Method for determining chemical components of fructus broussonetiae by liquid chromatography-mass spectrometry

Abstract

The invention discloses a method for determining chemical components of fructus broussonetiae, which adopts liquid chromatography-mass spectrometry for analysis, and comprises the following steps: adding a methanol aqueous solution with the volume concentration of more than 10% into a fructus broussonetiae sample, carrying out ultrasonic treatment, filtering, and detecting the subsequent filtrate, and the liquid chromatography conditions for detection are C18 chromatographic column, C18 chromatographic column, C18 chromatographic column, C18 chromatographic column, C18 chromatographic column, C18 chromatographic column, C18 chromatographic column, C18 chromatographic column, C18 chromatographic column, C18 chromatographic column, C18 chromatographic column, C18 chromatographic column and C18 chromatographic column; mobile phases: A: acetonitrile, and B: a formic acid aqueous solution with a volume concentration of 0.01-0.2%; the elution gradient is 0-5 min, 3% A; 5-13 min, 3-7% of A is obtained; 7-9% of A is selected in 13-28 min; in 28-35 min, 9% A is adopted, and the flow rate is 0.3-1.0 mL / min; the column temperature is 25-35 DEG C; and obtaining paper mulberry fruit component information according to a detection result. The chromatographic peak baseline obtained under the liquid phase body condition is stable, the peak pattern is sharp, and qualitative analysis of unknown compounds in fructus broussonetiae is facilitated.

Description

Technical Field

[0001] The invention relates to a method for measuring chemical components of papaya seeds by using liquid chromatography-mass spectrometry, and belongs to the field of chemical qualitative analysis of traditional Chinese medicines. Background Art

[0002] Cuscuta australis is the dried mature fruit of Broussonetia papyrifera (L.) Vent., a plant of the Moraceae family. It is harvested in autumn when the fruit is ripe, washed, dried, and the grayish white membranous persistent calyx and impurities are removed (Pang Suqiu, Wang Guoquan, Huang Baokang, et al. Study on the chemical composition of Cuscuta australis [J]. Chinese Medicinal Materials, 2009, 32(8): 1229-1231.). Cuscuta australis was first used as a medicinal material in the Ming Yi Bie Lu, where it was recorded as a top grade product with a sweet taste, cold nature, and non-toxicity. It has been recorded in all subsequent editions of the Pharmacopoeia of the People's Republic of China (Volume 1). Cuscuta australis is distributed in most parts of the country, and is mainly produced in Henan, Hubei, Hunan, Shanxi, Gansu, and other places. Cuscuta australis has the effects of nourishing the kidney and clearing the liver, improving eyesight and promoting diuresis. It is often used to treat symptoms such as soreness of the waist and knees, consumptive bone steaming, dizziness, cataracts, and edema (Xiong Shan, Ye Zuguang. Research progress on the chemical composition and pharmacological effects of Cuscuta australis [J]. Chinese Journal of Traditional Chinese Medicine Information, 2009, 16(5): 102-103.).

[0003] In recent years, domestic and foreign research on the natural active ingredients of Corydalis chinensis has focused on total flavonoids, pigments, polysaccharides, total saponins and Corydalis chinensis oil. Chi Yuxin et al. compared the amount of total flavonoids extracted from Corydalis chinensis and Broussonetia papyrifera leaves by ultrasonic extraction and Soxhlet extraction, and determined their content. The results showed that the total flavonoid content in Corydalis chinensis measured by ultrasonic extraction was significantly higher than that measured by Soxhlet extraction (Chi Yuxin, Dou Deqiang. Determination of total flavonoid content in Corydalis chinensis and Broussonetia papyrifera leaves [J]. Modern Chinese Medicine, 2008, 10 (11): 2.), but this method cannot confirm the specific structure of the compounds in Corydalis chinensis. Xiong Shan et al. degreased the seeds of Cucurbita chinensis with petroleum ether, and then used ZS-101 macroporous adsorption resin to remove most of the non-saponin components to obtain the total saponin and determined its content. The results showed that the average content of total saponin in Cucurbita chinensis was 0.48% by spectrophotometric method with perchloric acid colorimetry and absorbance at 407nm (Xiong Shan, Chen Yuwu, Ye Zuguang. Determination of total saponin content in Cucurbita chinensis [J]. Modern Chinese Medicine, 2009, 11(7):3.). This method also cannot confirm the specific structure of the compounds in Cucurbita chinensis. At present, the most studied extraction methods of chemical components in paper mulberry seeds are paper mulberry seed oil. Yuan Xiao et al. used supercritical carbon dioxide extraction technology to separate 60 fatty oil components from paper mulberry seeds (Yuan Xiao, Yuan Ping. Study on chemical components of paper mulberry seed oil extracted by supercritical carbon dioxide [J]. Chinese Herbal Medicine, 2005, 36(8):3.). Lin Wenqun et al. determined the content of palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid in paper mulberry seed fatty acids. The results showed that the essential fatty acids contained in paper mulberry seeds were significantly higher than those in other common edible oils (Lin Wenqun, Liu Jianqiu. Study on chemical components of paper mulberry seeds [J]. Subtropical Plant Science, 2000, 29(004):20-23.). These methods mainly analyzed and determined the components of paper mulberry seed oil. There is a lack of research on the determination of other active compounds with medicinal value in paper mulberry seeds in the existing technology. Therefore, there is an urgent need for a method that can effectively determine and identify the chemical components in Cuscuta australis seeds, so as to provide a certain research basis for the pharmacological research and quantitative detection and analysis of the active ingredients in Cuscuta australis seeds. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a detection method for effectively distinguishing and confirming unknown components in the extract of Cucurbitacin.

[0005] Technical solution: To solve the above technical problems, the present invention provides a method for detecting multiple components in paper mulberry seeds, using liquid chromatography coupled to mass spectrometry, comprising the following steps:

[0006] S1. Quantitatively weigh a sample of the seeds of the Chinese papaya, add a methanol aqueous solution having a volume concentration greater than 10% in proportion, and filter after ultrasonic treatment to obtain a filtrate;

[0007] S2. Detect the subsequent filtrate; the liquid chromatography conditions for the detection are: C18 chromatographic column; mobile phase: A: acetonitrile, B: formic acid aqueous solution with a volume concentration of 0.01% to 0.2%; elution gradient: 0 to 5 minutes, 3% A; 5 to 13 minutes, 3 to 7% A; 13 to 28 minutes, 7 to 9% A, 28 to 35 minutes, 9% A, flow rate of 0.3 to 1.0 mL / min; column temperature of 25 to 35° C.; mass spectrometry conditions for the detection are: electrospray ionization source ESI, capillary voltage 4000±200V, 3500±200V in positive and negative ion modes;

[0008] S3. Obtain information on the composition of the seeds of the paper mulberry tree according to the test results.

[0009] The chromatographic peak obtained under the liquid phase condition has a stable baseline and a sharp peak shape. The use of an aqueous formic acid solution of appropriate concentration in the mobile phase can ensure that the compounds to be tested are fully ionized, which is helpful for the qualitative analysis of the compounds.

[0010] Preferably, the Cuscuta australis sample is one of Cuscuta australis medicinal materials, Cuscuta australis decoction pieces, and Cuscuta australis formula granules, and the Cuscuta australis formula granules include Cuscuta australis medicinal material extract and excipients.

[0011] Preferably, the liquid chromatography injection volume is 0.5 to 6 μL, more preferably, the injection volume is 2 to 4 μL.

[0012] Preferably, in step S2, the mobile phase B is an aqueous formic acid solution with a volume concentration of 0.01% to 0.2%.

[0013] Preferably, the chromatographic column is one of Poroshell 120SB-C18 (150×4.6mm, 2.7μm), Poroshell 120Aq-C18 (150×4.6mm, 2.7μm) or CORTECST3 (150×4.6mm, 2.7μm) chromatographic columns, and more preferably, the chromatographic column is a CORTECST3 liquid chromatography column.

[0014] Preferably, the mass spectrometry detection conditions in step S2 also include: capillary outlet voltage 180V; cone voltage 70V; mass-to-charge ratio collection range m / z 50-1500; drying gas temperature 200-400°C, drying gas flow rate 1-15L / min; nebulization pressure 20-60psi, sampling speed 1spectra / s, sampling time 1000ms / spectra, positive ions use purine (121.050873) and HP-0921 (922.009798) for real-time mass number calibration, and nebulization pressure 6psi.

[0015] Preferably, step S3 includes the following steps: 1). Extracting the material peak from the mass spectrometry total ion current diagram to obtain an accurate molecular weight; 2). Searching the database according to the primary mass spectrometry ion fragment information of the compound peak to obtain the mass spectrometry fragment information matching the screened material in the database; 3). Combining and analyzing the adduct ion category, molecular weight error, retention time, relevant literature and the mass spectrometry fragmentation information obtained in step 2), to obtain the structure of the compound. In the present invention, the database is self-built data compiled based on the chemical components reported in the literature; the adduct ion categories include M+H, M+NH4, M+Na, M+K, M+Cl, M+COOH, etc.; the molecular weight error is an absolute value of less than 10ppm.

[0016] The results of the chemical composition of Cucurbita chinensis obtained using the above analysis method showed that a total of 17 chemical components were identified in the Cucurbita chinensis particles, including 6 nucleoside amino acid components, 5 organic acid components, 4 glycoside components, 1 cyclopentane component, and 1 other component.

[0017] Preferably, the ratio of the paper mulberry seed sample to the methanol aqueous solution is 0.1-1.0 g: 25-100 mL, and more preferably, the ratio of the paper mulberry seed sample to the methanol aqueous solution is 0.3 g: 50 mL.

[0018] Preferably, the step S1 further comprises: using a corresponding solvent to make up for the lost weight after the ultrasonic treatment.

[0019] Preferably, in order to optimize the liquid chromatography peak shape, the volume concentration of methanol in step S1 is 10% to 80%.

[0020] Preferably, in order to improve the response signal and take into account the analysis efficiency, the ultrasonic treatment time is 20 to 60 minutes, the power is 150 to 300 W, and the frequency is 20 to 50 kHz. More preferably, the ultrasonic treatment time is 20 to 45 minutes, the power is 250 W, and the frequency is 40 kHz. In the scheme, there is no special limitation on the ultrasonic method, and ultrasonic means known to those skilled in the art can be used.

[0021] Preferably, step S1 also includes centrifugation, the centrifugal speed is 10000-13000 rpm, and the centrifugal duration is 5-15 min; more preferably, the speed is 12000 rpm, and the duration is 10 min; the sample after centrifugation is filtered through a microporous filter membrane to obtain a filtrate, the pore size of the microporous filter membrane is 0.10-0.50 μm, and more preferably, the pore size of the microporous filter membrane is 0.22 μm.

[0022] Preferably, the liquid chromatography detector uses an ultraviolet detector (VWD) or a diode array detector (DAD), and the detection wavelength is 190 to 400 nm, more preferably 264 nm.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. It can effectively separate unknown components in Cuscuta australis; 2. The liquid chromatography-mass spectrometry technology adopted in the present invention does not require a reference substance. It only needs to directly compare the mass spectrum of the extracted components of the test sample with the retention time corresponding to each component on the instrument to know whether the sample contains the target compound; 3. A total of 17 chemical components were identified in the present invention. The method is time-saving, highly accurate, and has good repeatability, which provides a basis for further research on the material basis of the efficacy of Cuscuta australis and comprehensive quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the total ion current chromatogram (TIC) of elution time gradient 1 in Example 1;

[0025] Figure 2 It is the total ion current chromatogram (TIC) of elution time gradient 2 in Example 1;

[0026] Figure 3 It is the total ion current chromatogram (TIC) of elution time gradient 3 in Example 1;

[0027] Figure 4 Extracted ion chromatogram (EIC) of m / z 182.0732 in Example 2;

[0028] Figure 5 The primary mass spectrum (MS) of m / z 182.0732 in Example 2;

[0029] Figure 6 The primary mass spectrum (MS) of m / z 180.0666 in Example 2;

[0030] Figure 7 Extracted ion chromatogram (EIC) of m / z 193.0341 in Example 2;

[0031] Figure 8 The primary mass spectrum (MS) of m / z 193.0341 in Example 2;

[0032] Fig. 9 This is the primary mass spectrum (MS) of m / z 191.0199 in Example 2. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0034] 1. Instrument:

[0035] Agilent G6530 AccurateMass Q-TOF mass spectrometer (Agilent); Agilent MassHunter QualitativeAnalysis B.07.00 data acquisition and qualitative analysis software (Agilent); ME204E / 02 electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); KQ-250B ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); pure water system (Sartorius); AS165W centrifuge (Azovang (Shanghai) Trading Co., Ltd.). Methanol (chromatographic grade, Thermo); water is ultrapure water; formic acid (mass spectrometry grade, Aladdin); other reagents are analytical grade.

[0036] 2. Reagents and samples

[0037] Cuscuta australis formula granules were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd., product batch number 23040019.

[0038] Example 1: Determination of liquid chromatography detection conditions

[0039] 1. Preparation of samples of Corydalis chinensis

[0040] (1) Take an appropriate amount of mulberry seed granules, grind them into powder, take about 0.3 g, weigh them, put them in a stoppered conical flask, add 50 mL of 30% methanol, stopper it, weigh them, treat them with ultrasound (power 250 W, frequency 40 kHz) for 45 minutes, let them cool, weigh them again, make up the weight lost during the treatment with the corresponding solvent, and shake them well.

[0041] (2) Transfer to a centrifuge and centrifuge at 12,000 rpm for 10 minutes. Filter through a 0.22 μm filter membrane and take the filtrate to obtain the paper mulberry seed sample to be tested.

[0042] 2. Screening of detection conditions: CORTECS T3 liquid chromatography column (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm) was used; column temperature was 30°C; mobile phase A of the elution system was acetonitrile solution, and mobile phase B was 0.2% formic acid aqueous solution by volume; flow rate was 0.55 mL / min; injection volume was 3 μL, and detection wavelength was 248 nm; gradient elution was performed according to the provisions in Tables 1 to 3, and the liquid chromatograms detected were recorded. Figure 1 to Figure 3 .

[0043] Table 1: Investigation of elution time gradient 1

[0044] Time / minute Mobile phase A / % Mobile phase B / % 0～3 1 99 3～15 1→5 99→95 15～30 5→25 95→75

[0045] Table 2: Investigation of elution time gradient 2

[0046] Time / minute Mobile phase A / % Mobile phase B / % 0～5 5 95 5～20 10 95→90 20～30 10→20 90→80

[0047] Table 3: Investigation of elution time gradient 3

[0048] Time / minute Mobile phase A / % Mobile phase B / % 0～5 3 97 5～13 3→7 97→93 13～28 7→9 93→91 28～35 9 91

[0049] The results showed that the chromatographic peak baseline of time gradient 1 was uneven, and the chromatographic peaks obtained with time gradient 2 were few and of poor peak shape. The mass spectrum required by the present invention should show as many chromatographic peaks and chemical components in the paper mulberry seeds as possible. The chromatographic peaks obtained with time gradient 3 were rich in information and could comprehensively display the chemical components of the paper mulberry seeds. Therefore, time gradient 3 was finally selected as the gradient elution condition.

[0050] Example 2: Qualitative analysis of chemical composition of samples of Corydalis chinensis

[0051] 1. Prepare the test sample of Corydalis chinensis according to the method of Example 1

[0052] 2. Chromatography and mass spectrometry conditions:

[0053] Liquid chromatography conditions: a CORTECS T3 liquid chromatography column (column length: 150 mm, inner diameter: 4.6 mm, particle size: 2.7 μm) was used; the column temperature was 30°C; the mobile phase A of the elution system was acetonitrile solution, and the mobile phase B was a 0.2% by volume formic acid aqueous solution; the flow rate was 0.55 mL / min; the injection volume was 3 μL, the detection wavelength was 248 nm, and the gradient elution program in Table 3 of Example 1 was used.

[0054] Mass spectrometry conditions: double-jet ESI ion source, drying gas temperature 300℃, flow rate 8L / min, nebulizer pressure 35psi; capillary voltage 4000V in positive ion mode, capillary voltage 3500V in negative ion mode, capillary outlet voltage 180V, cone voltage 70V; high-resolution mode was used for data acquisition, mass-to-charge ratio acquisition range m / z50-1500, sampling speed 1spectra / s, sampling time 1000ms / spectra; purine (121.050873) and HP-0921 (922.009798) were used for real-time mass number calibration of positive ions; TFANH4 (112.985587) and HP-0921 (1033.988109) were used for real-time mass number calibration of negative ions, and nebulizer pressure 6psi.

[0055] 3. Compound identification and attribution

[0056] The Agilent MassHunter Qualitative Analysis B.07.00 software was used to analyze the mass spectrometry data of the formula granules of the seeds of the Chinese mulberry tree. According to the m / z value of each peak, the molecular ion peak, adduct ion and characteristic fragment ion were judged, and combined with the self-built database comparison, a total of 17 chemical components were identified, namely: citric acid, L-tyrosine, isoleucine, adenosine, guanosine, inosine, p-hydroxybenzoic acid-β-D-pyranose glycoside, (3,6-dimethoxy-2-nitro-benzoyl)-malonic acid diethyl ester, vanillic acid-4-O-β-D-glucoside, isocarpinic acid, pyridoxine-3-O-β-D-glucoside, tryptophan, neochlorogenic acid, p-hydroxybenzoic acid, broad-bract glycoside C, chlorogenic acid, and cryptochlorogenic acid. The specific results are shown in Table 4. Among them, nucleoside amino acids are one of the main components, with L-tyrosine ( Figure 4 ) as an example, in positive ion mode, the primary mass spectrometry quasi-molecular ion peak is m / z182.0732[M+H] + ( Figure 5 ), and m / z 204.0544 [M+Na] can also be observed + 、m / z220.0287[M+K] + The fragment peak ( Figure 5 ); In negative ion mode, the primary mass spectrometry quasi-molecular ion peak is m / z180.0666[MH] - ( Figure 6 ), and m / z 216.0457 [M+Cl] can also be observed - 、m / z220.0287[M+K] + The fragment peak ( Figure 6 ), based on molecular weight, ion fragment information, compound databases, and Chinese and English literature, it was inferred that the compound was L-tyrosine, with a molecular formula of C9H 11 NO3.

[0057] Organic acid compounds are also one of the main components of paper mulberry seeds, with citric acid ( Figure 7 ) as an example, in the positive ion mode, the primary mass spectrometry quasi-molecular ion peak is m / z193.0341[M+H] + ( Figure 8 ), and m / z 210.0608 [M+NH4] can also be observed + 、m / z215.0162[M+Na] + 、m / z230.9895[M+K] + The fragment peak ( Figure 8 ); In negative ion mode, the primary mass spectrometry quasi-molecular ion peak is m / z191.0198[MH] - ( Fig. 9), and m / z 226.9802 [M+Cl] can also be observed - The fragment peak ( Fig. 9 ).

[0058] Table 4: Analysis of chemical components in Cuscuta australis granules based on UPLC-QTOF-MS technology

[0059]

[0060]

[0061] Embodiment 3-4

[0062] Examples 3 and 4 are respectively a method for determining the chemical components of paper mulberry seeds by liquid chromatography-mass spectrometry. Their steps are basically the same as those of Example 2, and the only difference is that some parameters are different. For details, see Table 5:

[0063] Table 5 List of parameters in Examples 3 to 4

[0064]

[0065] The contents of other parts of Examples 3 to 4 are the same as those of Example 2, and the analysis results are also basically the same as those of Example 2, and will not be repeated here.

Claims

1. A method for determining the chemical components of paper mulberry seeds by liquid chromatography-mass spectrometry, characterized in that: The following steps are involved: S1. Quantitatively weigh a sample of the seeds of the Chinese papaya, add a methanol aqueous solution having a volume concentration greater than 10% in proportion, and filter after ultrasonic treatment to obtain a filtrate; S2, testing the subsequent filtrate; the liquid chromatography conditions for the testing are: C18 chromatographic column; Mobile phase: A: acetonitrile, B: formic acid aqueous solution with a volume concentration of 0.01% to 0.2%; elution gradient: 0 to 5 minutes, 3% A; 5 to 13 minutes, 3 to 7% A; 13 to 28 minutes, 7 to 9% A, 28 to 35 minutes, 9% A, flow rate of 0.3 to 1.0 mL / min; column temperature of 25 to 35° C.; mass spectrometry conditions for the detection are: electrospray ion source ESI, capillary voltage 4000±200V, 3500±200V in positive and negative ion modes; S3. According to the detection results in S2, obtain the composition information of the seeds of Corydalis chinensis.

2. The method for detecting multiple components in paper mulberry seeds according to claim 1, characterized in that: The ratio of the paper mulberry seed sample to the methanol aqueous solution is 0.1-1.0 g: 25-100 mL.

3. The method for detecting multiple components in paper mulberry seeds according to claim 1, characterized in that: The volume concentration of methanol in step S1 is 10% to 80%.

4. The method for detecting multiple components in paper mulberry seeds according to claim 1, characterized in that: There are a total of 17 components in the obtained paper mulberry seeds, including 6 nucleoside amino acid components, namely L-tyrosine, isoleucine, adenosine, guanosine, inosine, and tryptophan; 5 organic acid components, namely citric acid, neochlorogenic acid, p-hydroxybenzoic acid, chlorogenic acid, and cryptochlorogenic acid; 4 glycoside components, namely p-hydroxybenzoic acid-β-D-pyranose glucoside, vanillic acid-4-O-β-D-glucoside, pyridoxine-3-O-β-D-glucoside, and broad-bract chrysanthemum glycoside C; 1 iridoid component, namely cyperus glutaric acid; and 1 other component, namely (3,6-dimethoxy-2-nitro-benzoyl)-diethyl malonate.

5. The method for detecting multiple components in paper mulberry seeds according to claim 1, characterized in that: The mass spectrometry detection conditions in step S2 also include: mass-to-charge ratio acquisition range m / z 50-1500; drying gas temperature 100-500° C., drying gas flow rate 1-15 L / min; and atomization pressure 20-60 psi.

6. The method for detecting multiple components in paper mulberry seeds according to claim 1, characterized in that: The ultrasonic treatment time in step S1 is 20 to 60 minutes, the power is 150 to 300 W, and the frequency is 20 to 50 kHz.

7. The method for detecting multiple components in paper mulberry seeds according to claim 1, characterized in that: The Cuscuta australis sample is Cuscuta australis medicinal material, Cuscuta australis decoction pieces or Cuscuta australis formula granules.

8. The method for detecting multiple components in paper mulberry seeds according to claim 1, characterized in that: The chromatographic column is a CORTECS T3 chromatographic column.

9. The method for detecting multiple components in paper mulberry seeds according to claim 1, characterized in that: In step S2, the mobile phase B is a formic acid aqueous solution with a volume concentration of 0.01% to 0.2%.

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