A quality control method for Jindan Fuyan granules

Abstract

The present invention belongs to the technical field of Chinese medicine preparation detection, and specifically relates to a quality control method for Jindan Fuyan granules. The quality control method uses UHPLC-MS / MS-QAMS technology to determine the content of multiple components of Corydalis in Jindan Fuyan granules. A test solution and a reference solution are first prepared, and then the test solution and the reference solution are respectively measured by UHPLC-MS / MS, and the peak area is recorded. Corydalis butylene is used as an internal reference to calculate the relative correction factor and content of each component. The present invention uses UHPLC-MS / MS-QAMS to determine corydalis butylene by a one-measurement-multiple-evaluation method, and realizes indirect determination of multiple other related components. The method has low detection cost, high sensitivity, strong specificity, and short analysis time. It can well distinguish whether the Corydalis butylene slices used in the production of Jindan Fuyan granules are mixed with summer maltose, and provide a basis for quality monitoring of Jindan Fuyan granules.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine preparation detection, and particularly relates to a quality control method for Jindan Fuyan granules. Background Art

[0002] Chronic pelvic inflammatory disease is a common and frequently occurring disease in gynecology. The disease is protracted and the course of the disease is long, which poses a serious threat to women's health. The prescription of Jindan Fukang Granules is composed of 13 ingredients: Peony Root, Honeysuckle, Red Peony Root, Curcuma Atractylodes, Corydalis (vinegar-processed), Patrinia, Sargentodoxae, Prunus Persica, Angelica Sinensis, Myrrh (processed), Cinnamon Twig, Cyperus Rhizoma, and Coix Seed. It is mainly used to treat chronic pelvic inflammatory disease with internal damp-heat accumulation, with symptoms of lower abdominal and lumbar pain, or lower abdominal pain that refuses to be pressed, aggravated symptoms during menstruation or after fatigue, and heavy, thick, or fishy vaginal discharge. Among them, Corydalis ( Corydalis yanhusuo WTWang), also known as "Yuanhusuo", is pungent and bitter in taste, warm in nature, and enters the liver, heart, and spleen meridians. It has the effects of promoting blood circulation, promoting qi and relieving pain. It not only promotes the qi in the blood, but also promotes the blood in the qi. Therefore, it is a good medicine for promoting blood circulation, promoting qi and relieving pain. When matched with other medicines, it can achieve the effects of treating qi and blood, removing blood stasis and promoting qi, promoting qi and blood circulation, treating blood stasis and heat, and regulating qi and blood. The main active chemical components in Yanhusuo are alkaloid components represented by corydalis butyl, corydalis methyl, and papaverine. These components have good analgesic and anti-inflammatory effects, and play an important role in relieving the inflammatory response and various pains caused by chronic pelvic inflammatory disease. However, Chinese medicine is different from chemical synthetic drugs. The pharmacological effects of Chinese medicine are not produced by a certain chemical component, but often by a certain type of chemical component represented by multiple components or even several types of chemical components acting together through multiple targets. At present, the 2020 edition of the Chinese Pharmacopoeia only uses HPLC to determine the content of tetrahydropalmatine in Corydalis. However, the alkaloid active ingredients in Corydalis are far more than tetrahydropalmatine, including corydalis A, protopine, sea papaverine, tetrahydroafricana, etc. These alkaloid active ingredients work together to exert good anti-inflammatory and analgesic effects. The current quality standard of Jindan Fuyan Granules only has thin layer identification of tetrahydropalmatine as a single component, and no determination of the content of Corydalis. This method cannot achieve a comprehensive evaluation of the overall quality of Corydalis in the preparation by determining the content of multiple alkaloids in Corydalis.

[0003] Corydalis serrata Corydalis decumbens (Thunb.) The tuber of Pres., also known as "Tuyuanhu", also contains chemical components such as corydaline and protoopine. The overall alkaloid composition is very similar to that of Corydalis yanhusuo. If the Corydalis yanhusuo slices are mixed with corydalis yanhusuo slices, they cannot be identified by corydaline thin layer identification.

[0004] Therefore, through experimental research, it is necessary to compare the chemical composition of Corydalis yanhusuo and Xiatianwu, analyze the differences in their chemical composition, and develop a detection method that can quickly determine the content of effective alkaloids in Jindan Fuyan granules, and at the same time distinguish whether Corydalis yanhusuo slices in Jindan Fuyan granules are mixed with Xiatianwu, so as to achieve the purpose of controlling the quality of the Corydalis yanhusuo slices and preparations. Summary of the invention

[0005] The purpose of the present invention is to provide a quality control method for Jindan Fuyan granules. The method has the advantages of low inspection cost, high sensitivity, strong specificity, short analysis time, etc. The method can well evaluate the content of the effective alkaloids of Corydalis yanhusuo contained in the Jindan Fuyan granules, and at the same time can distinguish whether the Corydalis yanhusuo decoction pieces used in the production of Jindan Fuyan granules are mixed with Xiatianwu, thereby providing a basis for quality monitoring of Jindan Fuyan granules.

[0006] The present invention provides a quality control method for Jindan Fuyan granules. The quality control method adopts UHPLC-MS / MS-QAMS (liquid chromatography-mass spectrometry combined with one measurement and multiple evaluations) technology to determine the contents of multiple components of Corydalis yanhusuo in Jindan Fuyan granules, and specifically comprises the following steps:

[0007] (1) Preparation of Jindan Fuyan Granule Test Solution: Take an appropriate amount of Jindan Fuyan Granule, grind it into powder, accurately weigh 1.0 g, place it in a round-bottom flask, accurately add 50 mL of methanol, heat and reflux for 30 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 1 mL of the filtrate, place it in a 50 mL volumetric flask, add methanol to dilute to the mark, shake well, and filter through a 0.22 μm microporous filter membrane to obtain the solution;

[0008] (2) Preparation of reference solution: Accurately weigh 10.32 mg of hydroethyl coptisine, 10.08 mg of bisoprolol, 11.30 mg of tetrahydroafricanine, 10.87 mg of protopine, 11.36 mg of tetrahydropalmatine, 10.85 mg of papaverine, and 10.58 mg of tetrahydropalmatine, and place them in a 100 mL volumetric flask. Dissolve them in methanol and dilute to the mark. Shake well to obtain the reference solution.

[0009] (3) Determination: The reference solution was prepared into a series of standard solutions and analyzed by UHPLC-MS / MS. A standard curve was fitted with concentration as the horizontal axis and area as the vertical axis. The test solution was then determined by UHPLC-MS / MS, and the peak area was recorded. Corydalis butylene glycol was used as the internal reference to calculate the relative correction factor and content of each component.

[0010] Furthermore, in the above technical scheme, the chromatographic conditions of UHPLC are: using a C18 chromatographic column, acetonitrile as mobile phase A, 0.1% formic acid solution containing 5 mmol / L ammonium formate as mobile phase B, gradient elution, injection volume of 1 μL, flow rate of 0.3 mL / min, and column temperature of 40°C.

[0011] Furthermore, in the above technical solution, the C18 chromatographic column is any one of Waters BEH C18, Agilent ZORBAXSB C18, and Thermo Acclaim RSLC 120 C18.

[0012] Furthermore, in the above technical scheme, the gradient elution program is: 0 min, 10% mobile phase A, 90% mobile phase B; 0.3 min, 10% mobile phase A, 90% mobile phase B; 2 min, 25% mobile phase A, 75% mobile phase B; 8 min, 35% mobile phase A, 65% mobile phase B; 10 min, 60% mobile phase A, 40% mobile phase B; 10.1 min, 10% mobile phase A, 90% mobile phase B; 13 min, 10% mobile phase A, 90% mobile phase B.

[0013] Furthermore, in the above technical solution, the mass spectrometry conditions of MS / MS are: using an electrospray ionization source ESI + Mode, using multiple reaction ion monitoring (MRM) detection, the parameters used are as follows:

[0014] Spray voltage: 4.5 kV;

[0015] Air curtain air pressure: 25psi;

[0016] Spray gas pressure: 60psi;

[0017] Dry gas pressure: 55psi;

[0018] Collision gas: 6 psi;

[0019] Ion source temperature: 550°C.

[0020] Furthermore, in the above technical solution, the instrument used for the MS / MS is an Agilent 1290 UHPLC-ABSCIEX API4000 LC-MS instrument or a Waters ACQUITY UPLC-Xevo TQ-S LC-MS instrument.

[0021] Furthermore, in the above technical solution, the methanol used is analytical grade with a concentration of 100%.

[0022] Advantages compared to the prior art:

[0023] 1. The present invention adopts UHPLC-MS / MS-QAMS technology to determine tetrahydropalmatine, and utilizes the ion response ratio relationship between tetrahydropalmatine and other components in the mass spectrometer to achieve indirect determination of other multiple related components, saving a large amount of purchase funds for reference substances such as tetrahydroafricanine, papaverine, tetrahydropalmatine, hydrogenated berberine, bicuculline, and protopine in Corydalis, which can effectively reduce the detection cost.

[0024] 2. The UHPLC-MS / MS-QAMS method used in the present invention has high sensitivity and specificity, and can measure components with lower content in the analyte, while eliminating matrix interference. The analyte can be quickly determined by the qualitative ions and quantitative ions of the analyte, and does not need to be determined by relative retention time like the UHPLC-UV-QAMS (liquid chromatography combined with ultraviolet detector combined with one measurement and multiple evaluations) method, which is easy to cause misidentification and inspection errors.

[0025] 3. The analysis system established by the present invention has a short analysis time, and only 13 minutes are needed to complete the separation and detection work, which greatly saves time. At the same time, it has low inspection cost, high sensitivity, and strong specificity. It can quickly evaluate the content of multiple effective alkaloids in Jindan Fuyan Granules, and by detecting the characteristic components in Corydalis and Xiatianwu, it can well distinguish whether the Corydalis decoction pieces used in the production of Jindan Fuyan Granules are mixed with Xiatianwu, and it is highly practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a linear relationship diagram of hydrogen ethyl coptisine in Example 1 of the present invention;

[0027] Figure 2 This is a linear relationship diagram of bispyribac in Example 1 of the present invention;

[0028] Figure 3 This is a linear relationship diagram of tetrahydrotetramine in Example 1 of the present invention;

[0029] Figure 4 This is a linear relationship diagram of protopine in Example 1 of the present invention;

[0030] Figure 5 This is a linear relationship diagram of tetrahydropalmatine in Example 1 of the present invention;

[0031] Figure 6 It is the linear relationship diagram of papaverine in Example 1 of the present invention;

[0032] Figure 7 This is a linear relationship diagram of corydalis acetylcholine in Example 1 of the present invention;

[0033] Figure 8This is the MRM ion flow overlay of the positive sample of Jindan Fuyan granules prepared by the present invention;

[0034] Fig. 9 This is the MRM ion flow overlay diagram of the Jindan Fuyan granule sample of the present invention;

[0035] Fig.10 This is the MRM ion flow overlay diagram of the Corydalis yanhusuo decoction piece of the present invention;

[0036] Fig.11 This is the MRM ion flow superposition diagram of the summer-free decoction piece of the present invention;

[0037] Fig.12 The MRM ion flow diagram of hydrogenated ethyl coptisine and pyruvic acid in the chromatogram of the summer-free decoction piece of the present invention; wherein a is the MRM ion flow diagram of hydrogenated ethyl coptisine, and b is the MRM ion flow diagram of pyruvic acid;

[0038] Fig.13 The MRM ion flow diagram of bisporine and bikurin in the chromatogram of the Corydalis yanhusuo decoction piece of the present invention; wherein a is the MRM ion flow diagram of bisporine, and b is the MRM ion flow diagram of bikurin;

[0039] Fig.14 This is the MRM quantitative ion chromatogram of the test sample hydrogen ethyl coptisine of Jindan Fuyan Granules in Example 2 of the present invention;

[0040] Fig.15 This is the MRM quantitative ion chromatogram of Bikukulin, a test sample of Jindan Fuyan Granules in Example 2 of the present invention;

[0041] Fig.16 This is the MRM quantitative ion chromatogram of tetrahydrotetramine, a test sample of Jindan Fuyan Granules in Example 2 of the present invention;

[0042] Fig.17 This is the MRM quantitative ion flow chart of the original opium alkali of the Jindan Fuyan granules test sample in Example 2 of the present invention;

[0043] Fig.18 This is the MRM quantitative ion chromatogram of tetrahydropalmatine, a test product of Jindan Fuyan Granules in Example 2 of the present invention;

[0044] Fig.19 This is the MRM quantitative ion flow chart of the test sample papaverine of Jindan Fuyan granules in Example 2 of the present invention;

[0045] Fig. 20 This is the MRM quantitative ion chromatogram of the test product corydalis A in Jindan Fuyan granules in Example 2 of the present invention;

[0046] Fig.21The chromatogram of the mixed reference substance in Comparative Example 1 of the present invention; wherein 1 is hydrogen ethylcopper scutellarine, 2 is pyracanthaline, 3 is tetrahydroafricanine, 4 is protopine, 5 is papaverine and tetrahydropalmatine (the two test substances are not separated), and 6 is tetrahydropalmatine;

[0047] Fig. 22 This is the chromatogram of the Jindan Fuyan granules test sample in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0048] The above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation cases) can be combined with each other to form new or preferred technical solutions, but the present invention is not limited to these embodiments, and these embodiments do not limit the present invention in any way.

[0049] The experimental methods in the following examples are conventional methods unless otherwise specified. The preparations involved in the following examples are common commercial products unless otherwise specified and can be purchased from the market.

[0050] The present invention is further described in detail below in conjunction with the drawings and embodiments:

[0051] Experimental instruments and reagents:

[0052] 1. Instrument

[0053] Agilent 1290 liquid chromatograph (Agilent Technologies, USA); API 4000 mass spectrometer (AB SCIEX, USA); Sartorius Secura 125-1CN electronic balance (Sartorius Scientific Instruments Beijing Co., Ltd.); Mettler Toledo ML204 electronic balance (Mettler Toledo Instruments Shanghai Co., Ltd.).

[0054] 2. Drug test reagents

[0055] Tetrahydroafricanine (batch number: ER231223-06), glaucoma (batch number: VC240214-23), and tetrahydropalmatine (batch number: AF240126-04) were all from Stanford Chemicals, USA. Protopine (batch number: 110853-201003) and tetrahydropalmatine (batch number: 110726-201414) were purchased from the China Food and Drug Inspection Institute. Hydrogenated acetonitrile and bicuculline were made by Jiangxi Provincial Drug Inspection and Testing Institute. Acetonitrile (chromatographic grade, Sigma-Aldrich, USA) and water were ultrapure water prepared by Milli-Q. The other reagents were of analytical grade.

[0056] Three batches of Jindan Fuyan granules, all provided by Jiangxi Huatai Pharmaceutical Co., Ltd., numbered: J1-J3; three batches of Corydalis medicinal materials, numbered Y1-Y3, of which Y1 was produced in Dongyang, Zhejiang, and Y2-Y3 were produced in Pan'an, Zhejiang; three batches of Xiatianwu medicinal materials, numbered X1-X3, all produced in Yujiang, Jiangxi. The laboratory produced three batches of Jindan Fuyan granules according to the Jindan Fuyan granules prescription (in which Corydalis medicinal pieces were mixed with 10% Xiatianwu medicinal pieces, and the rest of the medicinal flavors remained unchanged) according to the Jindan Fuyan granules process, and produced three batches of Jindan Fuyan granules positive samples, numbered: JY1-JY3.

[0057] Example 1: Establishment of UHPLC-MS / MS-QAMS Detection Method and Methodological Study of Jindan Fuyan Granules

[0058] 1. Solution preparation

[0059] Reference solution: Accurately weigh 10.32 mg of hydrogenated berberine, 10.08 mg of bisoprolol, 11.30 mg of tetrahydrochalcone, 10.87 mg of protopine, 11.36 mg of corydalis, 10.85 mg of papaverine, and 10.58 mg of corydalis A reference substance, place them in a 100 mL volumetric flask respectively, add methanol to dissolve and dilute to the scale, shake well, and obtain (store in a refrigerator at 4 ℃).

[0060] Jindan Fuyan Granules test solution: Take Jindan Fuyan Granules, grind them into powder, accurately weigh 1.0g, put them into a round-bottom flask, accurately add 50mL of methanol, heat and reflux for 30min, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 1mL of the filtrate, put it into a 50mL volumetric flask, add methanol to dilute to the scale, shake well, and pass through a 0.22μm microporous filter membrane to obtain the solution.

[0061] Corydalis yanhusuo test solution: Take Corydalis yanhusuo medicinal material, crush it, pass it through a No. 4 sieve, accurately weigh 1.0g, put it in a round-bottom flask, accurately add 50mL of methanol, heat and reflux for 30min, cool, weigh again, make up the lost weight with methanol, shake well, accurately measure 1mL of the filtrate, put it in a 100mL volumetric flask, add methanol to dilute to the scale, shake well, and pass through a 0.22μm microporous filter membrane to obtain.

[0062] Xia Tian Wu test solution: Take Xia Tian Wu medicinal material, crush it, pass it through a No. 4 sieve, accurately weigh 0.10 g, put it in a round-bottom flask, accurately add 50 mL of methanol, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, accurately measure 1 mL of the filtrate, put it in a 100 mL volumetric flask, add methanol to dilute to the scale, shake well, and pass through a 0.22 μm microporous filter membrane to obtain.

[0063] Jindan Fuyan Granules positive sample test solution: Take the homemade Jindan Fuyan Granules positive sample, grind it, accurately weigh 1.0g, put it in a round-bottom flask, accurately add 50mL of methanol, heat and reflux for 30min, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 1mL of the filtrate, put it in a 50mL volumetric flask, add methanol to dilute to the scale, shake well, and pass through a 0.22μm microporous filter membrane to obtain.

[0064] 2. UPLC-MS / MS conditions

[0065] Chromatographic conditions: Agilent ZORBAX SB C18 (2.1×100mm, 1.8μm) column; mobile phase: acetonitrile (A)-0.1% formic acid solution (B) containing 5mmol / L ammonium formate, gradient elution; flow rate: 0.3mL / min; injection volume: 1μL; column temperature: 40℃. The gradient elution program is shown in Table 1.

[0066] Table 1 Gradient elution program

[0067]

[0068] Mass spectrometry conditions: electrospray ionization source ESI + Mode: multiple reaction ion monitoring (MRM); spray voltage: 4.5 kV; curtain gas pressure: 25 psi; spray gas pressure: 60 psi; drying gas pressure: 55 psi; collision gas: 6 psi; ion source temperature: 550 °C, qualitative ion (Q1), quantitative ion (Q3), declustering voltage (DP), collision energy (CE) are shown in Table 2.

[0069] Table 2 List of mass spectrometry parameters and retention time of each chromatographic peak

[0070]

[0071] Q3 Mass The ions marked with * are quantitative ions, and the ions marked with # are qualitative ions.

[0072] 3. Methodological research

[0073] 3.1 Linear relationship investigation: Accurately pipette an appropriate amount of the reference solution under "1. Preparation of solution", add methanol and gradually dilute it into a mixed reference solution containing 10, 50, 100, 500, 800, and 1000 ng / mL of hydrogenated beta-copper alkaloid, bikukulin, tetrahydroafricanine, protopine, tetrahydropalmatine, galangaline, and tetrahydropalmatine as the linear working solution. Inject 1 μL of each sample for testing according to the above UPLC-MS / MS conditions. Use AB SCIEX Analyst ®Software 1.6.3 used the peak area of ​​each alkaloid as the ordinate (Y) and the corresponding concentration (X) as the abscissa to fit the linear regression equation. The linear equations are shown in Table 3. The linear equations have good correlation coefficients in the range of 10 ng / mL-1000 ng / mL. The linear graphs are shown in Figures 1 to 7 shown.

[0074] Table 3 List of linear equations and correlation coefficients

[0075]

[0076] 3.2 Limit of quantification and limit of detection: Accurately draw an appropriate amount of reference solution under "1. Preparation of solution", add methanol and gradually dilute to the concentration of 0.5ng / mL of ethyl hydrogenated beta-copper alkaloid, bikukulin, tetrahydroafricanine, protopine, tetrahydropalmatine, galangaline, and tetrahydropalmatine, the signal-to-noise ratio of each peak is greater than 3:1, and when the concentration is 1.5ng / mL, the signal-to-noise ratio of each peak is greater than 10:1. Therefore, the instrument detection limit and quantification limit of the above 7 alkaloids are 0.5ng / mL and 1.5ng / mL respectively.

[0077] 3.3 Precision test: Take 500ng / mL mixed reference solution of hydrogenated beta-coptisine, bicuculline, tetrahydroafricanine, protopine, tetrahydropalmatine, galangaline, and tetrahydropalmatine, and analyze it 6 times in succession according to the above UPLC-MS / MS conditions. The RSD of the peak area of ​​each alkaloid measured 6 times was calculated respectively. The RSD of the above alkaloids were 1.75%, 1.30%, 1.51%, 1.48%, 1.53%, 1.68%, and 1.58%, respectively, and the RSD was less than 2%, indicating that the precision of the instrument met the requirements.

[0078] 3.4 Stability test: The treated sample solution was placed at room temperature, and the samples were analyzed at 0, 2, 4, 6, 8, 10 and 24 h. The RSDs of the peak areas of hydroethyl coptisine, bicyclosporin, tetrahydroafricanine, protopine, corydaline, papaverine and corydaline were calculated respectively. The results showed that the RSDs of the above alkaloids were 1.85%, 1.59%, 1.75%, 1.98%, 1.64%, 1.71% and 1.62%, respectively. The RSDs were all less than 2%, indicating that the sample solution had good stability within 24 h.

[0079] 3.5 Repeatability test: Take the Jindan Fuyan granule sample (No.: J1) and prepare 6 copies according to the test solution treatment method. Then use AB SCIEX Analyst ®Software 1.6.3 calculated the contents of hydrogenated coptisine, picrotoxin, tetrahydroafricanaine, protopine, tetrahydropalmatine, papaverine and corydalis. The results showed that hydrogenated coptisine was less than the limit of quantification (RSD: / ), picrotoxin was less than the limit of quantification (RSD: / ), the average content of tetrahydroafricanaine was 10.664μg / g (RSD: 1.92%), the average content of protopine was 9.4025μg / g (RSD: 2.20%), the average content of tetrahydropalmatine was 15.875μg / g (RSD: 2.01%), the average content of papaverine was 22.6851μg / g (RSD: 1.89%), and the average content of corydalis was 41.354μg / g (RSD: 2.02%). All the results met the requirements of repeatability determination.

[0080] 3.6 Recovery test: Take 6 portions of Jindan Fuyan granules sample of 0.5g (No.: J1), of which hydrogenated berberine and pyruvate were not detected, and added with methanol solution at the amount of 0.1μg of reference substance; after adding methanol solution of tetrahydroafricanine, protopine, corydaline, papaverine and corydaline A according to the amount of the test substance in the sample, prepare the sample according to the preparation method of the test sample solution, and calculate the recovery rate (%) = (actual measured amount - amount in the test sample) / amount added to the sample × 100%. The measured recovery results are shown in Tables 4 to 10.

[0081] Table 4 Recovery test results of hydrogenated ethyl coptisine

[0082]

[0083] Table 5 Results of the recovery test of pyruvate

[0084]

[0085] Table 6 Tetrahydrotetramine recovery test results

[0086]

[0087] Table 7 Results of the original opium base recovery test

[0088]

[0089] Table 8 Tetrahydropalmatine recovery test results

[0090]

[0091] Table 9 Results of the recovery test of papaverine

[0092]

[0093] Table 10. Test results of recovery rate of corydalis A

[0094]

[0095] 3.7 Establishment of relative correction factor

[0096] Selection of internal reference substances: Tetrahydroafricanine (batch number: ER231223-06), glaucoma (batch number: VC240214-23), and corydalis acetoin (batch number: AF240126-04) are imported reference substances; hydrogenated coptisine and bicuculline are made by Jiangxi Institute of Drug Inspection and Testing, which require complex extraction and identification processes. The above reference substances are not easy to obtain and are expensive. The cost of using them as internal reference substances is very high, so they are not suitable for selection. Protopine (batch number: 110853-201003) and corydalis acetoin (batch number: 110726-201414) were purchased from China Food and Drug Inspection Institute. Considering that the corydalis acetoin reference substance is relatively cheap and has good stability, and is also an important analgesic component in corydalis alkaloids. Therefore, corydalis acetoin was considered to be used as an internal standard to participate in the one-test-multiple-evaluation calculation, which saved the other 6 reference substances and was low in cost. At the same time, UHPLC-MS / MS technology has the advantages of simple pre-treatment, fast analysis speed, high sensitivity, strong specificity and good accuracy compared with HPLC-UV or UHPLC-UV technology. Therefore, UHPLC-MS / MS technology is used to carry out one test and multiple evaluations.

[0097] Previous studies have found that the concentration of each component has a good correlation with the peak area within the fitted linear range, and increases and decreases proportionally. Therefore, it is very feasible to use one test and multiple evaluations to determine the seven alkaloid components, and calculate according to the following formula.

[0098] F 相对 = F 内 / F 待 =(C 内 / A 内 ) / (C 待 / A 待 ) = (C 内 ×A 待 ) / (C 待 ×A 内 ),

[0099] F 内 , F 待 are the correction factors for the internal reference and the analyte,

[0100] C 内 , C待 are the concentrations of the internal reference and the reference substance to be tested, respectively.

[0101] A 内 , A 待 Peak area of ​​internal reference and reference substance of the substance to be tested,

[0102] F 相对 is the relative correction factor.

[0103] According to the formula, corydaline (No.: Y) was used as the internal standard to calculate the relative correction factors of hydrogenated coptisine (No.: 1), bicuculline (No.: 2), tetrahydroafricanine (No.: 3), protopine (No.: 4), papaverine (No.: 5), and corydaline (No.: 6).

[0104] At the same time, in order to verify the feasibility of the above method, three different chromatographic columns were used: Waters BEH C18 (2.1×100mm, 1.7μm), Agilent ZORBAX SB C18 (2.1×100mm, 1.8μm), Thermo Acclaim RSLC 120C18 (2.1×100mm, 2.2μm) and two different types of instruments: Agilent 1290 UHPLC-AB SCIEX API4000 LC-MS, Waters ACQUITY UPLC-Xevo TQ-S LC-MS to investigate the durability of the method. The results were measured under the above UPLC-MS / MS conditions as shown in Table 11. It can be seen from the data in Table 11 that the RSDs of the relative correction factors of the six components are all less than 3%, indicating that the relative correction factors of the remaining six components are durable when tetrahydropalmatine is used as the internal standard, and can be used in different chromatographic columns and LC-MS.

[0105] Table 11 Comparison of relative correction factors of 6 index components with tetrahydropalmatine (internal reference) under three chromatographic columns and two instrument conditions

[0106]

[0107] 3.8 Sample content determination

[0108] The external standard method used the linear regression equation of each alkaloid to calculate the content, and the one-measurement-multiple-evaluation relative correction factor used the average value of the relative correction factor to calculate the content.

[0109] The one-test-multiple-evaluation method and the external standard method were used to detect 7 alkaloids in 3 batches of Jindan Fuyan granules, 3 batches of Corydalis yanhusuo medicinal materials, 3 batches of Xiatianwu medicinal materials and 3 batches of self-made Jindan Fuyan granules positive samples. The results were shown in Table 12, Figures 8 to 13 As shown. From the data in Table 12, it can be seen that three batches of Jindan Fuyan granules and three batches of Yanhusuo medicinal materials contain tetrahydroafricanine, protopine, tetrahydropalmatine, sea papaverine, and corydalis, while hydrogen 2-type coptisine and pyruvicin are less than the quantitative limit; three batches of Xiatianwu medicinal materials contain hydrogen 2-type coptisine, pyruvicin, tetrahydroafricanine, protopine, and tetrahydropalmatine, while pyruvicin and corydalis are less than the quantitative limit; three batches of self-made Jindan Fuyan granules positive samples contain seven components, namely hydrogen 2-type coptisine, pyruvicin, tetrahydroafricanine, protopine, tetrahydropalmatine, sea papaverine, and corydalis, and hydrogen 2-type coptisine and pyruvicin are detected in Xiatianwu. Therefore, it can also be determined that the Yanhusuo decoction pieces in the three batches of self-made positive samples are mixed with Xiatianwu.

[0110] Table 12 Determination of the contents of seven alkaloids in Jindan Fuyan granules and self-made positive samples by one test and multiple evaluations

[0111]

[0112] From the above, we can know that the content of sciadipamine and sciadipamine in Corydalis yanhusuo is relatively high, but it only contains trace amounts of sciadipamine and pyruvicin (below the limit of quantification), and the content of sciadipamine and pyruvicin in Xiatianwu is relatively high, but it only contains trace amounts of sciadipamine and sciadipamine (both below the limit of quantification). Therefore, sciadipamine and pyruvicin can be used as distinguishing marker ingredients in Corydalis yanhusuo slices or prescriptions, and in preparations containing Corydalis yanhusuo mixed with Xiatianwu.

[0113] At the same time, from Figures 8 to 11 It can be seen that the seven components have good responses and peak shapes under the LC-MS method. Hydrogenated ethyl coptisine and pyruvic acid were detected in Xiatianwu decoction pieces, but glaucoma and corydalis were not detected; while glaucoma and corydalis were detected in Corydalis decoction pieces, but hydrogenated ethyl coptisine and pyruvic acid were not detected. While glaucoma and corydalis were detected in the homemade positive samples, hydrogenated ethyl coptisine and pyruvic acid were also detected, indicating that the Corydalis in the homemade positive samples was mixed with Xiatianwu decoction pieces. The Jindan Fuyan granules sample provided by the company was consistent with the Corydalis decoction pieces, and glaucoma and corydalis were detected, but hydrogenated ethyl coptisine and pyruvic acid were not detected. Therefore, this method can effectively identify whether the Corydalis decoction pieces in Jindan Fuyan granules are mixed with Xiatianwu decoction pieces.

[0114] Further, from Figure 12 to Figure 13 It can be seen that in summer, there is a large amount of hydrogenated ethyl coptisine ( Fig.12 a in, retention time 1.08min), Bikukulin ( Fig.12b in the sample, retention time 5.92min), while in the Corydalis yanhusuo slices, ethylhexanoate ( Fig.13 a in, retention time 1.08min), Bikukulin ( Fig.13 b in the sample, retention time 5.92min) are all very low (below the limit of quantification). If summer-free is added to the Corydalis yanhusuo slices, ethylcopper sinteringine and pyruvic acid can be detected in the sample. Therefore, it is further explained that ethylcopper sinteringine and pyruvic acid can be used as distinguishing marker components of summer-free mixed in Corydalis yanhusuo slices or prescriptions, and preparations containing Corydalis yanhusuo.

[0115] Example 2: Detection of alkaloids in Jindan Fuyan granules by UPLC-MS / MS

[0116] The preparation of the solution is the same as in Example 1.

[0117] An Agilent ZORBAX SB C18 (2.1×100 mm, 1.8 μm) chromatographic column was used and the UPLC-MS / MS conditions were the same as in Example 1 to detect the same batch of Jindan Fuyan granules.

[0118] Test results such as Figures 14 to 20 as well as Fig. 9 As shown in the figure, the quantitative ion channel of hydrogenated ethyl coptisine in the Jindan Fuyan granules was 192.2 / 148.9 (retention time: 1.08min), and the quantitative ion channel of pyracanthaline was 368.3 / 307.3 (retention time: 5.92min), both of which were below the limit of quantification; the quantitative ion channel of tetrahydroafricanine was 342.3 / 178.1 (retention time 6.29min), the quantitative ion channel of protopine was 354.3 / 188.0 (retention time 6.98min), the quantitative ion channel of tetrahydropalmatine was 356.2 / 192.0 (retention time 7.80min), the quantitative ion channel of papaverine was 356.3 / 325.1 (retention time 7.83min), and the quantitative ion channel of tetrahydropalmatine was 370.2 / 191.8 (retention time 8.88min).

[0119] It can be seen that the Jindan Fuyan granules contain tetrahydroafricanine, protopine, tetrahydropalmatine, papaverine and tetrahydropalmatine, but do not contain tetrahydropalmatine and bicuculline, and the product is not adulterated with summer. At the same time, although tetrahydropalmatine and papaverine have similar retention times, they do not interfere with each other in their respective ion channels, which is also the advantage of the UHPLC-MS / MS method compared with the UHPLC-UV method in terms of strong selectivity, good specificity and high sensitivity.

[0120] Example 3: Optimization of mobile phase

[0121] Since the seven components tested are all alkaloid components, the representative alkaloids proopioline, tetrahydropalmatine, and tetrahydropalmatine ion response and chromatographic peak tailing factor are used to evaluate the influence of the mobile phase. A mixed reference solution containing 250 ng / mL of the seven alkaloid reference substances is injected.

[0122] Chromatographic conditions: Agilent ZORBAX SB C18 (2.1×100 mm, 1.8 μm) chromatographic column was used; except for the mobile phase, other conditions were the same as those in Example 1.

[0123] Mobile phase (1): acetonitrile (A)-0.1% formic acid solution containing 5 mmol / L ammonium formate (B) gradient elution;

[0124] Mobile phase (2): acetonitrile (A)-0.1% formic acid solution (B) gradient elution;

[0125] Mobile phase (3): acetonitrile (A)-5 mmol / L ammonium formate solution (B) gradient elution;

[0126] Mobile phase (4): acetonitrile (A)-water (B) gradient elution;

[0127] Flow rate: 0.3 mL / min; injection volume: 1 μL; column temperature: 40°C.

[0128] The test results are shown in Table 13:

[0129] Table 13 Comparison of ion response values ​​and chromatographic peak symmetry factors of protopine, tetrahydropalmatine, and tetrahydropalmatine

[0130]

[0131] Ion response value: The ion response of mobile phase (1) is slightly smaller than that of mobile phase (2), but there is no significant difference between the two. The chromatographic peak symmetry factor of mobile phase (2) is worse than that of mobile phase (1), and the ion response of mobile phase (1) is much larger than that of mobile phase (3) and mobile phase (4).

[0132] Chromatographic peak symmetry factor: The mobile phase (1) is the best when its value is closest to 1 (the closer to 1, the more symmetrical the peak shape). Although the chromatographic peak symmetry factor of mobile phase (3) is comparable to that of mobile phase (1), its ion response is worse than that of mobile phase (1). The symmetry factors of mobile phases (2) and (4) are poor and cannot meet the analysis requirements.

[0133] From the above, it can be seen that mobile phase (1) is the best. The reason may be that formic acid is a good ionization aid, and ammonium formate can adjust the pH of the mobile phase to improve the chromatographic peak shape. Finally, when formic acid and ammonium formate were added to the mobile phase at the same time, the ion response and chromatographic peak shape met the requirements.

[0134] Comparative Example 1: Determination of alkaloids in Jindan Fuyan granules by UHPLC-UV method

[0135] Instruments: Agilent 1290 Infinity II ultra-high performance liquid chromatograph, Agilent liquid phase workstation, DAD detector; Sartorius Secura125-1CN electronic balance (Sartorius Scientific Instrument Beijing Co., Ltd.); MettlerToledo ML204 electronic balance (Mettler-Toledo Instrument Shanghai Co., Ltd.).

[0136] Preparation of reference solution: same as in Example 1.

[0137] Preparation of test solution: same as in Example 1.

[0138] Chromatographic conditions: Chromatographic column: Thermo Acclaim RSLC 120 C18; acetonitrile as mobile phase A, 0.1% formic acid solution containing 5 mmol / L ammonium formate as mobile phase B, gradient elution according to the program in Table 14; detection wavelength: 280 nm; flow rate: 0.3 mL / min.

[0139] Table 14 Elution program

[0140]

[0141] Accurately pipette 2 μL of reference solution and 2 μL of test solution, and measure according to the above chromatographic conditions. The chromatogram is as follows: Figure 21 to Figure 22 As shown in the figure, it can be seen that in the chromatogram of the reference substance, papaverine and tetrahydropalmatine were not separated; in the chromatogram of the test substance, the sensitivity of the analyte did not meet the requirements, and the chromatographic peak separation effect was poor, which would lead to inaccurate integration. Based on the above two reasons, the UHPLC-UV-QAMS method is not suitable as a detection method for the one-test-multiple-evaluation determination of 7 components in Jindan Fuyan Granules.

[0142] In summary, the present invention adopts UHPLC-MS / MS-QAMS to determine tetrahydropalmatine, and utilizes the proportional relationship between tetrahydropalmatine and other components, and adopts a one-test-multiple-evaluation method to realize the indirect determination of multiple other related components, thereby saving a large amount of purchase funds for reference substances, and the detection cost is low. The method has high sensitivity, strong specificity, and short analysis time. Hydroethyl coptisine and pyrimethamine are used as distinguishing marker components, and it can well distinguish whether the Corydalis yanusuo decoction pieces used in the production of Jindan Fuyan granules are mixed with summer syrup, thereby providing a basis for quality monitoring of Jindan Fuyan granules.

[0143] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A quality control method for Jindan Fuyan granules, characterized in that: The quality control method adopts UHPLC-MS / MS-QAMS technology to determine the content of multiple components of Corydalis in Jindan Fuyan granules, and specifically comprises the following steps: (1) Preparation of Jindan Fuyan granule test solution: Take an appropriate amount of Jindan Fuyan granules, grind them into powder, accurately weigh 1.0 g, put them into a round-bottom flask, accurately add 50 mL of methanol, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 1 mL of the filtrate, put it into a 50 mL volumetric flask, add methanol to dilute to the scale, shake well, and pass through a 0.22 μm microporous filter membrane to obtain; (2) Preparation of reference solution: Accurately weigh 10.32 mg of hydroethyl coptisine, 10.08 mg of bisoprolol, 11.30 mg of tetrahydroafricanine, 10.87 mg of protopine, 11.36 mg of tetrahydropalmatine, 10.85 mg of papaverine, and 10.58 mg of tetrahydropalmatine, and place them in a 100 mL volumetric flask. Dissolve them in methanol and dilute to the mark. Shake well to obtain the reference solution. (3) Determination: The reference solution was prepared into a series of standard solutions and analyzed by UHPLC-MS / MS. The concentration was used as the horizontal axis and the area was used as the vertical axis to fit a standard curve. The test solution was then measured by UHPLC-MS / MS, and the peak area was recorded. Corydalis butylene glycol was used as the internal reference to calculate the relative correction factor and content of each component. The chromatographic conditions of UHPLC are as follows: using a C18 chromatographic column, acetonitrile as mobile phase A, 0.1% formic acid solution containing 5 mmol / L ammonium formate as mobile phase B, gradient elution, injection volume of 1 μL, flow rate of 0.3 mL / min, column temperature of 40°C; the program of the gradient elution is as follows: 0 min, 10% mobile phase A, 90% mobile phase B; 0.3 min, 10% mobile phase A, 90% mobile phase B; 2 min, 25% mobile phase A, 75% mobile phase B; 8 min, 35% mobile phase A, 65% mobile phase B; 10 min, 60% mobile phase A, 40% mobile phase B; 10.1 min, 10% mobile phase A, 90% mobile phase B; 13 min, 10% mobile phase A, 90% mobile phase B; The MS / MS conditions included the use of an electrospray ionization source in ESI+ mode and detection using multiple reaction ion monitoring.

2. The quality control method of Jindan Fuyan granules according to claim 1, characterized in that: The C18 chromatographic column is any one of Waters BEH C18, Agilent ZORBAX SB C18, and Thermo Acclaim RSLC 120 C18.

3. The quality control method of Jindan Fuyan granules according to claim 1, characterized in that: The parameters used for the MS / MS were as follows: Spray voltage: 4.5 kV; Air curtain air pressure: 25psi; Spray gas pressure: 60psi; Dry gas pressure: 55psi; Collision gas: 6 psi; Ion source temperature: 550°C.

4. The quality control method of Jindan Fuyan granules according to claim 1, characterized in that: The instrument used for the MS / MS is an Agilent 1290 UHPLC-AB SCIEX API4000 liquid chromatography-mass spectrometer or a Waters ACQUITYUPLC-Xevo TQ-S liquid chromatography-mass spectrometer.

5. The quality control method of Jindan Fuyan granules according to claim 1, characterized in that: The methanol used was of analytical grade with a concentration of 100%.

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