CN106841457B - The measuring method of methaqualone and diazepam residual quantity in a kind of animal derived food - Google Patents
The measuring method of methaqualone and diazepam residual quantity in a kind of animal derived food Download PDFInfo
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- CN106841457B CN106841457B CN201710156822.2A CN201710156822A CN106841457B CN 106841457 B CN106841457 B CN 106841457B CN 201710156822 A CN201710156822 A CN 201710156822A CN 106841457 B CN106841457 B CN 106841457B
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- AAOVKJBEBIDNHE-UHFFFAOYSA-N diazepam Chemical compound N=1CC(=O)N(C)C2=CC=C(Cl)C=C2C=1C1=CC=CC=C1 AAOVKJBEBIDNHE-UHFFFAOYSA-N 0.000 title claims abstract description 113
- 229960003529 diazepam Drugs 0.000 title claims abstract description 112
- 229960002803 methaqualone Drugs 0.000 title claims abstract description 72
- JEYCTXHKTXCGPB-UHFFFAOYSA-N Methaqualone Chemical compound CC1=CC=CC=C1N1C(=O)C2=CC=CC=C2N=C1C JEYCTXHKTXCGPB-UHFFFAOYSA-N 0.000 title claims abstract description 70
- 238000000034 method Methods 0.000 title claims abstract description 39
- 235000013305 food Nutrition 0.000 title claims abstract description 21
- 241001465754 Metazoa Species 0.000 title claims abstract description 16
- 210000004185 liver Anatomy 0.000 claims abstract description 27
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 21
- 235000015277 pork Nutrition 0.000 claims abstract description 18
- 241000282894 Sus scrofa domesticus Species 0.000 claims abstract description 16
- 238000000746 purification Methods 0.000 claims abstract description 13
- 238000000605 extraction Methods 0.000 claims abstract description 9
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 9
- 238000005259 measurement Methods 0.000 claims abstract description 9
- 241000251468 Actinopterygii Species 0.000 claims abstract description 7
- 239000002904 solvent Substances 0.000 claims abstract description 3
- 150000002500 ions Chemical class 0.000 claims description 91
- 239000000523 sample Substances 0.000 claims description 69
- 239000012086 standard solution Substances 0.000 claims description 41
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 33
- 230000000155 isotopic effect Effects 0.000 claims description 33
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 16
- 239000012224 working solution Substances 0.000 claims description 15
- 239000012488 sample solution Substances 0.000 claims description 13
- 239000000945 filler Substances 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 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 8
- 235000019253 formic acid Nutrition 0.000 claims description 8
- 230000014759 maintenance of location Effects 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 239000007921 spray Substances 0.000 claims description 6
- 238000010813 internal standard method Methods 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000004949 mass spectrometry Methods 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 150000001768 cations Chemical class 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 230000004044 response Effects 0.000 claims description 3
- 238000013459 approach Methods 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 2
- 239000012085 test solution Substances 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 230000003595 spectral effect Effects 0.000 claims 1
- 241000282898 Sus scrofa Species 0.000 abstract description 27
- 238000011084 recovery Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 3
- 239000011159 matrix material Substances 0.000 abstract description 3
- 230000006978 adaptation Effects 0.000 abstract description 2
- 239000011812 mixed powder Substances 0.000 abstract 1
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 239000012496 blank sample Substances 0.000 description 26
- 241000276707 Tilapia Species 0.000 description 11
- 239000012530 fluid Substances 0.000 description 5
- 239000007789 gas Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 150000002576 ketones Chemical class 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 230000005059 dormancy Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000005554 hypnotics and sedatives Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
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
- G01N30/14—Preparation by elimination of some components
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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)
Abstract
The invention discloses the measuring methods of methaqualone and diazepam residual quantity in the animal-derived foods such as a kind of pork, the flesh of fish, pig liver and Ren sus domestica.It uses ethyl acetate for Extraction solvent, after the purification of mixed-powder pipe, is detected with liquid chromatography-tandem mass spectrometry instrument, inner mark method ration.Regression equation related coefficient of the present invention reaches 0.99 or more, and lower limit of measurement is 0.5 μ g/kg, and the rate of recovery on 0.5 μ of μ g/kg~5 g/kg addition concentration level is 90%~120%, relative standard deviation≤15% in laboratory.The method of the present invention is easy to operate, high sensitivity, strong antijamming capability, matrix wide adaptation range, and qualitative, quantitative science is accurate, suitable for the quick analysis of batch samples, can satisfy the technical requirements of China's interested regulatory authorities.
Description
Technical field
The invention belongs to the detection technique fields of food veterinary drug residue, and in particular to methaqualone and ground in animal-derived food
Dissolve the measuring method of residual quantity in west.
Background technique
Methaqualone and diazepam are clinically used as hypnotic sedative agent, belong to spiritual control medicine, and long-term use has additive
And dependence.In recent years, with the development of animal husbandry, there is criminal under the driving of economic interests, without authorization in livestock and poultry
It adds and uses in raising, to increase meat yield.In addition, during transportation using the effect that can also be played keep-alive, take care of yourself.Peace
The abuse of dormancy ketone and diazepam, will lead to the residual of drug in animal derived food, and the body for finally seriously affecting consumer is strong
Health.
Clear stipulaties diazepam allows to make to treat with but must not examine in animal food No. 235 bulletins of the Ministry of Agriculture, China
Out；Methaqualone is forbidden to use.The measuring method of methaqualone and diazepam residual quantity mainly has GB in animal-derived food at present
29697-2013 " diazepam and the how remaining measurement gas-chromatography-matter of methaqualone in national food safety standard animal food
Spectrometry " " measurement for exporting methaqualone residual quantity in animal derived food is efficient by (referred to as: contrast standard 1) and SN/T3039-2011
Liquid chromatography " (referred to as: contrast standard 2).But the former gas chromatography-mass spectrography is cumbersome, and interferes more, discomfort
Close the quick analysis of batch samples；10 μ g/kg of the latter's high effective liquid chromatography for measuring lower bound, is not achieved what supervision department assigned
1.0 μ g/kg of technical requirements, high performance liquid chromatography sensitivity is low, and matrix interference is big, without application value；It is specific below
Comparing result.In addition, being checked in documents 3: mass spectrum journal 2012,33 (1) " support by liquid chromatography-tandem mass spectrometry
Grow the residual quantity of trace methaqualone in fish ", in contrast, the present invention is easy to operate using QuEChERs purification pipe, without using
Organic solvent, it is convenient and environmentally friendly.
Summary of the invention
The purpose of the present invention is to provide the measuring methods of methaqualone and diazepam residual quantity in a kind of animal derived food.
To achieve the goals above, the invention adopts the following technical scheme:
The measuring method of methaqualone and diazepam residual quantity, step in a kind of food are as follows: take sample to be measured, extract, supernatant
Liquid is after QuEChERs purification pipe purification, and concentration volatilizes, and residue is to flow phased soln, then uses Liquid Chromatography-Tandem Mass Spectrometry
Detection, is finally quantified with internal standard method；The mobile phase is acetonitrile -5mmol/L formic acid, volume ratio 30:70；It is described
QuEChERs purify pipe the preparation method comprises the following steps: weighing 10 parts of C18Filler, 40 parts of NH2Filler and 1 part of PSA filler in centrifuge tube,
Mix to get.
The extraction process are as follows: in the sample to be measured, addition diazepam Isotopic Internal Standard object standard working solution, then plus
Enter ethyl acetate as Extraction solvent, be homogenized, mechanical shaking extraction, centrifugation, separation supernatant is spare.
The purification process are as follows: pipette extracting solution into QuEChERs purification pipe, vortex oscillation, centrifugation takes supernatant
Liquid to obtain the final product.
The standard curve of the inner mark method ration the preparation method comprises the following steps: accurate measure 100 μ g/L methaqualones and diazepam is mixed
Standardization working solution and 100 μ g/L diazepam Isotopic Internal Standard standard working solutions are appropriate, with the flowing phase dilution, are configured to pacify
Dormancy ketone and diazepam concentration are 0,0.2,0.5,1.0,2.0,5.0 and 10 μ g/L, and diazepam Isotopic Internal Standard concentration is 2.0 μ g/L
Series standard solution, for liquid chromatography-tandem mass spectrometry instrument measure；With methaqualone and diazepam and diazepam Isotopic Internal Standard
Characteristic ion mass chromatography peak area ratio is ordinate, and concentration of standard solution is abscissa, draws standard curve, seeks regression equation
And related coefficient.
The chromatographic condition of the liquid chromatogram are as follows: sample volume: 10 μ L；Column temperature: 40 DEG C；Mobile phase: A:5m mol/L formic acid
Solution；B: acetonitrile；Condition of gradient elution such as the following table 1:
Table 1: condition of gradient elution
The Mass Spectrometry Conditions are as follows: ion source: electric spray ion source；Scanning mode: cation scanning；Detection mode: more
Reaction monitoring；Spray voltage IS:4500V；Methaqualone qualitative ion pair and collision energy: 251.2 > 132.1, collision energy 35V；
251.2 > 91.1, collision energy 52V；Quota ion pair: 251.2 > 132.1；Diazepam qualitative ion pair and collision energy:
285.1 > 193.0, collision energy 45V；285.1 > 154.1, collision energy 38V；Quota ion pair: 285.1 > 154.1.
The qualitative method of the measuring method are as follows: pass through the retention time of sample chromatogram and the reservation of respective standard product
The characteristic ion of time, the characteristic ion of chromatographic peak and respective concentration standard solution chromatographic peak contrast qualitative；Sample and standard
The relative deviation of product retention time is not more than 5%；The relative abundance of sample characteristic ion is opposite with the fairly standard solution of concentration
Abundance is consistent, and relative abundance deviation range meets the following table 3 range, then can determine whether that there are corresponding measured objects in sample.
Table 3: qualitative, quota ion pair and collision energy
| Relative ion abundance % | > 50 | > 20~50 | > 10~20 | ≤10 |
| The relative deviation % of permission | ± 20% | ±25 | ±30 | ±50 |
The quantitative approach of the measuring method are as follows: sample solution and standard solution are taken, by internal standard method in terms of peak area ratio
It calculates, the concentration of methaqualone in test solution, diazepam is acquired by standard curve；Methaqualone in standard solution and sample solution,
The response of diazepam and diazepam Isotopic Internal Standard should all be within the range of linearity that instrument detects；
Standard curve calibration: byA and b are acquired, then
Methaqualone and diazepam residual quantity are calculated by formula 2 in sample:
In formula:
AsThe peak area of methaqualone and diazepam in _ _ _ _ standard solution；
A'isThe peak area of diazepam Isotopic Internal Standard in _ _ _ _ standard solution；
csThe concentration of methaqualone and diazepam in _ _ _ _ standard solution, unit are nanograms per milliliter；
c'isThe concentration of diazepam Isotopic Internal Standard in _ _ _ _ standard solution, unit are nanograms per milliliter；
The concentration of methaqualone and diazepam in c____ sample solution, unit are nanograms per milliliter；
cisThe concentration of diazepam Isotopic Internal Standard in _ _ _ _ sample solution, unit are nanograms per milliliter；
The peak area of methaqualone and diazepam in A____ sample；
AisThe peak area of diazepam Isotopic Internal Standard in _ _ _ _ sample；
For X____ for the residual quantity of methaqualone and diazepam in material of having a try, unit is ng/kg；
V____ dissolves the volume of residue, and unit is milliliter；
M____ expects quality for having a try, and unit is gram；
D____ extension rate；
Extension rate is 5 in this formula；Calculated result need to deduct blank value, the arithmetic average that measurement result is measured in parallel
Value indicates.
The food is animal derived food.
The animal derived food includes pork, the flesh of fish, pig liver or Ren sus domestica.
Beneficial effect:
The present invention provides in animal derived food for the first time, especially methaqualone in pork, the flesh of fish, pig liver and Ren sus domestica
With the detection method of diazepam residual quantity, art technology blank has been filled up.Regression equation related coefficient of the present invention reaches 0.99
More than, lower limit of measurement be 0.5 μ g/kg, 0.5 μ of μ g/kg~5 g/kg addition concentration level on the rate of recovery be 90%~
120%, relative standard deviation≤15% in laboratory.The method of the present invention is easy to operate, high sensitivity, strong antijamming capability, base
Matter wide adaptation range, qualitative, quantitative science is accurate, suitable for the quick analysis of batch samples, can satisfy China's regulator
The technical requirements of department.
Detailed description of the invention
Fig. 1: methaqualone standard solution characteristic ion mass chromatogram (0.5 μ g/L), (251.2 > 132.1)
Fig. 2: methaqualone standard solution characteristic ion mass chromatogram (0.5 μ g/L), (251.2 > 91.1)
Fig. 3: diazepam standard solution characteristic ion mass chromatogram (0.5 μ g/L), (285.1 > 193)
Fig. 4: diazepam standard solution characteristic ion mass chromatogram (0.5 μ g/L), (285.1 > 154.1)
Fig. 5: diazepam Isotopic Internal Standard standard solution characteristic ion mass chromatogram (2.0 μ g/L), (290.2 > 198.2)
Fig. 6: pork blank sample characteristic ion mass chromatogram, (251.2 > 132.1)
Fig. 7: pork blank sample characteristic ion mass chromatogram, (251.2 > 91.1)
Fig. 8: pork blank sample characteristic ion mass chromatogram, (285.1 > 193)
Fig. 9: pork blank sample characteristic ion mass chromatogram, (285.1 > 154.1)
Figure 10: pork blank sample characteristic ion mass chromatogram, (290.2 > 198.2)
Figure 11: pork blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(251.2>132.1)
Figure 12: pork blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(251.2>91.1)
Figure 13: pork blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(285.1>193)
Figure 14: pork blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(285.1>154.1)
Figure 15: pork blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(290.2>198.2)
Figure 16: Tilapia blank sample characteristic ion mass chromatogram, (251.2 > 132.1)
Figure 17: Tilapia blank sample characteristic ion mass chromatogram, (251.2 > 91.1)
Figure 18: Tilapia blank sample characteristic ion mass chromatogram, (285.1 > 193)
Figure 19: Tilapia blank sample characteristic ion mass chromatogram, (285.1 > 154.1)
Figure 20: Tilapia blank sample characteristic ion mass chromatogram, (290.2 > 198.2)
Figure 21: Tilapia blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(251.2>132.1)
Figure 22: Tilapia blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(251.2>91.1)
Figure 23: Tilapia blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(285.1>193)
Figure 24: Tilapia blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(285.1>154.1)
Figure 25: Tilapia blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(290.2>198.2)
Figure 26: Ren sus domestica blank sample characteristic ion mass chromatogram, (251.2 > 132.1)
Figure 27: Ren sus domestica blank sample characteristic ion mass chromatogram, (251.2 > 91.1)
Figure 28: Ren sus domestica blank sample characteristic ion mass chromatogram, (285.1 > 193)
Figure 29: Ren sus domestica blank sample characteristic ion mass chromatogram, (285.1 > 154.1)
Figure 30: Ren sus domestica blank sample characteristic ion mass chromatogram, (290.2 > 198.2)
Figure 31: Ren sus domestica blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(251.2>132.1)
Figure 32: Ren sus domestica blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(251.2>91.1)
Figure 33: Ren sus domestica blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(285.1>193)
Figure 34: Ren sus domestica blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(285.1>154.1)
Figure 35: Ren sus domestica blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(290.2>198.2)
Figure 36: pig liver blank sample characteristic ion mass chromatogram, (251.2 > 132.1)
Figure 37: pig liver blank sample characteristic ion mass chromatogram, (251.2 > 91.1)
Figure 38: pig liver blank sample characteristic ion mass chromatogram, (285.1 > 193)
Figure 39: pig liver blank sample characteristic ion mass chromatogram, (285.1 > 154.1)
Figure 40: pig liver blank sample characteristic ion mass chromatogram, (290.2 > 198.2)
Figure 41: pig liver blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(251.2>132.1)
Figure 42: pig liver blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(251.2>91.1)
Figure 43: pig liver blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(285.1>193)
Figure 44: pig liver blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(285.1>154.1)
Figure 45: pig liver blank adds methaqualone and diazepam sample characteristics mass of ion chromatogram (0.5 μ g/kg),
(290.2>198.2)
Figure 46: the characteristic ion mass chromatogram of non-purified blank pig liver sample, (251.2 > 132.1)
Figure 47: the characteristic ion mass chromatogram of cleaned blank pig liver sample, (251.2 > 132.1)
Figure 48: the characteristic ion mass chromatogram of non-purified blank pig liver sample, (251.2 > 91.1)
Figure 49: the characteristic ion mass chromatogram of cleaned blank pig liver sample, (251.2 > 91.1)
Figure 50: the characteristic ion mass chromatogram of non-purified blank pig liver sample, (285.1 > 193)
Figure 51: the characteristic ion mass chromatogram of cleaned blank pig liver sample, (285.1 > 193)
Figure 52: the characteristic ion mass chromatogram of non-purified blank pig liver sample, (285.1 > 154.1)
Figure 53: the characteristic ion mass chromatogram of cleaned blank pig liver sample, (285.1 > 154.1)
Specific embodiment
The specific technical solution of the present invention described further below, in order to which those skilled in the art is further understood that
The present invention, without constituting the limitation to its right.
Embodiment 1
1. principle
Sample is extracted with ethyl acetate, extracting solution through QuEChERs (Quick, Easy, Cheap, Effective,
Rugged, Safe) pipe purification is purified, concentration volatilizes, and residue is detected with flowing phased soln using liquid chromatography-tandem mass spectrometry instrument,
Inner mark method ration.
2. reagent and material
Reagent used below is analytical reagents in addition to especially indicating；Water is to meet level-one as defined in GB/T 6682
Water.
2.1 methaqualone standard solution: 0.1mg/mL, LGC company.
2.2 diazepam standard solution: 100 μ g/mL Cerilliant companies.
2.3 diazepam Isotopic Internal Standard object standard solution: deuterated diazepam (D5Diazepam) 1.0mg/mL
Cerilliant company.
2.4 acetonitriles: chromatographically pure, Fisher company.
2.5 ethyl acetate: Guangzhou Chemical Reagent Factory.
2.6 formic acid: chromatographically pure, Merck company.
2.7 C18Filler: LC-C1840-63 μm, CNW company.
2.8 NH2Filler: Zhen Xiang company.
2.9 PSA fillers: 40-63 μm, 60A, CNW company.
2.10 QuEChERs purification pipe: 100mg C is weighed18Filler, 400mg NH2Filler and 10mg PSA filler in
15mL plastic centrifuge tube mixes.
2.11 5mg/L methaqualones and diazepam hybrid standard intermediate fluid: accurate respectively to measure methaqualone and diazepam standard
Solution 0.5mL, with dilution in acetonitrile to scale, is configured to methaqualone and diazepam mixing that concentration is 5mg/L in 10mL measuring bottle
Standard intermediate fluid.
2.12 100 μ g/L methaqualones and diazepam hybrid standard working solution: precision measures the methaqualone and Di Xi of 5mg/L
Hybrid standard intermediate fluid 1.00mL is dissolved, in 50mL measuring bottle, with dilution in acetonitrile to scale, being configured to concentration is 100 μ g/L sleepings
Ketone and diazepam hybrid standard working solution.
2.13 10mg/L diazepam Isotopic Internal Standard standard intermediate fluids: precision measures diazepam Isotopic Internal Standard object standard
100 μ L of solution, in 10mL measuring bottle, with dilution in acetonitrile to scale, being configured to concentration is among 10mg/L diazepam internal standard standard
Liquid.
2.14 100 μ g/L diazepam Isotopic Internal Standard standard working solutions: precision measures 10mg/L diazepam Isotopic Internal Standard
Standard intermediate fluid 1.00mL, in 100mL measuring bottle, with dilution in acetonitrile to scale, being configured to concentration is the 100 same positions of μ g/L diazepam
Plain internal standard standard working solution.
3 instrument and equipments
3.1 liquid chromatography-tandem mass spectrometry instruments: UFLC-XR Shimadzu liquid chromatograph；The series connection of 3000 triple quadrupole bar of API
Mass spectrograph, the source ESI, AB Sciex company.
3.2 assay balances: sensibility reciprocal 0.00001g, Sartorius company.
3.3 balances: sensibility reciprocal 0.01g, Sartorius company.
3.4 meat tissue's bruishers: 200 type of GM, Retsch company.
3.5 vortex oscillators: MS 3basic, IKA company.
3.6 oscillators: Heidolph company.
3.7 tissue refiners: T 25, IKA company.
3.8 centrifuges: 3-30K, SIGMA company.
3.9 nitrogen evaporators: Biotage Turbo Vap.
3.10 filter membranes: 0.22 μm, nylon membrane.
The preparation and preservation of 4 samples
The preparation of 4.1 samples
Pork, Tilapia, pig liver and Ren sus domestica blank tissue are taken respectively, are rubbed, and make homogeneous.
--- the test sample after taking homogeneous, as material of having a try.
--- the blank sample after taking homogeneous, as blank sample.
--- the blank sample after taking homogeneous adds the standard working solution of suitable concentration, adds sample as blank.
The preservation of 4.2 samples
- 18 DEG C or less preservations.
5 determination steps
5.1 extracting
Sample 5g ± 0.05g that above-mentioned four kinds of meat is prepared is weighed respectively, is had in plug centrifuge tube in 50mL, is added 100 μ
100 μ L of g/L diazepam Isotopic Internal Standard standard working solution, adds anhydrous sodium sulfate powder 5g, adds ethyl acetate 10mL, 12000r/
Min is homogenized 30s, separately takes a 50mL centrifuge tube to add ethyl acetate 15mL, washing homogenate cutter head 10s, cleaning solution retains spare.Homogenate
Mechanical shaking extraction 10min afterwards, 4000r/min are centrifuged 5min, and supernatant is placed in another cleaning 50mL centrifuge tube.Residue washing
Mechanical shaking extraction is primary again for liquid, and 4000r/min is centrifuged 5min, and supernatant merges, and is uniformly mixed, to be clean.
5.2 purification
It pipettes in extracting solution 5mL to QuEChERs purification pipe, vortex oscillation 2min, 4000r/min are centrifuged 5min, supernatant
It is transferred to teat glass, 40 DEG C of nitrogen are blown to dry, add acetonitrile -5m mol/L formic acid (30+70, v/v) 1mL vortex oscillation 2min, fill
Divide dissolved residue, cross 0.22 μm of nylon leaching film, is measured for LC-MS/MS.
The preparation of 5.3 standard curves
Precision measures 100 μ g/L methaqualones and diazepam hybrid standard working solution and 100 μ g/L diazepam Isotopic Internal Standards
Appropriate standard working solution is diluted with acetonitrile -5m mol/L formic acid (30+70, v/v), is configured to methaqualone and diazepam concentration is
0,0.2,0.5,1.0,2.0,5.0 and 10 μ g/L, diazepam Isotopic Internal Standard concentration are the series standard solution of 2.0 μ g/L, are supplied
Liquid chromatography-tandem mass spectrometry instrument measurement.With the characteristic ion mass chromatography of methaqualone and diazepam and diazepam Isotopic Internal Standard
Peak area ratio is ordinate, and concentration of standard solution is abscissa, draws standard curve, asks regression equation and related coefficient.
5.4 measurement
5.4.1 liquid phase chromatogram condition
5.4.1.1 chromatographic column: Atlantis T3(4.6mm × 100mm, 3 μm of partial size).
5.4.1.2 mobile phase: A:5m mol/L formic acid solution；B: acetonitrile, gradient are shown in Table 1.
1 gradient elution program of table
5.4.1.3 column temperature: 40 DEG C.
5.4.1.4 sample volume: 10 μ L.
5.4.2 Mass Spectrometry Conditions
5.4.2.1 ion source: electric spray ion source.
5.4.2.2 scanning mode: cation scanning.
5.4.2.3 detection mode: multiple-reaction monitoring.
5.4.2.4 spray voltage: 4500V.
5.4.2.5 atomization gas: 10.
5.4.2.6 gas curtain gas: 9.
5.4.2.7 collision gas: 10.
5.4.2.8 auxiliary plus hot air temperature: 500 DEG C.
5.4.2.9 focus voltage: -110V.
5.4.2.10 entrance potential: -10V.
5.4.2.11 collision cell exit potential: -15V.
5.4.2.12 residence time: 0.1s.
5.4.2.13 qualitative, quota ion pair goes cluster voltage and collision energy to be shown in Table 2.
Table 2 is qualitative, quota ion pair and collision energy
5.4.3 measuring method
5.4.3.1 qualitative determination
By the retention time of sample chromatogram and the retention time of respective standard product, chromatographic peak characteristic ion with it is corresponding
The characteristic ion of concentration standard solution chromatographic peak contrasts qualitative.Sample and the relative deviation of standard items retention time are not more than
5%；The relative abundance of sample characteristic ion is consistent with the relative abundance of the fairly standard solution of concentration, and relative abundance deviation is no more than
The regulation of table 3 then can determine whether that there are corresponding measured objects in sample.
The tolerance range of 3 relative ion abundance of table
| Relative ion abundance % | > 50 | > 20~50 | > 10~20 | ≤10 |
| The relative deviation % of permission | ± 20% | ±25 | ±30 | ±50 |
5.4.3.2 quantitative determination
Sample solution and standard solution are taken, is calculated by internal standard method with peak area ratio.Peace in standard solution and sample solution
The response of dormancy ketone, diazepam and diazepam Isotopic Internal Standard should all be within the range of linearity that instrument detects.In above-mentioned chromatography-
Under Mass Spectrometry Conditions, characteristic ion mass chromatogram is shown in explanation in standard solution, blank sample solution and blank addition sample solution
Book attached drawing.
5.5 blank test
In addition to sample is not added, operation repetitive is carried out using identical step.
5.6 results calculate and statement
Standard curve calibration: byA and b are acquired, then
Methaqualone and diazepam residual quantity are calculated by formula (2) in sample:
In formula:
AsThe peak area of methaqualone and diazepam in _ _ _ _ standard solution；
A'isThe peak area of diazepam Isotopic Internal Standard in _ _ _ _ standard solution；
csThe concentration of methaqualone and diazepam in _ _ _ _ standard solution, unit are nanograms per milliliter (ng/mL)；
c'isThe concentration of diazepam Isotopic Internal Standard in _ _ _ _ standard solution, unit are nanograms per milliliter (ng/mL)；
The concentration of methaqualone and diazepam in c____ sample solution, unit are nanograms per milliliter (ng/mL)；
cisThe concentration of diazepam Isotopic Internal Standard in _ _ _ _ sample solution, unit are nanograms per milliliter (ng/mL)；
The peak area of methaqualone and diazepam in A____ sample；
AisThe peak area of diazepam Isotopic Internal Standard in _ _ _ _ sample；
For X____ for the residual quantity of methaqualone and diazepam in material of having a try, unit is ng/kg (μ g/kg)；
V____ dissolves the volume of residue, and unit is milliliter (mL)；
M____ expects quality for having a try, and unit is gram (g)；
D____ extension rate.
Extension rate is 5 in this formula.
Note: calculated result need to deduct blank value, and the arithmetic mean of instantaneous value that measurement result is measured in parallel indicates that retaining three has
Effect number.
5.7 detection method sensitivity, accuracy and precision
5.7.1 sensitivity
The determination of quantitative limit is determined according to the value of signal-to-noise ratio (S/N).In blank pork, the flesh of fish, pig liver and pig
The methaqualone and diazepam standard solution for adding 0.5 μ g/kg in kidney respectively, measure the ratio of its signal and noise, when S/N >=
Concentration when 10 and when the rate of recovery and relative standard deviation meet method for detecting residue requirement is quantitative limit.
Experimental result: quantifying for this method is limited to 0.5 μ g/kg.
5.7.2 accuracy
5.0g blank sample is accurately weighed in 50mL centrifuge tube, oneself is added and knows the series standard working solution of concentration, is made
It is respectively the tissue sample of 0.5,1.0,5.0 μ g/kg containing methaqualone and diazepam drug concentration, each concentration is done 6 in parallel, pressed
It is measured after sample pretreatment process processing, calculates recovery of standard addition.
Experimental result: the rate of recovery of this method on 0.5 μ of μ g/kg~5 g/kg addition concentration level is 90%~120%.
5.7.3 precision
5.0g blank sample is accurately weighed in 50mL centrifuge tube, oneself is added and knows the series standard working solution of concentration, is made
It is respectively the tissue sample of 0.5,1.0,5.0 μ g/kg containing methaqualone and diazepam drug concentration, each concentration is done 6 in parallel, pressed
It is measured after sample pretreatment process processing, calculates indoor relative standard deviation.
Experimental result: relative standard deviation≤15% in the laboratory of this method.
6. result:
This method is used for routine testing, has detected totally 180, pork, the flesh of fish, pig liver and Ren sus domestica sample, there has been no inspections
Sample out.
The method according to the invention is shown in the comparison result of contrast standard 1, contrast standard 2 and documents 3 respectively respectively
The following table 4,5,6.
Table 4: the comparison result of contrast standard 1 and the method for the present invention
Table 5: the Characteristic Contrast of contrast standard 2 and the application
Table 6: the Characteristic Contrast of documents 3 and the application
Clean-up effect of the invention is obvious, eliminates the chaff interferent in matrix, to interfere more blank pig liver sample
For, it is non-purified to see that Figure of description 46-53, comparing result are shown in Table 7 compared with cleaned same sample chromatogram.
Table 7: the non-purified characteristic ion mass chromatogram comparing result with cleaned same blank pig liver sample
Claims (9)
1. the measuring method of methaqualone and diazepam residual quantity in a kind of food, it is characterised in that step are as follows: take sample to be measured, mention
It takes, supernatant is after QuEChERs purification pipe purification, and concentration volatilizes, and residue is to flow phased soln, then uses liquid chromatography-tandem
Mass spectrography detection, is finally quantified with internal standard method；The mobile phase is acetonitrile -5m mol/L formic acid；The QuEChERs
Purify pipe the preparation method comprises the following steps: weighing 10 parts of C18Filler, 40 parts of NH2Filler and 1 part of PSA filler mix, i.e., in centrifuge tube
；The chromatographic condition of the liquid chromatogram are as follows: chromatographic column is Atlantis T3 chromatographic column；Sample volume: 10 μ L；Flow velocity 0.3mL/
min；Column temperature: 40 DEG C；Mobile phase: A:5m mol/L formic acid solution；B: acetonitrile；Condition of gradient elution: when 0.0min, mobile phase A
It is 70%, Mobile phase B 30%；When 3.0min, mobile phase A 90%, Mobile phase B 10%；When 9.0min, mobile phase A is
90%, Mobile phase B 10%；When 9.1min, mobile phase A 70%, Mobile phase B 30%；When 15.0min, mobile phase A is
70%, Mobile phase B 30%.
2. measuring method as described in claim 1, it is characterised in that: the extraction process are as follows: in the sample to be measured, add
Enter diazepam Isotopic Internal Standard object standard working solution, add ethyl acetate as Extraction solvent, be homogenized, mechanical shaking extraction, centrifugation,
It is spare to separate supernatant.
3. measuring method as described in claim 1, it is characterised in that: the purification process are as follows: pipette extracting solution to described
QuEChERs is purified in pipe, vortex oscillation, and centrifugation takes supernatant to obtain the final product.
4. measuring method as described in claim 1, it is characterised in that: the preparation method of the standard curve of the inner mark method ration
Are as follows: precision measures 100 μ g/L methaqualones and diazepam hybrid standard working solution and 100 μ g/L diazepam Isotopic Internal Standard standard works
It is appropriate to make liquid, with the flowing phase dilution, is configured to methaqualone and diazepam concentration is 0,0.2,0.5,1.0,2.0,5.0 and 10
μ g/L, diazepam Isotopic Internal Standard concentration are the series standard solution of 2.0 μ g/L, are measured for liquid chromatography-tandem mass spectrometry instrument；With
The characteristic ion mass chromatography peak area ratio of methaqualone and diazepam and diazepam Isotopic Internal Standard is ordinate, and standard solution is dense
Degree is abscissa, draws standard curve, asks regression equation and related coefficient.
5. measuring method as described in claim 1, it is characterised in that: the Mass Spectrometry Conditions are as follows: ion source: electron spray ion
Source；Scanning mode: cation scanning；Detection mode: multiple-reaction monitoring；Spray voltage IS:4500V；Methaqualone qualitative ion pair
And collision energy: 251.2 > 132.1, collision energy 35V；251.2 > 91.1, collision energy 52V；Quota ion pair: 251.2 >
132.1；Diazepam qualitative ion pair and collision energy: 285.1 > 193.0, collision energy 45V；285.1 > 154.1, collision energy
38V；Quota ion pair: 285.1 > 154.1.
6. measuring method as described in claim 1, it is characterised in that: the qualitative method of the measuring method are as follows: pass through examination
Expect the characteristic ion and respective concentration standard solution color of the retention time of chromatogram and the retention time of respective standard product, chromatographic peak
The characteristic ion of spectral peak contrasts qualitative；Sample and the relative deviation of standard items retention time are not more than 5%；Sample characteristic ion
Relative abundance it is consistent with the relative abundance of the fairly standard solution of concentration, relative abundance deviation range meet: relative ion abundance
Greater than 50%, the relative deviation of permission is ± 20%；Relative ion abundance be greater than 20% be less than or equal to 50%, permission relatively partially
Difference is ± 25%；Relative ion abundance is greater than 10% and is less than or equal to 20%, and the relative deviation of permission is ± 30%；Relative ion
Abundance is less than or equal to 10%, and the relative deviation of permission is ± 50%, then can determine whether that there are corresponding measured objects in sample.
7. measuring method as described in claim 1, it is characterised in that: the quantitative approach of the measuring method are as follows: take sample
Solution and standard solution calculate by internal standard method with peak area ratio, acquires methaqualone in test solution, diazepam by standard curve
Concentration；The response of methaqualone, diazepam and diazepam Isotopic Internal Standard in standard solution and sample solution should all be in instrument
Within the range of linearity of detection；
Standard curve calibration: byA and b are acquired, then
Methaqualone and diazepam residual quantity are calculated by formula 2 in sample:
In formula:
As--- --- in standard solution methaqualone and diazepam peak area；
A'is--- --- in standard solution diazepam Isotopic Internal Standard peak area；
cs--- --- in standard solution methaqualone and diazepam concentration, unit is nanograms per milliliter；
c'is--- --- in standard solution diazepam Isotopic Internal Standard concentration, unit is nanograms per milliliter；
The concentration of c --- --- methaqualone and diazepam in sample solution, unit is nanograms per milliliter；
cis--- --- in sample solution diazepam Isotopic Internal Standard concentration, unit is nanograms per milliliter；
The peak area of A --- --- methaqualone and diazepam in sample；
Ais--- --- in sample diazepam Isotopic Internal Standard peak area；
For X --- --- for the residual quantity of methaqualone and diazepam in material of having a try, unit is ng/kg；
V --- --- dissolves the volume of residue, and unit is milliliter；
M --- --- expects quality for having a try, and unit is gram；
D --- --- extension rate；
Extension rate is 5 in this formula；Calculated result need to deduct blank value, the arithmetic mean of instantaneous value table that measurement result is measured in parallel
Show.
8. measuring method as described in claim 1, it is characterised in that the food is animal derived food.
9. measuring method as claimed in claim 8, it is characterised in that the animal derived food includes pork, the flesh of fish, pork liver
Dirty or Ren sus domestica.
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