CN106908532B - The method for measuring aquatic products Malachite Green, quinolones, sulfa drugs - Google Patents
The method for measuring aquatic products Malachite Green, quinolones, sulfa drugs Download PDFInfo
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Abstract
The present invention relates to a kind of methods of measurement aquatic products Malachite Green, quinolones, sulfa drugs, including method and step to have: step 1, sample preparation；Step 2, extracting sample to be tested must be to upper press proof product；Step 3 determines chromatographic condition；Step 4 determines Mass Spectrometry Conditions；Step 5, the drafting of standard working curve；Step 6, sensitivity, accuracy and precision determination.Measurement simultaneously is extracted in concentration aquatic products matrix with/without color malachite green, quinolones, sulfa drugs the method for the present invention for the first time, solve the problems, such as that time-consuming, testing cost is high for traditional extracting respectively, Ion response value increases, sensitivity enhancing, detection limit is low, be truly realized it is qualitative, it is quantitative accurate, quick, efficient, sensitive, it can be used as the reliable detection means of three classes drug, batch samples be suitble to extract concentration.
Description
Technical field
The invention belongs to the application of liquid matter in analytical chemistry-matter connection instrument and ecological environment security technical field, especially one
The method that kind measures aquatic products Malachite Green, quinolones and sulfa drugs simultaneously using LC-MS instrument.
Background technique
As the raising and breeding environment of aquatic livestock intensive culture degree constantly deteriorate, aquatic animal disease is in
Type is more, wide coverage, the unfixed feature of disease time for now morbidity, cultivates dealer in order to avoid economy caused by disease is damaged
It loses, using a large amount of drug and disinfectant preparation, in addition replaced currently without special Medicines in Aquaculture, the status of light " anti-" weight " controlling ",
Medicament residue problem is more and more prominent, very big concern of the fish quality problem by government and society.Because malachite green,
Quinolones, sulfa drugs have has a broad antifungal spectrum, efficient, lower-price characteristic, become the preferred medicament of numerous raisers,
Therefore malachite green, quinolones, sulfa drugs become one of residual factor of the highest medicine of current recall rate, from protection consumer
The angle of health is set out, China has promulgated a series of national standards, harmful substance contents in clear stipulaties aquatic products
Limited Doses.Scholar has carried out the residual technical research of Aquatic Products by HPLC medicine first, has formulated SC/T3021-2004 respectively
Aquatic products are determined in liquid chromatogram measuring Malachite Green Residues in Aquatic Product, GB/T20361-2006 high performance liquid chromatography fluorescence detection
Malachite Green and crystal violet residual quantity, sulfonamides in No. 958 bulletin -12-2007 liquid chromatogram measuring aquatic products of the Ministry of Agriculture
Object residual quantity, quinolones medicament relict amount in No. 783 bulletin -2-2006 liquid chromatogram measuring aquatic products, with science and technology into
Step, i.e., after liquid chromatogram, the expanded application of LC-MS instrument, because it has many advantages, such as high sensitivity, qualitative accurate by quality inspection
Mechanism is approved that scholar, which has carried out, measures the residual technical research of aquatic products Chinese medicine using LC-MS instrument, has formulated GB/ respectively
T19857-2005 liquid matter-matter measurement aquatic products Malachite Green and crystal violet residual quantity, GB/T22951-2008 globe fish, eel
Measurement-Liquid Chromatography-Tandem Mass Spectrometry of 18 kinds of residual quantity of sulfonamide, GB/T20751-2006 sea eel and product in fish
In 15 kinds of quinolones medicament relict amounts measurement-Liquid Chromatography-Tandem Mass Spectrometry, No. 1077 bulletin -1-2008 of the Ministry of Agriculture
17 kinds of sulfamidos and measurement-Liquid Chromatography-Tandem Mass Spectrometry of 15 kinds of quinolones medicament relict amounts, in addition have in aquatic products
Scholar has also carried out the residual technical research DB34/T1421-2011 aquatic products Malachite Green of enzyme-linked immunization measurement medicine and its metabolism
Quick screening test-enzyme-linked immunization of object residual quantity, the survey of DB51/T1278-2011 fresh milk sulfa drug residue
Fixed-colloidal gold immunity chromatography, SN/T4535.1-2016 commercial kit detect quinolones method.In summary it detects
The advantages of technology, liquid chromatography is quantitatively accurate, the disadvantage is that being easy to be interfered by impurity, qualitative poor accuracy, liquid matter-matter method survey
Determine to be qualitative accurate compared with the advantage of liquid chromatography, small by impurity interference, insufficient place is instrument maintenance higher cost, enzyme-linked
Immunization measures and has the characteristics that specificity and sensibility height and quick, is generally used to quickly sieve, the disadvantage is that quantitative result
Accuracy is low.Currently, as Detection task amount increases, existing standard method Testing index is single, and detection is time-consuming, testing cost is high
The problem of it is increasingly prominent, to solve the above problems, two or several single Testing index, which are integrated into a detection method, to be become
The heat subject of current research.
Summary of the invention
The purpose of the present invention is for detection aquatic products Malachite Green, quinolones, the sulfa drugs prior art
Deficiency, and propose a kind of using LC-MS instrument while to measure the side of aquatic products Malachite Green, quinolones, sulfa drugs
Method.
The present invention solves its technical problem and adopts the following technical solutions to achieve:
A method of measurement aquatic products Malachite Green, quinolones, sulfa drugs, this method are joined using liquid matter
The detection that aquatic products are carried out with instrument, it is characterised in that as follows including method and step:
Step 1, sample preparation
Edible muscle parts sample is taken to be cut into no more than 0.5cm × 0.5cm × 0.5cm block detected aquatic products
Afterwards, it is placed in meat grinder to blend, the sample prepared saves under the conditions of being placed in -16 DEG C to -20 DEG C；
Step 2 is extracted
(1) the above-mentioned 5.00 ± 0.01g of sample prepared is weighed to be placed in 50mL centrifuge tube, be added 20 μ L mixing with/without
Color malachite green internal standard working solution, 50 μ L quinolones and sulfamido internal standard working solution, vortex 20s, avoid light place 20min are added
10g anhydrous sodium sulfate is vortexed and mixes, and 15mL, V is addedAcetonitrile: VConcentrated hydrochloric acidThe Acidifying acetonitrile of=1000:4 is homogenized 1min, ultrasonic extraction
10min, 6000r/min are centrifuged 5min, and supernatant is taken to cross neutral alumina column in 50mL pear shape bottle；
(2) 15mL ethyl acetate is added in the residue of aforesaid operations to repeat to extract 1 time, 6000r/min is centrifuged 5min,
Supernatant is incorporated in above-mentioned 50mL pear shape bottle；
(3) above-mentioned 50mL pear shape bottle is done in 40 DEG C of rotary evaporations of water-bath to close, 1mL constant volume solution is added, adds 2mL
N-hexane degreasing is crossed 0.2 μm of microfiltration membranes, is obtained to upper press proof product；
Step 3 determines chromatographic condition
Chromatographic column: Shim-packXR-ODS (75mm × 2.1mm), column temperature: 35 DEG C, sample room temperature: 10 DEG C, mobile phase
A: the ammonium acetate solution of the 5mmoL/L containing 0.1% formic acid, Mobile phase B: 0.1% formic acid-acetonitrile solution, sampling volume: 5.0 μ
L, flow velocity: 0.25mL/min, design parameter are shown in Table 1,
1 eluent gradient elution program of table
Step 4 determines Mass Spectrometry Conditions
(1) ionization mode: electric spray ion source (ESI), positive ion mode；
(2) ion source temperature: 600 DEG C；
(3) gas curtain gas: 20psi, collision gas: in, atomization gas: 60psi, auxiliary gas: 40psi, spray voltage: 5000v；
(4) scan pattern: multiple-reaction monitoring (MRM), reaction monitoring parent ion, daughter ion, orifice potential and collision energy
It is shown in Table 2；
2 target compound Mass Spectrometry Conditions of table
Step 5, the drafting of standard working curve
(1) it measures with/without color malachite green 100ng/mL standard work 8 liquid 0.05,0.1,0.2,0.5,1.0,2.0mL,
Measuring that 100ng/mL is deuterated to be mixed into working solution volumetric flask with/without color malachite green 0.2mL again, mobile phase is settled to 10.0mL,
External standard concentration is 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 20.0ng/mL, and internal standard concentration is
2ng/mL；1.0 μ g/mL sulfamidos, quinolones 0.1,0.2,0.5,1.0,1.5,2.0mL are measured, then are measured in 1.0 μ g/mL
Mark deuterated sulfanilamide (SN), quinolones takes 0.5mL, is mixed into working solution volumetric flask, mobile phase is settled to 10.0mL, and external standard concentration is
10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL are inside designated as 50ng/mL；
(2) inner mark method ration, test result are determined with/without color malachite green mass concentration in 0.1~100ng/mL model
With peak area in good linear relationship in enclosing, sulfamido and quinolones mass concentration are within the scope of 0.005~1 μ g/mL
It is in good linear relationship, linearly dependent coefficient r with peak area2It is all larger than 0.999；
Step 6, sensitivity, accuracy and precision determination
(1) sensitivity: under the sample volume as defined in this method and constant volume, being obtained by 3 times of signal-to-noise ratio computations, with/without
Malachite green detection is limited to 0.1 μ g/kg, and quinolone and sulfamido detection are limited to 0.5 μ g/kg；
(2) accuracy: its lower limit of quantitation (LOQ) is obtained with signal-to-noise ratio S/N > 10, is quantitatively limited to with/without color malachite green
0.25 μ g/kg, quinolone and sulfamido are quantitatively limited to 1.0 μ g/kg；
(3) precision: relative deviation≤15% in this method batch, relative deviation≤15% between batch.
Moreover, constant volume solution in (3) step of the step 2 are as follows: acetonitrile: 1mmol/L ammonium acetate solution contains 0.1% formic acid
=50:50.
The advantages and positive effects of the present invention are:
1, measurement simultaneously is extracted in concentration aquatic products matrix with/without color malachite green, quinolone the method for the present invention for the first time
Class, sulfa drugs solve the problems, such as that time-consuming, testing cost is high for traditional extracting respectively, are suitble to batch samples to extract dense
Contracting.
2, the present invention will be with/without color malachite green, quinolones, sulfonamides using AB5500QTrap LC-MS instrument
Object re-optimization obtains best mass spectrometry parameters, and Ion response value increases after optimization, sensitivity enhancing, and detection limit is low.
3, the present invention measures analysis using LC-MS instrument with/without color malachite green, quinolones, sulfonamides simultaneously
Object, hence it is evident that shorten compound minute, be truly realized it is qualitative, it is quantitative accurate, quick, efficient, sensitive, can be used as three classes drug
Reliable detection means.
4, the present invention fully considers the chemical property with/without color malachite green, quinolones, sulfa drugs, to difference
It extracts concentrated reagent to be screened, the final composite reagent for determining that extraction efficiency is high, the rate of recovery is stable, solve only with a kind of
Acidifying acetonitrile is as extraction reagent, the problem that sulfamido extraction effect is not good enough.
Detailed description of the invention
Fig. 1 is coloured malachite green second order ms figure in the present invention；
Fig. 2 is leucomalachite green second order ms figure in the present invention；
Fig. 3 is deuterated leucomalachite green second order ms figure in the present invention；
Fig. 4 is deuterated coloured malachite green second order ms figure in the present invention；
Fig. 5 is Norfloxacin second order ms figure in the present invention；
Fig. 6 is Ciprofloxacin second order ms figure in the present invention；
Fig. 7 is Enrofloxacin second order ms figure in the present invention；
Fig. 8 is deuterated Norfloxacin second order ms figure in the present invention；
Fig. 9 is deuterated Ciprofloxacin second order ms figure in the present invention；
Figure 10 is deuterated Enrofloxacin second order ms figure in the present invention；
Figure 11 is sulphadiazine second order ms figure in the present invention；
Figure 12 is sulphathiazole second order ms figure in the present invention；
Figure 13 is sulfamethyldiazine second order ms figure in the present invention；
Figure 14 is sulfamethazine second order ms figure in the present invention；
Figure 15 is bacteresulf second order ms figure in the present invention；
Figure 16 is sulfamethoxazole second order ms figure in the present invention；
Figure 17 is sulfadoxine second order ms figure in the present invention；
Figure 18 is sulfaquinoxaline second order ms figure in the present invention；
Figure 19 is deuterated fanasil second order ms figure in the present invention；
Figure 20 is deuterated madribon second order ms figure in the present invention.
Specific embodiment
The implementation of the invention is further described below, and following examples are merely illustrative and not limiting, cannot
It is limited the scope of protection of the present invention with this.
A method of measurement aquatic products Malachite Green, quinolones, sulfa drugs, this method are joined using liquid matter
The detection carried out with instrument to aquatic products, including method and step are as follows:
Step 1, sample preparation
All detected aquatic products only take edible muscle parts, and sample is cut into no more than 0.5cm × 0.5cm × 0.5cm
Block after, be placed in meat grinder and blend, the sample prepared saves under the conditions of being placed in -16 DEG C to -20 DEG C；
Wherein, frozen samples are first thawed at normal temperature before being prepared, and then carry out sample according to above-mentioned processing method
Preparation.
In specific implementation of the invention, for various fish: scaling, remove the peel, take muscle along back；For each seed shrimp,
Crab class: decaptitating, removes gutstring at decladding, takes muscle parts.
Step 2 is extracted
(1) it accurately weighs the above-mentioned 5.00 ± 0.01g of sample prepared to be placed in 50mL centrifuge tube, the accurate 20 μ L that are added are mixed
It closes with/without color malachite green internal standard working solution, 50 μ L quinolones and sulfamido internal standard working solution, vortex 20s, avoid light place
10g anhydrous sodium sulfate is added in 20min, is vortexed and mixes, and 15mL, V is addedAcetonitrile: VConcentrated hydrochloric acidThe Acidifying acetonitrile of=1000:4, homogenate
1min, ultrasonic extraction 10min, 6000r/min centrifugation 5min, takes supernatant to cross neutral alumina column in 50mL pear shape bottle；
(2) 15mL ethyl acetate is added in the residue of aforesaid operations to repeat to extract 1 time, 6000r/min is centrifuged 5min, on
Clear liquid is incorporated in above-mentioned 50mL pear shape bottle；
(3) above-mentioned 50mL pear shape bottle is done in 40 DEG C of rotary evaporations of water-bath to close, 1mL constant volume solution is added, adds 2mL
N-hexane degreasing is crossed 0.2 μm of microfiltration membranes, is obtained to upper press proof product；
Wherein, the constant volume solution are as follows: acetonitrile: 1mmol/L ammonium acetate solution contains 0.1% formic acid=50:50.
Step 3 determines chromatographic condition
Chromatographic column: Shim-packXR-ODS (75mm × 2.1mm)；Column temperature: 35 DEG C；Sample room temperature: 10 DEG C；Mobile phase
A: the ammonium acetate solution of the 5mmoL/L containing 0.1% formic acid；Mobile phase B: 0.1% formic acid-acetonitrile solution；Sampling volume: 5.0 μ
L；Flow velocity: 0.25mL/min, eluent gradient elution program are shown in Table 1,
Step 4 determines Mass Spectrometry Conditions
(1) ionization mode: electric spray ion source (ESI), positive ion mode；
(2) ion source temperature: 600 DEG C；
(3) gas curtain gas: 20psi, collision gas: in, atomization gas: 60psi, auxiliary gas: 40psi, spray voltage: 5000v；
(4) scan pattern: multiple-reaction monitoring (MRM), reaction monitoring parent ion, daughter ion, orifice potential and collision energy
It is shown in Table 2；
Step 5, the drafting of standard working curve
(1) it is accurate measure with/without color malachite green 100ng/mL standard working solution 0.05,0.1,0.2,0.5,1.0,
2.0mL, then the accurate 100ng/mL that measures are deuterated with/without color malachite green 0.2mL, are mixed into working solution volumetric flask, mobile phase is fixed
To hold to 10.0mL, external standard concentration is 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 20.0ng/mL,
Internal standard concentration is 2ng/mL；Accurate measurement 1.0 μ g/mL sulfamidos, quinolones 0.1,0.2,0.5,1.0,1.5,2.0mL, then
The deuterated sulfanilamide (SN) of 1.0 μ g/mL internal standard of correct amount, quinolones take 0.5mL, are mixed into working solution volumetric flask, mobile phase is settled to
10.0mL, external standard concentration are 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL, are inside designated as
50ng/mL；
(2) inner mark method ration, test result show: with/without color malachite green mass concentration in 0.1~100ng/mL model
With peak area in good linear relationship in enclosing, sulfamido and quinolones mass concentration are within the scope of 0.005~1 μ g/mL
It is in that good linear relationship is linear with peak area, linearly dependent coefficient r2It is all larger than 0.999；
Step 6, sensitivity, accuracy and precision determination
(1) sensitivity: under the sample volume as defined in this method and constant volume, being obtained by 3 times of signal-to-noise ratio computations, with/without
Malachite green detection is limited to 0.1 μ g/kg, and quinolone and sulfamido detection are limited to 0.5 μ g/kg；
(2) accuracy: its lower limit of quantitation (LOQ) is obtained with signal-to-noise ratio S/N > 10, is quantitatively limited to with/without color malachite green
0.25 μ g/kg, quinolone and sulfamido are quantitatively limited to 1.0 μ g/kg；
(3) precision: relative deviation≤15% in this method batch, relative deviation≤15% between batch.
The verifying of this detection method
Test does the mark-on of 3 horizontal groups, with/without peacock by the way of blank mark-on in aquatic products bare substrate
0.25 μ g/kg of malachite green, 1.25 μ g/kg, 10 μ g/kg, quinolone and each 2.5 μ g/kg of sulfamido, 12.5 μ g/kg, 100 μ g/kg,
Every group 6 are parallel, by being measured after above-mentioned processing, TIANZHU XINGNAO Capsul and relative standard deviation (n of the malachite green in blank sample
=6) 3 are shown in Table, the TIANZHU XINGNAO Capsul and relative standard deviation (n=6) table 4 of quinolone and sulfamido in blank sample；
TIANZHU XINGNAO Capsul and relative standard deviation (n=6) of 3 malachite green of table in blank sample
The TIANZHU XINGNAO Capsul and relative standard deviation (n=6) of 4 quinolone of table and sulfamido in blank sample
The results showed that can be reached with/without the recovery of standard addition of malachite green, quinolone, sulfamido in aquatic products
To testing requirements, illustrate this method accuracy with higher.Meanwhile relative standard deviation RSD is below 15%, shows result
In confidence interval, this method reliability with higher.
Embodiment 1, the selection of parent ion, daughter ion
This research is analyzed in aquatic products with Liquid Chromatography-Tandem Mass Spectrometry with/without color malachite green, quinolones and sulfanilamide (SN)
Class, select (ESI+) cation scanning mode can obtain more fragment informations, with molecular ion peak [M+H]+and secondly
Grade fragment is qualitative to the progress of its three classes compound, and is quantified with most strong secondary fragment.
With/without hydroxy functional group positively chargeable in color malachite green, quinolones and sulfamido, (ESI+) ion is selected
Change mode can be provided compared with the more fragment informations of (ESI+) positive ion mode, and sensitivity greatly improves, as shown in Figure 1.This
Method by 0.5 μ g/mL with/without color malachite green, quinolones and sulfamido and its internal standard solution, using peristaltic pump with 5.0 μ L/
Min speed continuous sample introduction mode carries out parent ion full scan in the positive-ion mode, determines respectively with/without color malachite green, 3 kinds
The parent ion of quinolone, 8 kinds of sulfanilamide (SN) and its deuterated internal standard compound.
Then the ion centered on respective parent ion, progress second order ms scanning select abundance opposite respectively respectively
Stronger two fragments characteristic ions form monitoring ion pair with parent ion and daughter ion, with multiple-reaction monitoring (MRM) mode pair
Object carries out qualitative and quantitative analysis, selects that abundance is most strong, glitch-free monitoring ion is as quota ion, secondary strong abundance ion
As qualitative ion, therefore the present invention has the characteristics that select specificity good.
Embodiment 2, sample pre-treatments condition
Sample pre-treatments
The present invention is to measure aquatic products Malachite Green and crystal violet residual quantity and agriculture with GB/T19857-2005 liquid matter matter
In the bulletin -1-2008 aquatic products of industry portion 1077 based on the measurement of 17 kinds of sulfamidos and 15 kinds of quinolones medicament relict amounts,
Integration further is optimized to two methods, by original two methods be integrated into compound extract and upper machine measurement can simultaneously into
A capable method.
1. sample weighs
It is weighed after the complete natural thaw of sample to be frozen, weighing should sample in four diagonal zones of meat gruel, and sampling should be kept away
Exempt to carry the water after thawing, guarantee the uniformity for weighing meat gruel, reduces the error that matrix does not mix generation.
2. extracting
It accurately weighs sample 5.00g (± 0.01g) to be placed in 50mL centrifuge tube, the accurate 20 μ L that are added are mixed with/colourless hole
Sparrow malachite green internal standard working solution, 50 μ L quinolones and sulfamido internal standard working solution, vortex 20s, avoid light place 20min.The first step is wanted
Guarantee that internal standard sufficiently diffuses in meat gruel, accomplishes that internal standard compound is synchronous with target compound as far as possible and extract.
10g anhydrous sodium sulfate is added, is vortexed and mixes, 15mL Acidifying acetonitrile (V is addedAcetonitrile: VConcentrated hydrochloric acid=1000:4), homogenate
1min, ultrasonic extraction 10min, 6000r/min centrifugation 5min, takes supernatant to cross neutral alumina column in 50mL pear shape bottle.The
Two steps first extract the abundant extraction that may insure with/without color malachite green, 3 kinds of quinolones with Acidifying acetonitrile.
It adds 15mL ethyl acetate in residue to repeat to extract 1 time, 6000r/min is centrifuged 5min, and supernatant is incorporated in
In 50mL pear shape bottle.Third step adopts the abundant extraction for being extracted with ethyl acetate and may insure 8 kinds of sulfanilamide (SN), and test result shows acid
Change acetonitrile to best with/without color malachite green, quinolones extraction effect, ethyl acetate is best to sulfamido extraction effect, because
This, test selection is first extracted with Acidifying acetonitrile, then is extracted with ethyl acetate the mode combined, allows three classes compound extraction effect
Reach best.
40 DEG C of rotary evaporations of water-bath are done to close, and acetonitrile is added: 1mmol/L ammonium acetate solution contains 0.1% formic acid=50:
2mL n-hexane degreasing is added in 501mL constant volume solution, 0.2 μm of microfiltration membranes is crossed, to upper machine.
The present invention will merge into primary extraction with/without color malachite green, quinolones, residual extract twice of sulfonamides, should
Extracting method step is simple, operation is time saving, it is good to save testing cost, recovering effect.
Embodiment 3, the determination of AB5500Q LC-MS instrument testing conditions
According to pertinent regulations in CAC and EU the 657/2002/EECth resolution, two pairs of ions is selected to carry out MRM monitoring
Meet testing requirements, while the selection of daughter ion mainly considers wherein parent ion and daughter ion according to the mass spectrogram of every kind of compound
It is chosen with architectural characteristic, and less in actual sample analysis mesostroma interference, point of various substances is determined according to mentioned above principle
After daughter ion, respectively using the molecular ion of various compounds as parent ion, relatively strong to its daughter ion progress full scan selection abundance,
Interfering lesser two pairs of daughter ions is qualitative ion, it is strongest it is a pair of be quota ion, finally with multiple-reaction monitoring (MRM) just from
Subpattern optimizes various mass spectrometry parameters.
Testing conditions:
Chromatographic condition: chromatographic column: Shim-packXR-ODS (75mm × 2.1mm)；Column temperature: 35 DEG C；Sample room temperature: 10
℃；Mobile phase A: the ammonium acetate solution of the 5mmoL/L containing 0.1% formic acid；Mobile phase B: 0.1% formic acid-acetonitrile solution；Sample introduction
Volume: 5.0 μ L；Flow velocity: 0.25mL/min, eluent gradient elution program are shown in Table 1.
Mass Spectrometry Conditions: (1) ionization mode: electric spray ion source (ESI), positive ion mode；Ion source temperature: 600 DEG C；
Gas curtain gas: 20psi；Collision gas: in；Atomization gas: 60psi；Assist gas: 40psi；Spray voltage: 5000v；Scan pattern: mostly anti-
It should monitor that (MRM, reaction monitoring parent ion, daughter ion, orifice potential and collision energy are shown in Table 2.
Embodiment 4, the rate of recovery and precision
Addition grouping: by the way of blank mark-on, 3 horizontal additions, addition are carried out in aquatic products bare substrate
With/without 0.25 μ g/kg of malachite green, 1.25 μ g/kg, 10 μ g/kg, each 2.5 μ g/kg of quinolone, sulfamido, 12.5 μ g/kg,
100 μ g/kg, each gradient 6 parallel.
Extract: accurately weighing sample 5.00g (± 0.01g) and be placed in 50mL centrifuge tube, it is accurate be added 20 μ L mixing with/without
Color malachite green internal standard working solution, 50 μ L quinolones and sulfamido internal standard working solution, vortex 20s, avoid light place 20min are added
10g anhydrous sodium sulfate is vortexed and mixes, and 15mL Acidifying acetonitrile (V is addedAcetonitrile: VConcentrated hydrochloric acid=1000:4), it is homogenized 1min, ultrasonic extraction
10min, 6000r/min are centrifuged 5min, and supernatant is taken to cross neutral alumina column in 50mL pear shape bottle.15mL is added in residue
Ethyl acetate repeats to extract 1 time, and 6000r/min is centrifuged 5min, and supernatant is incorporated in 50mL pear shape bottle, rotates in 40 DEG C of water-bath
Be evaporated to close dry, addition acetonitrile: 1mmol/L ammonium acetate solution contains 0.1% formic acid=50:501mL constant volume solution, and addition 2mL is just
Hexane degreasing crosses 0.2 μm of microfiltration membranes, to upper machine.The rate of recovery and relative standard deviation are shown in Table 5 and table 6.Test result shows: have/
The recovery of standard addition of no malachite green, quinolone, sulfamido in aquatic products can reach testing requirements, illustrate that this method has
Higher accuracy.Meanwhile relative standard deviation RSD is below 15%, show result in confidence interval, this method have compared with
High reliability.
TIANZHU XINGNAO Capsul and relative standard deviation (n=6) of 5 malachite green of table in blank sample
The TIANZHU XINGNAO Capsul and relative standard deviation (n=6) of 6 quinolone of table and sulfamido in blank sample
Embodiment 5, sensitivity, accuracy and precision
Under the sample volume as defined in method and constant volume, sensitivity obtains this method with/without hole by 3 times of signal-to-noise ratio computations
0.1 μ g/kg of sparrow malachite green detection limit, quinolone, sulfamido detection are limited to 0.5 μ g/kg.Accuracy obtains it with signal-to-noise ratio S/N > 10
Lower limit of quantitation (LOQ) is quantitatively limited to 0.25 μ g/kg with/without color malachite green, and quinolone, sulfamido are 1.0 μ g/kg；Precision:
Relative deviation≤15% in this method batch, relative deviation≤15% between batch.
Embodiment 6, the practical application of detection method
With the method for the present invention 90 samples of Ministry of Agriculture's city routine monitor are carried out with actually detected, wherein Leuco Malachite stone inspection
Extracting rate is 6%, and being greater than 1.0 μ g/kg by Ministry of Agriculture's regulation malachite green vestigial is positive sample, and practical qualification rate is 97%；Quinoline
Promise ketone recall rate is 46%, and wherein Enrofloxacin accounts for the 90% of detection, is greater than 100 μ by Ministry of Agriculture's regulation enrofloxacin residual
It is positive sample that g/kg or Ciprofloxacin and Norfloxacin total residual, which are greater than 100 μ g/kg, and practical qualification rate is 98%；Sulfanilamide (SN)
Class recall rate is 2%, and being greater than 100 μ g/kg by Ministry of Agriculture's regulation sulfamido residual total amount is positive sample, and practical qualification rate is
100%, the above testing result is consistent with adoption of existing standard testing result, it was demonstrated that the method for the present invention has practical application.
Claims (2)
1. a kind of method of measurement aquatic products Malachite Green, quinolones, sulfa drugs, this method are using LC-MS
The detection that instrument carries out aquatic products, it is characterised in that as follows including method and step:
Step 1, sample preparation
It takes edible muscle parts sample to be cut into no more than after 0.5cm × 0.5cm × 0.5cm block detected aquatic products, sets
It is blended in meat grinder, the sample prepared saves under the conditions of being placed in -16 DEG C to -20 DEG C；
Step 2 is extracted
(1) it weighs the above-mentioned 5.00 ± 0.01g of sample prepared to be placed in 50mL centrifuge tube, 20 μ L is added and are mixed with/colourless hole
Sparrow malachite green internal standard working solution, 50 μ L quinolones and sulfamido internal standard working solution, vortex 20s, avoid light place 20min, be added 10g without
Aqueous sodium persulfate is vortexed and mixes, and 15mL, V is addedAcetonitrile: VConcentrated hydrochloric acidThe Acidifying acetonitrile of=1000:4 is homogenized 1min, ultrasonic extraction 10min,
6000r/min is centrifuged 5min, and supernatant is taken to cross neutral alumina column in 50mL pear shape bottle；
(2) it adds 15mL ethyl acetate in the residue of aforesaid operations to repeat to extract 1 time, 6000r/min is centrifuged 5min, supernatant
Liquid is incorporated in above-mentioned 50mL pear shape bottle；
(3) by above-mentioned 50mL pear shape bottle in 40 DEG C of rotary evaporations of water-bath to close dry, 1mL constant volume solution is added, add 2mL just oneself
Alkane degreasing is crossed 0.2 μm of microfiltration membranes, is obtained to upper press proof product；
Step 3 determines chromatographic condition
Chromatographic column: Shim-pack XR-ODS75mm × 2.1mm, column temperature: 35 DEG C, sample room temperature: 10 DEG C, mobile phase A: contain
The ammonium acetate solution of the 5mmoL/L of 0.1% formic acid, Mobile phase B: 0.1% formic acid-acetonitrile solution, sampling volume: 5.0 μ L, stream
Speed: 0.25mL/min, gradient elution program:
Time are as follows: 0.1min, mobile phase A: Mobile phase B 98:2；
Time are as follows: 8.1min, mobile phase A: Mobile phase B 20:80；
Time are as follows: 10min, mobile phase A: Mobile phase B 20:80；
Time are as follows: 11min, mobile phase A: Mobile phase B 2:98；
Time are as follows: 13min, mobile phase A: Mobile phase B 2:98；
Time are as follows: 13.1min, mobile phase A: Mobile phase B 98:2；
Time are as follows: 15min, mobile phase A: Mobile phase B 98:2；
Step 4 determines Mass Spectrometry Conditions
(1) ionization mode: electric spray ion source ESI, positive ion mode；
(2) ion source temperature: 600 DEG C；
(3) gas curtain gas: 20psi, collision gas: in, atomization gas: 60psi, auxiliary gas: 40psi, spray voltage: 5000v；
(4) scan pattern: multiple-reaction monitoring MRM, reaction monitoring parent ion, daughter ion, orifice potential and collision energy are as follows；
Step 5, the drafting of standard working curve
(1) it measures with/without color malachite green 100ng/mL standard working solution 0.05,0.1,0.2,0.5,1.0,2.0mL, then measures
100ng/mL is deuterated to be mixed into working solution volumetric flask with/without color malachite green 0.2mL, and mobile phase is settled to 10.0mL, and external standard is dense
Degree is 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 20.0ng/mL, and internal standard concentration is 2ng/mL；
1.0 μ g/mL sulfamidos, quinolones 0.1,0.2,0.5,1.0,1.5,2.0mL are measured, then measure the 1.0 deuterated sulphurs of μ g/mL internal standard
Amine, quinolones take 0.5mL, are mixed into working solution volumetric flask, and mobile phase is settled to 10.0mL, external standard concentration be 10ng/mL,
20ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL are inside designated as 50ng/mL；
(2) inner mark method ration, test result are determined with/without color malachite green mass concentration within the scope of 0.1~100ng/mL
It is in good linear relationship with peak area, sulfamido and quinolones mass concentration are within the scope of 0.005~1 μ g/mL and peak
Area is in good linear relationship, linearly dependent coefficient r2It is all larger than 0.999；
Step 6, sensitivity, accuracy and precision determination
(1) it sensitivity: under the sample volume as defined in this method and constant volume, is obtained by 3 times of signal-to-noise ratio computations, with/without color hole
The detection of sparrow malachite green is limited to 0.1 μ g/kg, and quinolone and sulfamido detection are limited to 0.5 μ g/kg；
(2) accuracy: its lower limit of quantitation LOQ is obtained with signal-to-noise ratio S/N > 10, is quantitatively limited to 0.25 μ g/ with/without color malachite green
Kg, quinolone and sulfamido are quantitatively limited to 1.0 μ g/kg；
(3) precision: relative deviation≤15% in this method batch, relative deviation≤15% between batch.
2. the method for measurement aquatic products Malachite Green according to claim 1, quinolones, sulfa drugs, special
Sign is: constant volume solution in (3) step of the step 2 are as follows: acetonitrile: 1mmol/L ammonium acetate solution contains 0.1% formic acid=50:
50。
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| CN108918704A (en) * | 2018-06-04 | 2018-11-30 | 福建省纤维检验局 | A kind of detection method of leather and fur Malachite Green |
| CN111289659A (en) * | 2020-04-07 | 2020-06-16 | 广西壮族自治区水产科学研究院 | Rapid quantitative analysis method and kit for residual quantity of malachite green in aquatic products |
| CN112162052A (en) * | 2020-11-06 | 2021-01-01 | 深圳市格物正源质量标准系统有限公司 | Method for determining multiple residues of veterinary drugs in aquatic products |
| CN115078589B (en) * | 2022-07-15 | 2023-11-21 | 贵州省烟草科学研究院 | Chromatographic analysis method for amine additives in biodegradable mulch film |
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