CN116519844A - UPLC-MS/MS method for measuring 12 components in polygala tenuifolia fluid extract - Google Patents
UPLC-MS/MS method for measuring 12 components in polygala tenuifolia fluid extract Download PDFInfo
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- CN116519844A CN116519844A CN202310624088.3A CN202310624088A CN116519844A CN 116519844 A CN116519844 A CN 116519844A CN 202310624088 A CN202310624088 A CN 202310624088A CN 116519844 A CN116519844 A CN 116519844A
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- 239000000284 extract Substances 0.000 title claims abstract description 39
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- PMGMZCFZCYRJAG-UHFFFAOYSA-N tenuifoliside C Natural products COC1=C(O)C(OC)=CC(C=CC(=O)OCC2C(C(O)C(O)C(OC3(CO)C(C(O)C(CO)O3)OC(=O)C=CC=3C=C(OC)C(OC)=C(OC)C=3)O2)O)=C1 PMGMZCFZCYRJAG-UHFFFAOYSA-N 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 150000007964 xanthones Chemical class 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Physics & Mathematics (AREA)
- Immunology (AREA)
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a medicinal material quality detection method, in particular to a UPLC-MS/MS method for measuring 12 components in polygala tenuifolia fluid extract. The UPLC-MS/MS method comprises the following steps: s1: preparing a reference substance solution and an internal standard solution; s2: injecting the reference substance solution into UPLC-MS/MS for detection; s3: preparing a test sample solution, adding an internal standard solution into the test sample solution, detecting the test sample solution by adopting UPLC-MS/MS conditions which are the same as those of S2, and performing qualitative and quantitative analysis. The method is used for rapidly and qualitatively or quantitatively detecting 12 components (Siberian polygala tenuifolia sugar A5, siberian polygala tenuifolia sugar A6, 7-O-methyl mangiferin, polygala tenuifolia xanthone III, polygala tenuifolia glycoside A, polygala tenuifolia glycoside B, polygala tenuifolia glycoside C, 3,6' -sinapiyl sucrose, polygalagenin A, polygalasaponinXXVIII, tenuifolin and polygala tenuifolia saponin B) in the polygala tenuifolia fluid extract by a UPLC-MS/MS method, has short detection time and high sensitivity, and provides a reliable experimental basis for development and utilization of the polygala tenuifolia fluid extract and quality evaluation.
Description
Technical Field
The invention relates to a medicinal material quality detection method, in particular to a UPLC-MS/MS method for measuring 12 components in polygala tenuifolia fluid extract.
Background
The polygala tenuifolia fluid extract is carried in the part of pharmacopoeia of the people's republic of China, is prepared by the polygala tenuifolia through a percolation method, and mainly comprises oligosaccharide esters, xanthones, saponins, organic acids and the like, and is used for relieving cough with uncomfortable phlegm. The components of the polygala tenuifolia fluid extract are complex and various, the content measurement item is not established in the part of the pharmacopoeia of the people's republic of China, and the quality of the polygala tenuifolia fluid extract cannot be comprehensively evaluated.
The Chinese patent publication No. CN115754058A discloses a method for detecting tenuifolin in polygala tenuifolia fluid extract. The method comprises the steps of preparing a sample solution through alkaline hydrolysis; and then the sample solution and the reference solution are respectively subjected to high performance liquid chromatography detection, methanol is used as a mobile phase A phase, and 0.05% phosphoric acid aqueous solution is used as a mobile phase B phase during detection, so that tenuifolin in the polygala tenuifolia fluid extract after alkaline hydrolysis can be rapidly and qualitatively identified or quantitatively detected. However, the method can only detect the content of tenuifolin in the polygala tenuifolia fluid extract, and cannot comprehensively detect the quality of the polygala tenuifolia fluid extract.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to establish a method for simultaneously measuring 12 components such as Siberian polygala tenuifolia sugar A5, siberian polygala tenuifolia sugar A6, 7-O-methyl mangiferin, polygala tenuifolia xanthone III, polygala tenuifolia glycoside A, polygala tenuifolia glycoside B, polygala tenuifolia glycoside C, 3,6' -sinapioyl sucrose, polygalinin A, polygalasaponin XXVIII, tenuifolin, polygalin B and the like by using a UPLC-MS/MS method.
In order to solve the technical problems, the invention adopts the following technical scheme: a UPLC-MS/MS method for measuring 12 components in polygala tenuifolia fluid extract comprises the following steps:
s1: preparing a reference substance solution and an internal standard solution;
s2: injecting the reference substance solution into UPLC-MS/MS for detection;
s3: preparing a test sample solution, adding an internal standard solution into the test sample solution, detecting the test sample solution by adopting UPLC-MS/MS conditions which are the same as those of S2, and performing qualitative and quantitative analysis.
The invention has the beneficial effects that: according to the detection method, 12 components (sibiricose A5, sibiricose A6, 7-O-methyl mangiferin, polygalanone III, polygalacoside A, polygalacoside B, polygalacoside C, 3,6' -sinapioyl sucrose, polygalagenin A, polygalasaponinXXVIII, tenuifolin and polygalacoside B) in the polygala tenuifolia fluid extract are rapidly and qualitatively identified or quantitatively detected by a UPLC-MS/MS method, the detection time is short, the sensitivity is high, and a reliable experimental basis is provided for development and utilization of the polygala tenuifolia fluid extract and quality evaluation.
Drawings
FIG. 1 shows an MRM chromatogram of a control solution in accordance with an embodiment of the present invention;
FIG. 2 shows an MRM chromatogram of a sample solution according to an embodiment of the invention;
FIG. 3 shows a column of ACQUITYMRM chromatogram at BEH C18 (2.1X100 mm,1.7 μm);
FIG. 4 shows a chromatographic column Thermo scientificAccucore TM MRM chromatogram at AQ (2.1X105 mm,2.6 μm);
FIG. 5 shows the MRM chromatogram for a column of Thermo scientificAccucore RP-MS (4.6X105 mm,2.6 μm);
FIG. 6 shows the MRM chromatogram when the mobile phase is methanol-water;
FIG. 7 shows the MRM chromatogram when the mobile phase is methanol-0.1% formic acid water;
FIG. 8 shows the MRM chromatogram when the mobile phase is acetonitrile-water;
FIG. 9 shows the MRM chromatogram when the mobile phase is acetonitrile-0.1% formic acid water;
FIG. 10 shows a flow rate of 0.25mL min -1 MRM chromatograms at time;
FIG. 11 shows that the flow rate is 0.2mL min -1 MRM chromatograms at time;
FIG. 12 shows that the flow rate is 0.3mL min -1 MRM chromatograms at time;
FIG. 13 shows the MRM chromatogram at a column temperature of 30 ℃;
FIG. 14 shows the MRM chromatogram at a column temperature of 35 ℃;
FIG. 15 shows the MRM chromatogram at a column temperature of 40 ℃.
Detailed Description
In order to describe the technical contents, the achieved objects and effects of the present invention in detail, the following description will be made with reference to the embodiments in conjunction with the accompanying drawings.
The most critical concept of the invention is as follows: and (3) rapidly and qualitatively identifying or quantitatively detecting 12 components in the polygala tenuifolia fluid extract by using a UPLC-MS/MS method for determination.
The invention relates to a UPLC-MS/MS (ultra performance liquid chromatography-mass spectrometry) method for measuring 12 components in polygala tenuifolia fluid extract, which comprises the following steps:
s1: preparing a reference substance solution and an internal standard solution;
s2: injecting the reference substance solution into UPLC-MS/MS for detection;
s3: preparing a test sample solution, adding an internal standard solution into the test sample solution, detecting the test sample solution by adopting UPLC-MS/MS conditions which are the same as those of S2, and performing qualitative and quantitative analysis.
From the above description, the beneficial effects of the invention are as follows: the polygala tenuifolia fluid extract contains various components, such as sugar ester components, which have glycosidic bonds, high hydrophilicity and high polarity, and can show peaks in the reversed phase chromatographic column when the organic phase with a low proportion of 10-20 percent, and the peak-showing time is quick. The polarity of the mountain ketone component is medium, the peak is usually generated when 40-60% of the organic phase is in medium proportion, the polarity of the saponin component is small, the peak generation time is long, and the peak can be generated when 70% of the organic phase is in high proportion. Meanwhile, the polygala tenuifolia fluid extract contains various saponins, quantitative separation cannot be carried out in gradient elution, and the separation degree between peaks does not meet the quantitative requirement. Therefore, when HPLC or UPLC is performed, it takes a long time to separate these components, and the separation effect is poor, and the degree of separation is not satisfactory.
The UPLC-MS/MS has the characteristics of high analysis speed, strong specificity, high sensitivity, high accuracy, high stability, simultaneous analysis of multiple indexes and the like, can efficiently separate components, only measures the components to be measured, and cannot be influenced by other components with similar peaks.
Therefore, the method uses UPLC-MS/MS method to measure 12 components of polygala tenuifolia fluid extract, constructs quantitative method from three different types of chemical components of polygala tenuifolia fluid extract, optimizes chromatographic conditions, can independently detect required components, is not influenced by other components, has high separation speed and higher sensitivity, and comprehensively evaluates the quality of polygala tenuifolia fluid extract.
The detection method of the invention optimizes chromatographic conditions, and then rapidly and qualitatively identifies or quantitatively detects 12 components (Siberian polygala tenuifolia sugar A5, siberian polygala tenuifolia sugar A6, 7-O-methyl mangiferin, polygalanone III, polygalacoside A, polygalacoside B, polygalacoside C, 3,6' -sinapioyl sucrose, polygalacin A, polygalasaponinXXVIII (melon seed Jin Zaogan XXVIII), tenuifolin and polygalacoside B) in the polygala tenuifolia fluid extract by a UPLC-MS/MS method, thereby having short detection time, high sensitivity, good precision and repeatability, and providing reliable experimental basis for development, utilization and quality evaluation of the polygala tenuifolia fluid extract.
Further, in the chromatographic condition of S2, the chromatographic column is ACQUITYBEH C18, specification 2.1X100 mm,1.7 μm.
From the above description, ACQUITYBEH C18 The peak capacity is large (2.1X100 mm,1.7 μm), and the degree of separation is good.
Further, in the chromatographic condition of S2, the flow rate of the mobile phase is 0.25-0.3 mL/min.
Preferably, the flow rate of the mobile phase is 0.3mL/min.
From the above description, it is clear that the separation degree is good when the flow rate of the mobile phase is 0.25 to 0.3mL/min.
Further, in the chromatographic condition of S2, the column temperature is 30-40 ℃.
Preferably, the column temperature is 40 ℃.
As is clear from the above description, the column temperature conditions were changed, and the effect was not so great, but the degree of separation was good at a column temperature of 40 ℃.
Further, in the chromatographic condition of S2, acetonitrile is mobile phase a phase, and 0.1% (volume fraction) formic acid aqueous solution is mobile phase B phase.
From the above description, the use of acetonitrile-0.1% formic acid water system has the best peak capacity, and the chromatographic separation degree is better than other systems.
Further, in the chromatographic condition of S2, the gradient elution condition is:
0-4 min, the volume fraction of the mobile phase A is increased from 10% to 25%;
4-6 min, the volume fraction of the mobile phase A is increased from 25% to 45%;
6-8 min, increasing the volume fraction of the mobile phase A from 45% to 70%;
8-10 min, the volume fraction of the mobile phase A is increased from 70% to 100%;
10-10.2 min, the volume fraction of the mobile phase A is reduced from 100% to 10%;
10.2-12 min, the volume fraction of the mobile phase A phase is 10%.
Further, the preparation method of the reference substance solution comprises the following steps: respectively taking Siberian polygalase A5, siberian polygalase A6, 7-O-methyl mangiferin, polygalanone III, polygalacoside A, polygalacoside B, polygalacoside C, 3,6' -sinapioyl sucrose, polygalacin A, polygalasaponin XXVIII, tenuifolin and polygalacoside B, and adding 50% methanol solution to obtain reference solution.
Further, the preparation method of the internal standard solution comprises the following steps: respectively taking trans-2-hydroxy cinnamic acid, colchicoside and timosaponin BII as reference substances, and adding 50% methanol solution to obtain internal standard solution.
Further, the preparation method of the sample solution comprises the following steps: adding 50% methanol solution into radix Polygalae fluid extract, cooling, shaking, adding weight loss, and filtering with microporous membrane to obtain filtrate.
The reagents and instrumentation in the examples were as follows:
a DIONEX uitimite 3000 ultra-high performance liquid chromatography system tandem quadrupole-electrostatic orbital hydrazine mass spectrometer (samer femto-cell company) equipped with a HESI (electrospray ion source); MILLI-Q Direct16 ultrapure water preparation instrument (Millipore Co.); CPA225D one ten thousandth electronic balance (Sartorius company); XM-400UHP Xiaomei ultrasonic apparatus (Kunshan Xiaomei ultrasonic apparatus Co., ltd.).
The control polygala tenuifolia xanthone III (lot No. 111850-202006, content 96.5%), 3,6' -sinapioyl sucrose (lot No. 111848-202006, content 96.0%) and tenuifolia saponins (lot No. 111849-202106, content 97.3%) were purchased from Chinese food and drug assay institute. Tenuifoliside C (lot number: B21627), tenuifoliside B (lot number: B24057), tenuifoliside A (lot number: B21347), sibiricose A6 (lot number: B21335), sibiricose A5 (lot number: B21334), polygalasaponin XXVIII (lot number: B29582), onjisapin B (lot number: B21628), arilanin A (lot number: B27331), 7-O-Methylangin (lot number: B21843) were purchased from Shanghai Seiki Biotech Co. Methanol and acetonitrile were mass spectrum pure (sammer femto science and technology company), and formic acid was mass spectrum pure (Shanghai a Ding Shiji limited company). The polygala tenuifolia fluid extract is from Fujian Zhongyi pharmaceutical Co.
The first embodiment of the invention is as follows:
a UPLC-MS/MS method for measuring 12 components in polygala tenuifolia fluid extract comprises the following steps:
s1: preparing a reference substance solution: dividing intoTaking Siberian polygalase A5, siberian polygalase A6, 7-O-methyl mangiferin, polygalanone III, polygalacoside A, polygalacoside B, polygalacoside C, 3,6' -sinapioyl sucrose, polygalacin A, polygalasaponin XXVIII, tenuifolin and polygalacoside B, dissolving with 50% methanol solution to obtain 2 mg/mL solution -1 A single control solution;
preparing an internal standard solution: dissolving trans-2-hydroxy cinnamic acid, colchicoside and timosaponin BII reference powder in 50% methanol solution to obtain 2 mg/mL solution -1 Respectively and precisely measuring a proper amount of mother solution, placing the mother solution into a measuring flask, diluting the mother solution to a scale with 50% of methanol solution, and shaking the mother solution to obtain the mother solution with the concentration of 6, 12 and 800 ng.mL respectively -1 Is prepared from the internal standard mixture of (1).
S2: injecting the reference substance solution into an ultra-high performance liquid chromatography tandem mass spectrometer for detection;
the chromatographic conditions were as follows:
chromatographic column: ACQUITYBEH C18(2.1×100mm，1.7μm)；
Mobile phase: acetonitrile is a mobile phase A phase, and 0.1% formic acid water solution is a mobile phase B phase;
the gradient elution conditions were: 0-4 min,10% A25% A; 4-6 min,25% A45% A; 6-8 min,45% A70% A; 8-10 min,70% A100% A;10 to 10.2min,100 percent of A10 percent of A; 10.2-12 min,10% A;
flow rate: 0.3mL min -1 ；
Column temperature: 40 ℃;
sample injection amount: 2. Mu.L.
The mass spectrometry conditions were as follows:
ion mode: adopting a heating electrospray ion source (HESI) negative ion mode;
spray voltage: 3.0kV (-);
capillary temperature: 325 c,
auxiliary gas heating temperature: 350 ℃; the method comprises the steps of carrying out a first treatment on the surface of the
Sheath air flow rate (Arb): 45;
auxiliary gas flow rate (Arb): 10; the method comprises the steps of carrying out a first treatment on the surface of the
Purge gas flow rate (Arb): 1, a step of;
scanning mode: PRM scan mode.
The mass spectrum optimization parameters of each component to be detected and the internal standard are shown in table 1, and the MRM chromatogram of each component in the reference substance solution is shown in fig. 1.
TABLE 1
S3: preparing a test sample solution: taking 5 batches of traditional Chinese medicine fluid extracts of different batches, three batches of traditional Chinese medicine fluid extracts in parallel, precisely measuring 0.1mL of polygala tenuifolia fluid extract in each batch, placing the obtained product in a 50mL conical flask, adding 25mL of 50% methanol solution in volume fraction, weighing the obtained product, taking out the obtained product after ultrasonic treatment for 30min, shaking the obtained product after cooling, supplementing the weight loss, filtering the obtained product through a 0.22 mu m microporous filter membrane, and obtaining a subsequent filtrate to obtain a sample solution. The MRM chromatogram of each component in the test solution is shown in FIG. 2.
S4: the sample solution is taken and placed in a measuring flask, diluted 20 times by methanol solution with the volume fraction of 50%, internal standard solution is added according to the ratio of 1:1 (100:100, mu L), the sample solution is detected by adopting the same chromatographic and mass spectrum conditions as S2, the peak area is measured, the content of 12 components is calculated according to a standard curve, and the result is shown in Table 2.
TABLE 2
The second embodiment of the invention is as follows: linear regression equation and quantitative limit investigation;
precisely measuring the reference substance solution of each component to be measured in the first embodiment, and placing in a volumetric flask to obtainTo a concentration of 2.67, 1.69, 0.17, 2.91, 0.63, 5.48, 1.21, 1.57, 0.62, 25.14, 0.20, 62.81. Mu.g.mL, respectively -1 The reference substance mother liquor of (2) is diluted by methanol solution with the volume fraction of 50% step by step for 10, 25, 50, 100, 200 and 1000 times, and the equal volume of internal standard solution is added to obtain standard solutions with different concentrations. The peak areas of the components to be detected are obtained by analysis and measurement in an ultra-high performance liquid chromatography tandem mass spectrometer of the solutions with different concentrations of the reference substances, linear regression is carried out by taking the peak area ratio of the target component to the internal standard substance as an ordinate and the concentration of the target component as an abscissa, and the concentration of each target component at the signal-to-noise ratio S/N=10 and the S/N=3 is taken as a quantitative Limit (LOQ) and a detection Limit (LOD) respectively, and the values are shown in table 3.
TABLE 3 Table 3
The third embodiment of the invention is as follows: performing precision test;
the control solutions of reference example one were prepared at concentrations of 106.976, 67.748, 6.764, 116.428, 25.088, 219.088, 48.524, 62.936, 24.764, 1005.58, 8.00, 2512.248. Mu.g.mL, respectively -1 As the high-concentration mixed reference solution, the high-concentration mixed reference solution is diluted half and half to obtain the medium-concentration reference solution, and the high-concentration mixed reference solution is diluted half and half to obtain the low-concentration reference solution. Under the conditions of the chromatograph and the mass spectrum of the first embodiment, the daily precision is continuously measured for 6 times on the same day, the daily precision is continuously measured for 3 days, the peak area ratio of each reference substance and the internal standard is recorded, the RSD is calculated, the result is shown in the table 4, the daily precision RSD is less than 4.30%, and the daily precision is less than 4.64%, so that the instrument precision is good.
TABLE 4 Table 4
The fourth embodiment of the invention is as follows: stability test;
taking 0.1g of polygala tenuifolia fluid extract sample, preparing a sample solution according to the sample solution preparation method in the first embodiment, respectively carrying out sample injection on the prepared sample solution for 0, 2, 4, 8, 12, 24 and 48 hours to determine peak areas, recording the ratio of a reference substance to an internal standard peak area, and calculating RSD, wherein the results are shown in Table 5, and the RSD is smaller than 4.48%, so that the sample solution is stable within 48 hours.
The fifth embodiment of the invention is as follows: a repeatability test;
taking the same batch of polygala tenuifolia fluid extract samples, preparing 6 parts of sample solution simultaneously according to the sample solution preparation method of the first embodiment, respectively carrying out sample injection measurement, calculating the average content and RSD (reactive group detector), wherein the results are shown in Table 5, and the RSD is less than 4.66%, so that the method has good repeatability.
TABLE 5
The fifth embodiment of the invention is as follows: sample addition recovery rate test;
the known content of polygala tenuifolia fluid extract samples are precisely weighed, 9 parts are parallel, 3 kinds of reference substance solutions with the concentration of medium, low and high in the third embodiment are precisely added respectively, the sample solutions are prepared according to the sample solution preparation method in the first embodiment, sample injection measurement is carried out respectively, and the average recovery rate and RSD are calculated, and the result is shown in Table 6.
TABLE 6
The sixth embodiment of the invention is: screening for chromatographic conditions on the basis of example one (sample solution and chromatographic mass spectrometry conditions);
1. screening of different chromatographic columns
Compare nothingWith chromatographic column, ACQUITYBEH C18 (2.1X100 mm,1.7 μm); (FIG. 3), thermo scientificAccucore TM AQ (2.1X105 mm,2.6 μm); (FIG. 4), thermo scientificAccucore RP-MS (4.6X105 mm,2.6 μm) (FIG. 5).
The results are shown in FIGS. 3 to 5 (in the figures, reference numerals: 1. Siberian polygalase A5, 2. Siberian polygalase A6, 3.7-O-methyl mangiferin, 4. Polygalanone III, 5. Polygalacoside B, 6.3,6' -sinapiyl sucrose, 7. Yellow polygalacin A, 8. Polygalacoside A, 9. Polygalacoside C, 10.Polygalasaponin XXVIII;11. Tenuifolin, 12. Polygalacoside B)BEH C18 (2.1X100 mm,1.7 μm) has a large peak capacity and a good degree of separation.
2. Screening of different mobile phase systems
The 4 mobile phase systems of methanol-water (FIG. 6), methanol-0.1% formic acid water (FIG. 7), acetonitrile-water (FIG. 8), acetonitrile-0.1% formic acid water (FIG. 9) were compared.
The results are shown in FIGS. 6-9 (in the figures, reference numbers are 1. Siberian polygala tenuifolia sugar A5, 2. Siberian polygala tenuifolia sugar A6, 3.7-O-methyl mangiferin, 4. Polygalanone III, 5. Polygala tenuifolia glycoside B, 6.3,6' -sinigyl sucrose, 7. Yellow polygalacin A, 8. Polygala tenuifolia glycoside A, 9. Polygala tenuifolia glycoside C, 10.Polygalasaponin XXVIII;11. Tenuifolia saponin and 12. Polygala tenuifolia saponin B), and the acetonitrile-0.1% formic acid water system has the best peak capacity, and the chromatographic separation degree is better than other systems.
3. Screening of different flow rate conditions
Compare 0.25 mL/min -1 (FIG. 10), 0.2 mL/min -1 (FIG. 11), 0.3mL/min -1 (FIG. 12) different flow rate conditions.
The results are shown in FIGS. 10 to 12 (in the figures, the reference numerals are 1. Siberian polygalase A5, 2. Siberian polygalase A6, 3.7-O-methyl mangiferin, 4. Polygalanone III, 5. Polygalacoside B, 6.3,6' -sinapioyl sucrose, 7. Yellow polygalacin A, 8. Polygalacoside A;9. polygala tenuifolia glycoside C;10.Polygalasaponin XXVIII;11. tenuifolin; 12. polygalasaponin B) of 0.3 mL.min -1 The separation degree is better, and the flow rate is better than other flow rates in the chromatographic system.
4. Screening of different column temperature conditions
Different column temperature conditions were compared at 30 ℃ (fig. 13), 35 ℃ (fig. 14), 40 ℃ (fig. 15).
The results are shown in FIGS. 13 to 15 (reference numerals in the figures: 1. Siberian polygalase A5;2. Siberian polygalase A6; 3.7-O-methyl mangiferin; 4. Polygalanone III; 5. Polygalacoside B;6.3,6' -sinapyl sucrose; 7. Ghatti polygalacin A;8. Polygalacoside A;9. Polygalacoside C;10.Polygalasaponin XXVIII;11. Tenuifolin; 12. Polygalacoside B), and the column temperature conditions were changed with little influence on the results, but the degree of separation was good at 40℃and preferably 40 ℃.
In summary, the UPLC-MS/MS method for determining 12 components in the polygala tenuifolia fluid extract provided by the invention is used for rapidly and qualitatively identifying or quantitatively detecting 12 components (Siberian polygala tenuifolia sugar A5, siberian polygala tenuifolia sugar A6, 7-O-methyl mangiferin, polygala tenuifolia xanthone III, polygala tenuifolia glycoside A, polygala tenuifolia glycoside B, polygala tenuifolia glycoside C, 3,6' -sinapoyl sucrose, polygalagenin A, polygalasaponin XXVIII, tenuifolia saponin and polygala tenuifolia saponin B) in the polygala tenuifolia fluid extract by using the UPLC-MS/MS method, has short detection time and high sensitivity, and provides a reliable experimental basis for development, utilization and quality evaluation of the polygala tenuifolia fluid extract.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent changes made by the specification and drawings of the present invention, or direct or indirect application in the relevant art, are included in the scope of the present invention.
Claims (9)
1. A UPLC-MS/MS method for determining 12 ingredients in polygala tenuifolia fluid extract, comprising the steps of:
s1: preparing a reference substance solution and an internal standard solution;
s2: injecting the reference substance solution into UPLC-MS/MS for detection;
s3: preparing a test sample solution, adding an internal standard solution into the test sample solution, detecting the test sample solution by adopting UPLC-MS/MS conditions which are the same as those of S2, and performing qualitative and quantitative analysis.
2. The UPLC-MS/MS method according to claim 1, wherein in the chromatographic conditions of S2, the chromatographic column is an ACQUITYBEH C18, specification 2.1X100 mm,1.7 μm.
3. The UPLC-MS/MS method according to claim 1, wherein the flow rate of the mobile phase is 0.25 to 0.3mL/min in the chromatographic conditions of S2.
4. The UPLC-MS/MS method according to claim 1, wherein the column temperature is 30 to 40 ℃ in the chromatographic conditions of S2.
5. The UPLC-MS/MS method according to claim 1, characterized in that in the chromatographic conditions of S2 acetonitrile is mobile phase a phase and 0.1% formic acid aqueous solution is mobile phase B phase.
6. The UPLC-MS/MS method according to claim 1, characterized in that, among the chromatographic conditions of S2, the gradient elution conditions are:
0-4 min, the volume fraction of the mobile phase A is increased from 10% to 25%;
4-6 min, the volume fraction of the mobile phase A is increased from 25% to 45%;
6-8 min, increasing the volume fraction of the mobile phase A from 45% to 70%;
8-10 min, the volume fraction of the mobile phase A is increased from 70% to 100%;
10-10.2 min, the volume fraction of the mobile phase A is reduced from 100% to 10%;
10.2-12 min, the volume fraction of the mobile phase A phase is 10%.
7. The UPLC-MS/MS method according to claim 1, wherein the control solution is prepared by: respectively taking Siberian polygalase A5, siberian polygalase A6, 7-O-methyl mangiferin, polygalanone III, polygalacoside A, polygalacoside B, polygalacoside C, 3,6' -sinapioyl sucrose, polygalacin A, polygalasaponin XXVIII, tenuifolin and polygalacoside B, and adding 50% methanol solution to obtain reference solution.
8. The UPLC-MS/MS method according to claim 1, wherein the internal standard solution is prepared by the following steps: respectively taking trans-2-hydroxy cinnamic acid, colchicoside and timosaponin BII as reference substances, and adding 50% methanol solution to obtain internal standard solution.
9. The UPLC-MS/MS method according to claim 1, wherein the preparation method of the test sample solution is as follows: adding 50% methanol solution into radix Polygalae fluid extract, cooling, shaking, adding weight loss, and filtering with microporous membrane to obtain filtrate.
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