Method and application of simultaneous determination of multiple components in Qianggan capsules based on liquid chromatography-mass spectrometry

Abstract

The present invention relates to a method and application for determining the content of multiple components in a liver-strengthening capsule based on liquid chromatography-mass spectrometry technology. The contents of the liver-strengthening capsule are taken, ground, accurately weighed, a suitable amount is added, a solvent is added for extraction, centrifuged, shaken, filtered, and a filtrate is taken to obtain a sample to be tested; the sample to be tested is subjected to liquid chromatography-mass spectrometry, the stationary phase of the liquid phase is a C18 column, the mobile phase A is an aqueous solution containing 0.1±0.02% formic acid, the mobile phase B is acetonitrile, gradient elution is adopted, the flow rate is 0.3~0.5mL / min, the column temperature is 40±2°C, the mass spectrometer adopts positive and negative ion modes, an electrospray ion source, and the detection method adopts a multiple reaction ion monitoring mode. The detection method provided by the present invention has been verified by methodologies such as specificity and sensitivity, and has the characteristics of being sensitive, accurate, and reproducible. It provides a reliable method for product quality control and is conducive to improving patient medication safety.

Description

Technical Field

[0001] The present invention relates to the technical field of quality control of traditional Chinese medicines, and in particular to a method and application of simultaneously determining multiple components in a liver-strengthening capsule based on liquid chromatography-mass spectrometry. Background Art

[0002] Qianggan Capsules are derived from the classic prescription "Qianggan Decoction" in the "Compendium of Traditional Chinese Medicine - Prescriptions." Qianggan Capsules are an improved version of Qianggan Decoction, composed of 16 herbs, including Artemisia capillaris. They possess the effects of clearing heat and dampness, tonifying the spleen and nourishing blood, and invigorating qi and relieving depression. Clinically, they are primarily used to treat liver diseases such as chronic hepatitis, early-stage cirrhosis, fatty liver, and toxic hepatitis.

[0003] Currently, there are relatively many clinical studies on Qianggan Capsules, and the clinical efficacy is significant. However, there are few reports on the research of its chemical composition, and its material basis is still unclear. Determination of the content of multiple chemical components in Qianggan Capsules can effectively control its quality, thereby ensuring its safety and effectiveness in clinical use.

[0004] Qianggan Capsule is a traditional Chinese medicine compound with numerous medicinal flavors, complex ingredients, and a high number of active ingredients, especially low levels of some ingredients. In its current quality standard, for content determination, only a single ingredient, paeoniflorin, is used as a quantitative indicator. The content of a single indicator often cannot accurately reflect the quality of the compound, making it difficult to achieve true quality control. At the same time, the indicator ingredients of Artemisia capillaris and Isatis root, the main ingredients in Qianggan Capsule, are not controlled in the above method, which poses a hidden danger to the quality of Qianggan Capsule. Currently, there is an urgent need in the art for a multi-component content detection method for Qianggan Capsule to provide a basis for quality control of Qianggan Capsule. Summary of the Invention

[0005] The purpose of the present invention is to overcome the defects in the prior art and provide a method and application for simply and efficiently detecting multiple components of Qianggan Capsule based on liquid chromatography-mass spectrometry technology.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The present invention provides a method for simultaneously determining multiple components in Qianggan Capsules based on liquid chromatography-mass spectrometry technology, which comprises the following steps:

[0008] S1 Preparation of test sample: Take the contents of Qianggan Capsule, grind, accurately weigh an appropriate amount, add solvent to extract, centrifuge, shake well, filter, and take the filtrate to obtain the test sample;

[0009] Preparation of S2 standard curve:

[0010] Accurately weigh appropriate amounts of p-hydroxyacetophenone, protocatechuic aldehyde, salicylic acid, protocatechuic acid, phthalic acid, gallic acid, caffeic acid, danshensu, chlorogenic acid, neochlorogenic acid, rosmarinic acid, loganinic acid, isochlorogenic acid A, paeoniflorin, paeoniflorin, and salvianolic acid B reference substances to prepare reference substance solutions. Take a certain amount of each reference substance solution and mix them to obtain a negative ion compound mixture reference substance stock solution;

[0011] Accurately weigh appropriate amounts of GABA, L-proline, valine, L-leucine, 7-methoxycoumarin, ferulic acid, cytidine, liquiritigenin, isoliquiritigenin, adenosine, formononetin, dihydrotanshinone I, calycosin, tanshinone IIA, gentiopicroside, formononetin, calycosin, glycyrrhetinic acid, alismatinol A, 6'-O-β-D-glucosylgentiopicroside, astragaloside IV, and glycyrrhizic acid reference substances to prepare reference substance solutions, and mix a certain amount of each reference substance solution to obtain a stock solution of a mixed reference substance of a positive ion compound;

[0012] Dilute the reference substance stock solution to obtain mixed reference substance solutions of negative or positive ion compounds of different concentrations, test the prepared mixed reference substance solutions of each concentration according to the method of step S3, record the peak area, and establish a standard curve for each component;

[0013] S3 HPLC-QTRAP-MS / MS detection: The sample obtained in step S1 was subjected to high-performance liquid chromatography-mass spectrometry detection. The stationary phase of the high-performance liquid chromatography was a C18 chromatographic column with a filler particle size of 1.7 to 5 μm. The mobile phases were: A was a 0.1±0.02% formic acid aqueous solution, and B was acetonitrile, with a gradient elution. The flow rate was 0.3 to 0.5 mL min-1. The column temperature was 40±2°C. The mass spectrometer used positive and negative ion modes, an electrospray ion source, and multiple reaction monitoring.

[0014] Considering the significant differences in polarity among the active ingredients in Qianggan Capsules, a single solvent is difficult to efficiently extract all of them simultaneously. However, using the aforementioned extraction solvents ensures efficient extraction of multiple ingredients in Qianggan Capsules. Therefore, in some preferred embodiments of the present invention, the solvent selected in step S1 is a 10-100% methanol solution, preferably a 40-80% methanol solution, and more preferably a 50% methanol solution.

[0015] As some preferred embodiments of the present invention, the solubility-solid ratio during extraction in step S1 is 20-50 mL / g, and the concentration of the obtained extraction solution is more conducive to subsequent detection, and is further preferably 20 mL / g.

[0016] As some preferred embodiments of the present invention, the extraction in step S1 is ultrasonic extraction, the ultrasonic power is 200-300W, and the ultrasonic frequency is 30-50kHz.

[0017] Further preferably, the ultrasound is performed at 250W, 40kHz, and the ultrasonic extraction time is 20min.

[0018] As some preferred embodiments of the present invention, more optimal chromatographic conditions are as follows:

[0019] A C18 chromatographic column with a filler particle size of 1.7-5 μm was used; 2.6um F5 LC Column (100×2.1mm, 2.6μm);

[0020] Mobile phase: A is 0.1±0.02% formic acid aqueous solution, B is acetonitrile;

[0021] Flow rate: 0.4 mL min -1 ;

[0022] Column temperature: 40 ± 2 °C;

[0023] Injection volume: 5 μL.

[0024] Each injection was pre-equilibrated for 5 ± 0.5 min.

[0025] As some preferred embodiments of the present invention, in the gradient elution in step S2, the positive and negative mode gradient elution procedures are performed separately, and the procedures are as follows;

[0026] Gradient elution conditions in negative ion mode: 0-1 min, 98% A→85 A%; 1-6 min, 85% A→70 A%; 6-6.1 min, 70% A→2 A%; 6.1-7 min, 2% A→70 A%; 7-7.1 min, 70% A→98 A%; 7.1-8 min, 98% A.

[0027] Gradient elution conditions in positive ion mode: 0-1 min, 98% A→85 A%; 1-6 min, 85% A→70 A%; 6-6.1 min, 70% A→2 A%; 6.1-7 min, 2% A→70 A%; 7-7.1 min, 70% A→98 A%; 7.1-9 min, 98% A.

[0028] As some preferred embodiments of the present invention, the mass spectrometry conditions in step S3 are: source injection voltage (IS): -4500 / 5500 V; source temperature (TEM): 550-700°C; nebulizing gas pressure (GS1, N2): 55.0-65.0 psi; drying gas pressure (GS2, N2): 60.0-70.0 psi; curtain gas pressure (CUR, N2): 25.0-40.0 psi.

[0029] Further preferred mass spectrometry conditions are: ion source: electrospray ion source (ESI source); detection mode: multiple reaction ion monitoring (MRM) mode, using positive and negative ion modes; source injection voltage (IS): -4500 / 5500 V; source temperature (TEM): 650°C; nebulizing gas pressure (GS1, N2): 60.0 psi; drying gas pressure (GS2, N2): 65.0 psi; curtain gas pressure (CUR, N2): 35.0 psi.

[0030] As some preferred embodiments of the present invention, in step S3, the multiple reaction monitoring parameters are as follows:

[0031]

[0032]

[0033] As some preferred embodiments of the present invention, the multiple components of the liver-strengthening capsule are: hydroxyacetophenone; protocatechuic aldehyde; salicylic acid; protocatechuic acid; phthalic acid; gallic acid; caffeic acid; danshensu; chlorogenic acid; neochlorogenic acid; rosmarinic acid; loganinic acid; isochlorogenic acid A; paeoniflorin; paeoniflorin; salvianolic acid B; GABA; L-proline; valine; L-leucine; 7-methoxycoumarin; ferulic acid; cytidine; glycyrrhizin; isoliquiritigenin; adenosine; formononetin; dihydrotanshinone I; calycosin isoflavones; tanshinone IIA; gentiopicroside; formononetin; calycosin isoflavone glycosides; glycyrrhetinic acid; alismatinol A; 6′-O-β-D-glucosylgentiopicroside; astragaloside IV; glycyrrhizic acid.

[0034] As some preferred embodiments of the present invention, the concentration of the series of standard solutions in step S2 is 0.005-5.00 mg / mL.

[0035] Another aspect of the present invention provides application of the above-mentioned method for simultaneously determining multiple components in Qianggan Capsules based on liquid chromatography-mass spectrometry technology in the quality control or quality evaluation of Qianggan Capsules.

[0036] The beneficial effects of adopting the above technical solution are:

[0037] (1) The method provided by the present invention, using HPLC-QTRAP-MS / MS, can simultaneously and efficiently detect 39 ingredients in Qianggan Capsules. Furthermore, the index ingredients selected according to the present invention can objectively characterize the intrinsic quality of the compound, further clarifying the efficacy basis of the traditional Chinese medicine compound and providing a reference for the quality control of Qianggan Capsules.

[0038] (2) The method provided by the present invention can accurately determine the contents of 39 components in Qianggan Capsules by optimizing chromatographic conditions and mass spectrometry conditions and studying the fragmentation and collision conditions of parent ions and daughter ions. It has the advantages of fast analysis, high sensitivity, good stability, low detection limit and good repeatability.

[0039] (3) The method provided by the present invention, through the optimization of the above-mentioned chromatographic conditions, is more conducive to the separation of multiple components in Qianggan Capsules, especially the separation of isomers. Paeoniflorin and paeoniflorin are isomers that are difficult to separate by mass spectrometry, but the chromatographic conditions of the present invention can significantly improve their separation effect.

[0040] (4) The present invention provides an accurate, simple and rapid method for determining the content of multiple index components of Qianggan Capsules. The verification results show that the detection method established by the present invention has good precision (within-day precision RSD range of 1.0-5.1%), stability (RSD range of each component within 24 hours is 0.9-4.7%), repeatability (RSD range of 0.9-7.6%) and extraction recovery rate (the average recovery rate of each component ranges from 91.6-112.3%, RSD is 0.5-6.2%). Compared with the existing detection methods, it has obvious advantages and can be promoted and applied in the actual detection work of Qianggan Capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 This is the mass spectrum of 39 components of Qianggan Capsule;

[0043] Figure 2 Extracted ion chromatogram of the components determined for Qiangan Capsule;

[0044] Figure A is the extracted ion chromatogram of the sample in negative ion mode, Figure B is the extracted ion chromatogram of the mixed standard sample in negative ion mode, Figure C is the extracted ion chromatogram of the sample in positive ion mode, and Figure D is the extracted ion chromatogram of the mixed standard sample in positive ion mode. In the figure: 1. p-hydroxyacetophenone; 2. protocatechuic aldehyde; 3. salicylic acid; 4. protocatechuic acid; 5. phthalic acid; 6. gallic acid; 7. caffeic acid; 8. danshensu; 9. chlorogenic acid; 10. neochlorogenic acid; 11. rosmarinic acid; 12. loganinic acid; 13. isochlorogenic acid A; 14. paeoniflorin; 15. paeoniflorin; 16. salvianolic acid B; 17. GABA; 18. L-proline; 19. valine; 20. L-leucine; 21. 7- Methoxycoumarin; 22. Ferulic acid; 23. Cytidine; 24. Liquoricerin; 25. Isoliquiritigenin; 26. Adenosine; 27. Formononetin; 28. Dihydrotanshinone I; 29. Calycosin; 30. Tanshinone IIA; 31. Acaropicroside; 32. Gentianopicroside; 33. Formononetin; 34. Calycosin; 35. Glycyrrhetinic acid; 36. Alismatinol A; 37. 6′-O-β-D-glucosylgentiopicroside; 38. Astragaloside IV; 39. Glycyrrhizic acid. DETAILED DESCRIPTION

[0045] To make the objectives, technical solutions, and advantages of the present invention more apparent, the present invention provides a variety of embodiments. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0046] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0047] In this specific example, the instruments used are as follows:

[0048] An analytical balance (AG-135, MettLer Toledo, Switzerland) was used; a CORUI liquid chromatography system (Chengdu Ke Rui Technology Co., Ltd.) was used; a 3200-QTrap tandem quadrupole linear ion trap mass spectrometer equipped with a Turbo V ion source (AB, USA); a KW-250E ultrasonic cleaner (250 W, Kunzhou Ultrasonic Instrument Co., Ltd., Jiangsu); Analyst TF 1.7 data collection software and Peakview 2.2 Software (SCIEX, USA) were used for data processing. A D3024R high-speed refrigerated centrifuge was used (SCILOGEX, USA).

[0049] In this specific example, the reagents and drugs used are as follows:

[0050] Reference substances: calycosin (batch number 100162), 7-methoxycoumarin (batch number YJ0146-211025), gentiopicroside (batch number 102342), rosmarinic acid (batch number 100145), protocatechuic aldehyde (batch number 100063), glycyrrhizin (batch number 102654), paeoniflorin (purity>98%, batch number 102772), calycosin glucoside (purity>99%, batch number 103255), astragaloside IV (batch number 100730), p-hydroxyacetophenone (batch number Y J0138-211025), chlorogenic acid (purity >99%, batch number 103365), ferulic acid (purity >99.5%, batch number 103364), adenosine (purity 99.9%, batch number 200028-190502), loganin (purity 99.9%, batch number 200316-181103), danshensu (batch number YJ0157), and dihydrotanshinone I (batch number A0060). Unless otherwise noted, the purity of the other products was >98% and all were purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd. Glycyrrhizic acid (batch number HG4398S1), glycyrrhetinic acid (batch number HS21053B1), paeoniflorin (batch number HR2225W1), tanshinone ⅡA (batch number HR17531S1), gallic acid (batch number HR14726S1), salvianolic acid B (batch number HS19910B2), caffeic acid (batch number HR1531S1), 6'-O-β-D-glucosylgentiopicroside (batch number H / 1215W2), valine (batch number HR2559W2), L-proline, L-leucine Aminoacid (batch number HS3570V1), γ-aminobutyric acid (batch number L010L08), cytidine (batch number HR5936W2), protocatechuic acid (batch number HR1441S1), formononetin (batch number HR14515B1), formononetin (batch number HR4265W2), isoliquiritigenin (batch number HR14923S1), araliacine (batch number HR1624W5), phthalic acid (batch number HR5914W1), isochlorogenic acid A (batch number HR2219W9), and alismatol (batch number A HR16623S1), with a purity of >98%, were purchased from Baoji Chenguang Biological Co., Ltd.; salicylic acid (batch number B21505) was purchased from Shanghai Yuanye Technology Co., Ltd.; neochlorogenic acid (batch number DST180130-015), with a purity of >98%, was purchased from Chengdu Desite Biotechnology Co., Ltd.

[0051] Methanol and acetonitrile (chromatographic grade, TEDIA, USA); ultrapure water (Hangzhou Wahaha Co., Ltd.); formic acid (chromatographic grade, Diamond, USA). Liver-strengthening capsules were provided by Shijiazhuang Dongfang Pharmaceutical Co., Ltd., including liver-strengthening capsules (batch numbers: 01220635, 01220944, 01220945, 01221163, 01220943, 01221162, 01220942, 01220530, 01220946, 01220941); liver-strengthening capsule powder (batch numbers: 012211153, 012211154, 012211155, 012211156, 01220947, 01220948, 01220949, 01220950, 01220951, 01220952).

[0052] Example 1

[0053] The method for simultaneously determining multiple components in Qianggan Capsules based on LC-MS technology of the present invention comprises the following steps:

[0054] S1 Preparation of test solution

[0055] Take 10 capsules of Qiangan Capsules, remove the capsule shells, mix the contents, grind finely, take about 0.1g, accurately weigh, place in an EP tube, accurately add 2mL of 50% methanol, weigh the weight, ultrasonically extract for 20min, cool, weigh again, make up the lost weight with 50% methanol, shake well, centrifuge at 15000rpm for 5min, filter through a 0.22μm microporous membrane, and take the filtrate as the test solution.

[0056] S2 establishes a standard curve

[0057] Accurately weigh appropriate amounts of p-hydroxyacetophenone, protocatechuic aldehyde, salicylic acid, protocatechuic acid, phthalic acid, gallic acid, caffeic acid, danshensu, chlorogenic acid, neochlorogenic acid, rosmarinic acid, loganinic acid, isochlorogenic acid A, paeoniflorin, paeoniflorin, and salvianolic acid B reference substances to prepare reference substance solutions of certain concentrations. Take a certain amount of each reference substance solution and mix them to obtain a negative ion compound mixed reference substance stock solution.

[0058] Accurately weigh appropriate amounts of GABA, L-proline, valine, L-leucine, 7-methoxycoumarin, ferulic acid, cytidine, liquiritigenin, isoliquiritigenin; adenosine, formononetin, dihydrotanshinone I, calycosin, tanshinone IIA, gentiopicroside, formononetin, calycosin, glycyrrhetinic acid, alismatinol A, 6′-O-β-D-glucosylgentiopicroside, astragaloside IV, and glycyrrhizic acid reference substances to prepare reference substance solutions of certain concentrations, and mix a certain amount of each reference substance solution to obtain a mixed reference substance stock solution of positive ion compounds.

[0059] The concentrations of the stock solutions of the reference substances are as follows: hydroxyacetophenone 1.53 mg / ml; protocatechuic aldehyde 1.2 mg / ml; salicylic acid 1.75 mg / ml; protocatechuic acid 1.74 mg / ml; phthalic acid 1.6 mg / ml; gallic acid 3.76 mg / ml; caffeic acid 1.02 mg / ml; danshensu 3.2 mg / ml; chlorogenic acid 1.18 mg / ml; neochlorogenic acid 2.05 mg / ml; rosmarinic acid 1.17 mg / ml; loganinic acid 2.08 mg / ml; isochlorogenic acid A 1.12 mg / ml; paeoniflorin 1.72 mg / ml; paeoniflorin 2.46 mg / ml; salvianolic acid B 2.58 mg / ml; GABA 1.33 mg / ml; L-proline 0.54 mg / ml; valine 1.66 mg / ml; L-leucine 1.00 mg / ml; 7-methoxycoumarin 0.90 mg / ml; ferulic acid 1.13 mg / ml; cytidine 1.04 mg / ml; glycyrrhizin 0.67 mg / ml; isoliquiritigenin 0.525 mg / ml; adenosine 0.62 mg / ml; formononetin 0.60 mg / ml; dihydrotanshinone I 0.046 mg / ml; calycosin 0.5 mg / ml; tanshinone II A 0.025 mg / ml; picroside 1.2 mg / ml; gentiopicroside 5.14 mg / ml; formononetin 0.535 mg / ml; calycosin 0.2 mg / ml; glycyrrhetinic acid 0.57 mg / ml; alismatinol A 1.33 mg / ml; 6′-O-β-D-glucosylgentiopicroside 1.55 mg / ml; astragaloside IV 0.535 mg / ml; glycyrrhizic acid 2.04 mg / ml.

[0060] The concentrations of the components of the mixed reference substance are as follows: hydroxyacetophenone 7.65 μg / ml; protocatechuic aldehyde 6.00 μg / ml; salicylic acid 8.75 μg / ml; protocatechuic acid 5.22 μg / ml; phthalic acid 8.00 μg / ml; gallic acid 75.20 μg / ml; caffeic acid 5.10 μg / ml; danshensu 128.00 μg / ml; chlorogenic acid 59.00 μg / ml; neochlorogenic acid 82.00 μg / ml g / ml; rosmarinic acid 46.80μg / ml; loganinic acid 104.00μg / ml; isochlorogenic acid A 22.40μg / ml; paeoniflorin 86.00μg / ml; paeoniflorin 49.20μg / ml; salvianolic acid B 232.20μg / ml; GABA 3.99μg / ml; L-proline 27.00μg / ml; valine 8.30μg / ml; L-leucine 10.00μg / ml g / ml; 7-methoxycoumarin 8.95μg / ml; ferulic acid 5.65μg / ml; cytidine 10.40μg / ml; liquiritigenin 20.10μg / ml; isoliquiritigenin 2.625μg / ml; adenosine 31.00μg / ml; formononetin 24.00μg / ml; dihydrotanshinone I 2.3125μg / ml; calycosin isoflavone 10.00μg / ml; tanshinone IIA 1.25μg / ml; gentiopicroside 12.00μg / ml; gentiopicroside 205.60μg / ml; formononetin 1.605μg / ml; calycosin 9.50μg / ml; glycyrrhetinic acid 11.40μg / ml; alismatol A 13.30μg / ml; 6′-O-β-D-glucosylgentiopicroside 62.00μg / ml; astragaloside IV 5.35μg / ml; glycyrrhizic acid 81.60μg / ml.

[0061] Establish a standard curve for the reference substance

[0062] The HPLC-QTRAP-MS / MS method was used, and the specific detection conditions were as follows:

[0063] The chromatographic conditions are as follows:

[0064] use 2.6um F5 LC Column (100×2.1mm, 2.6μm), mobile phase: A is 0.1% formic acid aqueous solution, B is acetonitrile, flow rate: 0.4mL·min -1Gradient elution: In negative ion mode, gradient elution conditions were: 0–1 min, 98% A → 85% A; 1–6 min, 85% A → 70% A; 6–6.1 min, 70% A → 2% A; 6.1–7 min, 2% A → 70% A; 7–7.1 min, 70% A → 98% A; 7.1–8 min, 98% A. In positive ion mode, gradient elution conditions were: 0–1 min, 98% A → 85% A; 1–6 min, 85% A → 70% A; 6–6.1 min, 70% A → 2% A; 6.1–7 min, 2% A → 70% A; 7–7.1 min, 70% A → 98% A; 7.1–9 min, 98% A. Column temperature: 40°C. Injection volume: 5 μL. Pre-equilibration for 5 min was performed for each injection.

[0065] Mass spectrometry conditions are as follows:

[0066] Ion source: electrospray ionization (ESI); detection mode: multiple reaction monitoring (MRM); positive and negative ion modes; source spray voltage (IS): -4500 / 5500 V; source temperature (TEM): 650°C; nebulizer gas (GS1, N2): 60.0 psi; drying gas (GS2, N2): 65.0 psi; curtain gas (CUR, N2): 35.0 psi. Ion pairs monitored for 39 analytes, declustering potential (DP), and collision potential (CE) are shown in Table 1. The mass spectra are shown in Table 1. Figure 1 , extracted ion current diagram see Figure 2 .

[0067] Table 1 Retention time (RT), parent ion, daughter ion, declustering potential (DP), and collision potential (CE) of 39 components

[0068]

[0069]

[0070] The mixed reference solution of each concentration prepared in step S2 was tested according to the above-mentioned chromatographic and mass spectrometric conditions to obtain the extracted ion current chromatogram of the mixed reference solution of each concentration. The standard curve of each component was established with the peak area of each component as the y value and the concentration of each component as the x value.

[0071] S3 test sample content determination

[0072] The test solution prepared in step S1 was injected into a high performance liquid chromatography-mass spectrometer for analysis under the same chromatographic and mass spectrometric conditions as in step S3 to obtain an extracted ion current chromatogram of the test solution. The content of each component was calculated based on the peak area of each component and the standard curve of each component established in step S3.

[0073] Example 2 Methodology Verification

[0074] (1) Linear range, detection limit, and quantification limit

[0075] Take a series of standard solutions and, according to the method in Example 1, plot a standard curve with the reference substance concentration as the abscissa and the peak area as the ordinate, and perform linear regression to obtain a regression equation.

[0076] The reference solution was diluted gradually and assayed, with the amount of each substance at a signal-to-noise ratio (S / N) of 10 and S / N of 3 serving as the limit of quantification (LOQ) and limit of detection (LOD). The regression equations, linear ranges, LOQs, and LODs for each analyte are shown in Table 2.

[0077] Table 2 Linear range, detection limit and quantification limit of 39 components in Qiangan Capsule

[0078]

[0079]

[0080] Under the detection conditions of this example, the correlation coefficient of the standard curve of each component to be measured is r≥0.9914, indicating good linearity.

[0081] (2) Precision

[0082] Under the chromatographic and mass spectrometric conditions specified in Example 1, the same mixed reference solution was measured six times consecutively, the peak areas recorded, and the relative standard deviation (RSD) was calculated to evaluate instrument precision. Six samples of Qianggan Capsules from the same batch were processed according to the method described under "Test Solution Preparation." Parallel sample solutions were prepared, injected, and analyzed. The content of the measured components and RSD values were measured to assess the intra-day precision of the method. The inter-day precision of the method was assessed for three consecutive days. The results are shown in Table 3.

[0083] Table 3 Precision of 39 ingredients in Qiangan Capsule

[0084]

[0085]

[0086] The experimental results showed that the RSD of the instrument precision of the 39 test components was less than 7.6%, and the RSD of the intra-day and inter-day precision of the method were less than 5.1% and 9.0%, respectively. The results showed that the instrument repeatability was stable and the method had good precision.

[0087] (3) Sample recovery rate

[0088] Approximately 0.05 g of a Qianggan Capsule sample from the same batch with a known content was accurately weighed into six portions. A mixed reference solution containing similar contents was accurately added to each portion, ensuring a ratio of approximately 1:1 between the final reference solution and the content of each component in the test sample. Under the chromatographic and mass spectrometric conditions specified in Example 1, the content of the measured components in the sample solution was determined, and the recovery rate was calculated. The calculation formula is: Recovery rate (%) = (measured amount - original amount) / added amount × 100%. The results are shown in Table 4.

[0089] Table 4 Recovery rates of 39 components in Qiangan Capsules (n=6)

[0090]

[0091] The experimental results showed that the average recovery of the 39 test components ranged from 91.6% to 112.3%, with an RSD of 0.5% to 6.2%. The results showed that the method was accurate and reliable.

[0092] (4) Stability

[0093] A sample of Qianggan Capsule was taken, and a sample solution was prepared according to the method described in step S1 of Example 1. The sample was injected and analyzed at 0, 4, 6, 8, 12, and 24 hours, and the difference in the peak area of the measured component was compared with that at 0 hour. The RSD value was calculated to investigate the stability of the test solution. The results are shown in Table 5.

[0094] Table 5 Stability of 39 ingredients in Qianggan Capsule (n=3)

[0095]

[0096] The experimental results showed that the RSD of the peak areas of 39 test components in the test solution was less than 4.7%, indicating that the test solution had good stability within 24 hours.

[0097] Example 3 Test sample content determination

[0098] In this study, the contents of 39 components in 6 batches of Qiangan Capsules were determined. The results are shown in Table 6. The Qiangan Capsules samples were provided by Shijiazhuang Dongfang Pharmaceutical Co., Ltd.

[0099] Table 6 Determination results of 39 components in 6 batches of Qiangan Capsules

[0100]

[0101]

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for simultaneously determining multiple components in Qianggan Capsules based on liquid chromatography-mass spectrometry technology, characterized in that: It includes the following steps: S1 Preparation of test sample: Grind the contents of Qianggan Capsule, accurately weigh an appropriate amount, add solvent for extraction, centrifuge, shake well, filter, and collect the filtrate to obtain the test sample; S2 standard curve preparation: Accurately weigh appropriate amounts of p-hydroxyacetophenone, protocatechuic aldehyde, salicylic acid, protocatechuic acid, phthalic acid, gallic acid, caffeic acid, danshensu, chlorogenic acid, neochlorogenic acid, rosmarinic acid, loganinic acid, isochlorogenic acid A, paeoniflorin, paeoniflorin, and salvianolic acid B reference substances to prepare reference substance solutions. Take a certain amount of each reference substance solution and mix them to obtain a negative ion compound mixture reference substance stock solution; Accurately weigh appropriate amounts of GABA, L-proline, valine, L-leucine, 7-methoxycoumarin, ferulic acid, cytidine, liquiritigenin, isoliquiritigenin, adenosine, formononetin, dihydrotanshinone I, calycosin, tanshinone IIA, gentiopicroside, formononetin, calycosin, glycyrrhetinic acid, alismatinol A, 6'-O-β-D-glucosylgentiopicroside, astragaloside IV, and glycyrrhizic acid reference substances to prepare reference substance solutions, and mix a certain amount of each reference substance solution to obtain a stock solution of a mixed reference substance of a positive ion compound; Dilute the reference substance stock solution to obtain mixed reference substance solutions of negative or positive ion compounds of different concentrations, test the prepared mixed reference substance solutions of each concentration according to the method of step S3, record the peak area, and establish a standard curve for each component; S3 HPLC-QTRAP-MS / MS detection: The sample obtained in step S1 is subjected to high performance liquid chromatography-mass spectrometry detection, wherein the stationary phase of the high performance liquid chromatography is a C18 chromatographic column with a filler particle size of 1.7 to 5 μm; Mobile phase: A is 0.1±0.02% formic acid aqueous solution, B is acetonitrile, gradient elution; flow rate: 0.3~0.5 mL·min-1; column temperature: 40±2°C; the mass spectrometer uses positive and negative ion modes, an electrospray ion source, and multiple reaction monitoring. The solvent selected in step S1 is a 50% methanol solution; The solubility-solid ratio during extraction in step S1 is 20-50 mL / g; In the gradient elution in step S3, the positive and negative mode gradient elution procedures are performed separately, and the procedures are as follows; Gradient elution conditions in negative ion mode: 0-1 min, 98%A→85A%; 1-6 min, 85%A→70A%; 6-6.1 min, 70%A→2A%; 6.1-7 min, 2%A→70A%; 7-7.1 min, 70%A→98A%; 7.1-8 min, 98%A; Gradient elution conditions in positive ion mode: 0-1 min, 98%A→85A%; 1-6 min, 85%A→70A%; 6-6.1 min, 70%A→2A%; 6.1-7 min, 2%A→70A%; 7-7.1 min, 70%A→98A%; 7.1-9 min, 98%A; In step S3, the multiple reaction monitoring parameters are as follows: 。 2. The method for simultaneously determining multiple components in Qianggan Capsules based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The extraction in step S1 is ultrasonic extraction with an ultrasonic power of 200-300 W and an ultrasonic frequency of 30-50 kHz.

3. The method for simultaneously determining multiple components in Qianggan Capsules based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The mass spectrometry conditions in step S3 are as follows: source injection voltage (IS): -4500 / 5500 V; source temperature (TEM): 550-700°C; nebulizing gas pressure (GS1, N2): 55.0-65.0 psi; drying gas pressure (GS2, N2): 60.0-70.0 psi; and curtain gas pressure (CUR, N2): 25.0-40.0 psi.

4. The method for simultaneously determining multiple components in Qianggan Capsules based on liquid chromatography-mass spectrometry according to claim 1, characterized in that In step S3, the retention time of the compound in the multiple reaction monitoring is as follows: 。 5. The method for simultaneously determining multiple components in Qianggan Capsules based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The multiple components of the liver-strengthening capsule are: hydroxyacetophenone; protocatechuic aldehyde; salicylic acid; protocatechuic acid; phthalic acid; gallic acid; caffeic acid; danshensu; chlorogenic acid; neochlorogenic acid; rosmarinic acid; loganinic acid; isochlorogenic acid A; paeoniflorin; paeoniflorin; salvianolic acid B; GABA; L-proline; valine; L-leucine; 7-methoxycoumarin; ferulic acid; cytidine; glycyrrhizin; isoliquiritigenin; adenosine; formononetin; dihydrotanshinone I; calycosin isoflavones; tanshinone II A; gentiopicroside; formononetin; calycosin isoflavone glycosides; glycyrrhetinic acid; alismatinol A; 6′-O-β-D-glucosylgentiopicroside; astragaloside IV; glycyrrhizic acid.

6. The method for simultaneously determining multiple components in Qianggan Capsules based on liquid chromatography-mass spectrometry according to claim 1, characterized in that: The concentration of the series of standard solutions in step S2 is 0.005-5.00 mg / mL.

7. Use of the method for simultaneous determination of multiple components in Qianggan Capsules based on liquid chromatography-mass spectrometry according to any one of claims 1 to 6 in the quality control or quality evaluation of Qianggan Capsules.

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