CN117368360A - Method for simultaneously measuring residual amounts of gelsemium, koumine and koumine in edible tissues of pigs - Google Patents

Method for simultaneously measuring residual amounts of gelsemium, koumine and koumine in edible tissues of pigs Download PDF

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CN117368360A
CN117368360A CN202311374878.7A CN202311374878A CN117368360A CN 117368360 A CN117368360 A CN 117368360A CN 202311374878 A CN202311374878 A CN 202311374878A CN 117368360 A CN117368360 A CN 117368360A
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gelsemine
solution
sample
jian
solvent
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杨昆
邱荣超
胡佩佩
肖锐
曹俊杰
隆雪明
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Henan University of Animal Husbandry and Economy
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Henan University of Animal Husbandry and Economy
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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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • 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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    • 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
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material

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Abstract

The invention discloses a method for simultaneously measuring the residual quantity of gelsemine A, gelsemine and gelsemine in edible tissues of pigs, which comprises the following steps: preparing a reference substance solution by using gelsemine, koumine and an A Ma Jian standard substance, taking a pig edible tissue as a sample, performing ultrasonic extraction by using an acetonitrile solution containing formic acid to obtain an extract solution, concentrating, enriching by a solid phase extraction column, purifying, collecting an eluent, drying, redissolving, filtering to obtain a test substance solution, detecting the reference substance solution and the test substance solution by using an ultra-high liquid chromatography-mass spectrometry/mass spectrometry, and performing result treatment and quantitative analysis to obtain the residual quantity of the gelsemine, the koumine and the koumine in the pig edible tissue. The method utilizes the solid phase extraction-ultra-high liquid chromatography-mass spectrometry/mass spectrometry method, is simple to operate, has high sensitivity, is accurate and efficient, is environment-friendly, and has important significance for guaranteeing animal health and food safety.

Description

Method for simultaneously measuring residual amounts of gelsemium, koumine and koumine in edible tissues of pigs
Technical Field
The invention particularly relates to a method for simultaneously measuring the residual amounts of gelsemine, koumine and gelsemine in edible tissues of pigs.
Background
Gelsemium elegans is whole herb of the plant of the family loguaiaceae, also known as gelsemium elegans, kudzu vine, poison root, large tea medicine and the like, has bitter taste, is slightly pungent, is hot in nature and has large toxicity, and is mainly distributed in Zhejiang, fujian, guangdong, guangxi, hunan, guizhou, yunnan and other places. The application history of gelsemium elegans in China is very long, and the 'Shennong Bencao' records gelsemium elegans 'main golden sore, mastalgia, aversion to wind, cough with adverse qi rising, ghost'; the folk medicine has high toxicity, is mainly used for external application all the time, and can detumescence, detoxication, disinfestation and antipruritic; gelsemium has great toxicity to human, and it is reported that 10g of gelsemium stem and leaf or 2-8 g of gelsemium root can cause poisoning after being taken orally. However, small dosage of gelsemium elegans has the effect of promoting growth of some animals, such as people and livestock eating the gelsemium elegans in Tang Bencao, the sheep eating is fat and the pig eating can treat heat diseases; "Ben Cao gang mu" carried: "gelsemium elegans She Zhe is killed by people eating by mistake, and gelsemium elegans is large in fertilizer; the Chinese veterinary medicine in Guangdong is recorded in the following general herbal medicine: the gelsemium elegans can be taken regularly for pigs and sheep, so that the stomach can be invigorating, insecticidal and fattening can be achieved; the Guangxi traditional Chinese medicine is recorded in the description of 'feeding can promote appetite when the pig is poor in appetite'; thus, the gelsemium elegans is a relatively promising Chinese herbal medicine additive which can be developed into a Chinese herbal medicine additive for promoting animal growth.
In recent years, gelsemium elegans has been developed deeply at home and abroad, and the main component of gelsemium elegans, which is found to exert pharmacological action, is gelsemium elegans alkaloid; the gelsemium alkaloid is extracted, separated, purified and studied in structural aspect, and the highest content of the gelsemium alkaloid is found to be gelsemine, then gelsemine A, then gelsemine Heterophylla, and also contains indole alkaloids such as falcarinone, B, C, D and the like.
In order to ensure the edible safety of the edible tissues (muscles, livers and kidneys) of pigs and promote the development and application of Chinese herbal medicines at the same time so as to reduce the use of antibiotics for animals and further ensure the health and food safety of animals, a safety barrier is necessary to be arranged between the production of pigs and the supply of pork, and a reasonable drug holiday is set. The patent with publication number CN109655563A discloses a rapid detection method of gelsemium in an animal body, which comprises the following steps: taking toxic animal body fluid, preparing to-be-tested sample fluid, removing impurities from the to-be-tested sample fluid by using a deproteinizing agent, purifying, and then measuring by using a high performance liquid chromatography-mass spectrometry method. The patent with publication number CN108614052B discloses a method for simultaneously detecting gelsemine A, gelsemine and gelsemine in a biological sample based on two-dimensional liquid chromatography, which comprises the following steps: the method is suitable for qualitative and quantitative detection of gelsemine A, gelsemine B and gelsemine C in various biological samples (blood, urine and muscle), but the stability of 3 reference standard liquids and the influence of fluctuation of operation conditions on analysis results easily cause accuracy reduction.
At present, research on the rule of eliminating the residual of gelsemium component functional monomers in the edible tissues (muscles, livers and kidneys) of pigs is not seen, and a method for simultaneously and quantitatively analyzing trace gelsemium, gelsemium and gelsemium in the muscles, livers and kidneys of pigs by using internal standard substances is not seen.
Disclosure of Invention
Based on the defects of the prior art, the invention aims to provide a method for simultaneously measuring the residual quantity of gelsemine A, gelsemine and gelsemine in edible tissues of pigs, which is used for simultaneously and quantitatively analyzing trace gelsemine A, gelsemine and gelsemine in pig muscles, pig livers and pig kidneys by utilizing a solid phase extraction-ultra-high liquid chromatography-mass spectrometry/mass spectrometry method.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for simultaneously measuring the residual quantity of gelsemine, koumine and gelsemine in edible tissues of pigs comprises the following steps:
(1) Preparation of control solution
Respectively and accurately weighing gelsemine A, gelsemine and an A Ma Jian standard substance, dissolving and diluting with methanol to prepare each single reference substance stock solution, and storing at-30 to-15 ℃ in a dark place; then, a single reference substance stock solution of gelsemine, gelsemine and gelsemine is measured, and an aqueous solution containing methanol and formic acid is taken as a reference substance solvent to prepare a mixed reference substance standard solution with different concentrations through dilution and uniform mixing; measuring a single reference substance stock solution of the A Ma Jian, and diluting and uniformly mixing the single reference substance stock solution with a reference substance solvent to prepare an A Ma Jian reference substance standard solution with different concentrations;
(2) Preparation of test solutions
Sequentially adding an amantadine solution and an extracting solvent into a sample by taking a pig edible tissue as a sample and an acetonitrile solution containing formic acid as an extracting solvent, carrying out vortex for 20-40 s, carrying out ultrasonic treatment at 25-35 ℃ for 25-35 min, centrifuging for 4-6 min to obtain a primary extracting solution and residues, adding the extracting solvent into the residues, carrying out repeated vortex, ultrasonic treatment and centrifugal treatment to obtain a secondary extracting solution, combining the primary extracting solution and the secondary extracting solution, and centrifuging for 4-6 min to obtain a clear extracting solution;
drying the extract solution at 30+ -5deg.C under nitrogen flow or parallel evaporating to dryness to obtain solid extract; taking aqueous solution containing methanol and formic acid as an extract solvent, dissolving the solid extract by using the extract solvent, and swirling for 20-40 s to obtain liquid to be purified;
loading the liquid to be purified into an activated solid-phase extraction column, eluting with an aqueous solution containing methanol and formic acid, draining, eluting with methanol, collecting the eluent, and blow-drying the eluent under nitrogen flow to obtain a solid sample; taking an aqueous solution containing methanol and formic acid as a sample solvent, dissolving a solid sample by using the sample solvent, filtering by a filter membrane to obtain a sample solution, and filling the sample solution into a sample bottle for later use;
(3) Determination of the component to be measured in a sample
Taking the mixed reference substance standard solution prepared in the step (1) and the A Ma Jian reference substance standard solution, and sampling the sample solution prepared in the step (2), detecting and analyzing by using an ultra-high performance liquid chromatography tandem mass spectrometry in a multi-reaction monitoring mode, and quantifying the contents of gelsemine A, gelsemine and A Ma Jian in calculation according to the following formula;
the formula:
X＝(A×A is ×C s ×V)/(A s ×A i ×W)；
in the above formula:
x is the residual amount of gelsemine in the edible tissue sample of the pig to be detected, and the unit is mug/kg;
a is the peak area of gelsemine in the sample solution in the step (2);
A s peak area of gelsemine in the mixed reference standard solution in the step (1);
C s the concentration of gelsemine in the mixed reference standard solution in the step (1) is expressed in mug/L;
A i peak area of a Ma Jian added to the sample solution of step (2);
A is peak area of a Ma Jian in the a Ma Jian reference standard solution in the step (1);
v is the volume of the sample solution in the step (2), and the unit is mL;
w is the mass of the sample in the step (2), and the unit is g;
wherein the gelsemine refers to gelsemine A, gelsemine or gelsemine, A i Corresponding concentration of the A Ma Jian in the sample solution and A is The corresponding concentration of the A Ma Jian in the A Ma Jian reference standard solution is the same.
Preferably, the conditions for the ultra performance liquid chromatography in step (3) are: the chromatographic column is an XB-C18 chromatographic column, the temperature of the chromatographic column is 25-35 ℃, the mobile phase consists of a mobile phase A and a mobile phase B, an aqueous solution of formic acid with the volume concentration of 0.05-0.15% is used as the mobile phase A, acetonitrile is used as the mobile phase B, and gradient elution is carried out, wherein the flow rate is 0.2-0.4 mL/min.
Further, the chromatographic column adopts a Kinetex-XB-C18 chromatographic column, the specification of the chromatographic column is 100mm multiplied by 2.1mm, the aperture is 100A, and the particle size is 2.6 mu m; the gradient elution procedure was:
preferably, the mass spectrum is a tandem quadrupole ion trap mass spectrometer, and the mass spectrum measurement conditions are as follows: ESI is adopted as an electrospray ion source, MRM multi-reaction monitoring is adopted as a detection mode, a scanning mode is a positive ion mode, the electrospray voltage is 5000-6000V, and the ion source temperature is 450-550 ℃; the atomization air pressure is 48-52 psi, the auxiliary air pressure is 53-57 psi, the air curtain air pressure is 20-25 psi, the collision air pressure is 5-7 psi, the cluster removing voltage is 8-12V, and the deconvolution voltage is 13-17V.
Preferably, the concentration of the ama alkali solution in the step (2) is 190-210 mu g/L, and the adding amount of the ama alkali solution in each gram of sample is 47.5-52.5 mu L.
Preferably, the mass percentage of formic acid in the acetonitrile solution containing formic acid in the step (2) is 0.5-1.5%; when the extract solution is prepared, the addition amount of the extraction solvent is 4.5-5.5 mL per gram of sample based on the mass of the sample.
Preferably, the frequency of the ultrasonic wave in the step (2) is 95-105 Hz, and the rotating speed of the centrifugation is 5500-6500 rpm; when preparing the liquid to be purified, the volume of the extract solution is 1.8-2.2 times of the volume of the extract solvent.
Preferably, the solid phase extraction column in step (2) employsAn HLB 3cc (60 mg) Extraction Cartridges SPE column, the solid phase extraction column being sequentially activated with 3-4 mL of methanol and 3-4 mL of ultrapure water; and (3) when the liquid to be purified is loaded into the activated solid phase extraction column, the loading amount of the liquid to be purified is 4.5-5.5 mL.
Preferably, in the rinsing in the step (2), the usage amount of the rinsing liquid is 0.6-1 mL in terms of the loading volume of the liquid to be purified, and the usage amount of the rinsing liquid is 0.6-1 mL per mL of the liquid to be purified; and during elution, the dosage of the methanol is 1-1.6 mL of the eluent corresponding to each milliliter of the liquid to be purified according to the loading volume of the liquid to be purified.
Preferably, the reference substance solvent in the step (1), the extract solvent in the step (2), the sample solvent and the leacheate are prepared by uniformly mixing methanol and 0.05-0.15% formic acid aqueous solution according to the volume ratio of 10:88-92.
The gelsemine A, gelsemine and a Ma Jian related by the invention have the following structural formulas:
gelsemine (Gelsemine)
Gelsemine (Koumee)
Gelsemine (Humantenmine)
A Ma Jian (Ajmalice)
When the control solution is prepared, the purity of the gelsemium, the gelsemium and the al Ma Jian is more than 98 percent. The methanol, the acetonitrile and the formic acid used in the steps are chromatographic grade, and the water is ultrapure water; the filter membrane adopts a microporous filter membrane with the diameter of 0.22 mu m.
The technical scheme of the invention mainly comprises the following steps:
1) Pretreatment of edible tissues (muscle, liver and kidney) of pigs, comprisingSelection and addition of an internal standard, determination of a solution of 0.1% methanol in formic acid-water (10:90, v/v) in an acidic environment for 1% formic acid production, and developmentHLB 3cc (60 mg) Extraction Cartridges SPE column purification procedure;
2) Sample introduction (UPLC-MS/MS);
3) Electrospray (ESI) ionization was performed, and Multiplex Reaction (MRM) monitored for ion pair quantitative/confirmatory analysis;
4) The target analyte compounds described above include: gelsemine, koumine and gelsemine;
5) The extraction solvent uses chromatographic grade or ultrapure water, so that the interference of other impurities in the common organic solvent is avoided;
6) The UPLC-MS/MS method comprises the following steps: chromatographic column: a KinetexXB-C18100A chromatographic column with the specification of 100mm multiplied by 2.1mm and the particle size of 2.6 μm; the flow rate is: 0.30mL/min; mobile phase: phase a was 0.1% aqueous formic acid and phase B was acetonitrile, time gradient elution procedure: 0 to 2.00min,10 percent of acetonitrile, 2.00 to 6.00min,10 to 65 percent of acetonitrile, 6 to 8.00min,65 percent of acetonitrile, 8.01 to 10.00min,10 percent of acetonitrile; sample injection amount: 10. Mu.L; chromatographic column temperature: 25-35 ℃. Electrospray ion source (ESI): scanning positive ions, wherein the electrospray voltage is 5500V; ion source temperature: 500 ℃; the atomization air pressure is as follows: 50psi; the auxiliary air pressure is as follows: 55psi; the air pressure of the air curtain is as follows: 20-25 psi; the collision air pressure is: 6.0psi; the cluster removal voltage is 10V; the deconvolution voltage is: 15V;
7) Qualitative validation analysis method: processing the standard sample by the same method as the actual sample, and recording the retention time of each substance;
8) In the quantitative analysis, an internal standard method is adopted for quantification;
9) In the invention, when a chromatographic column is selected, a Phenomenex Kinetex C column (100 multiplied by 2.1mm,2.6 mu m) is selected, and three equivalent chromatographic columns of Inertsil ODS-3 (150 multiplied by 2.1mm,4 mu m) and ZORBAX Eclipse C18 (150 multiplied by 2.1mm,3.5 mu m) are tested under the condition of 0.1 percent of formic acid-water and acetonitrile mobile phase; the result shows that the Kinetex C18 column can ensure that gelsemine, koumine, gelsemine and A Ma Jian are well separated, and the peak shape is symmetrical and sharp;
10 Optimization of extraction solvent: in the experiment, methanol, acetonitrile, 1% hydrochloric acid-water, mclvaine (4.0), methanol-water (1:1, v/v), acetonitrile-water (1:1, v/v), formic acid/ammonium acetate (the addition amounts are respectively 0.1%, 0.5% and 1%) and acetonitrile are selected as extraction solvents for extraction, and the extraction efficiencies of all the extraction agents are compared; the result shows that the 1% formic acid-acetonitrile is a good extraction solvent, and can ensure that the recovery rate of gelsemine A, gelsemine and gelsemine is high;
11 Solid phase extraction cartridge selection): according to the molecular structure characteristics of gelsemine, gelsemine and gelsemine, the experiment adopts C18 solid phase extraction columns (SPE columns) of different manufacturers, and the purifying treatment effects of HLB, varian Bond Elute Plexa C18 and clearart ODS C18 are compared with the extraction efficiency of each column; the result shows that the HLB purifying treatment effect is good for gelsemine, koumine and gelsemine, and the standard recovery rate result basically meets the requirement;
12 The method has the following advantages in determining the residual amounts of gelsemine, koumine and gelsemine in edible tissues (pig muscle, liver and kidney) of pigs: first, the invention can detect the trace amount of gelsemine, koumine and gelsemine existing in pork, pig liver and kidney. In the investigation process of the invention, the detection limit of the analyzed gelsemium, gelsemium and gelsemium is 0.5 mug/L. And secondly, the pretreatment process of the method is simple and effective, the treatment time is short, the reagent dosage is small, and the method is an environment-friendly pretreatment technology.
In summary, the invention utilizes solid phase extraction-ultra-high liquid chromatography-mass spectrometry/mass spectrometry to measure gelsemine, koumine and gelsemine in edible pig tissues (muscle, liver and kidney), establishes a method for simultaneously measuring the residual amount of the gelsemine, the koumine and the gelsemine in the edible pig tissues, ultrasonically extracts a sample by using an acetonitrile solution containing formic acid, weathers and concentrates nitrogen, purifies by a solid phase extraction column, uses a Kinetex XB-C18100A chromatographic column for separation under the gradient elution condition, and adopts a multi-reaction monitoring mode (MRM scan) to compare information with retention time and quantitative/qualitative ion pairs to carry out quantitative/confirmatory analysis on a target compound.
Compared with the prior art, the invention has the following beneficial effects: according to the invention, the A Ma Jian is adopted as an internal standard for quantification, and because the A Ma Jian has similar structure, similar polarity, stable performance and no existence in a sample, and does not cross react with gelsemine, the detection result is not interfered, the A Ma Jian is cheap and easy to obtain, and is more environment-friendly and economical, and therefore, the A Ma Jian is finally preferred as the internal standard. According to the invention, according to the structural characteristics of the components to be detected and impurity components in the edible tissues of pigs, the conditions of ultra-high performance liquid chromatography and mass spectrometry are optimized through a large number of experiments, and pure substance A Ma Jian reference substances which are not existing in samples and have stable performance, low cost and easy acquisition are adopted as internal standard substances.
Drawings
FIG. 1 is a schematic diagram of an analysis flow of a sample of the present invention;
FIG. 2 is a second order mass spectrum of gelsemine A as described in example 1;
FIG. 3 is a second order mass spectrum of gelsemine as described in example 1;
FIG. 4 is a secondary mass spectrum of gelsemine as described in example 1;
fig. 5 is a total ion flow diagram of the control solutions of gelsemine, gelsemine and a Ma Jian concentration of 1 μg/L described in example 1, which are gelsemine (5.25 min), gelsemine (5.70 min), gelsemine (5.92 min) and a Ma Jian (6.70 min) in order from left to right according to the time axis.
The upper letters and the horizontal and vertical coordinates in fig. 3-5 are all automatically generated by the instrument, and have no influence on the determination of the technical scheme and the display of the technical effect.
Detailed Description
In order to make the technical objects, technical solutions and advantageous effects of the present invention more apparent, the technical solutions of the present invention will be further described with reference to specific examples, which are intended to illustrate the present invention but are not to be construed as limiting the present invention, and specific techniques or conditions are not specified in the examples, and are performed according to techniques or conditions described in the literature in the art or according to the product specifications.
The apparatus used in the following examples included: AB Sciex4500 ultra performance liquid chromatography tandem quadrupole ion trap mass spectrometer (AB SCIEX), PL203 electronic analytical balance (mertrex), ME204E one ten thousandth balance (mertrex), MX-F vortex mixer (SCILOGEX), X1R centrifuge (Thermo), BX 3200HP sonicator (new Miao), pipette gun: 100. Mu.L, 200. Mu.L, 1000. Mu.L (Eppendorf).
The materials and samples for use in the following examples include: the gelsemine, gelsemine seed, gelsemine and a Ma Jian standard substance are purchased from Shanghai standard biological limited company respectively, and the purity is more than 98 percent; methanol, acetonitrile (LC/MS grade), formic acid (LC/MS grade), ultrapure water (Millipore); the muscle, liver and kidney of the edible pig tissue are from Liuyang of Wufeng corporation.
A method for simultaneously measuring the residual quantity of gelsemine, koumine and gelsemine in edible tissues of pigs comprises the following steps:
(1) Preparation of control solution
Respectively and accurately weighing gelsemine A, gelsemine and an A Ma Jian standard substance, dissolving and diluting with methanol to prepare each single reference substance stock solution, and storing at-30 to-15 ℃ in a dark place; then, a single reference substance stock solution of gelsemine, gelsemine and gelsemine is measured, an aqueous solution containing methanol and formic acid (methanol: 0.1% formic acid-water (10:90, v/v)) is taken as a reference substance solvent, and mixed reference substance standard solutions with different concentrations are prepared through dilution and uniform mixing; measuring a single reference substance stock solution of the A Ma Jian, and diluting and uniformly mixing the single reference substance stock solution with a reference substance solvent to prepare an A Ma Jian reference substance standard solution with different concentrations;
(2) Preparation of test solutions
(2.1) extraction
Taking pig edible tissue as a sample, taking an acetonitrile solution (1.0% formic acid-acetonitrile) containing formic acid as an extraction solvent, sequentially adding 190-210 mug/L of amantadine solution (the adding amount of the amantadine solution in each gram of the sample is 47.5-52.5 mug) and the extraction solvent (the adding amount of the amantadine solution in each gram of the sample is 4.5-5.5 mL), carrying out vortex 20-40 s, carrying out ultrasonic treatment at 25-35 ℃ for 25-35 min (the frequency is 95-105 Hz), centrifuging for 4-6 min (the rotating speed is 5500-6500 rpm) to obtain primary extract and residues, adding the extraction solvent (the adding amount of the amantadine solution in each gram of the sample is 4.5-5.5 mL), carrying out repeated vortex, ultrasonic treatment and centrifugal treatment to obtain secondary extract, and carrying out centrifugal treatment after the primary extract and the secondary extract are combined, and carrying out centrifugal treatment for 4-6 min to obtain clear extract solution;
(2.2) concentrating
Drying the extract solution at 30+ -5deg.C under nitrogen flow or parallel evaporating to dryness to obtain solid extract; taking aqueous solution containing methanol and formic acid as an extract solvent, dissolving the solid extract by using the extract solvent, and swirling for 20-40 s to obtain liquid to be purified; wherein the volume of the extract solution is 1.8-2.2 times of the volume of the extract solvent;
(2.3) purification
Taking outActivating an SPE column by using 2-4 mL of methanol and 2-4 mL of ultrapure water in sequence through an HLB 3cc (60 mg) Extraction Cartridges SPE column, loading 4.5-5.5 mL of to-be-purified liquid into the activated SPE column, eluting with 3-5 mL of aqueous solution containing methanol and formic acid (methanol: 0.1% formic acid-water (10:90, v/v)) as eluent, draining, eluting with 5-8 mL of methanol, collecting eluent, and blow-drying the eluent under nitrogen flow to obtain a solid test product; taking aqueous solution containing methanol and formic acid (methanol: 0.1% formic acid-water (10:90, v/v)) as sample solvent, dissolving solid sample with the sample solvent, and passing through 0:filtering with 22 μm microporous membrane to obtain sample solution, and filling into sample bottle;
(3) Determination of the component to be measured in a sample
Taking the mixed reference substance standard solution prepared in the step (1) and the A Ma Jian reference substance standard solution, and sampling the sample solution prepared in the step (2), and performing detection analysis by using an ultra-high performance liquid chromatography-tandem mass spectrometry under a multi-reaction monitoring mode, wherein parameters are set as follows:
(3.1) UPLC Condition
The chromatographic column adopts a Kinetex-XB-C18 chromatographic column, the specification of the chromatographic column is 100mm multiplied by 2.1mm, the aperture is 100A, and the particle size is 2.6 mu m; the sample injection amount is 10 mu L; the chromatographic column temperature is 25-35 ℃, the mobile phase consists of a mobile phase A and a mobile phase B, an aqueous solution of formic acid with the volume concentration of 0.05-0.15% is taken as the mobile phase A, acetonitrile is taken as the mobile phase B, and gradient elution is carried out, wherein the gradient elution comprises the following procedures:
the method comprises the steps of carrying out a first treatment on the surface of the The flow rate is 0.2-0.4 mL/min;
(3.2) MS conditions
ESI is adopted as an electrospray ion source, MRM multi-reaction monitoring is adopted as a detection mode, a scanning mode is a positive ion mode, the electrospray voltage is 5000-6000V, and the ion source temperature is 450-550 ℃; the atomization air pressure is 48-52 psi, the auxiliary air pressure is 53-57 psi, the air curtain air pressure is 20-25 psi, the collision air pressure is 5-7 psi, the cluster removing voltage is 8-12V, and the deconvolution voltage is 13-17V;
the contents of gelsemine, koumine, gelsemine and a Ma Jian in the calculation by the following formula were quantified:
the formula:
X＝(A×A is ×C s ×V)/(A s ×A i ×W)；
in the above formula:
x is the residual amount of gelsemine in the edible tissue sample of the pig to be detected, and the unit is mug/kg;
a is the peak area of gelsemine in the sample solution in the step (2);
A s peak area of gelsemine in the mixed reference standard solution in the step (1);
C s the concentration of gelsemine in the mixed reference standard solution in the step (1) is expressed in mug/L;
A i peak area of a Ma Jian added to the sample solution of step (2);
A is peak area of a Ma Jian in the a Ma Jian reference standard solution in the step (1);
v is the volume of the sample solution in the step (2), and the unit is mL;
w is the mass of the sample in the step (2), and the unit is g;
wherein the gelsemine refers to gelsemine A, gelsemine or gelsemine, A i Corresponding concentration of the A Ma Jian in the sample solution and A is The corresponding concentration of the A Ma Jian in the A Ma Jian reference standard solution is the same.
Example 1
A method for simultaneously measuring the residual quantity of gelsemine, koumine and gelsemine in edible tissues of pigs comprises the following steps:
(1) Preparation of control solution
Respectively accurately weighing 10mg of gelsemine A, 10mg of gelsemine seed, 10mg of gelsemine hexyl and 10mg of gelsemine A Ma Jian standard substances, placing into a 10mL volumetric flask, dissolving and diluting with methanol, and fixing the volume to prepare each single reference substance stock solution with the concentration of 1mg/mL, and storing at the temperature of minus 20 ℃ in a dark place;
then, a single reference substance stock solution of gelsemine, gelsemine and gelsemine is measured, an aqueous solution containing methanol and formic acid (methanol: 0.1% formic acid-water (10:90, v/v)) is taken as a reference substance solvent, and mixed reference substance standard solutions with different concentrations are prepared by dilution and uniform mixing, wherein the concentration series are 0.5 mug/L, 1.0 mug/L, 5.0 mug/L, 10.0 mug/L, 20.0 mug/L, 50.0 mug/L and 100.0 mug/L;
measuring a single reference substance stock solution of the A Ma Jian, diluting and uniformly mixing the single reference substance stock solution with a reference substance solvent to prepare reference substance standard solutions of the A Ma Jian with different concentrations, wherein the concentration series are 0.5 mug/L, 1.0 mug/L, 5.0 mug/L, 10.0 mug/L and 20.0 mug/L;
(2) Preparation of test solutions
(2.1) extraction
Taking pig edible tissue as a sample, taking an acetonitrile solution (1.0% formic acid-acetonitrile) containing formic acid as an extraction solvent, sequentially adding 100 mu L of ama alkaline solution (with the concentration of 200 mu g/L) and 10mL of the extraction solvent into 2.00g of the sample, carrying out vortex for 30s, carrying out ultrasonic treatment at 30 ℃ for 30min (with the ultrasonic frequency of 100 Hz), centrifuging for 5min (with the rotating speed of 6000 rpm) to obtain primary extract and residues, adding 10mL of the extraction solvent into the residues, carrying out vortex, ultrasonic treatment and centrifuging treatment repeatedly to obtain secondary extract, combining the primary extract and the secondary extract, and centrifuging for 5min (with the rotating speed of 6000 rpm) to obtain clear extract solution;
(2.2) concentrating
Accurately transferring 10mL of the extract solution into a parallel nitrogen blowing pipe by using a 10.0mL pipette or a pipettor, and drying or evaporating the extract solution in parallel under nitrogen flow at 30 ℃ to obtain a solid extract; taking aqueous solution containing methanol and formic acid as an extract solvent, dissolving the solid extract by using 5mL of the extract solvent, and swirling for 30s to obtain liquid to be purified;
(2.3) purification
Taking outActivating an SPE column by using 3mL of methanol and 3mL of ultrapure water in sequence through an HLB 3cc (60 mg) Extraction Cartridges SPE column, loading 5mL of to-be-purified liquid into the activated SPE column, taking 4mL of aqueous solution containing methanol and formic acid (methanol: 0.1% formic acid-water (10:90, v/v)) as eluent, eluting, pumping, eluting by using 6.5mL of methanol, collecting eluent, and blow-drying the eluent under a nitrogen flow to obtain a solid test product; taking aqueous solution containing methanol and formic acid (methanol: 0.1% formic acid-water (10:90, v/v)) as a sample solvent, dissolving solid sample with 1mL of the sample solvent, filtering with a 0.22 μm microporous filter membrane to obtain sample solution, and filling into a sample bottle (1.5 mL liquid phase sample injection vial);
(3) Determination of the component to be measured in a test sample
Taking the mixed reference substance standard solution prepared in the step (1) and the A Ma Jian reference substance standard solution, and sampling the sample solution prepared in the step (2), and performing detection analysis by using an ultra-high performance liquid chromatography-tandem mass spectrometry under a multi-reaction monitoring mode, wherein parameters are set as follows:
(3.1) UPLC Condition
The chromatographic column adopts a Kinetex-XB-C18 chromatographic column, the specification of the chromatographic column is 100mm multiplied by 2.1mm, the aperture is 100A, and the particle size is 2.6 mu m; the sample injection amount is 10 mu L; the chromatographic column temperature is 25-35 ℃, the mobile phase consists of a mobile phase A and a mobile phase B, an aqueous solution of formic acid with the volume concentration of 0.1% is used as the mobile phase A, acetonitrile is used as the mobile phase B, and gradient elution is carried out, wherein the gradient elution procedure is as follows:
the method comprises the steps of carrying out a first treatment on the surface of the The flow rate is 0.3mL/min;
(3.2) MS conditions
ESI is adopted as an electrospray ion source, MRM multi-reaction monitoring is adopted as a detection mode, a scanning mode is a positive ion mode, 5500V is adopted as an electrospray voltage, and the temperature of the ion source is 500 ℃; the atomization air pressure is 50psi, the auxiliary air pressure is 55psi, the air curtain air pressure is 25psi, the collision air pressure is 6psi, the declustering voltage is 10V, and the deconvolution voltage is 15V;
the contents of gelsemine, koumine, gelsemine and a Ma Jian in the calculation by the following formula were quantified:
the formula:
X＝(A×A is ×C s ×V)/(A s ×A i ×W)；
in the above formula:
x is the residual amount of gelsemine in the edible tissue sample of the pig to be detected, and the unit is mug/kg;
a is the peak area of gelsemine in the sample solution in the step (2);
A s peak area of gelsemine in the mixed reference standard solution in the step (1);
C s the concentration of gelsemine in the mixed reference standard solution in the step (1) is expressed in mug/L;
A i peak area of a Ma Jian added to the sample solution of step (2);
A is peak area of a Ma Jian in the a Ma Jian reference standard solution in the step (1);
v is the volume of the sample solution in the step (2), and the unit is mL;
w is the mass of the sample in the step (2), and the unit is g;
wherein, the gelsemine refers to gelsemine, koumine or gelsemine;
A i corresponding concentration of the A Ma Jian in the sample solution and A is The corresponding concentration of the A Ma Jian in the A Ma Jian reference standard solution is the same (namely, according to the addition amount of the Amaranthin solution in the sample, the concentration of the A Ma Jian in the sample solution is calculated to be C i Detecting the peak area of the A Ma Jian in the sample solution as A i Re-detection of concentration C i The peak area of the A Ma Jian in the A Ma Jian reference standard solution is A is Or calculating peak area A of the A Ma Jian in the A Ma Jian reference standard solution according to the A Ma Jian standard curve is )。
In the above steps, the analysis flow of the sample is shown in fig. 1, and the process is as follows:
1) 2.00+ -0.05 g of sample was added to a 50mL polytetrafluoroethylene centrifuge tube with plug;
2) 100. Mu.L of Amaranthaceae alkali solution with the concentration of 200. Mu.g/L is added;
3) 10mL of 1% formic acid-acetonitrile solution was added and vortexed for 30s;
4) Carrying out ultrasonic treatment at 30 ℃ for 30min, wherein the ultrasonic frequency is 100Hz;
5) Taking out from the ultrasonic machine;
6) Centrifuging at 6000rpm for 5min;
7) Taking out from the centrifugal machine;
8) Separating out primary extract and residue;
9) Adding 10mL of 1% formic acid-acetonitrile solution into the residue, repeatedly vortex, ultrasonically treating and centrifuging, and separating out secondary extract;
10 Combining the primary extract and the secondary extract, wherein the solution becomes turbid, and the solution becomes clear after centrifugation at 6000rpm for 5min by using a centrifuge, namely an extract solution;
11 Accurately transferring 10mL of the extract solution with a 10.0mL pipette or pipette;
12 Drying with nitrogen flow at 30 ℃;
13 5mL of methanol and 0.1% formic acid-water (10:90, v/v) are used for dissolution, and vortex is carried out for 30s to obtain liquid to be purified;
14 TakingAn HLB 3cc (60 mg) Extraction Cartridges SPE column, activating an SPE column with 3mL of methanol and 3mL of ultrapure water in sequence, loading 5mL of the solution to be purified into the activated SPE column, eluting with 4mL of methanol and 0.1% formic acid-water (10:90, v/v), draining, and eluting with 6.5mL of methanol;
15 Collecting the eluent;
16 Drying the eluent under nitrogen flow;
17 1mL of methanol, 0.1% formic acid-water (10:90, v/v), and passing through a microporous filter membrane;
18 Filling the sample into a sample bottle for standby;
19 And (3) absorbing 10 mu L of the solution by using an automatic microsyringe to perform UPLC-MS/MS analysis, and finally quantifying the contents of the gelsemine, the gelsemine and the a Ma Jian in the liquid sample to be detected by calculating the respective peak areas, so as to calculate the residual quantity of the gelsemine, the gelsemine and the gelsemine in the sample.
The results and analysis were as follows:
the mass spectrometry parameters are shown in table 1.
Table 1 mass spectrometry parameters of gelsemium, koumine and gelsemium and aj Ma Jian
Note that: * Quantitative ion pairs
The secondary mass spectrograms of the gelsemine, the gelsemine and the gelsemine are shown in figures 2-4, the reference standard solution and the 1.0 mug/L A Ma Jian reference standard solution are mixed together for detection, and the total ion flow diagram of the gelsemine, the gelsemine and the Amara alkali solution is shown in figure 5.
Because of matrix effect in tandem mass spectrometry detection, the inhibition rates of matrix effect of different substances in different tissues are inconsistent, and standard curves after correction corresponding to an internal standard method are inconsistent. In order to more accurately quantify, separate measurement and calculation are carried out on different substances of different tissues; the peak areas of the quantitative ions of gelsemine, gelsemine and gelsemine are taken as the ordinate, the corresponding concentration values of the standard solution are taken as the abscissa, the internal standard is used for correction, the standard curve is fitted, and the regression equation and the correlation coefficient are calculated, wherein the specific steps are as follows:
(1) Establishing a gelsemine A, gelsemine B and gelsemine hexyl standard curve according to the peak areas corresponding to the mixed reference standard solutions with concentration series of 0.5 mug/L, 1.0 mug/L, 5.0 mug/L, 10.0 mug/L, 20.0 mug/L, 50.0 mug/L and 100.0 mug/L;
(2) An A Ma Jian standard curve is established according to the peak areas corresponding to the A Ma Jian reference standard solutions with concentration series of 0.5 mug/L, 1.0 mug/L, 5.0 mug/L, 10.0 mug/L and 20.0 mug/L;
(3) Taking samples of pig muscle, pig liver and pig kidney which do not contain gelsemine, gelsemine and gelsemine, adding gelsemine, gelsemine and gelsemine according to a certain concentration, adding a certain concentration of alpha Ma Jian (before detection, adding internal standard substances with the same concentration in the same batch for each sample to be detected so as to eliminate the loss of the sample in the treatment process), detecting according to the analysis flow undergone by the samples, and reusing a formula (X= (A multiplied by A) is ×C s ×V)/(A s ×A i X W)) was calculated and the results are shown in table 2.
TABLE 2 Linear Range, linear equation and correlation coefficient
Taking samples of pig muscle, pig liver and pig kidney which do not contain gelsemine, gelsemine and gelsemine, respectively carrying out an addition and recovery experiment, determining accuracy and daily/daytime precision, adding standard solutions with different concentrations into the samples, carrying out the experiment according to the method, and carrying out sample injection determination by using UPLC-MS/MS. The average recovery rate in the range of 3 addition levels of low, medium and high was expressed as accuracy by the recovery rate, each concentration was measured in parallel 6 times and the daytime precision was measured for 5 consecutive days, and the results are shown in tables 3 to 5.
TABLE 3 accuracy and precision of gelsemium A in pig muscle, liver and kidney
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TABLE 4 accuracy and precision of gelsemins in porcine muscle, liver and kidney
TABLE 5 accuracy and precision of gelsemium elegans in pig muscle, liver and kidney
From tables 3 to 5, it can be seen that gelsemine, koumine and gelsemine are in the edible tissues (muscle, liver and kidney) of pigs, the accuracy (n=6) is between 80 and 120 percent within the addition recovery range (1.0 to 50 ng/mL), the daily precision and the daily precision are less than or equal to 15 percent, and the method meets the standards formulated by national residue detection methodology investigation, and is higher in accuracy, good in precision and stable in performance.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that any modifications, equivalents, modifications, and the like, which would occur to persons skilled in the art without departing from the principles of the present invention, are intended to be included within the scope of the present invention.

Claims (10)

1. The method for simultaneously measuring the residual amounts of gelsemine, koumine and gelsemine in the edible tissues of pigs is characterized by comprising the following steps:
(1) Preparation of control solution
Respectively and accurately weighing gelsemine A, gelsemine and an A Ma Jian standard substance, dissolving and diluting with methanol to prepare each single reference substance stock solution, and storing at-30 to-15 ℃ in a dark place; then, a single reference substance stock solution of gelsemine, gelsemine and gelsemine is measured, and an aqueous solution containing methanol and formic acid is taken as a reference substance solvent to prepare a mixed reference substance standard solution with different concentrations through dilution and uniform mixing; measuring a single reference substance stock solution of the A Ma Jian, and diluting and uniformly mixing the single reference substance stock solution with a reference substance solvent to prepare an A Ma Jian reference substance standard solution with different concentrations;
(2) Preparation of test solutions
Sequentially adding an amantadine solution and an extracting solvent into a sample by taking a pig edible tissue as a sample and an acetonitrile solution containing formic acid as an extracting solvent, carrying out vortex for 20-40 s, carrying out ultrasonic treatment at 25-35 ℃ for 25-35 min, centrifuging for 4-6 min to obtain a primary extracting solution and residues, adding the extracting solvent into the residues, carrying out repeated vortex, ultrasonic treatment and centrifugal treatment to obtain a secondary extracting solution, combining the primary extracting solution and the secondary extracting solution, and centrifuging for 4-6 min to obtain a clear extracting solution;
drying the extract solution at 30+ -5deg.C under nitrogen flow or parallel evaporating to dryness to obtain solid extract; taking aqueous solution containing methanol and formic acid as an extract solvent, dissolving the solid extract by using the extract solvent, and swirling for 20-40 s to obtain liquid to be purified;
loading the liquid to be purified into an activated solid-phase extraction column, eluting with an aqueous solution containing methanol and formic acid, draining, eluting with methanol, collecting the eluent, and blow-drying or parallel evaporating the eluent under nitrogen flow to obtain a solid sample; taking an aqueous solution containing methanol and formic acid as a sample solvent, dissolving a solid sample by using the sample solvent, filtering by a filter membrane to obtain a sample solution, and filling the sample solution into a sample bottle for later use;
(3) Determination of the component to be measured in a sample
Taking the mixed reference substance standard solution prepared in the step (1) and the A Ma Jian reference substance standard solution, and sampling the sample solution prepared in the step (2), detecting and analyzing by using an ultra-high performance liquid chromatography tandem mass spectrometry in a multi-reaction monitoring mode, and quantifying the contents of gelsemine A, gelsemine and A Ma Jian in calculation according to the following formula;
the formula:
X＝(A×A is ×C s ×V)/(A s ×A i ×W)；
in the above formula:
x is the residual amount of gelsemine in the edible tissue sample of the pig to be detected, and the unit is mug/kg;
a is the peak area of gelsemine in the sample solution in the step (2);
A s peak area of gelsemine in the mixed reference standard solution in the step (1);
C s the concentration of gelsemine in the mixed reference standard solution in the step (1) is expressed in mug/L;
A i peak area of a Ma Jian added to the sample solution of step (2);
A is peak area of a Ma Jian in the a Ma Jian reference standard solution in the step (1);
v is the volume of the sample solution in the step (2), and the unit is mL;
w is the mass of the sample in the step (2), and the unit is g;
wherein the gelsemine refers to gelsemine A, gelsemine or gelsemine, A i Corresponding concentration of the A Ma Jian in the sample solution and A is The corresponding concentration of the A Ma Jian in the A Ma Jian reference standard solution is the same.
2. The method for simultaneously determining the residual amounts of gelsemine and gelsemine in edible tissues of pigs according to claim 1, wherein the conditions for the ultra performance liquid chromatography determination in the step (3) are as follows: the chromatographic column is an XB-C18 chromatographic column, the temperature of the chromatographic column is 25-35 ℃, the mobile phase consists of a mobile phase A and a mobile phase B, an aqueous solution of formic acid with the volume concentration of 0.05-0.15% is used as the mobile phase A, acetonitrile is used as the mobile phase B, and gradient elution is carried out, wherein the flow rate is 0.2-0.4 mL/min.
3. The method for simultaneously determining the residual amounts of gelsemine and gelsemine in edible tissues of pigs according to claim 2, which is characterized in that: the chromatographic column adopts a Kinetex-XB-C18 chromatographic column, the specification of the chromatographic column is 100mm multiplied by 2.1mm, the aperture is 100A, and the particle size is 2.6 mu m; the gradient elution procedure was:
。
4. the method for simultaneously determining the residual amounts of gelsemine and gelsemine in edible tissues of pigs according to claim 1, which is characterized in that: the mass spectrum adopts a tandem quaternary rod ion trap mass spectrometer, and the mass spectrum measurement conditions are as follows: ESI is adopted as an electrospray ion source, MRM multi-reaction monitoring is adopted as a detection mode, a scanning mode is a positive ion mode, the electrospray voltage is 5000-6000V, and the ion source temperature is 450-550 ℃; the atomization air pressure is 48-52 psi, the auxiliary air pressure is 53-57 psi, the air curtain air pressure is 20-25 psi, the collision air pressure is 5-7 psi, the cluster removing voltage is 8-12V, and the deconvolution voltage is 13-17V.
5. The method for simultaneously determining the residual amounts of gelsemine and gelsemine in edible tissues of pigs according to claim 1, which is characterized in that: the concentration of the ama alkali solution in the step (2) is 190-210 mu g/L, and the adding amount of the ama alkali solution in each gram of sample is 47.5-52.5 mu L.
6. The method for simultaneously determining the residual amounts of gelsemine and gelsemine in edible tissues of pigs according to claim 5, which is characterized in that: the mass percentage of formic acid in the acetonitrile solution containing formic acid in the step (2) is 0.5-1.5%; when the extract solution is prepared, the addition amount of the extraction solvent is 4.5-5.5 mL per gram of sample based on the mass of the sample.
7. The method for simultaneously determining the residual amounts of gelsemine and gelsemine in edible tissues of pigs according to claim 6, which is characterized in that: the frequency of the ultrasonic wave in the step (2) is 95-105 Hz, and the rotating speed of the centrifugation is 5500-6500 rpm; when preparing the liquid to be purified, the volume of the extract solution is 1.8-2.2 times of the volume of the extract solvent.
8. The method for simultaneously determining the residual amounts of gelsemine and gelsemine in edible tissues of pigs according to claim 7, which is characterized in that: the solid phase extraction column in the step (2) adoptsAn HLB 3cc 60mg Extraction Cartridges SPE column, wherein the solid phase extraction column is sequentially activated by 3-4 mL of methanol and 3-4 mL of ultrapure water; and (3) when the liquid to be purified is loaded into the activated solid phase extraction column, the loading amount of the liquid to be purified is 4.5-5.5 mL.
9. The method for simultaneously determining the residual amounts of gelsemine and gelsemine in edible tissues of pigs according to claim 8, which is characterized in that: when the leaching in the step (2) is carried out, the using amount of the leaching solution is calculated by the loading volume of the liquid to be purified, and the using amount of the leaching solution corresponding to each milliliter of the liquid to be purified is 0.6-1 mL; and during elution, the dosage of the methanol is 1-1.6 mL of the eluent corresponding to each milliliter of the liquid to be purified according to the loading volume of the liquid to be purified.
10. The method for simultaneously determining the residual amounts of gelsemine and gelsemine in edible tissues of pigs according to any one of claims 1 to 9, characterized in that: the reference substance solvent in the step (1), the extract solvent in the step (2), the test substance solvent and the leacheate are prepared by uniformly mixing methanol and 0.05-0.15% formic acid aqueous solution according to the volume ratio of 10:88-92.
CN202311374878.7A 2023-10-23 2023-10-23 Method for simultaneously measuring residual amounts of gelsemium, koumine and koumine in edible tissues of pigs Pending CN117368360A (en)

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