CN111735892A - Method for measuring multiple diuretics in animal derived food - Google Patents
Method for measuring multiple diuretics in animal derived food Download PDFInfo
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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
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The application discloses a method for detecting the content of diuretic in animal derived food, which comprises the following steps: step 1, homogenizing the animal-derived food; step 2, sampling according to a preset amount from the homogenized animal-derived food; step 3, uniformly mixing the sample with a matched amount of an extracting agent, performing centrifugal treatment, and taking supernatant; step 4, mixing the obtained supernatant with a purifying agent in a matched amount, carrying out secondary centrifugation treatment, taking all the supernatant and nitrogen to be blown to be nearly dry, adding a water-0.1% formic acid methanol solution to dissolve residues, and filtering the obtained solution to be used as a solution to be detected; and 5, injecting the solution to be detected into a liquid chromatogram-tandem mass spectrometer for detection, recording a chromatogram, and performing diuretic determination based on the chromatogram and the mass spectrum. By adopting the technical scheme, the sensitivity of the diuretic detection method is improved, and the high-sensitivity detection of the diuretic in the animal-derived food is realized.
Description
Technical Field
The application relates to the field of food safety detection, in particular to a method for determining multiple diuretics in pork, beef, mutton, milk and eggs.
Background
Diuretics are commonly used drugs in clinical practice and are widely used for the treatment of diuresis, detumescence, hypotension, cerebral edema and the like. In recent years, in livestock breeding and disease treatment, diuretics are used by many farmers, and therefore, they may remain in many animal-derived foods. Diuretics, when introduced into the body, can directly or indirectly cause changes in renal function and histology, resulting in decreased renal blood flow and decreased glomerular filtration rate through mechanisms such as decreased extracellular volume, altered tone of the ball and ball arterioles, and ball-tube feedback. It can also cause acute interstitial nephritis, renal tubule obstruction, etc., and chronic change of renal calculus, urinary sediment abnormality and low potassium nephropathy caused by long-term intake of diuretic, and has strong nephrotoxic effect. The detection method of the diuretic mainly adopts the high performance liquid chromatography and liquid chromatography-mass spectrometry combined technology, and the detection objects mainly comprise urine and blood of human bodies and animals.
At present, the import and export industry standards exist in China to detect the diuretic in the animal-derived food, but the detection limit is higher, and along with the emphasis of people on food safety, a detection method with lower detection limit must be continuously developed to adapt to the requirement on food safety detection; in the literature, studies on the metabolic mechanism and physiological toxicity of diuretics in organisms as drugs and the influence on the biological functions or partial functions of organisms are emphasized, and few studies on detection methods are made, and qualitative or quantitative analysis studies on diuretics in blood or urine are mostly made.
Due to the use of diuretics in the breeding industry at present, the detection and monitoring of the diuretics in animal derived foods are not sustainable.
Disclosure of Invention
The applicant finds that, when carrying out a detection experiment on a diuretic in animal-derived food, on one hand, the detection accuracy of the detection method in the existing literature is seriously interfered by an animal-derived food body during the detection of the animal-derived food, and the existing method is not applicable when being applied to the detection of the diuretic in the animal-derived food, and on the other hand, based on the current national conditions, the detection of the existing diuretic can ensure the basic safety of the food, but the detection limit is still relatively high, so that the future monitoring requirement on the using condition of the diuretic is difficult to meet.
In view of the above problems, the present invention provides a method capable of simultaneously and accurately detecting low detection limits of 10 diuretics in pork, beef, mutton, milk and eggs.
A method for detecting the content of diuretic in animal derived food is characterized by comprising the following steps:
and 5, injecting the solution to be detected into a liquid chromatogram-tandem mass spectrometer for detection, recording a chromatogram, and performing diuretic determination based on the chromatogram and the mass spectrum.
Preferably, the chromatographic conditions of the liquid chromatography-tandem mass spectrometer are:
a) a chromatographic column: c18A column, 150mm × 2.1.1 mm (inside diameter), particle size 3.5 μm, or equivalent;
b) sample introduction amount: 10 mu L of the solution;
c) mobile phase: a is water, B is 0.1% methanoic acid solution, and gradient elution is carried out.
Preferably, the scavenger comprises 150mg of anhydrous magnesium sulfate, 50mg of PSA, 50mg of C18And 10mg of graphitized carbon black.
Preferably, the scavenger comprises 500mg of anhydrous magnesium sulfate, 30mg of PSA, 30mg of C18And 10mg of graphitized carbon black.
PreferablyThe purifying agent comprises 150mg of anhydrous magnesium sulfate, 30mg of PSA and 30mg of C18And 5mg of graphitized carbon.
Preferably, the diuretic comprises hydrochlorothiazide, chlorothiazide, triamterene, probenecid, chlorthalidone, acetazolamide, furosemide, sulfa, spironolactone and canrenone.
Preferably, the method further comprises adding an internal standard solution during detection.
Preferably, the extractant comprises acetonitrile.
Preferably, when the solution to be detected is injected into the liquid chromatography-tandem mass spectrometer for detection, a gradient elution process is included, and the gradient elution process is as follows:
the beneficial effect of this application is: the method can effectively extract and detect the diuretic from pork, beef, mutton, milk and eggs, and further accurately detect the content of the diuretic in a sample to be detected by a liquid chromatography-tandem mass spectrometer, thereby improving the sensitivity, simplicity, reliability and batch detection capability of the diuretic detection method, reducing the detection limit by nearly one magnitude and realizing high-sensitivity detection of the diuretic in animal-derived food.
Drawings
The advantages of the above and/or additional aspects of the present application will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a schematic flow chart of a method for measuring diuretic content according to one embodiment of the present application;
FIG. 2 is a graph comparing different extractants to diuretic extraction under otherwise identical conditions;
FIG. 3 is a graph comparing the efficiency of pork purification by different purifiers under otherwise identical conditions;
Detailed Description
In order that the above objects, features and advantages of the present application can be more clearly understood, the present application will be described in further detail with reference to the accompanying drawings and detailed description. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, however, the present application may be practiced in other ways than those described herein, and therefore the scope of the present application is not limited by the specific embodiments disclosed below.
The detection method of the present invention can detect the following 10 diuretics.
Hydrochlorothiazide, alternative name: hydrochlorothiazide; english name: (ii) hydrochlorhiazide; CAS number: 58-93-5; the molecular formula is as follows: c7H8ClN3O4S2(ii) a Molecular weight: 297.74. the characteristics are as follows: white crystalline powder, odorless, slightly bitter taste; insoluble in water, chloroform and diethyl ether, soluble in acetone, and slightly soluble in ethanol; it is soluble in sodium hydroxide solution, but is easily hydrolyzed.
Chlorothiazide, english name: chlorothiazide; CAS number: 58-94-6; the molecular formula is as follows: c7H6ClN3O4S2(ii) a Molecular weight: 295.72. as diuretics and antihypertensive agents.
Triamterene, english name: triamterene; CAS number: 396-01-0; the molecular formula is as follows: c12H11N7(ii) a Molecular weight: 253.26. yellow solid, very slightly soluble in glacial acetic acid, hardly soluble in water, ethanol, chloroform. No or almost no odor, and no odor. Is a pressure-sensitive epithelial sodium ion channel (ENaC) blocker and has diuretic effect.
Probenecid, english name: probenecid; CAS number: 57-66-9; the molecular formula is as follows: c13H19NO4S; molecular weight: 285.36. a white crystalline powder, which is a potent selective transient receptor potential vanilloid receptor channel 2(TRPV2) agonist.
Chlorthalidone, english name: chlorothalidone; CAS number: 66258-76-2; the molecular formula is as follows: c14H11ClN2O4S; molecular weight: 338.8. white to light yellow-white crystalline powder, tasteless. Mainly combined with erythrocyte carbonic anhydrase, and are slower in excretion and metabolism.
Acetazolamide, english name: acetazolamide; CAS number: 59-66-5; the molecular formula is as follows: c4H6N4O3S2(ii) a Molecular weight: 222.25. the characteristics are as follows: white needle crystal or crystalline powder, no odor, and slightly bitter taste. Is slightly soluble in boiling water, slightly soluble in water and ethanol, hardly soluble in chloroform or diethyl ether, and easily soluble in ammonia solution. Is mainly used for treating glaucoma, mild cardiac edema and the like. It is combustible and can release toxic nitrogen oxide and sulfur oxide smoke by heating and decomposing.
Furosemide, alternative name: furananilic acid, furosemide and diuretic are effective sulfonamides acting on the medullary part of the ascending branch of the loop. English name: furosemide; CAS number: 54-31-9; the molecular formula is as follows: c12H11ClN2O5S; molecular weight: 330.74. due to its illicit use for masking other drug tests, furosemide is classified as an illicit drug by the world's anti-stimulant body. The characteristics are as follows: white or off-white crystalline powder, odorless, and almost tasteless. Is soluble in acetone, methanol and dimethylformamide, slightly soluble in ethanol and insoluble in water.
Refined sulfanilamide, alternative name: 4-amino-6-chloro-1, 3-benzenedisulfonamide; english name: 4-amino-6-chlorobenzene-1, 3-disulphonamide; CAS number: 121-30-2; the molecular formula is as follows: c6H8ClN3O4S2(ii) a Molecular weight: 285.73. the characteristics are as follows: crystalline, sparingly soluble in water, is an intermediate of the drug hydrochlorothiazide.
Spironolactone, alternative name: spironolactone, the english name: spironolactone; CAS number: 52-01-7; the molecular formula is as follows: c24H32O4S; molecular weight: 416.57. the characteristics are as follows: is white or off-white fine crystalline powder, slightly bitter, odorless or slightly thiol-smelling. It is very soluble in chloroform, not easy to be water, soluble in ethanol, and soluble in benzene or ethyl acetate.
Canrenone, english name: canrenone; CAS number: 976-71-6; the molecular formula is as follows: c22H28O3(ii) a Molecular weight: 340.46, respectively; the characteristics are as follows: yellowish or milky crystalline powder; can be used for treating heart failure edema and hepatic cirrhosis ascites.
In the process of research on the detection of the diuretic, the applicant finds that different treatments are required to realize accurate detection of the diuretic for different food sources and different diuretics, and a slight difference in treatment links can greatly reduce the accuracy of the detection of the diuretic.
Therefore, in the case of detecting a diuretic in an animal-derived food sample, the applicant has conducted a large number of experiments and optimized the experiments based on the relevant standards, and proposed a method for detecting more than 10 kinds of diuretics in an animal-derived food with high sensitivity.
As shown in fig. 1, the method for detecting the content of diuretic in animal-derived food provided in this embodiment includes:
pretreatment is an important link in the process of detecting the diuretic, and whether the pretreatment is proper or not determines whether the diuretic can be detected or not and the detection accuracy.
Homogenizing pork, beef, mutton, etc. in a knife grinder, mixing, dividing into two parts, and respectively placing into clean containers with marks. Cleaning fresh eggs, removing shells, homogenizing with a knife grinder, mixing, dividing into two parts, and respectively placing into a cleaning container.
Weighing a predetermined amount of sample, such as 2g, placing in a 50mL centrifuge tube, adding an extracting agent (such as 10mL acetonitrile and 2g potassium dihydrogen phosphate), shaking for 30min, centrifuging at 9000r/min for 5min, taking out the supernatant, adding 10mL of a purifying agent, repeatedly extracting once, centrifuging, combining the supernatants in a 50mL centrifuge tube, and purifying.
The results of tests on methanol, ethyl acetate, acetonitrile and the like as extraction solvents and comparison of the peak areas of 10 diuretics when 20. mu.g/kg is added show that the extraction of matrix impurities by methanol and ethyl acetate is much interfered, and the effect of acetonitrile is better extraction efficiency than methanol and ethyl acetate (see figure 2), so that the applicant adopts acetonitrile as the extraction solvent.
specifically, in order to reduce the influence of other substances in a sample to be detected on the detection of the content of the diuretic, the purifying agent is selected, the recovery rate of the diuretic is set as a survey index, and the effect of various purifying agents is tested.
The addition amount of each purifying agent was optimized according to the orthogonal experiment of pork, beef, mutton, milk and egg matrix shown in table 1 (see fig. 3 for pork results), and the 2 nd group of test results shown in fig. 3 all showed better purifying effect on the extraction of 10 kinds of diuretics from pork. And then selecting the optimal purifying agent for beef, mutton, milk and egg samples according to orthogonal experiment results, and finally determining that the purifying agent is prepared from 150mg of anhydrous magnesium sulfate, 50mg of PSA and 50mg of C for the pork, beef and mutton samples18And 10mg of graphitized carbon black, and for milk, 500mg of anhydrous magnesium sulfate, 30mg of PSA, 30mg of C as a purifying agent18And 10mg of graphitized carbon black, and for eggs, 150mg of anhydrous magnesium sulfate, 30mg of PSA, and 30mg of C as purificant18And 5mg of graphitized carbon.
The applicant finds that the detection limit can be obviously reduced and the detection precision can be improved by adopting the purifying agent proportioning mode. Especially for the triamterene, the detection limit can be reduced by dozens of times, so that the inventor obtains the low detection limit for the first time, and the method has important reference significance for further reducing the detection limit of the diuretic in the future.
TABLE 1 orthogonal test Condition Table
| Test group number | Magnesium sulfate Anhydrous (mg) | PSA(mg) | C18(mg) | Graphitized carbon black (mg) |
| 1 | 150 | 30 | 30 | 5 |
| 2 | 150 | 50 | 50 | 10 |
| 3 | 150 | 80 | 80 | 15 |
| 4 | 300 | 30 | 50 | 15 |
| 5 | 300 | 50 | 80 | 5 |
| 6 | 300 | 80 | 30 | 10 |
| 7 | 500 | 30 | 80 | 10 |
| 8 | 500 | 50 | 30 | 15 |
| 9 | 500 | 80 | 50 | 5 |
And 3, taking the purified product as a solution to be detected, and carrying out liquid chromatography detection.
Injecting the solution to be detected into a liquid chromatogram-tandem mass spectrometer for detection, and recording a chromatogram, wherein the chromatographic conditions of the liquid chromatogram-tandem mass spectrometer are as follows:
a) a chromatographic column: c18A column, 150mm × 2.1.1 mm (inside diameter), particle size 3.5 μm, or equivalent;
b) sample introduction amount: 10 mu L of the solution;
c) mobile phase: a is water, B is 0.1% methanoic acid solution, gradient elution, elution procedure is shown in Table 2.
TABLE 2 gradient elution procedure for liquid chromatography
d) Column flow rate: 0.3 mL/min;
e) column temperature: at 40 ℃.
Conditions of Mass Spectrometry
The mass spectrometric detection conditions are shown in table 3.
Table 310 Mass spectrometric detection conditions for diuretics
According to the steps, the detection limits of 10 diuretics in pork, beef, mutton, eggs and milk are respectively 1.0 mu g/kg of hydrochlorothiazide, 1.0 mu g/kg of chlorothiazide, 0.1 mu g/kg of triamterene, 2.0 mu g/kg of probenecid, 2.0 mu g/kg of chlorthalidone, 2.0 mu g/kg of acetazolamide, 2.0 mu g/kg of furosemide, 2.0 mu g/kg of sulfaspermine, 2.0 mu g/kg of spironolactone and 2.0 mu g/kg of canrenone by continuously reducing the concentration of the diuretics.
Pork, beef, mutton, eggs and milk which are measured to be free of hydrochlorothiazide, chlorothiazide, methotrexate, probenecid, chlorthalidone, acetazolamide, furosemide, sulfa, spironolactone and canrenone are weighed, 10 diuretics with high, medium and low concentration levels are respectively added, the accuracy of the verification method for the standard addition recovery rate is examined, the specific condition is shown in table 4, the standard addition recovery condition is good, and the method is proved to have better accuracy.
TABLE 5 spiking recovery test results
Comparative example:
this example was compared with the diuretic detection industry standard SN/T5167-2019, in which acetonitrile extraction and QuEChERS purification were used, and other conditions were the same as those of the inventive example. In this comparative example, which was a methotrexate sample purified with 150mg anhydrous magnesium sulfate, 50mg PSA, 50mg C18 and 7.5mg GCB, the detection limit was reduced from 2.0. mu.g/kg to 0.1. mu.g/kg, indicating that this example is a detection method with a lower detection limit.
Although the present application has been disclosed in detail with reference to the accompanying drawings, it is to be understood that such description is merely illustrative and not restrictive of the application of the present application. The scope of the present application is defined by the appended claims and may include various modifications, adaptations, and equivalents of the invention without departing from the scope and spirit of the application.
Claims (9)
1. A method for detecting the content of diuretic in animal derived food is characterized by comprising the following steps:
step 1, homogenizing the animal-derived food;
step 2, sampling according to a preset amount from the homogenized animal-derived food;
step 3, uniformly mixing the sample with a matched amount of an extracting agent, performing centrifugal treatment, and taking supernatant;
step 4, mixing the obtained supernatant with a matched amount of a purifying agent, carrying out secondary centrifugation treatment, taking all the supernatant and nitrogen to be blown to be nearly dry, adding a water-methanoic acid solution to dissolve residues, and filtering the obtained solution to be used as a solution to be detected;
and 5, injecting the solution to be detected into a liquid chromatogram-tandem mass spectrometer for detection, recording a chromatogram, and performing diuretic determination based on the chromatogram and the mass spectrum.
2. The method of claim 1, wherein the level of diuretic is determined by measuring the level of diuretic in the animal derived food,
the chromatographic conditions of the liquid chromatogram-tandem mass spectrometer are as follows:
a) a chromatographic column: c18A column, 150mm × 2.1.1 mm (inside diameter), particle size 3.5 μm, or equivalent;
b) sample introduction amount: 10 mu L of the solution;
c) mobile phase: a is water, B is 0.1% methanoic acid solution, and gradient elution is carried out.
3. The method for detecting the content of diuretic in animal-derived food as claimed in claim 1, which is used for detecting the content of diuretic in pork, beef and mutton,
the purifying agent comprises 150mg of anhydrous magnesium sulfate, 50mg of PSA and 50mg of C18And 10mg of graphitized carbon black.
4. The method for detecting the content of diuretic in animal-derived food as claimed in claim 1, which is used for detecting the content of diuretic in milk,
the purifying agent comprises 500mg of anhydrous magnesium sulfate, 30mg of PSA and 30mg of C18And 10mg of graphitized carbon black.
5. The method for detecting the content of diuretic in animal-derived food according to claim 1, which is used for detecting the content of diuretic in egg,
the purifying agent comprises 150mg of anhydrous magnesium sulfate, 30mg of PSA and 30mg of C18And 5mg of graphitized carbon black.
6. The method of claim 4, wherein the level of diuretic is determined by measuring the level of diuretic in the animal derived food,
the diuretic comprises hydrochlorothiazide, chlorothiazide, triamterene, probenecid, chlorthalidone, acetazolamide, furosemide, refined sulfanilamide, spironolactone and canrenone.
7. The method of claim 1, further comprising adding an internal standard solution during the measurement.
8. The method of claim 1, wherein the extraction reagent comprises acetonitrile.
9. The method for detecting the content of the diuretic in the animal-derived food according to claim 1, wherein the step of injecting the solution to be detected into the liquid chromatography-tandem mass spectrometer for detection comprises a gradient elution process, wherein the gradient elution process comprises:
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| CN113588828A (en) * | 2021-07-30 | 2021-11-02 | 西安市食品药品检验所(西安市药品不良反应监测中心) | Method for simultaneously detecting forty-eight stimulants in animal-derived food |
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| CN112858516A (en) * | 2021-01-25 | 2021-05-28 | 石家庄海关技术中心 | Method for rapidly analyzing residual quantity of diuretic in animal-derived food |
| CN112858516B (en) * | 2021-01-25 | 2022-10-04 | 石家庄海关技术中心 | Method for rapidly analyzing residual quantity of diuretic in animal-derived food |
| CN113588828A (en) * | 2021-07-30 | 2021-11-02 | 西安市食品药品检验所(西安市药品不良反应监测中心) | Method for simultaneously detecting forty-eight stimulants in animal-derived food |
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