, the content of gallic acid was 2.644-4.049 mg / g; the content of protocatechuic acid was 0.1175-0.1846 mg / g; the content of neochlorogenic acid was 0.3662-0.5921 mg / g; the content of chlorogenic acid was 1.354-1.609 mg / g; the content of cryptochlorogenic acid was 0.4363-0.6689 mg / g ; Loganin content is 3.640~6.008mg / g; Corilagin content is 0.9905~2.316mg / g; Loganin content is 0.6843~1.003mg / g; Isochlorogenic acid B content is 0.7053~0.8372mg / g; Isochlorogenic acid A content is 0.3942~0.5321mg / g; Isochlorogenic acid C content is 0.6495~0.8 388mg / g; the content of angelica alcohol A is 0.8528-1.245mg / g; the content of dihydroanthellol acetate is 0.7487-0.8916mg / g; the content of osthole is 1.567-1.777mg / g; the content of dihydroanthellol angelica ester is 0.2017-0.2359mg / g; the content of dipsaponin VI is 9.652-18.24mg / g.

[0112] Example 2

[0113] The embodiment of the present invention provides a method for constructing a characteristic fingerprint of Qufeng Zhitong Capsule, comprising the following steps:

[0114] S 1. Preparation of reference solution

[0115] Take appropriate amount of 15 kinds of reference substances, weigh accurately, dissolve gallic acid, protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and loganic acid reference substances in 10% methanol aqueous solution (v / v) to prepare reference substance stock solutions with concentrations of 3.620 mg / mL, 0.3228 mg / mL, 0.7140 mg / mL, 1.804 mg / mL, 1.012 mg / mL, and 3.618 mg / mL, respectively; dissolve corilagin, loganin, isochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C in 50% methanol aqueous solution (v / v) to prepare reference substance stock solutions with concentrations of 3.620 mg / mL, 0.3228 mg / mL, 0.7140 mg / mL, 1.804 mg / mL, 1.012 mg / mL, and 3.618 mg / mL, respectively. The reference substances of angelica alcohol, dihydroanthenoyl acetate, osthole and dihydroanthenoyl angelate were dissolved in methanol to prepare reference substance stock solutions with concentrations of 1.612 mg / mL, 1.618 mg / mL, 0.4980 mg / mL, 0.1996 mg / mL and 0.7980 mg / mL, respectively; the reference substances of angelica alcohol, dihydroanthenoyl acetate, osthole and dihydroanthenoyl angelate were dissolved in methanol to prepare reference substance stock solutions with concentrations of 1.086 mg / mL, 1.003 mg / mL, 1.974 mg / mL and 0.3204 mg / mL, respectively. Take an appropriate amount of the stock solution to prepare a mixed reference solution with concentrations of 24.89 μg / mL of gallic acid, 1.332 μg / mL of protocatechuic acid, 3.034 μg / mL of neochlorogenic acid, 7.442 μg / mL of chlorogenic acid, 3.036 μg / mL of cryptochlorogenic acid, 33.92 μg / mL of loganin, 10.68 μg / mL of corilagin, 7.686 μg / mL of loganin, 2.490 μg / mL of isochlorogenic acid B, 1.747 μg / mL of isochlorogenic acid C, 3.192 μg / mL of angelicol A, 6.269 μg / mL of dihydroanthenoyl acetate, 10.86 μg / mL of osthole, and 1.602 μg / mL of dihydroanthenoyl angelate.

[0116] S2. Take 28 batches of Qufengzhitong capsules and prepare test solutions respectively. The preparation method is as follows: accurately weigh 0.5g of the content of Qufengzhitong capsules into a 25mL volumetric flask, add an appropriate amount of 75% methanol aqueous solution (v / v), shake well, ultrasonically extract at 60℃ for 30min, place at room temperature, add 75% methanol aqueous solution (v / v) to the scale, shake well, take 2mL and centrifuge for 10min, take 1mL of supernatant, add 2mL of water to dilute, and obtain the Qufengzhitong capsule test solution. Use Waters ACQUITY UPLC H-ClassPLUS ultra-high performance liquid chromatograph to measure the above-mentioned reference solution and the test solution, record the fingerprint spectrum, import all the fingerprint spectrum into the traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system software, select the common characteristic chromatographic peaks in the fingerprint spectrum of different batches of Qufengzhitong capsules as characteristic fingerprint peaks, generate standard reference fingerprint spectrum, and calculate the relative retention time and relative peak area of ​​each common characteristic fingerprint peak; the chromatographic conditions are:

[0117] Column: ACQUITY BEH C 18 (2.1×100mm, 1.7μm);

[0118] Column temperature: 40°C;

[0119] Injection volume: 2 μL;

[0120] Flow rate: 0.3 mL / min;

[0121] The detection wavelengths are as follows:

[0122]

[0123]

[0124] Mobile phase: Phase A is 0.1% formic acid aqueous solution, phase B is methanol;

[0125] The gradient elution conditions were:

[0126]

[0127] According to the determination results of the relative retention time of each compound in the fingerprint of 28 batches of Qufeng Zhitong Capsules, 15 chromatographic peaks with large peak area, good peak shape and separation were selected as characteristic fingerprint peaks. By comparing with the chromatogram of the reference substance, the characteristic fingerprint peaks were identified as gallic acid (peak 1), protocatechuic acid (peak 2), neochlorogenic acid (peak 3), chlorogenic acid (peak 4), cryptochlorogenic acid (peak 5), loganin (peak 6), corilagin (peak 7), loganin (peak 8), isochlorogenic acid B (peak 9), isochlorogenic acid A (peak 10), isochlorogenic acid C (peak 11), angelicol A (peak 12), dihydroanthenoyl acetate (peak 13), osthole (peak 14), and dihydroanthenoyl angelic acid (peak 15). Further, 15 common characteristic fingerprint peaks are preferably determined to generate a standard control fingerprint spectrum, with peak 6 as the reference peak. The relative retention time and relative peak area of ​​the 15 common chromatographic peaks are shown in Tables 8 and 9;

[0128] Table 8 Relative retention time results of common peaks in fingerprints of 28 batches of Qufengzhitong Capsules

[0129]

[0130] Table 9 Relative peak area results of common peaks in the fingerprints of 28 batches of Qufeng Zhitong Capsules

[0131]

[0132] The characteristic fingerprints of 28 batches of Qufengzhitong capsule samples are as follows Figure 5 As shown;

[0133] The characteristic fingerprint of Qufengzhitong capsule is as follows Figure 6 shown.

[0134] Test Example 2

[0135] The test example of the present invention provides a methodological investigation of the construction method of the characteristic fingerprint of Qufeng Zhitong Capsule in Example 2:

[0136] (1) Precision test

[0137] The Qufengzhitong Capsule test solution was prepared according to the method of step S2 in Example 2, and the same sample solution was accurately pipetted and injected 6 times, and the fingerprint was recorded. The relative retention time RSD of each common chromatographic peak was less than 0.08%, and the relative peak area RSD of the main chromatographic peak was less than 2.07%.

[0138] (2) Reproducibility test

[0139] According to the method of step S2 in Example 2, 6 samples of Qufeng Zhitong Capsule test solution were prepared in parallel, and the above test solution was determined by ultra-high performance liquid chromatography, and the chromatographic conditions were the same as step S2 in Example 2, and the fingerprint spectrum was recorded. The relative retention time RSD of each main chromatographic peak was less than 0.17%, and the relative peak area RSD of the main common chromatographic peak was less than 2.11%, indicating that the detection method has good reproducibility.

[0140] (3) Stability test

[0141] The Qufeng Zhitong Capsule test solution was prepared according to the method of step S2 in Example 2, and stored at room temperature. The same sample solution was accurately aspirated at 0, 2, 4, 6, 8, 10, and 12 hours, and the fingerprint was recorded. The relative retention time RSD of each main chromatographic peak was less than 0.46%, and the relative peak area RSD of the common chromatographic peak was less than 2.12%, indicating that the test solution had good stability within 12 hours at room temperature.

[0142] (4) Calculation of similarity of fingerprints of Qufengzhitong capsules

[0143] Take 28 batches (S1-S28) of Qufeng Zhitong Capsules and prepare test solutions according to the operation of step S2. Perform ultra-high performance liquid chromatography determination in sequence according to the chromatographic conditions of step S2, record the fingerprints, and import all the fingerprints into the Chinese medicine chromatographic fingerprint similarity evaluation system software (2012 version) to calculate the similarity. The results show that the similarities of the 28 batches of Qufeng Zhitong Capsules (S1-S28) are 0.995, 0.999, 0.9 The similarity results showed that the similarity of 28 batches of Qufeng Zhitong Capsules was above 0.9.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the contents of multiple components in Qufengzhitong capsules, characterized in that: The method comprises the following steps: extracting the contents of Qufeng Zhitong Capsule with methanol aqueous solution to obtain a test solution, taking the Qufeng Zhitong Capsule test solution and a reference solution for UPLC detection, wherein the reference substances include gallic acid, protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, loganinic acid, corilagin, loganin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angelicol A, dihydroanthenoyl acetate, cyperus saponin VI, osthole, and dihydroanthenoyl angelic acid; The chromatographic conditions of the UPLC detection are: using C 18 Bonded silica gel chromatographic column; 0.1% formic acid aqueous solution v / v , as mobile phase A, methanol as mobile phase B, and the linear gradient elution conditions are: Dipsacoside VI was detected alone at 211 nm, and the other 15 reference components were detected at wavelengths of 239-325 nm, with a flow rate of 0.3 mL / min and a column temperature of 35-45°C. The component and content information was obtained based on the UPLC test results.

2. The method for determining the contents of multiple components in Qufeng Zhitong Capsules according to claim 1, characterized in that: The relationship between the detection wavelength and the reference substance is: Dipsacoside VI was detected separately at 211 nm.

3. The method for determining the contents of multiple components in Qufeng Zhitong Capsules according to claim 1, characterized in that: The specific operation of extracting the contents of the Qufeng Zhitong Capsule with a methanol aqueous solution is as follows: using a 25% to 100% methanol aqueous solution v / v , ultrasonically extract the contents of the Qufeng Zhitong capsule.

4. The method for determining the contents of multiple components in Qufeng Zhitong Capsules according to claim 3, characterized in that: 75% methanol aqueous solution v / v, Ultrasonic extraction of the contents of Qufengzhitong capsules.

5. The method for determining the contents of multiple components in Qufeng Zhitong Capsules according to claim 3, characterized in that: The temperature of the ultrasonic extraction is 25-60° C., and the time of the ultrasonic extraction is 20-40 min.

6. The method for determining the contents of multiple components in Qufeng Zhitong Capsules according to claim 3, characterized in that: The mass volume ratio of the content of the Qufeng Zhitong capsule to the methanol aqueous solution is 1:25-125g:mL.

7. A method for constructing a fingerprint of Qufengzhitong Capsule, characterized in that: The following steps are involved: S1. Prepare a reference solution of the index component to be tested in Qufeng Zhitong Capsule; S2. Take different batches of Qufeng Zhitong Capsules, extract the contents respectively with methanol-water solution to obtain test sample solutions of different batches of Qufeng Zhitong Capsules, measure the reference solution and the test sample solution with the UPLC method in the content determination method described in any one of claims 1 to 6, record the fingerprints, import all the fingerprints into the Chinese medicine chromatographic fingerprint similarity evaluation system software, select the common characteristic chromatographic peaks in the fingerprints of different batches of Qufeng Zhitong Capsules as characteristic fingerprint peaks, generate standard control fingerprints, and calculate the relative retention time and relative peak area of ​​each characteristic fingerprint peak.

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