CN108982712A - Salmeterol, datro, the method for Ao Dateluo in a kind of measurement pork - Google Patents
Salmeterol, datro, the method for Ao Dateluo in a kind of measurement pork Download PDFInfo
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- 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
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Abstract
The present invention provides a kind of salmeterol in measurement pork, datro, the method for Ao Dateluo, including method and step has: step 1, sample preparation；Step 2 extracts sample to be tested；Step 3, S3 liquid chromatography-tandem mass instrument testing conditions are determining and detect；Step 4, the drafting of standard working curve；Step 5 calculates Ao Dateluo in pork, datro, the content of salmeterol according to the liquid chromatogram of solution to be measured in S2.The method of the present invention measures salmeterol in pork, datro, Ao Dateluo simultaneously for the first time, it solves at present to the detection blank of this salmeterol, datro, Ao Dateluo in pork, Ion response value is high, high sensitivity, detection limit is low, it is truly realized qualitative, quantitative accurate, quick, efficient, sensitive, can be used as the reliable detection means of three kinds of drugs in pork.
Description
Technical field
The invention belongs to technical field of food detection, reach more particularly to salmeterol, the indenes in a kind of measurement pork
The method of special sieve, Ao Dateluo.
Background technique
Its beta 2 receptor for passing through excited airway smooth muscle and mast cell film surface of β 2 receptor agonist, diastole air flue are smooth
Flesh, the permeability for reducing capilary, increases air flue at the release for reducing mast cell and basophilic granulocyte degranulation and its medium
Asthma symptoms are alleviated in swing of epithelial cilia etc..Such drug is more, can be divided into short-acting (effect maintains 4~6 hours) and long-acting
(maintaining 12h) β 2 receptor agonist, wherein salmeterol, datro, Ao Dateluo are common kinds.
The unreasonable use of salmeterol, datro, Ao Dateluo be easy to cause medicament residue, while left drug may
Human health is caused it is potentially hazardous, so must be discontinued on time to guarantee animal product quality safety, while according to beast
The regulation of medicine national standard and professional standard is monitored point salmeterol, datro, Ao Dateluo in animal tissue
Analysis.But due to the physicochemical properties of veterinary drug difference, differ greatly, in addition in test sample other compositions interference, to dynamic
Quickly analysis detection constitutes very big difficulty simultaneously for salmeterol, datro, Ao Dateluo residual in object tissue.Sha Meite
The method of sieve, datro, Ao Dateluo currently without national standard method, in document, it is also desirable to use multiple and different methods pair
These compounds are detected, and operating procedure is complicated, and testing cost is high, therefore are needed to develop a kind of general detection method and be used for
The detection of daily sample.
Summary of the invention
The purpose of the present invention is to overcome above-mentioned the deficiencies in the prior art, provide a kind of Sha Meite measured in pork
Sieve, datro, the method for Ao Dateluo.
To achieve the above object, the present invention adopts the following technical solutions:
Salmeterol, datro, the method for Ao Dateluo in a kind of measurement pork, include the following steps:
S1 sample preparation:
It takes edible muscle samples to be cut into small pieces (go allowance for bark, bone, internal organ, fat) tested pork, is placed in meat grinder rubbing, system
The sample got ready saves under the conditions of being placed in -16 DEG C ~ -20 DEG C；
S2 is extracted:
(1) the pork sample of rubbing is placed in centrifuge tube, sodium acetate buffer is added and mixes well, then plus β-grape alditol glucoside
Enzyme, after mixing, 30 ~ 50 DEG C of water-bath hydrolysis；
(2) sample to be tested covers oscillation, and centrifugation takes supernatant in centrifuge tube, and perchloric acid solution is added, and covers oscillation, will be complete
Portion's supernatant is transferred in centrifuge tube, adjusts pH value to 8 ~ 13 with sodium hydroxide solution；
(3) ethyl acetate is added in water phase, covers the mixing that is vortexed, and vibrates 5 ~ 15min, is centrifuged 3 ~ 10min, takes organic phase in centrifuge tube
In, nitrogen is blown；
Ethyl acetate is added in water phase, covers the mixing that is vortexed, and vibrates 5 ~ 15min, is centrifuged 3 ~ 10min, takes organic phase in same centrifugation
Guan Zhong, nitrogen are blown；
Water phase be added t-butyl methyl ether, cover be vortexed mix, vibrate 5 ~ 15min, be centrifuged 3 ~ 10min, take organic phase in it is same from
In heart pipe, nitrogen is blown, drying；
(4) sodium acetate buffer is added, covers and is vortexed；
(5) cation exchange pillar is connected to vacuum apparatus, is successively activated with first alcohol and water；S2 (4) is obtained residual
Slag solution upper prop successively washs pillar with water, 2% methanol aqueous solution, methanol and thoroughly drains, finally washed with ammonium hydroxide methanol solution
Ingredient to be measured on de- pillar, flow control is in 0.5 ~ 1mL/min；Eluent is dried with nitrogen under 35 ~ 45 DEG C of water-baths；
(6) mobile phase is added to redissolve, solution is transferred in centrifuge tube, 1000r/min is centrifuged 5 ~ 20min, with liquid-transfering gun pipette tips
Take supernatant into micro-sampling bottle；
S3 liquid chromatography-tandem mass instrument testing conditions are determining and detect:
Chromatographic condition are as follows: ACQUITY UPLC BEH C18(2.1mm × 100mm), gradient elution；Mobile phase A is containing 0.1% first
The ammonium acetate solution of the 5mmoL/L of acid, Mobile phase B are 0.1% formic acid-acetonitrile solution；40 DEG C of chromatogram column temperature, sampling volume 5
μ L, flow velocity 0.3mL/min；
Mass Spectrometry Conditions are as follows: ESI cation scan pattern, capillary voltage are respectively 3kV, 400 DEG C of source temperature, remove solvent temperature degree:
400 DEG C, solvent gas argon gas flow velocity: 800L/h is removed, vertebral foramen gas nitrogen flow rate: 50 L/h, more reaction detection mode detections；
S4 draws standard working curve:
Ao Dateluo, datro, salmeterol hybrid standard stock solution are measured, different hybrid standard work is configured to
Liquid, quantified by external standard method draw standard curve, obtain Ao Dateluo, datro, salmeterol according to its concentration and peak area
Standard curve；
S5 is calculated:
According to Ao Dateluo, datro, the peak area of salmeterol and its correspondence in the liquid chromatogram of solution to be measured in S2
Standard items standard curve, calculate Ao Dateluo in pork, datro, the content of salmeterol.
As an optimization, salmeterol, datro, the method for Ao Dateluo in a kind of measurement pork, including it is as follows
Step:
S1 sample preparation:
It takes edible muscle samples to be cut into 2cm × 2cm fritter (go allowance for bark, bone, internal organ, fat) tested pork, is placed in Minced Steak
Machine rubs, and the sample prepared saves under the conditions of being placed in -16 DEG C ~ -20 DEG C；
S2 is extracted:
(1) the pork sample of rubbing is placed in centrifuge tube, sodium acetate buffer is added and mixes well, then plus β-grape alditol glucoside
Enzyme, after mixing, 37 DEG C of water-baths hydrolyze 12h；
(2) sample to be tested covers oscillation, and centrifugation takes supernatant in centrifuge tube, and perchloric acid solution is added, and covers oscillation, will be complete
Portion's supernatant is transferred in centrifuge tube, adjusts pH value to 11 with 1mol/L sodium hydroxide solution；
(3) ethyl acetate is added in water phase, covers the mixing that is vortexed, and vibrates 10min, is centrifuged 5min, takes organic phase in centrifuge tube, nitrogen
It blows；
Ethyl acetate is added in water phase, covers the mixing that is vortexed, and vibrates 10min, is centrifuged 5min, takes organic phase in same centrifuge tube,
Nitrogen is blown；
T-butyl methyl ether is added in water phase, covers the mixing that is vortexed, and vibrates 10min, is centrifuged 5min, takes organic phase in same centrifuge tube
In, nitrogen is blown, drying；
(4) sodium acetate buffer is added, covers vortex 1min；
(5) cation exchange pillar is connected to vacuum apparatus, is successively activated with first alcohol and water；S2 (4) is obtained residual
Slag solution upper prop successively washs pillar with water, 2% methanol aqueous solution, methanol and thoroughly drains, finally with 5% ammonium hydroxide methanol solution
The ingredient to be measured on pillar is eluted, flow control is in 0.5mL/min；Eluent is dried with nitrogen under 40 DEG C of water-baths；
(6) mobile phase is added to redissolve, solution is transferred in centrifuge tube, 1000r/min is centrifuged 10min, is taken with liquid-transfering gun pipette tips
Supernatant is into micro-sampling bottle；
S3 liquid chromatography-tandem mass instrument testing conditions are determining and detect:
Chromatographic condition are as follows: ACQUITY UPLC BEH C18(2.1mm × 100mm), gradient elution；Mobile phase A is containing 0.1% first
The ammonium acetate solution of the 5mmoL/L of acid, Mobile phase B are 0.1% formic acid-acetonitrile solution；40 DEG C of chromatogram column temperature, sampling volume 5
μ L, flow velocity 0.3mL/min；
Mass Spectrometry Conditions are as follows: ESI cation scan pattern, capillary voltage are respectively 3kV, 400 DEG C of source temperature, remove solvent temperature degree:
400 DEG C, solvent gas argon gas flow velocity: 800L/h is removed, vertebral foramen gas nitrogen flow rate: 50 L/h, more reaction detection mode detections；
S4 draws standard working curve:
Ao Dateluo, datro, salmeterol hybrid standard stock solution are measured, different hybrid standard work is configured to
Liquid, quantified by external standard method draw standard curve, obtain Ao Dateluo, datro, salmeterol according to its concentration and peak area
Standard curve；
S5 is calculated:
According to Ao Dateluo, datro, the peak area of salmeterol and its correspondence in the liquid chromatogram of solution to be measured in S2
Standard items standard curve, calculate Ao Dateluo in pork, datro, the content of salmeterol.
As an optimization, measuring Ao Dateluo, datro, salmeterol hybrid standard stock solution in the S4, match
It is set to the hybrid standard work of 1.0ng/mL, 2.0 ng/mL, 5.0 ng/mL, 10.0 ng/mL, 20.0 ng/mL, 50.0 ng/mL
Make liquid, quantified by external standard method.
Compared with prior art, the beneficial effects of the present invention are:
The method of the present invention measures salmeterol in pork, datro, Ao Dateluo simultaneously for the first time, solves at present in pork
This salmeterol, datro, the detection blank of Ao Dateluo, Ion response value is high, and high sensitivity, detection limit is low, really does
To qualitative, quantitative accurate, quick, efficient, sensitive, the reliable detection means of three kinds of drugs in pork can be used as.
Specific embodiment
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without creative efforts belongs to the model that the present invention protects
It encloses.
Embodiment 1
Salmeterol in a kind of measurement pork, datro, the method for Ao Dateluo, 3 kinds of beta-receptor agonists, this method is to answer
The detection carried out with LC-MS instrument to pork, includes the following steps:
S1 sample preparation
It takes edible muscle samples to cut 2cm × 2cm fritter (go allowance for bark, bone, internal organ etc.) tested pork, is placed in meat grinder and chews
Broken, the sample prepared saves under the conditions of being placed in -16 DEG C；
S2 is extracted
(1) sample of the 2.00g through smashing to pieces is weighed in 50 mL centrifuge tubes, and 8mL sodium acetate buffer is added and mixes well, then plus
50 μ L β-grape alditol glucoside enzyme, after mixing, 37 DEG C of water-baths hydrolyze 12h；
(2) sample to be tested covers oscillation 15min, is centrifuged 5min(8000r/min), it takes 4mL supernatant in centrifuge tube, is added
0.1mol/L perchloric acid solution 5mL covers 5 ~ 10min of oscillation, whole supernatants (about 10mL) is transferred in 50mL centrifuge tube,
PH value is adjusted to 11 with the sodium hydroxide solution of 1mol/L；
(3) water phase be added 10mL ethyl acetate, cover be vortexed mix, vibrate 10min, be centrifuged 5min, take organic phase in 15mL from
In heart pipe, nitrogen is blown；
Water phase be added 5mL ethyl acetate, cover be vortexed mix, vibrate 10min, be centrifuged 5min, take organic phase in same 15mL from
In heart pipe, nitrogen is blown；
8mL t-butyl methyl ether is added in water phase, covers the mixing that is vortexed, and vibrates 10min, is centrifuged 5min, takes organic phase in same 15mL
In centrifuge tube, nitrogen is blown, drying；
(4) 5mL 0.2mol/L sodium acetate buffer is added, covers vortex 1min；
(5) cation exchange pillar is connected to vacuum apparatus, is successively activated with 3mL methanol and 3mL water.By above-mentioned residue
Solution upper prop successively washs pillar with 2mL water, 2mL2% methanol aqueous solution, 2mL methanol and thoroughly drains, and finally uses 2mL5% ammonia
Water beetle alcoholic solution elutes the ingredient to be measured on pillar, and in 0.5mL/min, eluent nitrogen under 40 DEG C of water-baths is blown flow control
It is dry.
(6) 200 μ L mobile phases are accurately added to redissolve, solution is transferred in 1.5 mL centrifuge tubes, 10min is centrifuged
(1000r/min) takes supernatant into micro-sampling bottle, to upper machine with 200 μ L pipette tips.
S3 liquid chromatography-tandem mass instrument testing conditions are determining and detect:
Chromatographic condition are as follows: ACQUITY UPLC BEH C18(2.1mm × 100mm), gradient elution；Mobile phase A is containing 0.1% first
The ammonium acetate solution of the 5mmoL/L of acid, Mobile phase B are 0.1% formic acid-acetonitrile solution；40 DEG C of chromatogram column temperature, sampling volume 5
μ L, flow velocity 0.3mL/min；Design parameter is shown in Table 1.
1 eluent gradient elution program of table
Mass Spectrometry Conditions are as follows: ESI cation scan pattern, capillary voltage are respectively 3kV, 400 DEG C of source temperature, remove solvent temperature degree:
400 DEG C, solvent gas argon gas flow velocity: 800L/h is removed, vertebral foramen gas nitrogen flow rate: 50 L/h, more reaction detection mode detections；More reactions
Detection pattern detection, reaction monitoring parent ion, daughter ion and collision energy are shown in Table 2；
2 target compound Mass Spectrometry Conditions of table
| Compound name | Parent ion | Daughter ion | Collision energy |
| Salmeterol | 416.30 | 91.13 | 40 |
| Datro | 393.24 | 117.15 | 44 |
| Ao Dateluo | 387.15 | 121.17 | 38 |
The drafting of S4 standard working curve
Ao Dateluo, datro, salmeterol hybrid standard stock solution are measured, external standard concentration is 1ng/mL, 2.0 ng/
ML, 5.0 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, quantified by external standard method, test result determine linearly dependent coefficient
R2 is all larger than 0.999.
S5 is calculated:
According to Ao Dateluo, datro, the peak area of salmeterol and its correspondence in the liquid chromatogram of solution to be measured in S2
Standard items standard curve, calculate Ao Dateluo in pork, datro, the content of salmeterol.
(1) sensitivity: taking as defined in this method is had a characteristic that using the test result that the application method obtains
Under sample volume and constant volume, it must be detected by 3 times of signal-to-noise ratio computations and be limited to 0.5 μ g/kg；(2) accuracy: with signal-to-noise ratio S/N > 10
Obtaining its lower limit of quantitation (LOQ) is 1.0 μ g/kg；(3) precision: relative deviation≤10% in this method batch, relative deviation between batch≤
10%。
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (3)
1. salmeterol, datro, the method for Ao Dateluo in a kind of measurement pork, which is characterized in that including walking as follows
It is rapid:
S1 sample preparation:
It takes edible muscle samples to be cut into small pieces (go allowance for bark, bone, internal organ, fat) tested pork, is placed in meat grinder rubbing, system
The sample got ready saves under the conditions of being placed in -16 DEG C ~ -20 DEG C；
S2 is extracted:
(1) the pork sample of rubbing is placed in centrifuge tube, sodium acetate buffer is added and mixes well, then plus β-grape alditol glucoside
Enzyme, after mixing, 30 ~ 50 DEG C of water-bath hydrolysis；
(2) sample to be tested covers oscillation, and centrifugation takes supernatant in centrifuge tube, and perchloric acid solution is added, and covers oscillation, will be complete
Portion's supernatant is transferred in centrifuge tube, adjusts pH value to 8 ~ 13 with sodium hydroxide solution；
(3) ethyl acetate is added in water phase, covers the mixing that is vortexed, and vibrates 5 ~ 15min, is centrifuged 3 ~ 10min, takes organic phase in centrifuge tube
In, nitrogen is blown；
Ethyl acetate is added in water phase, covers the mixing that is vortexed, and vibrates 5 ~ 15min, is centrifuged 3 ~ 10min, takes organic phase in same centrifugation
Guan Zhong, nitrogen are blown；
Water phase be added t-butyl methyl ether, cover be vortexed mix, vibrate 5 ~ 15min, be centrifuged 3 ~ 10min, take organic phase in it is same from
In heart pipe, nitrogen is blown, drying；
(4) sodium acetate buffer is added, covers and is vortexed；
(5) cation exchange pillar is connected to vacuum apparatus, is successively activated with first alcohol and water；S2 (4) is obtained residual
Slag solution upper prop successively washs pillar with water, 2% methanol aqueous solution, methanol and thoroughly drains, finally washed with ammonium hydroxide methanol solution
Ingredient to be measured on de- pillar, flow control is in 0.5 ~ 1mL/min；Eluent is dried with nitrogen under 35 ~ 45 DEG C of water-baths；
(6) mobile phase is added to redissolve, solution is transferred in centrifuge tube, 1000r/min is centrifuged 5 ~ 20min, with liquid-transfering gun pipette tips
Take supernatant into micro-sampling bottle；
S3 liquid chromatography-tandem mass instrument testing conditions are determining and detect:
Chromatographic condition are as follows: ACQUITY UPLC BEH C18(2.1mm × 100mm), gradient elution；Mobile phase A is containing 0.1% first
The ammonium acetate solution of the 5mmoL/L of acid, Mobile phase B are 0.1% formic acid-acetonitrile solution；40 DEG C of chromatogram column temperature, sampling volume 5
μ L, flow velocity 0.3mL/min；
Mass Spectrometry Conditions are as follows: ESI cation scan pattern, capillary voltage are respectively 3kV, 400 DEG C of source temperature, remove solvent temperature degree:
400 DEG C, solvent gas argon gas flow velocity: 800L/h is removed, vertebral foramen gas nitrogen flow rate: 50 L/h, more reaction detection mode detections；
S4 draws standard working curve:
Ao Dateluo, datro, salmeterol hybrid standard stock solution are measured, different hybrid standard work is configured to
Liquid, quantified by external standard method draw standard curve, obtain Ao Dateluo, datro, salmeterol according to its concentration and peak area
Standard curve；
S5 is calculated:
According to Ao Dateluo, datro, the peak area of salmeterol and its correspondence in the liquid chromatogram of solution to be measured in S2
Standard items standard curve, calculate Ao Dateluo in pork, datro, the content of salmeterol.
2. salmeterol, datro, the method for Ao Dateluo in a kind of measurement pork according to claim 1,
It is characterized in that,
S1 sample preparation:
It takes edible muscle samples to be cut into 2cm × 2cm fritter (go allowance for bark, bone, internal organ, fat) tested pork, is placed in Minced Steak
Machine rubs, and the sample prepared saves under the conditions of being placed in -16 DEG C ~ -20 DEG C；
S2 is extracted:
(1) the pork sample of rubbing is placed in centrifuge tube, sodium acetate buffer is added and mixes well, then plus β-grape alditol glucoside
Enzyme, after mixing, 37 DEG C of water-baths hydrolyze 12h；
(2) sample to be tested covers oscillation, and centrifugation takes supernatant in centrifuge tube, and perchloric acid solution is added, and covers oscillation, will be complete
Portion's supernatant is transferred in centrifuge tube, adjusts pH value to 11 with 1mol/L sodium hydroxide solution；
(3) ethyl acetate is added in water phase, covers the mixing that is vortexed, and vibrates 10min, is centrifuged 5min, takes organic phase in centrifuge tube, nitrogen
It blows；
Ethyl acetate is added in water phase, covers the mixing that is vortexed, and vibrates 10min, is centrifuged 5min, takes organic phase in same centrifuge tube,
Nitrogen is blown；
T-butyl methyl ether is added in water phase, covers the mixing that is vortexed, and vibrates 10min, is centrifuged 5min, takes organic phase in same centrifuge tube
In, nitrogen is blown, drying；
(4) sodium acetate buffer is added, covers vortex 1min；
(5) cation exchange pillar is connected to vacuum apparatus, is successively activated with first alcohol and water；S2 (4) is obtained residual
Slag solution upper prop successively washs pillar with water, 2% methanol aqueous solution, methanol and thoroughly drains, finally with 5% ammonium hydroxide methanol solution
The ingredient to be measured on pillar is eluted, flow control is in 0.5mL/min；Eluent is dried with nitrogen under 40 DEG C of water-baths；
(6) mobile phase is added to redissolve, solution is transferred in centrifuge tube, 1000r/min is centrifuged 10min, is taken with liquid-transfering gun pipette tips
Supernatant is into micro-sampling bottle；
S3 liquid chromatography-tandem mass instrument testing conditions are determining and detect:
Chromatographic condition are as follows: ACQUITY UPLC BEH C18(2.1mm × 100mm), gradient elution；Mobile phase A is containing 0.1% first
The ammonium acetate solution of the 5mmoL/L of acid, Mobile phase B are 0.1% formic acid-acetonitrile solution；40 DEG C of chromatogram column temperature, sampling volume 5
μ L, flow velocity 0.3mL/min；
Mass Spectrometry Conditions are as follows: ESI cation scan pattern, capillary voltage are respectively 3kV, 400 DEG C of source temperature, remove solvent temperature degree:
400 DEG C, solvent gas argon gas flow velocity: 800L/h is removed, vertebral foramen gas nitrogen flow rate: 50 L/h, more reaction detection mode detections；
S4 draws standard working curve:
Ao Dateluo, datro, salmeterol hybrid standard stock solution are measured, different hybrid standard work is configured to
Liquid, quantified by external standard method draw standard curve, obtain Ao Dateluo, datro, salmeterol according to its concentration and peak area
Standard curve；
S5 is calculated:
According to Ao Dateluo, datro, the peak area of salmeterol and its correspondence in the liquid chromatogram of solution to be measured in S2
Standard items standard curve, calculate Ao Dateluo in pork, datro, the content of salmeterol.
3. salmeterol, datro, the method for Ao Dateluo in a kind of measurement pork according to claim 1,
It is characterized in that, Ao Dateluo, datro, salmeterol hybrid standard stock solution is measured in the S4, is configured to 1.0ng/
The hybrid standard working solution of mL, 2.0 ng/mL, 5.0 ng/mL, 10.0 ng/mL, 20.0 ng/mL, 50.0 ng/mL, external standard
Standard measure.
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|---|---|---|---|---|
| CN111380968A (en) * | 2018-12-29 | 2020-07-07 | 天津药业研究院有限公司 | Method for detecting content of odaterol and related substances |
| CN111624292A (en) * | 2020-07-10 | 2020-09-04 | 武汉伯瑞恒医药科技有限公司 | Method for determining concentration of indacaterol glycopyrronium bromide in serum |
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