Method for qualitatively analyzing small molecule compounds in Xinnaoning capsules by using UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) technology

Abstract

The invention relates to the technical field of medicine analysis and detection, in particular to a method for qualitatively analyzing small molecule compounds in Xinnaoning capsules by using a UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) technology. The small molecule compounds comprise polar small molecule compounds and weak polar small molecule compounds. The method comprises the following steps: preparing a test solution; carrying out UPLC / Q-TOF MS detection; and performing qualitative analysis. Through qualitative analysis of polar and weak polar small molecule compounds in the Xinnaoning capsule, a material basic component library is established, and a theoretical basis and technical support are provided for more comprehensive and effective control of product quality.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis and detection, and particularly relates to a method for qualitatively analyzing small molecule compounds in Xinnaoning Capsule by UPLC-MS technology. Background Art

[0002] As an exclusive product, Xinnaoning Capsule has been approved by the Pharmacopoeia Commission of the People's Republic of China and is first included in the first part of the Chinese Pharmacopoeia (2015 Edition). It is a Chinese patent medicine preparation made from Ginkgo biloba leaves, Buxus microphylla Sieb. et Zucc., Salvia miltiorrhiza Bunge, Litsea lancilimba Merr., and Allium macrostemon Bunge as the main raw materials. It has the functions of promoting blood circulation, regulating qi, and dredging collaterals to relieve pain. Clinically, it is used for chest bi-syndrome, headache, and dizziness caused by qi stagnation and blood stasis, manifested as chest stuffiness and stabbing pain, palpitation and uneasiness, dizziness and vertigo, etc., as well as those with the above symptoms in coronary heart disease and cerebral arteriosclerosis. It is widely used clinically.

[0003] In the prior art, the invention patent application with the publication number of CN104007216A discloses a quality standard detection method for Xinnaoning Capsule, which adds the qualitative identification of Allium macrostemon Bunge and Buxus microphylla Sieb. et Zucc. by thin layer chromatography and the quantitative detection of salvianolic acid B in Salvia miltiorrhiza Bunge by high performance liquid chromatography on the basis of the existing quality standard. This method is stable, reliable, and highly specific, improving the control of product quality. However, Xinnaoning Capsule is made from five kinds of traditional Chinese medicines, and its chemical components are up to hundreds. The types of active ingredients detected by the detection method disclosed in the above patent are relatively few, and it is impossible to achieve more comprehensive and effective quality control of Xinnaoning Capsule. At present, there is no relevant literature report on the systematic qualitative research of the compound components in Xinnaoning Capsule in China.

[0004] Based on the above problems, the present invention provides a method for qualitatively analyzing small molecule compounds in Xinnaoning Capsule by UPLC-MS technology. By qualitatively analyzing the polar and weakly polar small molecule compounds in Xinnaoning Capsule, it aims to provide a theoretical basis and technical support for achieving more comprehensive and effective control of product quality. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for qualitatively analyzing small molecule compounds in Xinnaoning Capsule by UPLC-MS technology.

[0006] To achieve the above purpose, the technical scheme adopted by the present invention is as follows:

[0007] In the method for qualitatively analyzing small molecule compounds in Xinnaoning Capsule by UPLC-MS technology of the present invention, the small molecule compounds include polar small molecule compounds and weakly polar small molecule compounds, and the method comprises the following steps:

[0008] S1, preparing a test solution:

[0009] S11, Preparation of test solution for qualitative analysis of polar small molecule compounds: Weigh the content of Xinnaoning capsules, add an appropriate amount of methanol, perform ultrasonic treatment, filter, and obtain the solution.

[0010] S12, Preparation of test solution for qualitative analysis of weakly polar small molecule compounds: Weigh the content of Xinnaoning capsules, add an appropriate amount of water, perform ultrasonic treatment, and filter to obtain the extract; take an appropriate amount of the extract, add petroleum ether for extraction, retain the aqueous solution, evaporate the extraction solution to dryness under reduced pressure, dissolve the residue in an appropriate amount of methanol to obtain the petroleum ether extract solution; take the aqueous solution retained in the previous step, add chloroform for extraction, retain the aqueous solution, evaporate the extraction solution to dryness under reduced pressure, dissolve the residue in an appropriate amount of methanol to obtain the chloroform extract solution; take the aqueous solution retained in the previous step, add ethyl acetate for extraction, retain the aqueous solution, evaporate the extraction solution to dryness under reduced pressure, dissolve the residue in an appropriate amount of methanol to obtain the ethyl acetate extract solution; take the aqueous solution retained in the previous step, add water-saturated n-butanol for extraction, evaporate the extraction solution to dryness under reduced pressure, dissolve the residue in an appropriate amount of methanol to obtain the n-butanol extract solution.

[0011] S2, UPLC / Q-TOF MS detection:

[0012] Inject each test solution prepared in step S1 into a liquid chromatography-tandem mass spectrometry instrument for UPLC / Q-TOF MS detection to obtain the total ion chromatograms of each sample in positive and negative modes.

[0013] S3, Qualitative analysis:

[0014] With the aid of PeakView software, perform structural analysis on each spectral peak through the secondary scanning fragment ions in positive and negative modes of UPLC / Q-TOF MS to determine the name, molecular formula, and structural formula of the compound.

[0015] Preferably, the specific process for preparing the test solution for qualitative analysis of polar small molecule compounds in step S1 of the present invention is: accurately weigh 1-1.5 g of the content of Xinnaoning capsules, add 10-20 mL of methanol, weigh, perform ultrasonic treatment for 30-60 min, make up the weight with methanol, and filter through a microporous membrane to obtain the solution.

[0016] More preferably, the specific process for preparing the test solution for qualitative analysis of polar small molecule compounds in step S1 of the present invention is: accurately weigh 1 g of the content of Xinnaoning capsules, add 10 mL of methanol, weigh, perform ultrasonic treatment for 30 min, make up the weight with methanol, and filter through a microporous membrane to obtain the solution.

[0017] Preferably, the specific process for preparing the test sample solution for qualitative analysis of weakly polar small molecule compounds in step S1 of the present invention is as follows: Weigh 4 - 6 g of the content of Xinnaoning capsules, add 40 - 60 mL of water, perform ultrasonic treatment for 30 - 60 min, and obtain an extract after filtration; accurately measure 40 - 50 mL of the extract, add 80 - 100 mL of petroleum ether for extraction, extract continuously 4 times, retain the aqueous solution, combine the extraction solutions and rotary evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up the volume to 1 mL to obtain the petroleum ether extract solution; take the aqueous solution retained in the previous step, add 80 - 100 mL of chloroform for extraction, extract continuously 4 times, retain the aqueous solution, combine the extraction solutions and rotary evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up the volume to 10 mL to obtain the chloroform extract solution; take the aqueous solution retained in the previous step, add 80 - 100 mL of ethyl acetate for extraction, extract continuously 4 times, retain the aqueous solution, combine the extraction solutions and rotary evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up the volume to 25 mL to obtain the ethyl acetate extract solution; take the aqueous solution retained in the previous step, add 80 - 100 mL of water-saturated n-butanol for extraction, extract continuously 4 times, combine the extraction solutions and rotary evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up the volume to 10 mL to obtain the n-butanol extract solution.

[0018] More preferably, the specific process for preparing the test sample solution for qualitative analysis of weakly polar small molecule compounds in step S1 of the present invention is as follows: Weigh 5 g of the content of Xinnaoning capsules, add 50 mL of water, perform ultrasonic treatment for 30 min, and obtain an extract after filtration; accurately measure 40 mL of the extract, add 80 mL of petroleum ether for extraction, extract continuously 4 times, retain the aqueous solution, combine the extraction solutions and rotary evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up the volume to 1 mL to obtain the petroleum ether extract solution; take the aqueous solution retained in the previous step, add 80 mL of chloroform for extraction, extract continuously 4 times, retain the aqueous solution, combine the extraction solutions and rotary evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up the volume to 10 mL to obtain the chloroform extract solution; take the aqueous solution retained in the previous step, add 80 mL of ethyl acetate for extraction, extract continuously 4 times, retain the aqueous solution, combine the extraction solutions and rotary evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up the volume to 25 mL to obtain the ethyl acetate extract solution; take the aqueous solution retained in the previous step, add 80 mL of water-saturated n-butanol for extraction, extract continuously 4 times, combine the extraction solutions and rotary evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up the volume to 10 mL to obtain the n-butanol extract solution.

[0019] The boiling range of the petroleum ether described in the present invention is 60 - 90 °C.

[0020] The chromatographic conditions for UPLC / Q-TOF MS detection in step S2 of the present invention are as follows:

[0021] The chromatographic column is C 18Column, with a specification of 250 mm × 4.6 mm, 5 μm; column temperature is 30 °C; mobile phase flow rate is 0.8 mL / min; injection volume is 5 μL; acetonitrile is used as mobile phase A and 0.1% formic acid water is used as mobile phase B for gradient elution, and the elution program is as follows:

[0022] From 0 to 17 min, the volume ratio of mobile phase A to mobile phase B changes from 3:97 to 15:85;

[0023] From 17 to 28 min, the volume ratio of mobile phase A to mobile phase B changes from 15:85 to 21:79;

[0024] From 28 to 38 min, the volume ratio of mobile phase A to mobile phase B is 21:79;

[0025] From 38 to 56 min, the volume ratio of mobile phase A to mobile phase B changes from 21:79 to 39:61;

[0026] From 56 to 63 min, the volume ratio of mobile phase A to mobile phase B changes from 39:61 to 47:53;

[0027] From 63 to 69 min, the volume ratio of mobile phase A to mobile phase B changes from 47:53 to 60:40;

[0028] From 69 to 80 min, the volume ratio of mobile phase A to mobile phase B changes from 60:40 to 80:20;

[0029] From 80 to 90 min, the volume ratio of mobile phase A to mobile phase B changes from 80:20 to 85:15;

[0030] From 90 to 95 min, the volume ratio of mobile phase A to mobile phase B is 85:15.

[0031] The mass spectrometry conditions for the UPLC / Q-TOF MS detection described in step S2 of the present invention are as follows:

[0032] First stage: scanning time 0.25 s; acquisition time 95 min; acquisition range 100 - 20000 Da; nebulizing gas flow rate 50 mL / min; desolvation gas flow rate 50 mL / min; curtain gas flow rate 35 mL / min; desolvation gas temperature 500 °C; ion spray voltage is 4500 V in negative mode and 5500 V in positive mode; declustering voltage is ±100 V; collision energy is ±10 V; dynamic background subtraction mode;

[0033] Level 2: Scanning time 0.1 s; acquisition time 95 min; acquisition range 50 - 2000 Da; atomization gas flow rate 50 mL / min; desolvation gas flow rate 50 mL / min; curtain gas flow rate 35 mL / min; desolvation gas temperature 500 °C; ion spray voltage 4500 V in negative mode and 5500 V in positive mode; declustering voltage ±100 V; collision energy ±40 V, collision energy spread ±20 V; ion release delay 74; ion beam width 25; dynamic background subtraction mode.

[0034] Advantages of the present invention:

[0035] By qualitatively analyzing the polar and weakly polar small molecule compounds in Xinnaoning capsules and establishing a library of the basic components of the substances, the present invention provides a theoretical basis and technical support for more comprehensive and effective control of product quality. Description of the Drawings

[0036] Figure 1 It is the total ion current chromatogram of UPLC-MS (positive mode) for polar small molecule compounds in Xinnaoning capsules;

[0037] Figure 2 It is the total ion current chromatogram of UPLC-MS (negative mode) for polar small molecule compounds in Xinnaoning capsules;

[0038] Figure 3 It is the total ion current chromatogram of UPLC-MS (petroleum ether - positive mode) for weakly polar small molecule compounds in Xinnaoning capsules;

[0039] Figure 4 It is the total ion current chromatogram of UPLC-MS (petroleum ether - negative mode) for weakly polar small molecule compounds in Xinnaoning capsules.

[0040] Figure 5 It is the total ion current chromatogram of UPLC-MS (chloroform - positive mode) for weakly polar small molecule compounds in Xinnaoning capsules;

[0041] Figure 6 It is the total ion current chromatogram of UPLC-MS (chloroform - negative mode) for weakly polar small molecule compounds in Xinnaoning capsules;

[0042] Figure 7 It is the total ion current chromatogram of UPLC-MS (ethyl acetate - positive mode) for weakly polar small molecule compounds in Xinnaoning capsules;

[0043] Figure 8 It is the total ion current chromatogram of UPLC-MS (ethyl acetate - negative mode) for weakly polar small molecule compounds in Xinnaoning capsules;

[0044] Figure 9Total ion current chromatogram of weakly polar small molecule compounds in Xinnaoning Capsules by UPLC-MS (n-butanol - positive mode);

[0045] Figure 10 Total ion current chromatogram of weakly polar small molecule compounds in Xinnaoning Capsules by UPLC-MS (n-butanol - negative mode). Specific embodiments

[0046] The technical solutions of the present invention will be described in detail below in conjunction with specific embodiments. The following embodiments are only for explanation and illustration, and do not constitute a limitation to the technical solutions of the present invention.

[0047] Example 1

[0048] Qualitative analysis of polar small molecule compounds in Xinnaoning Capsules is carried out as follows:

[0049] S1 Preparation of test solution

[0050] Accurately weigh 1 g of the content of Xinnaoning Capsules, add 10 mL of methanol, weigh, ultrasonically treat for 30 min, make up the weight with methanol, and filter through a 0.45 μm microporous filter membrane to obtain.

[0051] S2 UPLC / Q-TOF MS detection

[0052] Inject the test solution prepared in the above steps into a liquid chromatography tandem mass spectrometer for UPLC / Q-TOF MS detection to obtain the total ion current chromatogram of the sample. The chromatographic and mass spectrometric conditions for UPLC / Q-TOF MS detection are as follows:

[0053] 1) Chromatographic conditions:

[0054] The chromatographic column is Phenomenex Luna C 18 column, with a specification of 250 mm × 4.6 mm, 5 μm; the column temperature is 30 °C; the mobile phase flow rate is 0.8 mL / min; the injection volume is 5 μL; acetonitrile is used as mobile phase A and 0.1% formic acid water is used as mobile phase B for gradient elution. The elution program is as follows:

[0055] 0 - 17 min, the volume ratio of mobile phase A to mobile phase B changes from 3:97 to 15:85;

[0056] 17 - 28 min, the volume ratio of mobile phase A to mobile phase B changes from 15:85 to 21:79;

[0057] 28 - 38 min, the volume ratio of mobile phase A to mobile phase B is 21:79;

[0058] 38 - 56 min, the volume ratio of mobile phase A to mobile phase B changes from 21:79 to 39:61;

[0059] From 56 to 63 min, the volume ratio of mobile phase A to mobile phase B changes from 39:61 to 47:53;

[0060] From 63 to 69 min, the volume ratio of mobile phase A to mobile phase B changes from 47:53 to 60:40;

[0061] From 69 to 80 min, the volume ratio of mobile phase A to mobile phase B changes from 60:40 to 80:20;

[0062] From 80 to 90 min, the volume ratio of mobile phase A to mobile phase B changes from 80:20 to 85:15;

[0063] From 90 to 95 min, the volume ratio of mobile phase A to mobile phase B is 85:15;

[0064] 2) Mass spectrometry conditions:

[0065] First stage: Scanning time 0.25 s; Acquisition time 95 min; Acquisition range 100 - 20000 Da; Nebulizing gas flow rate 50 mL / min; Desolvation gas flow rate 50 mL / min; Curtain gas flow rate 35 mL / min; Desolvation gas temperature 500 °C; Ion spray voltage is 4500 V in negative mode and 5500 V in positive mode; Declustering voltage is ±100 V; Collision energy is ±10 V; Dynamic background subtraction mode;

[0066] Second stage: Scanning time 0.1 s; Acquisition time 95 min; Acquisition range 50 - 2000 Da; Nebulizing gas flow rate 50 mL / min; Desolvation gas flow rate 50 mL / min; Curtain gas flow rate 35 mL / min; Desolvation gas temperature 500 °C; Ion spray voltage is 4500 V in negative mode and 5500 V in positive mode; Declustering voltage is ±100 V; Collision energy is ±40 V, collision energy spread is ±20 V; Ion release delay 74; Ion beam width 25; Dynamic background subtraction mode;

[0067] S3 Qualitative analysis:

[0068] With the aid of PeakView software, the chromatographic peaks are structurally analyzed by combining the secondary scan fragment ions in the positive and negative modes of UPLC / Q - TOF MS with information such as literature reports, relative ratio of isotope peaks, and chemical bond cleavage rules to determine the name, molecular formula, and structural formula of the compound.

[0069] Example 2

[0070] Qualitative analysis of weakly polar small - molecule compounds in Xinnaoning capsules is as follows:

[0071] S1 Preparation of test solution

[0072] Weigh 5 g of the content of Xinnaoning Capsules, add 50 mL of water, and perform ultrasonic treatment for 30 min. After filtration, obtain the extract. Accurately measure 40 mL of the extract, add 80 mL of petroleum ether (boiling range 60 - 90 °C) for extraction, and perform continuous extraction 4 times. Retain the aqueous solution, combine the extraction solutions and evaporate to dryness under reduced pressure. Dissolve the residue in methanol and dilute to 1 mL to obtain the petroleum ether extract solution. Take the aqueous solution retained in the previous step, add 80 mL of chloroform for extraction, and perform continuous extraction 4 times. Retain the aqueous solution, combine the extraction solutions and evaporate to dryness under reduced pressure. Dissolve the residue in methanol and dilute to 10 mL to obtain the chloroform extract solution. Take the aqueous solution retained in the previous step, add 80 mL of ethyl acetate for extraction, and perform continuous extraction 4 times. Retain the aqueous solution, combine the extraction solutions and evaporate to dryness under reduced pressure. Dissolve the residue in methanol and dilute to 25 mL to obtain the ethyl acetate extract solution. Take the aqueous solution retained in the previous step, add 80 mL of water-saturated n-butanol for extraction, and perform continuous extraction 4 times. Evaporate the n-butanol extraction solution to dryness under reduced pressure. Dissolve the residue in methanol and dilute to 10 mL to obtain the n-butanol extract solution.

[0073] S2 UPLC / Q-TOF MS detection

[0074] Inject each of the test solution prepared in the above steps into a liquid chromatography-tandem mass spectrometry instrument for UPLC / Q-TOF MS detection to obtain the total ion current chromatogram of each sample. The chromatographic and mass spectrometric conditions for UPLC / Q-TOF MS detection are as follows:

[0075] 1) Chromatographic conditions:

[0076] The chromatographic column is Phenomenex Luna C 18 column, with a specification of 250 mm × 4.6 mm, 5 μm; the column temperature is 30 °C; the flow rate of the mobile phase is 0.8 mL / min; the injection volume is 5 μL; use acetonitrile as mobile phase A and 0.1% formic acid water as mobile phase B for gradient elution. The elution program is as follows:

[0077] 0 - 17 min, the volume ratio of mobile phase A to mobile phase B changes from 3:97 to 15:85;

[0078] 17 - 28 min, the volume ratio of mobile phase A to mobile phase B changes from 15:85 to 21:79;

[0079] 28 - 38 min, the volume ratio of mobile phase A to mobile phase B is 21:79;

[0080] 38 - 56 min, the volume ratio of mobile phase A to mobile phase B changes from 21:79 to 39:61;

[0081] 56 - 63 min, the volume ratio of mobile phase A to mobile phase B changes from 39:61 to 47:53;

[0082] From 63 to 69 min, the volume ratio of mobile phase A to mobile phase B changes from 47:53 to 60:40;

[0083] From 69 to 80 min, the volume ratio of mobile phase A to mobile phase B changes from 60:40 to 80:20;

[0084] From 80 to 90 min, the volume ratio of mobile phase A to mobile phase B changes from 80:20 to 85:15;

[0085] From 90 to 95 min, the volume ratio of mobile phase A to mobile phase B is 85:15;

[0086] 2) Mass spectrometry conditions:

[0087] First stage: scanning time 0.25 s; acquisition time 95 min; acquisition range 100 - 20000 Da; nebulizing gas flow rate 50 mL / min; desolvation gas flow rate 50 mL / min; curtain gas flow rate 35 mL / min; desolvation gas temperature 500 °C; ion spray voltage is 4500 V in negative mode and 5500 V in positive mode; declustering voltage is ±100 V; collision energy is ±10 V; dynamic background subtraction mode;

[0088] Second stage: scanning time 0.1 s; acquisition time 95 min; acquisition range 50 - 2000 Da; nebulizing gas flow rate 50 mL / min; desolvation gas flow rate 50 mL / min; curtain gas flow rate 35 mL / min; desolvation gas temperature 500 °C; ion spray voltage is 4500 V in negative mode and 5500 V in positive mode; declustering voltage is ±100 V; collision energy is ±40 V, and the collision energy dispersion is ±20 V; ion release delay 74; ion beam width 25; dynamic background subtraction mode;

[0089] S3 Qualitative analysis:

[0090] With the aid of PeakView software, the structural analysis of each spectral peak is carried out by combining the fragment ions of the second-stage scan in the positive and negative modes of UPLC / Q-TOF MS with information such as literature reports, relative ratios of isotope peaks, and chemical bond cleavage rules to determine the name, molecular formula, and structural formula of the compound.

[0091] In order to further verify the reliability of the present invention, the inventors conducted a series of experiments on the basis of the previous quantitative analysis and fingerprint analysis, as follows:

[0092] 1. Experimental instruments and materials

[0093] 1.1 Experimental instruments

[0094] Waters UPLC high performance liquid chromatograph (Waters Corporation, USA); AB Sciex 5600 Triple TOF liquid chromatography-mass spectrometry instrument (AB Sciex Company, USA); Millipore Elix 5 ultrapure water instrument (Millipore Corporation, USA); KQ-500E ultrasonic processor (Kunshan Ultrasonic Equipment Co., Ltd.).

[0095] 1.2 Experimental reagents

[0096] Methanol, acetonitrile, and formic acid are all chromatographically pure and are from Merck KGaA, Germany.

[0097] 1.3 Experimental materials

[0098] Xinnaoning capsules (batch number: 20181032, provided by Guizhou Jingcheng Pharmaceutical Co., Ltd.); the medicinal materials in the formula of Xinnaoning capsules.

[0099] 2. Qualitative analysis of polar small molecule compounds in Xinnaoning capsules

[0100] 2.1 Preparation of test solution

[0101] Precisely weigh 1 g of the content of Xinnaoning capsules, add 10 mL of methanol, weigh, ultrasonically treat for 30 min, make up the weight, and filter through a 0.45 μm microporous filter membrane to obtain the solution.

[0102] 2.2 UPLC-MS conditions

[0103] (1) Chromatographic conditions

[0104] Phenomenex Luna C 18 Column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (mobile phase A) - 0.1% formic acid in water (mobile phase B), and the gradient elution program is shown in Table 1; flow rate: 0.8 mL / min; column temperature: 30 °C; injection volume: 5 μL.

[0105] Table 1 Gradient elution program

[0106]

[0107] Note: Equilibrate with 3% acetonitrile for 15 min before the column.

[0108] (2) Mass spectrometry conditions

[0109] Level 1: Scanning time 0.25 s; acquisition time 95 min; acquisition range 100 - 20000 Da; nebulizing gas flow rate 50 mL / min; desolvation gas flow rate 50 mL / min; curtain gas flow rate 35 mL / min; desolvation gas temperature 500 °C; ion spray voltage 4500 V in negative mode and 5500 V in positive mode; declustering voltage ±100 V; collision energy ±10 V; dynamic background subtraction mode.

[0110] Level 2: Scanning time 0.1 s; acquisition time 95 min; acquisition range 50 - 2000 Da; nebulizing gas flow rate 50 mL / min; desolvation gas flow rate 50 mL / min; curtain gas flow rate 35 mL / min; desolvation gas temperature 500 °C; ion spray voltage 4500 V in negative mode and 5500 V in positive mode; declustering voltage ±100 V; collision energy ±40 V, collision energy spread ±20 V; ion release delay 74; ion beam width 25; dynamic background subtraction mode.

[0111] 2.3 Determination results

[0112] 2.3.1 Total ion chromatogram of UPLC-MS determination

[0113] Precisely pipette 5 μL of the test solution, and detect it by UPLC / Q-TOF MS to obtain the LC-MS total ion chromatograms of polar small molecule compounds in Xinnaoning in positive and negative modes (as shown in Figure 1 、 Figure 2 ).

[0114] 2.3.2 Qualitative results

[0115] For qualitative analysis, with the aid of PeakView software (AB Sciex), the structures of each peak were analyzed by combining the fragment ions of the second-level scanning in positive and negative modes of UPLC / Q-TOF MS with information such as literature reports, relative ratios of isotope peaks, and chemical bond cleavage rules. The results are shown in Table 2.

[0116] Table 2 Qualitative results of polar small molecule compounds in Xinnaoning capsules

[0117]

[0118]

[0119]

[0120] The results show that:

[0121] (1) The main molecular ion peaks obtained from the UPLC-MS detection of Xinnaoning are [M + H] + and [M - H] -peaks, and there are also [M+NH 4 + 、[M+COOH] - plasma peaks for structural analysis.

[0122] (2) A total of 90 compounds were co-identified, including 6 amino acids (Aminobutyric Acid, (+)-L-Alliin, Arginine, Pyroglutamic Acid, L-Phenylalanine, L-Cystine), 4 polysaccharides (Raffinose, Maltotetraose, Gentiobiose, Lupeose), and 4 nucleosides (2-Deoxy-D-Ribose, Adenosine, Guanosine, Thymidine).

[0123] (3) Among the identified compounds, 39 components are from Salvia miltiorrhiza, and 30 of them are unique components of Salvia miltiorrhiza in the formula. The results show that the identified compounds from Salvia miltiorrhiza are mainly phenolic acids of Salvia miltiorrhiza, and the responses of salvianolic acid A and B are the highest.

[0124] (4) Among the identified compounds, 13 components are from Allium macrostemon Bunge, and 3 of them ((+)-L-Alliin, 3',5-Dihydroxy-4'-Methoxyflavone-7-o-α-Rhamnosesyl(1.6)-β-d-Glucoside, Macrostemonoside F) are unique components of Allium macrostemon Bunge in the formula.

[0125] (5) Among the identified compounds, 26 components are from Buxus microphylla Sieb. et Zucc., and 18 of them are unique components of Buxus microphylla Sieb. et Zucc., mainly flavonoids.

[0126] (6) Among the identified compounds, 33 components are from Ginkgo biloba L., and 20 of them are unique components of Ginkgo biloba L., including Ginkgetin, Anacardic Acid C, and various Ginkgolide characteristic components.

[0127] (7) Among the identified compounds, 5 components are from Litsea lancifolia (Roxb.) Pers., and (1R,2R,5S)-5-isopropyl-2-methylcyclohex-3-ene-1,2,-diol and 4-Hydroxycryptone are unique components of Litsea lancifolia (Roxb.) Pers., mainly phenolic acids.

[0128] 3. Qualitative analysis of weakly polar small molecule compounds in Xinnaoning Capsules ​

[0129] 3.1 Preparation of Test Solution

[0130] Precisely weigh 5 g of the content in Xinnaoning Capsules (batch number: 20181032), add 50 mL of water, ultrasonically treat for 30 min, and filter; precisely measure 40 mL of the extract, add 80 mL of petroleum ether (boiling range 60 - 90 °C) for extraction, extract continuously for 4 times, retain the aqueous solution, combine the extraction solutions and rotary evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up the volume to 1 mL to obtain the petroleum ether extract solution; take the retained aqueous solution, add 80 mL of chloroform for extraction, extract continuously for 4 times, retain the aqueous solution, combine the extraction solutions and rotary evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up the volume to 10 mL to obtain the chloroform extract solution; take the retained aqueous solution, add 80 mL of ethyl acetate for extraction, extract continuously for 4 times, retain the aqueous solution, combine the extraction solutions and rotary evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up the volume to 25 mL to obtain the ethyl acetate extract solution; take the retained aqueous solution, add 80 mL of n-butanol saturated with water for extraction, extract continuously for 4 times, combine the extraction solutions and rotary evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up the volume to 10 mL to obtain the n-butanol extract solution.

[0131] 3.2 UPLC-MS Conditions

[0132] The chromatographic and mass spectrometric conditions are the same as those in "2.2 UPLC-MS Conditions".

[0133] 3.3 Determination Results

[0134] 3.3.1 UPLC-MS Analysis Results of Petroleum Ether Fraction

[0135] Precisely pipette 5 μL of the test solution, detect it by UPLC / Q-TOF MS, and obtain the total ion current chromatograms of the petroleum ether fraction in Xinnaoning Capsules in positive and negative modes, as shown in Figure 3 、 Figure 4 .

[0136] For qualitative analysis, with the aid of PeakView software (AB Sciex), the structures of each peak are analyzed by combining the secondary scanning fragment ions in positive and negative modes of UPLC / Q-TOF MS with information such as literature reports, relative ratios of isotope peaks, and chemical bond cleavage rules. The results are shown in Table 3 below.

[0137] Table 3 Qualitative Results of Weakly Polar Small Molecule Compounds (Petroleum Ether Fraction) in Xinnaoning Capsules

[0138]

[0139]

[0140]

[0141] Note: Compounds with the suffix “-qt” indicate the aglycones formed by the loss of a glycosidic bond from the corresponding compounds.

[0142] The results showed that:

[0143] (1) The main molecular ion peaks obtained by UPLC-MS analysis of the petroleum ether fraction of Xinnaoning were [M+H] + and [M-H] - , and there were also [M+NH4] + , [M+COOH] - and other ionic peaks for structural analysis.

[0144] (2) A total of 76 compounds were qualitatively analyzed, including 2 amino acids (Arginine, Pyroglutamic Acid) and 3 polysaccharides (Raffinose, Maltotetraose, Gentiobiose).

[0145] (3) Among the qualitatively analyzed compounds, 41 were from Salvia miltiorrhiza, and 37 of them were unique to Salvia miltiorrhiza in the formula. The results showed that the qualitatively analyzed compounds from Salvia miltiorrhiza were mainly phenolic acids of Salvia miltiorrhiza.

[0146] (4) Among the qualitatively analyzed compounds, 7 were from Allium macrostemon. Except for Gentiobiose, Pyroglutamic Acid, and Methyl Oleate, the other 4 were unique to Allium macrostemon in the formula.

[0147] (5) Among the qualitatively analyzed compounds, 6 were from Litsea lancifolia. Except for Gentiobiose, the other 5 were unique to this medicinal material.

[0148] (6) Among the qualitatively analyzed compounds, 13 were from Buxus microphylla Sieb. et Zucc. Except for Gentiobiose and Scoparol, the other 12 were unique to Buxus microphylla Sieb. et Zucc. and were mainly flavonoids.

[0149] (7) Among the qualitatively analyzed compounds, 17 were from Ginkgo biloba L. Among them, 13 were unique to Ginkgo biloba L., including characteristic components such as ginkgolide J, Hydroginkgolinic acid, Anacardic acid C, and Anacardic acid D.

[0150] 3.3.2 Results of UPLC-MS analysis of the chloroform fraction

[0151] Precisely aspirate 5 μL of the test sample solution and detect it by UPLC-Q / TOF MS. The total ion chromatograms of the chloroform fraction of Xinnaoning Capsule in positive and negative modes are as follows Figures 5 to 6 shown.

[0152] For qualitative analysis, with the assistance of PeakView software (AB Sciex), the structural analysis of each spectral peak was carried out by combining the fragment ions obtained from the second-level scanning in the positive and negative modes of UPLC / Q-TOF MS with information such as literature reports, relative ratios of isotope peaks, and chemical bond cleavage rules. The results are shown in Table 4 below.

[0153] Table 4 Qualitative Results of Weakly Polar Small Molecule Compounds (Chloroform Fraction) in Xinnaoning Capsule

[0154]

[0155]

[0156]

[0157] The results show that:

[0158] (1) The main molecular ion peaks obtained from the UPLC-MS detection of the chloroform fraction of Xinnaoning are [M+H] + and [M-H] - , and there are also plasma peaks such as [M+NH4] + , [M+COOH] - for structural analysis.

[0159] (2) A total of 54 compounds were qualitatively identified, including 4 amino acids (Arginine, Pyroglutamic Acid, Histidine, Homoarginine), 3 polysaccharide (Raffinose, Maltotetraose, Gentiobiose) components, and 2 nucleoside (Guanine, Thymine) components.

[0160] (3) Among the qualitatively identified compounds, 20 components are from Salvia miltiorrhiza, and 16 of them are unique components of Salvia miltiorrhiza in the formula. The results show that the qualitatively identified compounds of Salvia miltiorrhiza are mainly phenolic acids and quinones of Salvia miltiorrhiza.

[0161] (4) Among the identified compounds, 9 components are from Allium macrostemon Bunge. Except for Pyroglutamic acid, 4-Hydroxybenzoic Acid, and Melilotoside, the remaining 6 components are unique to Allium macrostemon Bunge in the formula. Among them, 2 components (N-Feruloyltyramine and 4-hydroxy-4,7-dimethyl-1-tetralone) are consistent with the monomer components isolated by Shanghai Jiao Tong University.

[0162] (5) Among the identified compounds, 5 components are from Litsea lancifolia (Roxb.) Pers. Except for Citric acid, the remaining 4 components are unique to this medicinal material.

[0163] (6) Among the identified compounds, 8 components are from Buxus microphylla Sieb. et Zucc. Except for Citric acid, Inosine, and Luteolin, 5 of these components are unique to Buxus microphylla Sieb. et Zucc.

[0164] (7) Among the identified compounds, 22 components are from Ginkgo biloba L. Among them, 16 components are unique to Ginkgo biloba L., including characteristic components such as Ginkgolide A, Ginkgolide M, Ginkgolide J (ginkgolides A, M, J), Anacardic Acid C (ginkgolic acid C), Ginkgetin, and Isoginkgetin.

[0165] 3.3.3 UPLC-MS analysis results of the ethyl acetate fraction

[0166] Precisely pipette 5 μL of the test solution and detect it by UPLC-Q / TOF MS to obtain the total ion current chromatograms of the ethyl acetate fraction of Xinnaoning Capsule in positive and negative modes as Figures 7 to 8 shown.

[0167] For qualitative analysis, with the aid of PeakView software (AB Sciex), the structural analysis of each peak is carried out by combining the fragment ions obtained from the second-level scanning in positive and negative modes of UPLC / Q-TOF MS, literature reports, relative ratios of isotope peaks, and the rules of chemical bond cleavage. The results are shown in Table 5 below.

[0168] Table 5 Qualitative results of weakly polar small molecule compounds (ethyl acetate fraction) in Xinnaoning Capsule

[0169]

[0170]

[0171]

[0172] The results showed that:

[0173] (1) The main molecular ion peaks obtained by UPLC-MS detection of the ethyl acetate fraction of Xinnaoning were [M+H] + and [M-H] - , and there were also [M+NH4] + , [M+COOH] - and other ion peaks for structural analysis.

[0174] (2) A total of 71 compounds were qualitatively determined, including 4 amino acids (Arginine, Pyroglutamic Acid, Homoarginine, L-Cystine), 3 polysaccharide components (Raffinose, Maltotetraose, Gentiobiose), and 3 nucleoside components (Sulcatone, Uridine, Adenosine).

[0175] (3) Among the qualitatively determined compounds, 37 components were from Salvia miltiorrhiza, and 28 of them were unique components of Salvia miltiorrhiza in the formula, mainly phenolic acids of Salvia miltiorrhiza.

[0176] (4) Among the qualitatively determined compounds, 7 components were from Allium macrostemon Bunge, and Propyl trisulfide and XB-25 were unique components of Allium macrostemon Bunge in the formula.

[0177] (5) Among the qualitatively determined compounds, 4 components were from Litsea lancifolia (Roxb.) Pers., and (E)-Arachidin II and Leukotriene D5 were unique components of this medicinal material.

[0178] (6) Among the qualitatively determined compounds, 18 components were from Buxus microphylla Sieb. et Zucc., and 8 of them were unique components of Buxus microphylla Sieb. et Zucc.

[0179] (7) Among the qualitatively determined compounds, 26 components were from Ginkgo biloba L., and 15 of them were unique components of Ginkgo biloba L., including characteristic components such as Ginkgolide A, Ginkgolide B, Ginkgolide C (ginkgolides A, B, C), and Anacardic Acid C (ginkgolic acid C).

[0180] 3.3.4 UPLC-MS analysis results of the n-butanol fraction

[0181] Precisely pipette 5 μL of the test solution, and detect it by UPLC-Q / TOF MS. The total ion current chromatograms of the n-butanol fraction of Xinnaoning capsules in positive and negative modes are as Figures 9 to 10 shown.

[0182] Qualitative analysis was carried out with the assistance of PeakView software (AB Sciex). The structural analysis of each spectral peak was performed by combining the secondary scanning fragment ions in the positive and negative modes of UPLC / Q-TOF MS with information such as literature reports, relative ratios of isotope peaks, and chemical bond cleavage rules. The results are shown in Table 6 below.

[0183] Table 6 Qualitative Results of Weakly Polar Small Molecule Compounds (n-Butanol Fraction) in Xinnaoning Capsules

[0184]

[0185]

[0186]

[0187] The results showed that:

[0188] (1) The main molecular ion peaks obtained from the UPLC-MS detection of the n-butanol fraction of Xinnaoning were [M+H] + and [M-H] - , and there were also plasma peaks such as [M+NH4] + , [M+COOH] - for structural analysis.

[0189] (2) A total of 62 compounds were qualitatively identified, including 3 amino acids (Arginine, Pyroglutamic Acid, L-Cystine), 1 polysaccharide (Raffinose) component, and 4 nucleoside (Uridine, Adenosine, Guanosine, Thymine) components.

[0190] (3) Among the qualitatively identified compounds, 29 components were from Salvia miltiorrhiza, and 22 of them were unique components of Salvia miltiorrhiza in the formula, mainly Salvia phenolic acids.

[0191] (4) Among the qualitatively identified compounds, 9 components were from Allium macrostemon Bunge, and 3 components, namely 3',5-Dihydroxy-4'-methoxyflavone-7-o-α-rhamnosesyl(1.6)-β-d-glucoside, Trigofoenoside A, and Macrostemonoside F, were unique components of Allium macrostemon Bunge in the formula.

[0192] (5) Among the qualitatively identified compounds, 2 non-characteristic components, Aminobutyric acid and Citric acid, were from Litsea lancifolia (Roxb.) Pers.

[0193] (6) Among the identified compounds, 16 components are from *Buxus microphylla* Sieb. et Zucc., and 12 of them are unique to *Buxus microphylla* Sieb. et Zucc.

[0194] (7) Among the identified compounds, 23 components are from *Ginkgo biloba* L., and 15 of them are unique to *Ginkgo biloba* L.

[0195] Although the present invention has been described in detail in the foregoing with general descriptions, specific embodiments and experiments, some modifications or improvements can be made on the basis of the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection of the present invention.

Claims

1. A method for qualitatively analyzing small molecule compounds in Xinnaoning Capsules using UPLC-MS technology, wherein the small molecule compounds include polar small molecule compounds and weakly polar small molecule compounds, characterized in that: The steps include: S1, prepare the test solution: S11, preparation of test solution for qualitative analysis of polar small molecule compounds: weigh the contents of Xinnaoning capsules, add appropriate amount of methanol, ultrasonically treat, filter, and obtain; S12, preparation of test solution for qualitative analysis of weakly polar small molecule compounds: weigh the contents of Xinnaoning Capsules, add appropriate amount of water, ultrasonically treat, and filter to obtain an extract; take an appropriate amount of the extract, add petroleum ether for extraction, retain the water, evaporate the extract under reduced pressure to dryness, and add an appropriate amount of methanol to dissolve the residue to obtain a petroleum ether extract solution; take the water retained in the previous step, add chloroform for extraction, retain the water, evaporate the extract under reduced pressure to dryness, and add an appropriate amount of methanol to dissolve the residue to obtain a chloroform extract solution; take the water retained in the previous step, add ethyl acetate for extraction, retain the water, evaporate the extract under reduced pressure to dryness, and add an appropriate amount of methanol to dissolve the residue to obtain an ethyl acetate extract solution; take the water retained in the previous step, add water-saturated n-butanol for extraction, evaporate the extract under reduced pressure to dryness, and add an appropriate amount of methanol to dissolve the residue to obtain an n-butanol extract solution; S2, UPLC / Q-TOF MS detection: Inject each test sample solution prepared in step S1 into a liquid chromatography tandem mass spectrometer for UPLC / Q-TOF MS detection to obtain a total ion current diagram of each sample in positive and negative modes; S3, qualitative analysis: With the help of PeakView software, the fragment ions were scanned by the secondary mode in the positive and negative modes of UPLC / Q-TOF MS to analyze the structure of each peak and determine the name, molecular formula and structural formula of the compound.

2. The method according to claim 1, characterized in that The specific process for preparing the test solution for qualitative analysis of polar small molecule compounds described in step S1 is: accurately weigh 1-1.5 g of the content of Xinnaoning Capsule, add 10-20 mL of methanol, weigh, ultrasonically treat for 30-60 minutes, make up the weight with methanol, and filter with a microporous filter membrane to obtain.

3. The method according to claim 2, characterized in that The specific process of preparing the test solution for qualitative analysis of polar small molecule compounds described in step S1 is: accurately weigh 1 g of the content of Xinnaoning Capsule, add 10 mL of methanol, weigh, ultrasonically treat for 30 minutes, make up the weight with methanol, and filter with a microporous filter membrane to obtain.

4. The method according to claim 1, characterized in that: The specific process for preparing the test solution for qualitative analysis of weakly polar small molecule compounds described in step S1 is as follows: weigh 4-6 g of the contents of Xinnaoning Capsule, add 40-60 mL of water, ultrasonically treat for 30-60 min, and filter to obtain an extract; accurately measure 40-50 mL of the extract, add 80-100 mL of petroleum ether for extraction, extract continuously for 4 times, retain the water, combine the extracts and evaporate to dryness under reduced pressure, dissolve the residue in methanol and make up to 1 mL to obtain a petroleum ether extract solution; take the water retained in the previous step, add 80-100 mL of chloroform for extraction, extract continuously for 4 times, and the water The mixture was retained, the extracts were combined and evaporated to dryness under reduced pressure, and the residue was dissolved in methanol and fixed to 10 mL to obtain a chloroform extract solution; the aqueous solution retained in the previous step was added with 80-100 mL of ethyl acetate for extraction, and the extraction was continued for 4 times. The aqueous solution was retained, the extracts were combined and evaporated to dryness under reduced pressure, and the residue was dissolved in methanol and fixed to 25 mL to obtain an ethyl acetate extract solution; the aqueous solution retained in the previous step was added with 80-100 mL of water-saturated n-butanol for extraction, and the extraction was continued for 4 times. The extracts were combined and evaporated to dryness under reduced pressure, and the residue was dissolved in methanol and fixed to 10 mL to obtain a n-butanol extract solution.

5. The method according to claim 4, characterized in that The specific process for preparing the test solution for qualitative analysis of weakly polar small molecule compounds described in step S1 is as follows: weigh 5 g of the contents of Xinnaoning Capsule, add 50 mL of water, ultrasonically treat for 30 min, and filter to obtain an extract; accurately measure 40 mL of the extract, add 80 mL of petroleum ether for extraction, extract 4 times continuously, retain the water, combine the extracts and evaporate to dryness under reduced pressure, dissolve the residue in methanol and make the volume to 1 mL to obtain a petroleum ether extract solution; take the water retained in the previous step, add 80 mL of chloroform for extraction, extract 4 times continuously, retain the water, combine the extracts The liquid was taken and evaporated to dryness under reduced pressure, and the residue was dissolved in methanol and fixed to 10 mL to obtain a chloroform extract solution; the aqueous solution retained in the previous step was added with 80 mL of ethyl acetate for extraction, and the extraction was continued for 4 times, the aqueous solution was retained, the extracts were combined and evaporated to dryness under reduced pressure, and the residue was dissolved in methanol and fixed to 25 mL to obtain an ethyl acetate extract solution; the aqueous solution retained in the previous step was added with 80 mL of water-saturated n-butanol for extraction, and the extraction was continued for 4 times, the extracts were combined and evaporated to dryness under reduced pressure, and the residue was dissolved in methanol and fixed to 10 mL to obtain a n-butanol extract solution.

6. The method according to any one of claims 1, 4 or 5, characterized in that: The boiling range of the petroleum ether is 60-90°C.

7. The method according to claim 1, characterized in that The chromatographic conditions for UPLC / Q-TOF MS detection in step S2 are: The chromatographic column is C 18 Column, specification: 250mm×4.6mm, 5μm; column temperature: 30℃; mobile phase flow rate: 0.8mL / min; injection volume: 5μL; acetonitrile as mobile phase A, 0.1% formic acid water as mobile phase B, gradient elution, elution program: From 0 to 17 min, the volume ratio of mobile phase A to mobile phase B changed from 3:97 to 15:85; From 17 to 28 min, the volume ratio of mobile phase A to mobile phase B changed from 15:85 to 21:79; 28-38 min, the volume ratio of mobile phase A to mobile phase B was 21:79; From 38 to 56 min, the volume ratio of mobile phase A to mobile phase B changed from 21:79 to 39:61; From 56 to 63 min, the volume ratio of mobile phase A to mobile phase B changed from 39:61 to 47:53; From 63 to 69 min, the volume ratio of mobile phase A to mobile phase B changed from 47:53 to 60:40; From 69 to 80 min, the volume ratio of mobile phase A to mobile phase B was changed from 60:40 to 80:20; From 80 to 90 min, the volume ratio of mobile phase A to mobile phase B changed from 80:20 to 85:15; 90-95 min, the volume ratio of mobile phase A to mobile phase B was 85:

15.

8. The method according to claim 1, characterized in that The mass spectrometry conditions for UPLC / Q-TOF MS detection in step S2 are as follows: Level 1: Scan time 0.25s; Acquisition time 95min; Acquisition range 100-20000Da; Nebulizer gas flow rate 50mL / min; Desolvation gas flow rate 50mL / min; Curtain gas flow rate 35mL / min; Desolvation gas temperature 500℃; Ion spray voltage 4500V in negative mode and 5500V in positive mode; Declustering voltage ±100V; Collision energy ±10V; Dynamic background subtraction mode; Level 2: scanning time 0.1s; acquisition time 95min; acquisition range 50-2000Da; nebulizer gas flow rate 50mL / min; desolvation gas flow rate 50mL / min; curtain gas flow rate 35mL / min; desolvation gas temperature 500℃; ion spray voltage 4500V in negative mode and 5500V in positive mode; declustering voltage is ±100V; collision energy is ±40V, and collision energy dispersion is ±20V; ion release delay 74; ion beam width 25; dynamic background subtraction mode.

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