CN113390997A - Method for simultaneously and quantitatively detecting matrine and oxymatrine in tea - Google Patents
Method for simultaneously and quantitatively detecting matrine and oxymatrine in tea Download PDFInfo
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- CN113390997A CN113390997A CN202110681432.3A CN202110681432A CN113390997A CN 113390997 A CN113390997 A CN 113390997A CN 202110681432 A CN202110681432 A CN 202110681432A CN 113390997 A CN113390997 A CN 113390997A
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- ZSBXGIUJOOQZMP-UHFFFAOYSA-N Isomatrine Natural products C1CCC2CN3C(=O)CCCC3C3C2N1CCC3 ZSBXGIUJOOQZMP-UHFFFAOYSA-N 0.000 title claims abstract description 45
- ZSBXGIUJOOQZMP-JLNYLFASSA-N Matrine Chemical compound C1CC[C@H]2CN3C(=O)CCC[C@@H]3[C@@H]3[C@H]2N1CCC3 ZSBXGIUJOOQZMP-JLNYLFASSA-N 0.000 title claims abstract description 45
- 229930014456 matrine Natural products 0.000 title claims abstract description 45
- XVPBINOPNYFXID-JARXUMMXSA-N 85u4c366qs Chemical compound C([C@@H]1CCC[N@+]2(CCC[C@H]3[C@@H]21)[O-])N1[C@@H]3CCCC1=O XVPBINOPNYFXID-JARXUMMXSA-N 0.000 title claims abstract description 36
- 229930015582 oxymatrine Natural products 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 35
- 241001122767 Theaceae Species 0.000 title claims abstract 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 66
- 239000000243 solution Substances 0.000 claims abstract description 51
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 17
- 238000000605 extraction Methods 0.000 claims abstract description 17
- 150000002500 ions Chemical class 0.000 claims abstract description 16
- 229960001701 chloroform Drugs 0.000 claims abstract description 11
- 238000001819 mass spectrum Methods 0.000 claims abstract description 11
- 238000012544 monitoring process Methods 0.000 claims abstract description 10
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims abstract description 9
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 9
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000010931 gold Substances 0.000 claims abstract description 9
- 229910052737 gold Inorganic materials 0.000 claims abstract description 9
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 claims abstract description 9
- SOSWVVMGPYTPKV-UHFFFAOYSA-N acetic acid;azane;formic acid Chemical compound [NH4+].OC=O.CC([O-])=O SOSWVVMGPYTPKV-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- 238000010828 elution Methods 0.000 claims abstract description 8
- 238000010812 external standard method Methods 0.000 claims abstract description 7
- 238000002390 rotary evaporation Methods 0.000 claims abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 5
- 238000003113 dilution method Methods 0.000 claims abstract description 5
- 239000011159 matrix material Substances 0.000 claims description 22
- 239000000126 substance Substances 0.000 claims description 13
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
- 239000012086 standard solution Substances 0.000 claims description 12
- 239000011550 stock solution Substances 0.000 claims description 12
- 238000005303 weighing Methods 0.000 claims description 11
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 9
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 8
- 239000005695 Ammonium acetate Substances 0.000 claims description 8
- 229940043376 ammonium acetate Drugs 0.000 claims description 8
- 235000019257 ammonium acetate Nutrition 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 7
- 238000005173 quadrupole mass spectroscopy Methods 0.000 claims description 7
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 235000019253 formic acid Nutrition 0.000 claims description 6
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 6
- 238000005516 engineering process Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000004949 mass spectrometry Methods 0.000 claims description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
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- 238000002604 ultrasonography Methods 0.000 claims description 3
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- 238000001514 detection method Methods 0.000 abstract description 15
- 239000000575 pesticide Substances 0.000 abstract description 8
- 229930013930 alkaloid Natural products 0.000 abstract description 3
- 150000003797 alkaloid derivatives Chemical class 0.000 abstract description 3
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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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
- G01N30/62—Detectors specially adapted therefor
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Abstract
The invention discloses a method for simultaneously and quantitatively detecting matrine and oxymatrine in tea, which comprises the steps of adding ammonia water into a tea sample, taking trichloromethane as an extraction reagent, carrying out ultrasonic extraction, and carrying out rotary evaporation until the tea sample is nearly dry; adding methanol to a constant volume, preparing a standard curve solution by a stepwise dilution method, finally separating by adopting a Hypersil GOLD C18 column, performing gradient elution by taking methanol and ammonium acetate formic acid solution as mobile phases, wherein the flow rate is 0.2mL/min, the column temperature is 30 ℃, the column temperature mass spectrum is detected by adopting an electrospray positive ion source and a multi-reaction monitoring mode, and the quantification is performed by an external standard method. The method has the advantages of simple pretreatment, high accuracy and precision, high practicability and high detection limit of both the matrine and the oxymatrine of 0.01mg/kg, can realize item detection of the matrine and the oxymatrine in various tea leaves and enhances the monitoring of alkaloid pesticide risks.
Description
Technical Field
The invention relates to a method for simultaneously and quantitatively detecting matrine and oxymatrine in tea, belonging to the technical field of analytical chemistry.
Background
The botanical pesticide has low toxicity to human and livestock, is easy to degrade in environment, has little residue or no residue, and the like, and becomes a research hotspot of plant protection workers of various countries since the 20 th century and the 80 th year. The botanical pesticide is mainly used for vegetables, tea, Chinese medicinal materials, landscaping, cotton and fruits. Especially, chemical synthetic pesticides are forbidden in the production technical rules of organic tea, and plant-derived pesticides become ideal prevention and treatment agents for organic tea gardens. The matrine is a water-soluble alkaloid extracted from roots, stems and the like of radix sophorae flavescentis by a strict process, is a natural plant-derived broad-spectrum pesticide, has stomach toxicity and contact killing effects, and has ideal control effects on aphids, aleyrodid, cabbage caterpillars, tea caterpillars, diamond back moths, tea geometrids, lesser leafhoppers and the like. Can be mixed with common chemical synthetic pesticide, shows obvious synergistic effect, greatly reduces the dosage of the chemical pesticide and reduces the pollution of the chemical synthetic pesticide to the environment.
Under the regulations of european union regulations (EC) No 834/2007 and (EC) No 889/2008 on the production and labeling of organic products, the use of matrine or matrine-containing products in organic agricultural inputs will No longer be accepted by the european union since 6 and 20 days 2017. Because the income of the domestic tea is obviously higher than that of the foreign tea, and the quantity and limit requirements of the domestic tea are loose from the pesticide residue requirements of countries and regions such as European Union, Japan and the like, the tea grower often does not strictly use the pesticide according to the requirements of imported countries in the planting process. In 2019, 9 months and 9 days, according to the information of a rapid early warning system (RASFF) of food and feed of European Union, Spain refuses export black tea of China to enter the country, and the reason is that the content of the matrine which cannot be used is detected to be 0.029 mg/kg. Therefore, the research on a suitable detection method has important significance for guaranteeing the tea export in China.
The existing methods for measuring matrine mainly comprise a thin-layer scanning method, a capillary electrophoresis method, a microfluidic chip non-contact conductivity detection method, a high performance liquid chromatography, a gas chromatography, a chromatography-mass spectrometry method and the like. The thin-layer scanning method and the capillary electrophoresis method for determining the matrine have the disadvantages of complicated separation steps, long analysis time and poor interference resistance; the matrine content in the matrine injection is detected by adopting a microfluidic chip non-contact conductivity technology, the detection limit is 1.0 mug/mL, and the low-content detection requirement cannot be met; the high performance liquid chromatography and the gas chromatography are mainly applied to the determination of the content of matrine in the medicine at present. Therefore, the research on a method which is simple in treatment and is suitable for simultaneously detecting the residual quantity of matrine and oxymatrine in tea leaves is an urgent problem to be solved.
Disclosure of Invention
In view of this, the present invention aims to provide a method for simultaneously and quantitatively detecting matrine and oxymatrine in tea, which can overcome the defects of the prior art.
The purpose of the invention is realized by the following technical scheme:
a method for simultaneously and quantitatively detecting matrine and oxymatrine in tea comprises the following steps:
s1, extracting the sample,
quantitatively weighing a tea sample, adding ammonia water into the sample, taking trichloromethane as an extraction reagent, extracting by adopting ultrasonic waves, and performing rotary evaporation until the sample is nearly dry to obtain a standard substance;
s2, preparing a standard curve solution,
adding methanol into the standard substance to a constant volume, and preparing a standard curve solution by adopting a stepwise dilution method;
s3, detecting the residual quantity of matrine and oxymatrine in the standard curve solution by adopting a high performance liquid chromatography-tandem triple quadrupole mass spectrometry technology.
In the step s1, weighing 0.50g of tea sample, adding 0.5mL of ammonia water, standing for 30min, adding 20mL of chloroform, performing ultrasound for 30min, and repeating for at least 2 times; then 0.5g of anhydrous sodium sulfate is added and mixed evenly, and the whole solution is steamed to be nearly dry in a rotating way.
In the aforementioned step s2, the step of,
(1) weighing quantitative standard substances respectively, preparing into 1.0mg/mL standard stock solution with methanol, and storing at low temperature;
(2) taking a proper amount of each standard stock solution, and diluting the stock solution into a mixed standard solution of 1.0mg/L by using 5% methanol;
(3) diluting the sample blank matrix solution step by step to prepare a standard curve solution with the concentration of 2-100 ng/mL;
(4) filtering each standard curve solution through a filter membrane with the particle size of not more than 0.22 mu m, and detecting on a machine.
In the step s2, the mixed standard solutions are diluted step by step with the blank matrix solution to prepare standard curve solutions with the concentrations of 2, 5, 10, 20, 50 and 100 ng/mL.
In the step s2, a proper amount of blank tea leaf sample is weighed, processed according to the method for extracting the sample in the step s1, and then 5% methanol is used for constant volume, so that a blank matrix solution is obtained.
In the aforementioned step s2, the standard stock solution was stored in an environment of-20 ℃.
In the step s3, separating by adopting a Hypersil GOLD C18 column, performing gradient elution by using a mobile phase A and a mobile phase B, detecting by adopting an electrospray positive ion source and a multi-reaction monitoring mode by adopting a column temperature mass spectrum, and quantifying by adopting an external standard method;
wherein the mobile phase A is methanol, and the mobile phase B is ammonium acetate formic acid solution.
The ammonium acetate-formic acid solution was a 5mmol/L aqueous ammonium acetate solution, and 0.1% formic acid was contained in the 5mmol/L aqueous ammonium acetate solution.
In the foregoing step s3, the measurement conditions of the hplc-tandem triple quadrupole mass spectrometry are as follows:
(1) the high performance liquid chromatography is Hypersil GOLD C18, the specification is column length 100mm, column inner diameter 2.1mm, particle size 1.9 μm;
the column temperature is 30 ℃,
flow rate: 0.2 mL/min;
sample introduction amount: 10 mu L of the solution;
mobile phase conditions are shown in table 1:
TABLE 1 HPLC gradient elution
Operating time: 8 min;
(2) the mass spectrum conditions are as follows:
adopting an electrospray positive ion mode;
the scanning mode is as follows: monitoring multiple reactions;
temperature of the drying gas: 300 ℃;
flow rate of drying gas: 3L/min;
the flow rate of the sheath gas: 15L/min;
the capillary temperature is 350 ℃;
spraying voltage: 3200V;
the mass spectrum parameters in the multiple reaction monitoring mode are shown in Table 2
TABLE 2 Mass Spectrometry parameters in multiple reaction monitoring modes
Compared with the prior art, the method for simultaneously and quantitatively detecting the matrine and the oxymatrine in the tea disclosed by the invention has the advantages that after ammonia water is added into a tea sample, chloroform is used as an extraction reagent, ultrasonic extraction is carried out, and rotary steaming is carried out until the tea sample is nearly dry; adding methanol to a constant volume, preparing a standard curve solution by a stepwise dilution method, finally separating by adopting a Hypersil GOLD C18 column, performing gradient elution by taking methanol and ammonium acetate formic acid solution as mobile phases, wherein the flow rate is 0.2mL/min, the column temperature is 30 ℃, the column temperature mass spectrum is detected by adopting an electrospray positive ion source and a multi-reaction monitoring mode, and the quantification is performed by an external standard method. The result shows that the matrine and oxymatrine have good linear relation in the mass concentration range of 2-100ng/mL, and the correlation coefficient (r)2) Not less than 0.995, the average recovery rate under the high, medium and low 3 addition levels is 90.6-116.2%, the relative standard deviation (n is 6) is 2.7-5.4%, and the detection limits are 0.01mg/kg, so that the method is suitable for simultaneously detecting the residual quantity of matrine and oxymatrine in tea.
The invention has the beneficial effects that:
(1) the method is simple in pretreatment aiming at the tea sample, only the extracting solution needs to be added for ultrasonic extraction, and the operation is time-saving, labor-saving, convenient and efficient;
(2) the detection low limit is determined by adopting a blank matrix to gradually dilute the standard solution and an external standard method, the accuracy and the precision are high, and the detection low limits of the matrine and the oxymatrine are both 0.01 mg/kg;
(3) the residual quantity of matrine and oxymatrine in the tea leaves is detected by using a high performance liquid chromatography-triple quadrupole mass spectrometry technology, the pretreatment, chromatography and mass spectrometry conditions are optimized, an addition recovery experiment is carried out, the method is feasible, the result is reliable, and the detection lower limit can reach the requirement of 0.01 mg/kg;
(4) the practicability is strong, the project detection of matrine and oxymatrine in various tea leaves can be realized, and the risk monitoring of alkaloid pesticides is enhanced.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.
Drawings
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings, in which:
FIG. 1 is a total ion flow diagram of chloroform direct extraction after ammoniation;
fig. 2 is a total ion flow diagram for water direct extraction.
FIG. 3 is a total ion flow graph of an ammoniated water extraction.
FIG. 4 is ion chromatogram and ion fragment diagram of matrine extraction.
FIG. 5 is ion chromatogram and ion fragment diagram of oxymatrine extraction.
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the preferred embodiments are illustrative of the invention only and are not limiting upon the scope of the invention.
A method for simultaneously and quantitatively detecting matrine and oxymatrine in tea comprises the following steps:
s1, extracting the sample,
quantitatively weighing a tea sample, adding ammonia water into the sample, taking trichloromethane as an extraction reagent, extracting by adopting ultrasonic waves, and performing rotary evaporation until the sample is nearly dry to obtain a standard substance;
s2, preparing a standard curve solution,
adding methanol to the standard substance to a constant volume, and preparing a standard curve solution by adopting a stepwise dilution method;
s3, detecting the residual quantity of matrine and oxymatrine in the standard curve solution by adopting a high performance liquid chromatography-tandem triple quadrupole mass spectrometry technology.
In step s1, weighing 0.50g of tea sample, adding 0.5mL of ammonia water, standing for 30min, adding 20mL of chloroform, performing ultrasound for 30min, and repeating for at least 2 times; then 0.5g of anhydrous sodium sulfate is added and mixed evenly, and the whole solution is steamed to be nearly dry in a rotating way.
Preferably, the rotary evaporation equipment for rotary evaporation of the tea sample is an R300 rotary evaporator of Switzerland Qi company.
In a step s2, the method is carried out,
(1) weighing quantitative standard substances respectively, preparing into 1.0mg/mL standard stock solution with methanol, and storing at low temperature;
(2) taking a proper amount of each standard stock solution, and diluting the stock solution into a mixed standard solution of 1.0mg/L by using 5% methanol;
(3) diluting the sample blank matrix solution step by step to prepare a standard curve solution with the concentration of 2-100 ng/mL;
(4) filtering each standard curve solution through a filter membrane with the particle size of not more than 0.22 mu m, and detecting on a machine.
In particular, the standard stock solution can be stored in a-20 ℃ refrigerator.
Specifically, each mixed standard solution is diluted step by using a blank matrix solution to prepare a standard curve solution with the concentration of 2, 5, 10, 20, 50 and 100 ng/mL.
Weighing a proper amount of blank tea leaf sample, processing according to the sample extraction method in the step s1, and then adding 5% methanol to constant volume to obtain a blank matrix solution.
In step s3, separating by adopting a Hypersil GOLD C18 column, performing gradient elution by using a mobile phase A and a mobile phase B, detecting by adopting a column temperature mass spectrum, an electrospray positive ion source and a multi-reaction monitoring mode, and quantifying by an external standard method; wherein the mobile phase A is methanol, and the mobile phase B is ammonium acetate formic acid solution.
The ammonium acetate formic acid solution was a 5mmol/L aqueous ammonium acetate solution, and the 5mmol/L aqueous ammonium acetate solution contained 0.1% formic acid.
Preferably, the high performance liquid chromatography is prepared as U3000 high performance liquid chromatograph of the american siemer flyer; the mass spectrometer was an Access MAX triple quadrupole mass spectrometer from semer airlines usa.
In step s3, the determination conditions of the HPLC-tandem triple quadrupole mass spectrometry are as follows:
(1) the high performance liquid chromatography is Hypersil GOLD C18, the specification is column length 100mm, column inner diameter 2.1mm, particle size 1.9 μm;
the column temperature is 30 ℃,
flow rate: 0.2 mL/min;
sample introduction amount: 10 mu L of the solution;
mobile phase conditions are shown in table 1:
TABLE 1 HPLC gradient elution
Operating time: 8 min;
(2) the mass spectrum conditions are as follows:
adopting an electrospray positive ion mode;
the scanning mode is as follows: monitoring multiple reactions;
temperature of the drying gas: 300 ℃;
flow rate of drying gas: 3L/min;
the flow rate of the sheath gas: 15L/min;
the capillary temperature is 350 ℃;
spraying voltage: 3200V;
the mass spectrum parameters in the multiple reaction monitoring mode are shown in Table 2
TABLE 2 Mass Spectrometry parameters in multiple reaction monitoring modes
Results and analysis
Referring to fig. 1-3, three methods are respectively adopted to extract matrine and oxymatrine from folium Camelliae sinensis
Extraction method 1: extracting with pure water;
extraction method 2: extracting with pure water after ammoniation;
extraction method 3: extracting trichloromethane after ammoniation;
through comparison, the direct extraction effect of pure water is poor, the effect is improved after ammonia water is added, and the effect is optimal when chloroform after ammoniation is used for extraction.
The packing and particle size of the chromatographic column can affect the separation degree of the components to be measured, wherein the results of comparison of three different types of chromatographic columns show that:
adopting ACQUITY UPLC HSS T3 with specification of 100 × 2.1mm and 1.8 μm chromatographic column, wherein the peak top of the target component is bifurcated, and the separation degree is poor;
separating with chromatographic column of 100 × 2.1mm and 1.7 μm, with wide peak and poor peak shape of target component;
the Hypersil GOLD C18 chromatographic column with the specification of 100 × 2.1mm and 1.9 μm has good retention capacity for target components, good peak shape and high separation degree.
Referring to fig. 4-5, the organic phase was compared with methanol and acetonitrile, and the results showed that methanol was used as the mobile phase, which showed higher response values than acetonitrile; the aqueous phase is compared by 0.1% formic acid aqueous solution and 5mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid), and the result shows that the extraction ion chromatography, ion fragment peak type and separation degree of matrine and oxymatrine are better by selecting methanol-5 mmol/L ammonium acetate (containing 0.1% formic acid) as the mobile phase.
Comparing the target response value of the negative matrix standard solution with the target response value of the pure solvent standard solution at the same concentration, and when the ratio is 1, indicating that no matrix effect exists; a ratio > 1 indicates matrix enhancement; when the ratio is less than 1, the matrix inhibition effect is shown; when the ratio is between 0.8 and 1.2, the matrix effect is generally considered to be weak and can be ignored. Selecting a negative tea sample, preparing a matrix sample solution obtained by the treatment of the corresponding steps into a matrix mixed standard solution with the concentration of 10 mu g/L, and detecting the matrix mixed standard solution with the same concentration prepared from pure methanol. The calculated ratio of the matrine is 20, the ratio of the oxymatrine is 3.7, and the result shows that the response value of the target substance is greatly influenced by matrix interference. Therefore, the blank matrix is adopted to dilute the standard solution for external standard method quantification so as to reduce the influence of matrix effect.
In the experiment, the blank matrix is gradually added to the lowest concentration which can be detected by the instrument, and the relative standard deviation of the parallel measured value of the concentration in a period of time is ensured to be lower, so that the detection lower limit of the method is determined. The result shows that the detection lower limit of the matrine and the oxymatrine is 0.01 mg/kg. The linear range, correlation coefficient, detection limit and regression equation of matrine and oxymatrine are shown in table 3:
TABLE 3 Linear Range, correlation coefficient, detection limits and regression equation for matrine and oxymatrine
Recovery and precision were determined at three levels of 10, 20, 100 μ g/kg added to the blank and the results are shown in table 4:
table 4 matrine and oxymatrine recovery rates with relative standard deviation [ n ═ 6)
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention without departing from the technical spirit of the present invention are within the scope of the present invention.
Claims (9)
1. A method for simultaneously and quantitatively detecting matrine and oxymatrine in tea is characterized by comprising the following steps:
s1, extracting the sample,
quantitatively weighing a tea sample, adding ammonia water into the sample, taking trichloromethane as an extraction reagent, extracting by adopting ultrasonic waves, and performing rotary evaporation until the sample is nearly dry to obtain a standard substance;
s2, preparing a standard curve solution,
adding methanol into the standard substance to a constant volume, and preparing a standard curve solution by adopting a stepwise dilution method;
s3, detecting the residual quantity of matrine and oxymatrine in the standard curve solution by adopting a high performance liquid chromatography-tandem triple quadrupole mass spectrometry technology.
2. The method for simultaneously and quantitatively detecting matrine and oxymatrine in tea leaves according to claim 1, wherein: in step s1, weighing 0.50g of tea sample, adding 0.5mL of ammonia water, standing for 30min, adding 20mL of chloroform, performing ultrasound for 30min, and repeating for at least 2 times; then 0.5g of anhydrous sodium sulfate is added and mixed evenly, and the whole solution is steamed to be nearly dry in a rotating way.
3. The method for simultaneously and quantitatively detecting matrine and oxymatrine in tea leaves according to claim 1, wherein: in a step s2, the method is carried out,
(1) weighing quantitative standard substances respectively, preparing into 1.0mg/mL standard stock solution with methanol, and storing at low temperature;
(2) taking a proper amount of each standard stock solution, and diluting the stock solution into a mixed standard solution of 1.0mg/L by using 5% methanol;
(3) diluting the sample blank matrix solution step by step to prepare a standard curve solution with the concentration of 2-100 ng/mL;
(4) filtering each standard curve solution through a filter membrane with the particle size of not more than 0.22 mu m, and detecting on a machine.
4. The method for simultaneously and quantitatively detecting matrine and oxymatrine in tea leaves according to claim 3, wherein: and diluting each mixed standard solution step by using a blank matrix solution to prepare standard curve solutions with the concentrations of 2, 5, 10, 20, 50 and 100 ng/mL.
5. The method for simultaneously and quantitatively detecting matrine and oxymatrine in tea leaves according to claim 4, wherein: weighing a proper amount of blank tea leaf sample, processing according to the sample extraction method in the step s1, and then adding 5% methanol to constant volume to obtain a blank matrix solution.
6. The method for simultaneously and quantitatively detecting matrine and oxymatrine in tea leaves according to claim 5, wherein: the standard stock solution was stored in an environment at-20 ℃.
7. The method for simultaneously and quantitatively detecting matrine and oxymatrine in tea leaves according to claim 1, wherein: in step s3, separating by adopting a Hypersil GOLD C18 column, carrying out gradient elution by using a mobile phase A and a mobile phase B, detecting by adopting an electrospray positive ion source and a multi-reaction monitoring mode by adopting a column temperature mass spectrum, and quantifying by adopting an external standard method;
wherein the mobile phase A is methanol, and the mobile phase B is ammonium acetate formic acid solution.
8. The method for simultaneously and quantitatively detecting matrine and oxymatrine in tea leaves according to claim 6, wherein: the ammonium acetate formic acid solution was a 5mmol/L aqueous ammonium acetate solution and contained 0.1% formic acid in the 5mmol/L aqueous ammonium acetate solution.
9. The method for simultaneously and quantitatively detecting matrine and oxymatrine in tea leaves according to claim 6, wherein: in step s3, the conditions for measuring the hplc-tandem triple quadrupole mass spectrometry are as follows:
(1) the high performance liquid chromatography is Hypersil GOLD C18, the specification is column length 100mm, column inner diameter 2.1mm, particle size 1.9 μm;
the column temperature is 30 ℃,
flow rate: 0.2 mL/min;
sample introduction amount: 10 mu L of the solution;
mobile phase conditions are shown in table 1:
TABLE 1 HPLC gradient elution
Operating time: 8 min;
(2) the mass spectrum conditions are as follows:
adopting an electrospray positive ion mode;
the scanning mode is as follows: monitoring multiple reactions;
temperature of the drying gas: 300 ℃;
flow rate of drying gas: 3L/min;
the flow rate of the sheath gas: 15L/min;
the capillary temperature is 350 ℃;
spraying voltage: 3200V;
the mass spectrum parameters in the multiple reaction monitoring mode are shown in Table 2
TABLE 2 Mass Spectrometry parameters in multiple reaction monitoring modes
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