CN111208231A - Method for simultaneously detecting six illegally added compounds in plant beverage - Google Patents
Method for simultaneously detecting six illegally added compounds in plant beverage Download PDFInfo
- Publication number
- CN111208231A CN111208231A CN202010063716.1A CN202010063716A CN111208231A CN 111208231 A CN111208231 A CN 111208231A CN 202010063716 A CN202010063716 A CN 202010063716A CN 111208231 A CN111208231 A CN 111208231A
- Authority
- CN
- China
- Prior art keywords
- solution
- detection
- plant beverage
- volume fraction
- simultaneously detecting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 39
- 150000001875 compounds Chemical class 0.000 title claims abstract description 34
- 235000013361 beverage Nutrition 0.000 title claims abstract description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 63
- 239000000243 solution Substances 0.000 claims abstract description 61
- 238000001514 detection method Methods 0.000 claims abstract description 59
- 239000000523 sample Substances 0.000 claims abstract description 31
- 239000007788 liquid Substances 0.000 claims abstract description 23
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 claims abstract description 22
- OROGSEYTTFOCAN-DNJOTXNNSA-N codeine Chemical compound C([C@H]1[C@H](N(CC[C@@]112)C)C3)=C[C@H](O)[C@@H]1OC1=C2C3=CC=C1OC OROGSEYTTFOCAN-DNJOTXNNSA-N 0.000 claims abstract description 22
- 239000012085 test solution Substances 0.000 claims abstract description 16
- 239000004098 Tetracycline Substances 0.000 claims abstract description 14
- 229960002180 tetracycline Drugs 0.000 claims abstract description 14
- 235000019364 tetracycline Nutrition 0.000 claims abstract description 14
- 229930101283 tetracycline Natural products 0.000 claims abstract description 14
- 150000003522 tetracyclines Chemical class 0.000 claims abstract description 14
- 239000013558 reference substance Substances 0.000 claims abstract description 12
- AKJDEXBCRLOVTH-UHFFFAOYSA-N Carbetapentane citrate Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O.C=1C=CC=CC=1C1(C(=O)OCCOCCN(CC)CC)CCCC1 AKJDEXBCRLOVTH-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000004100 Oxytetracycline Substances 0.000 claims abstract description 11
- 229960004126 codeine Drugs 0.000 claims abstract description 11
- 229960003276 erythromycin Drugs 0.000 claims abstract description 11
- OROGSEYTTFOCAN-UHFFFAOYSA-N hydrocodone Natural products C1C(N(CCC234)C)C2C=CC(O)C3OC2=C4C1=CC=C2OC OROGSEYTTFOCAN-UHFFFAOYSA-N 0.000 claims abstract description 11
- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229960000282 metronidazole Drugs 0.000 claims abstract description 11
- 229960000625 oxytetracycline Drugs 0.000 claims abstract description 11
- IWVCMVBTMGNXQD-PXOLEDIWSA-N oxytetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3[C@H](O)[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-PXOLEDIWSA-N 0.000 claims abstract description 11
- 235000019366 oxytetracycline Nutrition 0.000 claims abstract description 11
- IWVCMVBTMGNXQD-UHFFFAOYSA-N terramycin dehydrate Natural products C1=CC=C2C(O)(C)C3C(O)C4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000005303 weighing Methods 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 238000001914 filtration Methods 0.000 claims abstract description 7
- 238000001819 mass spectrum Methods 0.000 claims abstract description 7
- 239000012088 reference solution Substances 0.000 claims abstract description 7
- 239000012488 sample solution Substances 0.000 claims abstract description 7
- 239000012982 microporous membrane Substances 0.000 claims abstract description 3
- 239000003960 organic solvent Substances 0.000 claims abstract description 3
- 150000002500 ions Chemical class 0.000 claims description 18
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
- 239000012528 membrane Substances 0.000 claims description 9
- 238000010828 elution Methods 0.000 claims description 8
- 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
- 235000019253 formic acid Nutrition 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims description 5
- 238000000132 electrospray ionisation Methods 0.000 claims description 5
- 230000005526 G1 to G0 transition Effects 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 4
- IAWPVNMEXASPFO-UHFFFAOYSA-N C(CCCCCCCCCCCCCCCCC)[SiH3].[C] Chemical group C(CCCCCCCCCCCCCCCCC)[SiH3].[C] IAWPVNMEXASPFO-UHFFFAOYSA-N 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000001471 micro-filtration Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 238000000605 extraction Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 2
- 239000011159 matrix material Substances 0.000 description 15
- 241000196324 Embryophyta Species 0.000 description 12
- 239000012086 standard solution Substances 0.000 description 11
- 239000003814 drug Substances 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000007865 diluting Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 235000013305 food Nutrition 0.000 description 7
- 235000015092 herbal tea Nutrition 0.000 description 7
- 238000004949 mass spectrometry Methods 0.000 description 7
- 238000011084 recovery Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000001816 cooling Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000011097 chromatography purification Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 238000002137 ultrasound extraction Methods 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 238000012935 Averaging Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000012496 blank sample Substances 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000011287 therapeutic dose Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000000955 prescription drug Substances 0.000 description 1
- 230000000506 psychotropic effect Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011158 quantitative evaluation Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- VLYWMPOKSSWJAL-UHFFFAOYSA-N sulfamethoxypyridazine Chemical compound N1=NC(OC)=CC=C1NS(=O)(=O)C1=CC=C(N)C=C1 VLYWMPOKSSWJAL-UHFFFAOYSA-N 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
Landscapes
- 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)
Abstract
The invention provides a method for simultaneously detecting six illegally added compounds in plant beverage, which comprises the following steps: (1) preparation of a test solution: extracting the sample with methanol solution, and filtering with microporous membrane to obtain sample solution; (2) preparation of control solutions: precisely weighing tetracycline, oxytetracycline, codeine, pentoxyverine citrate, metronidazole and erythromycin reference substances, and dissolving with organic solvent to a constant volume to obtain a reference substance solution; (3) and (3) detection: and respectively injecting the test solution and the reference solution into a liquid chromatography-mass spectrometer for detection. The method optimizes the optimal chromatographic mass spectrum condition and the optimal extraction and purification condition, has good linear relation among tetracycline, oxytetracycline, codeine, pentoxyverine citrate, erythromycin and metronidazole, has good sensitivity, accuracy and precision, and is suitable for qualitative and quantitative detection of six components such as tetracycline in the plant beverage.
Description
Technical Field
The invention belongs to the field of detection and analysis, and particularly relates to a method for simultaneously detecting six illegally added compounds in a plant beverage.
Background
The current laws and regulations stipulate that no medicine must be added to the food produced and managed. At present, herbal tea prepared and sold on site is taken as common food management, so that medicines cannot be added. In the current market competition and commercial environment that attracts the return rate of purchases, illegal addition of compounds is often prohibited in on-site herbal tea selling operations.
In 2019, the Guangdong province market supervision and management bureau arranges to carry out special plant beverage (herbal tea) risk monitoring, and tetracycline, oxytetracycline, codeine, pentoxyverine citrate, metronidazole, erythromycin and the like are respectively detected from the samples of Zhujia, Guangdong and Yuexi. The six compounds are all brought into the prescription drug management in China, but the national standard for detecting the components in the plant beverage (herbal tea) is still absent.
The risk monitoring results show that the detection rate of the six ingredients in the specially collected herbal tea sample is 7.8% (8/103). The herbal tea is used as an old non-first-line medicine, a psychotropic medicine, a central inhibitory medicine and the like, is illegally added into common food herbal tea, has a large potential safety risk, and has urgent need in supervision, and related detection methods are urgently needed to be supplemented.
Disclosure of Invention
The invention aims to provide a method for simultaneously detecting six illegally added compounds in plant beverage.
The technical scheme adopted by the invention is as follows:
a method for simultaneously detecting six illegally added compounds in plant beverage comprises the following steps:
(1) preparation of a test solution: extracting the sample with methanol solution, and filtering with microporous membrane to obtain sample solution;
(2) preparation of control solutions: precisely weighing tetracycline, oxytetracycline, codeine, pentoxyverine citrate, metronidazole and erythromycin reference substances, and dissolving with organic solvent to a constant volume to obtain a reference substance solution;
(3) and (3) detection: and respectively injecting the test solution and the reference solution into a liquid chromatography-mass spectrometer for detection.
Preferably, in the detecting step, the detection is performed by a liquid chromatography-mass spectrometer under the following chromatographic conditions:
stationary phase: a chromatographic column using carbon octadecyl silane bonded silica gel as a filler;
mobile phase: phase A: formic acid solution; phase B: formic acid acetonitrile solution;
flow rate: 0.25-0.40 mL/min;
and (3) an elution mode: gradient elution.
Preferably, in the detecting step, the detection is performed by a liquid chromatography-mass spectrometer under the following chromatographic conditions:
sample introduction amount: 2-10 μ L;
stationary phase: phenomenex Kinetex C18 chromatographic column with filler particle diameter of 2.6um and diameter and length of 2.1mm and 100mm respectively;
mobile phase: phase A: a formic acid solution with a volume fraction of 0.1%; phase B: 0.1 percent of formic acid acetonitrile solution by volume fraction;
the elution gradient was: 0.0min-6.5min, the volume fraction of the mobile phase A is changed from 95% to 5%, and the volume fraction of the mobile phase B is changed from 5% to 95%; 6.5min-7.5min, the volume fraction of the mobile phase A is 5%, and the volume fraction of the mobile phase B is 95%; 7.6min-10.0min, the volume fraction of the mobile phase A is 95%, and the volume fraction of the mobile phase B is 5%;
flow rate: 0.3 mL/min;
column temperature: 35-40 ℃.
Preferably, in the detecting step, the detection is performed by a liquid chromatography-mass spectrometer tandem mass spectrometer under the following mass spectrometry conditions:
an ionization mode: carrying out electrospray ionization;
the scanning mode is as follows: a positive ion;
the detection mode is as follows: detecting multiple reactive ions;
ionization voltage: 4.0kV-5.0 kV;
air curtain pressure: 241.3kPa-310.3 kPa;
collision gas pressure: 48kPa to 69 kPa;
ionization temperature: 400-500 ℃;
auxiliary heating gas pressure: 320kPa to 380 kPa.
Preferably, in the detecting step, the detection is performed by a liquid chromatography-mass spectrometer tandem mass spectrometer under the following mass spectrometry conditions:
an ionization mode: carrying out electrospray ionization;
detection mode: a positive ion;
the detection mode is as follows: detecting multiple reactive ions;
ionization voltage: 4.5 kV;
air curtain pressure: 275.6 kPa;
collision gas pressure: 62 kPa;
ionization temperature: 450 deg.C
Auxiliary heating gas pressure: 345 kPa.
Preferably, in the step of preparing the reference solution, each reference is dissolved in methanol to a constant volume, so as to prepare the reference solution with a mass concentration of 0.5-2.0 mg/mL.
Preferably, in the step of preparing the reference solution, each reference is added to the same container to prepare a mixed reference solution.
Preferably, in the step of preparing the sample solution, the microfiltration membrane is an organic membrane having a diameter of 0.22 μm.
The invention has the beneficial effects that:
(1) the method optimizes the optimal chromatographic mass spectrum condition and the optimal extraction and purification condition, has good linear relation among tetracycline, oxytetracycline, codeine, pentoxyverine citrate, erythromycin and metronidazole, has good sensitivity, accuracy and precision, and is suitable for qualitative and quantitative detection of 6 components such as tetracycline in plant beverages.
(2) According to the conditions of the chromatogram and the mass spectrum of the method, the detection limit of the method of the liquid sample is 0.25mg/kg, and the quantitative limit of the method is 0.5 mg/kg; the method detection limit of the solid sample is 2.5mg/kg, the method quantitative limit is 5.0mg/kg, and the requirement of illegal addition inspection can be met.
Drawings
FIG. 1 is a reference chart of a multi-reaction detection scan of tetracycline.
FIG. 2 is a reference chart of a multi-reaction detection scan of oxytetracycline.
Figure 3 is a reference map spectrum of a multiple reaction detection scan for codeine.
FIG. 4 is a reference chart of a multi-reaction detection scan of pentoxyverine citrate.
FIG. 5 is a reference chart of a multi-reaction detection scan of metronidazole.
FIG. 6 is a reference chart of a multi-reaction detection scan of erythromycin.
Fig. 7 is a total ion flow graph of a blank sample.
Fig. 8 is a total ion flow graph of negative sample addition mixed standard intermediate solution.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. The following is a description of preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The apparatus and reagents in the following examples are as follows:
1. the instrument comprises the following steps:
LC-MS (liquid chromatography-mass spectrometer): AB Sciex Triple Quad 5500;
a chromatographic column: phenomenex kinex Kinetex C18, 2.6um, 2.1 x 100 mm);
a water purifier: MILLIPORE Mili-Q Advantage A10;
balance: sartorius BS323S, METTLER MS205 DU;
numerical control ultrasonic cleaner: KQ-500DE, ultrasonic instruments Inc. of Kunshan.
2. Reagent testing:
methanol: carrying out chromatographic purification;
acetonitrile: carrying out chromatographic purification;
formic acid: carrying out chromatographic purification;
the source information of each control is shown in table 1:
TABLE 1 Source information of each control
| Name (R) | Manufacturer of the product | Batch number | Content (wt.) |
| Tetracycline derivatives | NATIONAL INSTITUTES FOR FOOD AND DRUG CONTROL | 130306-201419 | 96.9％ |
| Oxytetracycline | NATIONAL INSTITUTE FOR THE CONTROL OF PHARMACEUTICAL AND BIOLOGICAL PRODUCT | 130487-200703 | 88.8％ |
| Pentoxyverine citrate | NATIONAL INSTITUTES FOR FOOD AND DRUG CONTROL | 100432-201803 | 99.8％ |
| Codeine | A ChemTek Inc | S032785 | 99.8％ |
| Erythromycin | NATIONAL INSTITUTES FOR FOOD AND DRUG CONTROL | 130307-201417 | 92.8％ |
| Metronidazole | NATIONAL INSTITUTES FOR FOOD AND DRUG CONTROL | 100191-201808 | 100％ |
3. Conditions of the apparatus
3.1 liquid chromatography conditions:
sample introduction amount: 2 mu L of the solution;
mobile phase: phase A: 0.1% formic acid solution (volume fraction); phase B: 0.1% formic acid acetonitrile solution (volume fraction);
flow rate: 0.3 mL/min;
column temperature: 35 ℃;
the gradient elution procedure is shown in table 2:
table 2 gradient elution procedure (volume fraction,%)
3.2 Mass Spectrometry conditions:
an ionization mode: carrying out electrospray ionization;
detection mode: a positive ion;
the detection mode is as follows: detecting multiple reactive ions;
ionization voltage: 4.5 kV;
air curtain pressure: 275.6 kPa;
collision gas pressure: 62 kPa;
ionization temperature: 450 deg.C
For comparison, the control solutions and the test solutions in the following examples were prepared as follows:
preparation of control solutions:
standard stock solutions: precisely weighing 10mg of each of tetracycline, oxytetracycline, codeine, pentoxyverine citrate, metronidazole and erythromycin, respectively placing the 10mg into a10 mL volumetric flask, dissolving the 10mg into methanol (with chromatographic purity), diluting the solution to a scale, and preparing a standard storage liquid with the concentration of 1mg/mL, and storing the standard storage liquid at the temperature of minus 18 ℃ in a dark place.
Mixing standard intermediate solution: precisely sucking 0.25mL of each of the standard stock solutions (1mg/mL) of tetracycline, oxytetracycline, codeine, pentoxyverine citrate, metronidazole and erythromycin, placing the standard stock solutions into a same 25mL volumetric flask, diluting the volumetric flask to a scale with methanol (pure chromatogram), shaking up to prepare a mixed standard solution with the concentration of 10 mu g/mL, and storing the mixed standard solution at 4 ℃ in a dark place.
Preparation of a test solution:
vegetable beverage (liquid): weighing 1.00g (accurate to 0.01g) of plant type beverage, placing the plant type beverage in a 50mL volumetric flask, adding about 40mL of 50% methanol solution in volume fraction, carrying out ultrasonic extraction for 30min, cooling to room temperature, fixing the volume to scale with the 50% methanol solution in volume fraction, shaking up, filtering with a 0.22 mu m organic filter membrane, loading on a machine, diluting properly according to actual concentration to a standard curve linear range, and analyzing by a liquid chromatography-mass spectrometer.
Vegetable beverage (solids): weighing 0.10g (accurate to 0.001g) of plant type beverage, placing the plant type beverage in a 50mL volumetric flask, adding about 40mL of 50% methanol aqueous solution in volume fraction, ultrasonically extracting for 30min, cooling to room temperature, fixing the volume to the scale with the 50% methanol aqueous solution in volume fraction, shaking up, filtering with a 0.22 mu m organic filter membrane, loading on a machine, diluting properly according to actual concentration to the linear range of a standard curve, and analyzing by a liquid chromatography-mass spectrometer.
Under the above chromatographic mass spectrometry conditions, the reference values of mass spectrometry parameters of each compound (solution concentration 50ng/mL) are shown in Table 3:
the reference spectrum of the multi-reaction detection scan of each compound is shown in figures 1-6:
TABLE 3 reference values for mass spectral parameters of the respective compounds
Qualitative detection
And detecting the test solution and the mixed standard solution according to the chromatographic mass spectrometry conditions, and if the mass chromatographic peaks of the compounds in the detected test solution are consistent with those of the mixed standard solution, and in the spectrogram of the test solution with the background subtracted, the relative abundance of each qualitative ion is compared with the spectrogram of the mixed standard solution obtained under the same conditions with the similar concentration, and the error does not exceed the specified range shown in the table 4, determining that the corresponding compounds exist in the sample.
TABLE 4 extent of variation in relative abundance of qualitative ions in test solutions
| Relative ion abundance | >50 | 20-50 | 10-20 | <10 |
| Allowable relative deviation% | ±20 | ±25 | ±30 | ±50 |
Quantitative detection
Mixing standard solutions: accurately sucking a proper amount of the mixed standard intermediate solution, diluting the mixed standard intermediate solution by using a methanol solution with the volume fraction of 50%, shaking up, and preparing into series of standard solutions with the concentrations of 10ng/mL, 20ng/mL, 50ng/mL, 80ng/mL, 100ng/mL and 200ng/mL in sequence.
Detecting the mixed standard solution according to the chromatographic mass spectrometry conditions, and drawing a standard working curve by taking the peak area as a vertical coordinate and the concentration of the mixed standard solution as a horizontal coordinate to obtain a table 5; the results show that the compounds have a good linear relationship in the corresponding linear range.
TABLE 5 regression equation, correlation coefficient and Linear Range for six Compounds
And detecting the test solution, and quantifying the test solution by using a standard working curve. The quantitative calculation formula is as follows:
wherein, X is the content of the compound component in the test sample, and the unit is as follows: mg/kg;
c is the concentration of the tested compound component solution obtained on the standard working curve, and the unit is as follows: ng/mL;
v is the constant volume of the test solution, and the unit is as follows: mL;
m is the sample weighing of the sample, and the unit is as follows: g.
blank values should be subtracted from the calculation.
Example (b): detection limit and quantitation limit determination
The mixed standard solution was added to the negative sample (vegetable beverage sample not containing the above six compounds, the same applies hereinafter) stepwise, and the concentration at S/N.gtoreq.3 was selected as the detection limit and the concentration at S/N.gtoreq.10 was selected as the quantification limit. The results of the detection limit and the quantitative limit calculation for each compound component are shown in table 6:
TABLE 6 detection limit and quantitative limit results for each compound component
The results show that the detection limit of the liquid sample by the method is 0.25mg/kg, and the quantitative limit of the method is 0.5 mg/kg; the method detection limit of the solid sample is 2.5mg/kg, the method quantitative limit is 5.0mg/kg, and the requirement of illegal addition inspection can be met. The relationship between the method quantitation limit of the six compound components and the therapeutic dose is detailed in table 6, and the results show that the method quantitation limit of the six compound components can reach 1/100 of the therapeutic dose, and the method has sufficient sensitivity.
Detection example 1: matrix effect detection
There are two main methods for evaluating the matrix effect: (1) post column sampling method established by Bonfiglio et al; (2) the post-extraction labeling method proposed by Matuszewski et al. The method (1) is a method capable of rapidly and qualitatively determining whether a certain substrate has a substrate effect, but has the defect that quantitative evaluation cannot be carried out. And (2) comparing the response values of various compounds in the matrix of the standard-added sample and the blank solution of the standard-added sample, not only can qualitative analysis be carried out, but also the influence value of the matrix effect can be quantitatively analyzed (SSE, the calculation formula is as follows), so that the matrix effect of the plant beverage is evaluated by adopting the method (2). The mixed standard solution is diluted into a sample matrix solution standard curve containing tetracycline, oxytetracycline, codeine, pentoxyverine citrate, erythromycin and metronidazole and a blank solution standard curve with the concentration gradient of 10ng/mL, 20ng/mL, 50ng/mL, 80ng/mL, 100ng/mL and 200ng/mL by using a negative sample matrix solution and a methanol solution with the volume fraction of 50%, a standard curve is established by an external standard method, and the ratio of the slope of the sample matrix solution standard curve to the slope of the blank solvent standard curve is the matrix effect SSE of the sample. The evaluation results of each substrate are shown in table 7:
TABLE 7 evaluation results of substrate Effect of different substrates
Detection example 2: accuracy and precision detection
Accurately sucking a proper amount of the mixed standard intermediate solution, diluting with a methanol solution with the volume fraction of 50%, shaking up, and preparing into a solution with the concentration of 1 ug/mL.
And (3) liquid matrix recovery detection: respectively weighing 3 parts of 1g of negative sample solution, placing the solution in 3 50mL volumetric flasks, precisely weighing 0.25mL and 0.50mL of the 1ug/mL solution and 0.25mL of the 10ug/mL mixed standard intermediate solution, adding the corresponding volumetric flasks, adding 40mL of 50% methanol solution, performing ultrasonic extraction at 40 ℃ for 30min, cooling to room temperature, diluting the 50% methanol solution to a constant volume, passing through a 0.22 mu m organic filter membrane, taking the filtrate, sequentially preparing solutions with three levels of low concentration, medium concentration and high concentration, performing accuracy detection, introducing samples for 6 times at each concentration level under the condition of the chromatographic mass spectrum, and averaging the detection values, wherein the results are shown in Table 8:
table 8 liquid matrix recovery and relative standard deviation for six compound components
As can be seen from Table 8, the average recovery rate of the six compound components in the liquid matrix is 66.1-108.6%, the precision is 0.0-10.7%, and the method has good accuracy and precision.
And (3) solid matrix recovery detection: respectively weighing 3 parts of 0.2g of negative sample solution, placing the negative sample solution in 3 50mL volumetric flasks, respectively precisely weighing 0.25mL and 0.50mL of the 1ug/mL solution and 0.25mL of the 10ug/mL mixed standard intermediate solution, adding the corresponding volumetric flasks, adding 40mL of 50% methanol solution, performing ultrasonic extraction at 40 ℃ for 30min, cooling to room temperature, fixing the volume of the 50% methanol solution to a scale, passing through a 0.22 mu m organic filter membrane, taking filtrate, sequentially preparing solutions with three levels of low concentration, medium concentration and high concentration for accuracy detection, performing sample injection for 6 times at each concentration level under the condition of the chromatographic mass spectrum, and averaging the detection values, wherein the results are shown in Table 9:
TABLE 9 solid matrix recovery and relative standard deviation for six compound components
As can be seen from Table 9, the results show that the average recovery rate of the six components such as tetracycline in the solid matrix is 71.5-119.7%, the precision is 0.4-14.6%, and the method has good accuracy and precision.
Detection example 3: specificity of the assay
Determination of the addition of mixed standard intermediate solutions to the negative samples: taking a negative sample, placing the negative sample in a 50mL volumetric flask, adding 0.1mL of 10 mug/mL mixed standard intermediate solution, dissolving the negative sample by 40mL of 50% methanol solution in volume fraction, carrying out ultrasonic treatment for 30min, cooling to room temperature, fixing the volume to scale by 50% methanol solution in volume fraction, shaking up, filtering by a 0.22 mu m filter membrane, taking filtrate, and analyzing by a high performance liquid chromatography-tandem mass spectrometer.
Blank negative sample determination: the negative samples were run as described above except that no mixed standard intermediate solution was added.
The total ion flow graph of the negative sample added with the mixed standard intermediate solution and the total ion flow graph of the blank sample are shown in figures 7-8, and the graphs show that the total ion flow graph of the negative sample added with the mixed standard intermediate solution has no interference and the method has good specificity.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. A method for simultaneously detecting six illegally added compounds in plant beverage comprises the following steps:
(1) preparation of a test solution: extracting the sample with methanol solution, and filtering with microporous membrane to obtain sample solution;
(2) preparation of control solutions: precisely weighing tetracycline, oxytetracycline, codeine, pentoxyverine citrate, metronidazole and erythromycin reference substances, and dissolving with organic solvent to a constant volume to obtain a reference substance solution;
(3) and (3) detection: and respectively injecting the test solution and the reference solution into a liquid chromatography-mass spectrometer for detection.
2. The method for simultaneously detecting six illegally added compounds in a plant beverage according to claim 1, wherein: in the detection step, the detection of the liquid chromatography-mass spectrometer is carried out according to the following chromatographic conditions:
stationary phase: a chromatographic column using carbon octadecyl silane bonded silica gel as a filler;
mobile phase: phase A: formic acid solution; phase B: formic acid acetonitrile solution;
flow rate: 0.25-0.40 mL/min;
and (3) an elution mode: gradient elution.
3. The method for simultaneously detecting six illegally added compounds in a plant beverage according to claim 2, wherein: in the detection step, the detection of the liquid chromatography-mass spectrometer is carried out according to the following chromatographic conditions:
sample introduction amount: 2-10 μ L;
stationary phase: phenomenex KinetexC18 chromatographic column with filler particle diameter of 2.6um and diameter and length of 2.1mm and 100mm respectively;
mobile phase: phase A: a formic acid solution with a volume fraction of 0.1%; phase B: 0.1 percent of formic acid acetonitrile solution by volume fraction;
the elution gradient was: 0.0min-6.5min, the volume fraction of the mobile phase A is changed from 95% to 5%, and the volume fraction of the mobile phase B is changed from 5% to 95%; 6.5min-7.5min, the volume fraction of the mobile phase A is 5%, and the volume fraction of the mobile phase B is 95%; 7.6min-10.0min, the volume fraction of the mobile phase A is 95%, and the volume fraction of the mobile phase B is 5%;
flow rate: 0.3 mL/min;
column temperature: 35-40 ℃.
4. The method for simultaneously detecting six illegally added compounds in a plant beverage according to any one of claims 1 to 3, wherein: in the detection step, the detection of the liquid chromatogram-mass spectrometer is carried out according to the following mass spectrum conditions:
an ionization mode: carrying out electrospray ionization;
the scanning mode is as follows: a positive ion;
the detection mode is as follows: detecting multiple reactive ions;
ionization voltage: 4.0kV-5.0 kV;
air curtain pressure: 241.3kPa-310.3 kPa;
collision gas pressure: 48kPa to 69 kPa;
ionization temperature: 400-500 ℃;
auxiliary heating gas pressure: 320kPa to 380 kPa.
5. The method for simultaneously detecting six illegally added compounds in plant beverage according to claim 4, wherein: in the detection step, the detection of the liquid chromatogram-mass spectrometer is carried out according to the following mass spectrum conditions:
an ionization mode: carrying out electrospray ionization;
detection mode: a positive ion;
the detection mode is as follows: detecting multiple reactive ions;
ionization voltage: 4.5 kV;
air curtain pressure: 275.6 kPa;
collision gas pressure: 62 kPa;
ionization temperature: 450 deg.C
Auxiliary heating gas pressure: 345 kPa.
6. The method for simultaneously detecting six illegally added compounds in a plant beverage according to claim 1, wherein: in the preparation step of the reference substance solution, each reference substance is dissolved by methanol to a constant volume, so as to prepare the reference substance solution with the mass concentration of 0.5-2.0 mg/mL.
7. The method for simultaneously detecting six illegally added compounds in a plant beverage according to claim 6, wherein the method comprises the following steps: in the preparation step of the reference substance solution, each reference substance is fixed to the same container to prepare a mixed reference substance solution.
8. The method for simultaneously detecting six illegally added compounds in a plant beverage according to claim 1, wherein: in the step of preparing the test solution, the microfiltration membrane is an organic filtration membrane with a diameter of 0.22 μm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010063716.1A CN111208231B (en) | 2020-01-20 | 2020-01-20 | Method for simultaneously detecting six illegally added compounds in plant beverage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010063716.1A CN111208231B (en) | 2020-01-20 | 2020-01-20 | Method for simultaneously detecting six illegally added compounds in plant beverage |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111208231A true CN111208231A (en) | 2020-05-29 |
| CN111208231B CN111208231B (en) | 2022-07-22 |
Family
ID=70789780
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010063716.1A Active CN111208231B (en) | 2020-01-20 | 2020-01-20 | Method for simultaneously detecting six illegally added compounds in plant beverage |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111208231B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107102075A (en) * | 2017-04-20 | 2017-08-29 | 广东产品质量监督检验研究院 | A kind of method of illegal addition pappy shell in quick discriminating herbal tea |
| CN107677743A (en) * | 2017-09-28 | 2018-02-09 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | A kind of method of chlorphenamine maleate and other chemicalses in detection herbal beverage |
-
2020
- 2020-01-20 CN CN202010063716.1A patent/CN111208231B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107102075A (en) * | 2017-04-20 | 2017-08-29 | 广东产品质量监督检验研究院 | A kind of method of illegal addition pappy shell in quick discriminating herbal tea |
| CN107677743A (en) * | 2017-09-28 | 2018-02-09 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | A kind of method of chlorphenamine maleate and other chemicalses in detection herbal beverage |
Non-Patent Citations (5)
| Title |
|---|
| CHEN Y. S. ET AL.: "Pharmaceutical residues in tidal surface sediments of three rivers in southeastern China at detectable and measurable levels", 《ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH》 * |
| LARISSA J. M. JANSEN ET AL.: "The analysis of tetracyclines, quinolones, macrolides, lincosamides, pleuromutilins, and sulfonamides in chicken feathers using UHPLC-MS/MS in order to monitor antibiotic use in the poultry sector", 《ANALYTICAL AND BIOANALYTICAL CHEMISTRY》 * |
| 温家欣 等: "高效液相色谱法同时快速测定凉茶中11种非法添加化学药物", 《分析测试学报》 * |
| 胡佳哲 等: "高效液相色谱-串联质谱法测定凉茶中19种非法添加药物", 《分析试验室》 * |
| 韩婉清 等: "固相萃取-超高效液相色谱-串联质谱测定凉茶中15种植物源性兴奋剂和外源性药物", 《分析化学》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111208231B (en) | 2022-07-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2508531C2 (en) | Method of preparation of specimens for analysis and cartridge for it | |
| CN101290306A (en) | Milk and milk product tetracycline antibiotic residual quantity checking method | |
| CN114791470A (en) | Method for detecting polymyxin in blood by high performance liquid chromatography-tandem mass spectrometry and application | |
| CN111650302B (en) | Separation detection method for isomers of octylphenol and nonylphenol | |
| CN105486775B (en) | A method for detecting the contents of various components in Yougui Pills | |
| CN108760920B (en) | Method for determining residual quantity of cyazofamid and metabolites thereof based on HPLC-MSMS method | |
| CN108872425A (en) | The method that hydrophilic Interaction Chromatography-tandem mass spectrometry quickly measures honey streptomycin and dihydrostreptomycin | |
| CN111208231B (en) | Method for simultaneously detecting six illegally added compounds in plant beverage | |
| CN117929600A (en) | Detection method of 3-piperidine formamide | |
| CN115728408B (en) | Method for simultaneously determining seven artemisinin related compounds in artemisia annua | |
| CN102590364A (en) | A method for simultaneous detection of fumonisins B1 and B2 in traditional Chinese medicines in different matrices | |
| CN116973492A (en) | Method for analyzing content of polyalcohol in fermented grains | |
| CN110940755A (en) | Method for detecting 4 tadalafil substances in food | |
| CN113866305A (en) | Method for rapidly and accurately analyzing theanine in fresh tea leaves based on liquid chromatography-mass spectrometry technology | |
| CN113533594A (en) | Method and kit for determining tranexamic acid content | |
| CN112611813A (en) | Method for testing genotoxic impurities of Sacubitril valsartan sodium starting material | |
| CN111337611A (en) | Method for detecting malachite green, leucomalachite green, crystal violet and leucocyte crystal violet in aquatic products | |
| CN113447593B (en) | Detection method of nafil substances | |
| CN116008450B (en) | Method for detecting genotoxic impurities in pregabalin | |
| CN110988200A (en) | Analysis method of imidazole residue in recombinant human teriparatide for injection | |
| CN119104645B (en) | A method for simultaneous detection of 6 GLP-1RA drugs | |
| CN117451903B (en) | Method for simultaneously detecting 14 organic acids in fruit wine | |
| CN114577943B (en) | Method for indirectly measuring aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder | |
| CN119936272B (en) | Method for detecting N-nitrosoambroxol in medicine by LC-MS/MS | |
| CN115389673B (en) | Method for determining genotoxic impurities in ranitidine hydrochloride by liquid chromatography-mass spectrometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |