CN106908532A - Determine aquatic products Malachite Green, quinolones, the method for sulfa drugs - Google Patents
Determine aquatic products Malachite Green, quinolones, the method for sulfa drugs Download PDFInfo
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Abstract
Have the present invention relates to a kind of method for determining aquatic products Malachite Green, quinolones, sulfa drugs, including method and step：Step one, sample preparation；Step 2, extracting detected sample must treat press proof product；Step 3, determines chromatographic condition；Step 4, determines Mass Spectrometry Conditions；Step 5, the drafting of standard working curve；Step 6, the determination of sensitivity, the degree of accuracy and precision.The inventive method first simultaneously determine be extracted concentration aquatic products matrix in with/without color malachite green, quinolones, sulfa drugs, solve the problems, such as that traditional to extract time-consuming, testing cost respectively high, Ion response value increases, sensitivity strengthens, detection limit is low, be truly realized it is qualitative, it is quantitative accurate, quick, efficient, sensitive, can be adapted to batch samples and extract concentration as the reliable detection means of three class medicines.
Description
Technical field
Application and ecological environment security technical field, particularly one the invention belongs to liquid matter in analytical chemistry-matter connection instrument
Plant the method that application liquid matter-matter combined instrument determines aquatic products Malachite Green, quinolones and sulfa drugs simultaneously.
Background technology
Raising and breeding environment with aquatic livestock intensive culture degree constantly deteriorate, and aquatic animal disease is in
Species is more for now morbidity, wide coverage, the unfixed feature of disease time, the economic damage that cultivation dealer causes in order to avoid disease
Lose, using substantial amounts of medicine and disinfectant preparation, add and replace currently without special Medicines in Aquaculture, the present situation of light " anti-" weight " controlling ",
Medicament residue problem is increasingly protruded, and fish quality problem receives the very big concern of government and society.Because malachite green,
Quinolones, sulfa drugs have has a broad antifungal spectrum, efficiently, lower-price characteristic, the first-selected medicament as numerous raisers,
Therefore malachite green, quinolones, sulfa drugs turn into one of current residual factor of recall rate highest medicine, from protection consumer
Healthy angle 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, and SC/T 3021- have been formulated respectively
2004 liquid chromatogram measuring Malachite Green Residues in Aquatic Product, GB/T 20361-2006 high performance liquid chromatography fluoroscopic examination is determined
Aquatic products Malachite Green and crystal violet residual quantity, sulphur in No. 958 bulletin -12-2007 liquid chromatogram measuring aquatic products of the Ministry of Agriculture
Sulfonamides residues amount, quinolones medicament relict amount in No. 783 bulletin -2-2006 liquid chromatogram measuring aquatic products, with section
After the progress of skill, i.e. liquid chromatogram, the liquid matter-expanded application of matter combined instrument, because it has sensitivity high, qualitative accurate etc. excellent
Point is approved that scholar has carried out using liquid matter-matter combined instrument measure residual technical research of aquatic products Chinese medicine, makes respectively by quality inspection organization
Determine GB/T 19857-2005 liquid matter-matter and determine aquatic products Malachite Green and crystal violet residual quantity, GB/T 22951-2008
18 kinds of measure-Liquid Chromatography-Tandem Mass Spectrometries of residual quantity of sulfonamide, GB/T 20751-2006 in globe fish, eel
15 kinds of measure-Liquid Chromatography-Tandem Mass Spectrometries of quinolones medicament relict amount in eel and product, No. 1077 public affairs of the Ministry of Agriculture
Accuse 17 kinds of sulfamidos and 15 kinds of measure-liquid chromatography-tandem mass spectrometries of quinolones medicament relict amount in -1-2008 aquatic products
Method, the scholar having in addition has also carried out ELISA and has determined the residual technical research DB34/T 1421-2011 aquatic products Malachites of medicine
Quick screening test-the ELISA of malachite green and its metabolite residue amount, DB51/T 1278-2011 fresh milk sulfamidos
Measure-the colloidal gold immunity chromatography of medicament residue, SN/T 4535.1-2016 commercial kits detection quinolones method.
In summary detection technique, the advantage of liquid chromatography is quantitatively accurate, has the disadvantage easily to be disturbed by impurity, the qualitative degree of accuracy
Difference, it is qualitative accurate that liquid matter-matter method is determined compared with the advantage of liquid chromatography, is disturbed small by impurity, and not enough place is instrument dimension
Shield it is relatively costly, ELISA determine and with specificity and sensitiveness it is high and quick the characteristics of, be generally used to quickly sieve,
Have the disadvantage that quantitative result accuracy is low.Currently, as Detection task amount is increased, existing standard method Testing index is single, detection
Time-consuming, testing cost problem high is increasingly highlighted, and to solve the above problems, two or several single Testing index is integrated into one
Individual detection method turns into the heat subject of current research.
The content of the invention
The purpose of the present invention is directed to detection aquatic products Malachite Green, quinolones, sulfa drugs prior art
Deficiency, and propose a kind of application liquid matter-matter combined instrument and determine aquatic products Malachite Green, quinolones, sulfa drugs simultaneously
Method.
The present invention solves its technical problem and takes following technical scheme to realize：
A kind of measure aquatic products Malachite Green, quinolones, the method for sulfa drugs, the method be using liquid matter-
The detection that matter combined instrument is carried out to aquatic products, it is characterised in that as follows including method and step：
Step one, sample preparation
Detected aquatic products are taken into the block that edible muscle parts sample is cut into no more than 0.5cm × 0.5cm × 0.5cm
Afterwards, it is placed in meat grinder to blend, the sample for preparing is preserved under the conditions of being placed in -16 DEG C to -20 DEG C；
Step 2, extracts
(1) the above-mentioned 5.00 ± 0.01g of sample for preparing is weighed to be placed in 50mL centrifuge tubes, add 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 sulfates, are vortexed and mix, and add 15mL, VAcetonitrile：VConcentrated hydrochloric acid=1000:4 Acidifying acetonitrile, is homogenized 1min, ultrasonic extraction
10min, 6000r/min are centrifuged 5min, take supernatant and cross neutral alumina column in 50mL pear shape bottles；
(2) add 15mL ethyl acetate in the residue of aforesaid operations to repeat to extract 1 time, 6000r/min centrifugation 5min,
Supernatant is incorporated in above-mentioned 50mL pear shape bottles；
(3) above-mentioned 50mL pear shape bottles are added into 2mL in 40 DEG C of rotary evaporations of water-bath near dry, addition 1mL constant volume solution
N-hexane degreasing, crosses 0.2 μm of microfiltration membranes, must treat press proof product；
Step 3, determines chromatographic condition
Chromatographic column：Shim-pack XR-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：5.0μ
L, flow velocity：0.25mL/min, design parameter is shown in Table 1,
The eluent gradient elution program of table 1
Step 4, determines Mass Spectrometry Conditions
(1) ionization mode：Electric spray ion source (ESI), positive ion mode；
(2) ion source temperature：600℃；
(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, taper hole voltage and collision energy
It is shown in Table 2；
The target compound Mass Spectrometry Conditions of table 2
Step 5, the drafting of standard working curve
(1) measure with/without color malachite green 100ng/mL standards 8 liquid 0.05,0.1,0.2,0.5,1.0,2.0mL of work,
Measure 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 is measured in 1.0 μ g/mL
Mark deuterated sulfanilamide (SN), quinolones and take 0.5mL, be mixed into working solution volumetric flask, mobile phase is settled to 10.0mL, 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, result of the test, it is determined that with/without color malachite green mass concentration in 0.1~100ng/mL models
With peak area in good linear relationship in enclosing, sulfamido and quinolones mass concentration are in the range of 0.005~1 μ g/mL
It is in good linear relationship, linearly dependent coefficient r with peak area2It is all higher than 0.999；
Step 6, the determination of sensitivity, the degree of accuracy and precision
(1) sensitivity：Under the sample volume and constant volume of our law regulation, obtained by 3 times of signal-to-noise ratio computations, with/without
Peacock
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) degree of 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.
And, constant volume solution is in (3) step of the step 2：Acetonitrile:1mmol/L ammonium acetate solutions contain 0.1% formic acid
=50:50.
Advantages and positive effects of the present invention are：
1st, the inventive method first simultaneously determine be extracted concentration aquatic products matrix in with/without color malachite green, quinolone
Class, sulfa drugs, solve the problems, such as that time-consuming, testing cost is high for traditional extracting respectively, and suitable batch samples extract dense
Contracting.
2nd, matter-matter combined instrument will be with/without color malachite green, quinolones, sulfanilamide (SN) using AB5500Q Trap liquid for the present invention
Class medicine re-optimization, obtains optimal mass spectrometry parameters, and Ion response value increases after optimization, and sensitivity enhancing, detection limit is low.
3rd, the present invention determines analysis using liquid matter-matter combined instrument with/without color malachite green, quinolones, sulfamido simultaneously
Medicine, 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 class medicines
The reliable detection means of thing.
4th, the present invention take into full account with/without color malachite green, quinolones, sulfa drugs chemical property, to difference
Extract concentrated reagent to be screened, finally determine the composite reagent that extraction efficiency is high, the rate of recovery is stable, solve only with one kind
Acidifying acetonitrile is used as extracts reagent, the problem that sulfamido extraction effect is not good enough.
Brief description of the drawings
Fig. 1 is coloured malachite green second order mses figure in the present invention；
Fig. 2 is leucomalachite green second order mses figure in the present invention；
Fig. 3 is deuterated leucomalachite green second order mses figure in the present invention；
Fig. 4 is deuterated coloured malachite green second order mses figure in the present invention；
Fig. 5 is Norfloxacin second order mses figure in the present invention；
Fig. 6 is Ciprofloxacin second order mses figure in the present invention；
Fig. 7 is Enrofloxacin second order mses figure in the present invention；
Fig. 8 is deuterated Norfloxacin second order mses figure in the present invention；
Fig. 9 is deuterated Ciprofloxacin second order mses figure in the present invention；
Figure 10 is deuterated Enrofloxacin second order mses figure in the present invention；
Figure 11 is sulphadiazine second order mses figure in the present invention；
Figure 12 is sulphathiazole second order mses figure in the present invention；
Figure 13 is sulfamethyldiazine second order mses figure in the present invention；
Figure 14 is sulfamethazine second order mses figure in the present invention；
Figure 15 is bacteresulf second order mses figure in the present invention；
Figure 16 is sulfamethoxazole second order mses figure in the present invention；
Figure 17 is sulfadoxine second order mses figure in the present invention；
Figure 18 is sulfaquinoxaline second order mses figure in the present invention；
Figure 19 is deuterated fanasil second order mses figure in the present invention；
Figure 20 is deuterated madribon second order mses figure in the present invention.
Specific embodiment
Present invention implementation is further described below, following examples are descriptive, are not limited, it is impossible to
Protection scope of the present invention is limited with this.
A kind of measure aquatic products Malachite Green, quinolones, the method for sulfa drugs, the method be using liquid matter-
The detection that matter combined instrument is carried out to aquatic products, including method and step is as follows：
Step one, 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 for preparing be placed in -16 DEG C to -20 DEG C under the conditions of preserve；
Wherein, frozen samples are first thawed at normal temperatures before being prepared, and then carry out sample according to above-mentioned processing method
Prepare.
In specific implementation of the invention, for various fish：Scale, remove the peel, muscle is taken along back；For each seed shrimp,
Crab class：Decaptitate, shell, remove gutstring, take muscle parts.
Step 2, extracts
(1) the above-mentioned 5.00 ± 0.01g of sample for preparing accurately is weighed to be placed in 50mL centrifuge tubes, it is accurate to add 20 μ L to mix
Close 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, adds 10g anhydrous sodium sulfates, is vortexed and mixes, and adds 15mL, VAcetonitrile：VConcentrated hydrochloric acid=1000:4 Acidifying acetonitrile, homogenate
1min, ultrasonic extraction 10min, 6000r/min centrifugation 5min, take supernatant and cross neutral alumina column in 50mL pear shape bottles；
(2) add 15mL ethyl acetate in the residue of aforesaid operations to repeat to extract 1 time, 6000r/min centrifugation 5min, on
Clear liquid is incorporated in above-mentioned 50mL pear shape bottles；
(3) above-mentioned 50mL pear shape bottles are added into 2mL in 40 DEG C of rotary evaporations of water-bath near dry, addition 1mL constant volume solution
N-hexane degreasing, crosses 0.2 μm of microfiltration membranes, must treat press proof product；
Wherein, the constant volume solution is：Acetonitrile:1mmol/L ammonium acetate solutions contain 0.1% formic acid=50:50.
Step 3, determines chromatographic condition
Chromatographic column：Shim-pack XR-ODS(75mm×2.1mm)；Column temperature：35℃；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；Sampling volume：5.0μ
L；Flow velocity：0.25mL/min, design parameter is shown in Table 1,
The eluent gradient elution program of table 1
Step 4, determines Mass Spectrometry Conditions
(1) ionization mode：Electric spray ion source (ESI), positive ion mode；
(2) ion source temperature：600℃；
(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, taper hole voltage and collision energy
It is shown in Table 2；
The target compound Mass Spectrometry Conditions of table 2
Step 5, the drafting of standard working curve
(1) it is accurate measure with/without color malachite green 100ng/mL standard working solutions 0.05,0.1,0.2,0.5,1.0,
2.0mL, then accurately measure that 100ng/mL is deuterated to be mixed into working solution volumetric flask with/without color malachite green 0.2mL, mobile phase is fixed
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；1.0 μ g/mL sulfamidos, quinolones 0.1,0.2,0.5,1.0,1.5,2.0mL accurately are measured, then
The deuterated sulfanilamide (SN) of μ g/mL internal standards of correct amount 1.0, quinolones take 0.5mL, are mixed into working solution volumetric flask, and mobile phase is settled to
10.0mL, external standard concentration is 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL, is inside designated as
50ng/mL；
(2) inner mark method ration, result of the test shows：With/without color malachite green mass concentration in 0.1~100ng/mL models
With peak area in good linear relationship in enclosing, sulfamido and quinolones mass concentration are in the range of 0.005~1 μ g/mL
It is in linear good linear relationship, linearly dependent coefficient r with peak area2It is all higher than 0.999；
Step 6, the determination of sensitivity, the degree of accuracy and precision
(1) sensitivity：Under the sample volume and constant volume of our law regulation, 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) degree of 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 checking of this detection method
Experiment does 3 mark-ons of horizontal group, with/without peacock by the way of blank mark-on in aquatic products bare substrate
The μ g/kg of malachite green 0.25,1.25 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 parallel, is determined by after above-mentioned treatment, TIANZHU XINGNAO Capsul and relative standard deviation (n of the malachite green in blank sample
=6) it is shown in Table 3, quinolone and TIANZHU XINGNAO Capsul and relative standard deviation (n=6) table 4 of the sulfamido in blank sample；
TIANZHU XINGNAO Capsul and relative standard deviation (n=6) of the malachite green of table 3 in blank sample
The quinolone of table 4 and TIANZHU XINGNAO Capsul and relative standard deviation (n=6) of the sulfamido in blank sample
Test result indicate that：Can be reached with/without the recovery of standard addition of malachite green, quinolone, sulfamido in aquatic products
Required to detection, illustrate that this method has accuracy higher.Meanwhile, relative standard deviation RSD is below 15%, shows result
In confidential interval, this method has reliability higher.
Embodiment 1, parent ion, the selection of daughter ion
This research with Liquid Chromatography-Tandem Mass Spectrometry analysis aquatic products in with/without color malachite green, quinolones and sulfanilamide (SN)
Class, selection (ESI+) cation scan mode can obtain more patch informations, its with molecular ion peak [M+H]+and secondly
Level fragment pair and is quantified with most strong secondary fragment thirdly class compound carries out qualitative.
With/without color malachite green, quinolones and hydroxy functional group positively chargeable in sulfamido, from (ESI+) ion
Change pattern can be provided and greatly improved compared with the more patch informations of (ESI+) positive ion mode, and sensitivity, 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 modes carry out parent ion full scan in the positive-ion mode, determine 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 respectively using respective parent ion as central ion, second order mses scanning is carried out, selects abundance relative respectively
Two stronger fragments characteristic ions, with parent ion and daughter ion composition monitoring ion pair, with multiple-reaction monitoring (MRM) pattern pair
Object carries out qualitative and quantitative analysis, selection abundance is most strong, glitch-free monitoring ion as quota ion, secondary strong abundance ion
The characteristics of having selection selectivity good as qualitative ion, therefore the present invention.
Embodiment 2, sample pre-treatments condition
Sample pre-treatments
The present invention is to determine aquatic products Malachite Green and crystal violet residual quantity and agriculture with GB/T 19857-2005 liquid matter matter
In the bulletin -1-2008 aquatic products of industry portion 1077 based on 17 kinds of sulfamidos and 15 kinds of measure of quinolones medicament relict amount,
Further two methods are optimized with integration, compound extraction is integrated into by original two methods and upper machine measure can be while be entered
A capable method.
1. sample is weighed
Weighed after the complete naturally to thaw of sample to be frozen, weighing should should keep away in the four of meat gruel diagonal zones samplings, sampling
Exempt to carry the water after thawing, it is ensured that weigh the uniformity of meat gruel, reduce the error that matrix does not mix generation.
2. extract
Accurately sample 5.00g (± 0.01g) is weighed to be placed in 50mL centrifuge tubes, it is accurate to add 20 μ L to be 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 will
Guarantee internal standard is fully diffused in meat gruel, is accomplished that internal standard compound is synchronous with target compound as far as possible and is extracted.
10g anhydrous sodium sulfates are added, is vortexed and is mixed, add 15mL Acidifying acetonitriles (VAcetonitrile：VConcentrated hydrochloric acid=1000:4), it is homogenized
1min, ultrasonic extraction 10min, 6000r/min centrifugation 5min, take supernatant and cross neutral alumina column in 50mL pear shape bottles.The
Two steps first extract the abundant extraction that may insure with/without color malachite green, 3 kinds of quinolones with Acidifying acetonitrile.
Add 15mL ethyl acetate in residue to repeat to extract 1 time, 6000r/min centrifugation 5min, supernatant is incorporated in
In 50mL pear shape bottles.3rd step is extracted using ethyl acetate and may insure 8 kinds of abundant extractions of sulfanilamide (SN), and result of the test shows, acid
Change acetonitrile to most preferably, ethyl acetate is optimal to sulfamido extraction effect with/without color malachite green, quinolones extraction effect, because
This, experiment selection is first extracted with Acidifying acetonitrile, then extracts the mode that is combined with ethyl acetate, allows three class compound extraction effects
Reach optimal.
40 DEG C of rotary evaporations of water-bath add acetonitrile near dry:1mmol/L ammonium acetate solutions contain 0.1% formic acid=50:
501mL constant volume solution, adds 2mL n-hexane degreasings, crosses 0.2 μm of microfiltration membranes, treats machine.
The present invention will be merged into and once extracted with/without color malachite green, quinolones, the residual extraction twice of sulfonamides, should
Extracting method step is simple, it is time saving to operate, it is good to save testing cost, recovering effect.
Embodiment 3, the determination of AB5500Q liquid matter-matter combined instrument testing conditions
According to pertinent regulations in No. 657/2002/EEC resolution of CAC and EU, two pairs of ions of selection carry out MRM monitorings
Satisfaction detection is required, while the mass spectrogram of the main consideration of the selection of daughter ion wherein parent ion and daughter ion according to every kind of compound
Chosen with architectural characteristic, and it is less in actual sample analysis mesostroma interference, determine dividing for various materials according to mentioned above principle
After daughter ion, the molecular ion with various compounds is as parent ion respectively, its daughter ion is carried out full scan choose abundance it is relatively strong,
Less two pairs of daughter ions are disturbed for qualitative ion, most strong a pair are quota ion, finally with multiple-reaction monitoring (MRM) just from
Subpattern optimizes various mass spectrometry parameters.
Testing conditions：
Chromatographic condition：Chromatographic column：Shim-pack XR-ODS(75mm×2.1mm)；Column temperature：35℃；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：5.0μL；Flow velocity：0.25mL/min, design parameter is shown in Table 1.
The eluent gradient elution program of table 1
Tab.1 Elutionprocess ofthe separation
Mass Spectrometry Conditions：(1) ionization mode：Electric spray ion source (ESI), positive ion mode；Ion source temperature：600℃；
Gas curtain gas:20psi；Collision gas:In；Atomization gas:60psi；Auxiliary gas:40psi；Spray voltage:5000v；Scan pattern：It is more anti-
Should monitor that (MRM, reaction monitoring parent ion, daughter ion, taper hole voltage and collision energy are shown in Table 2.
The target compound Mass Spectrometry Conditions of table 2
Tab.2 Mass spectrometric conditions for monitoring oftarget compounds
Embodiment 4, the rate of recovery and precision
Addition packet：By the way of blank mark-on, 3 additions of level are carried out in aquatic products bare substrate, added
With/without the μ g/kg of malachite green 0.25,1.25 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 is parallel.
Extract：It is accurate to weigh sample 5.00g (± 0.01g) and be placed in 50mL centrifuge tubes, it is accurate add 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 sulfates, are vortexed and mix, and add 15mL Acidifying acetonitriles (VAcetonitrile：VConcentrated hydrochloric acid=1000:4) 1min, ultrasonic extraction, are homogenized
10min, 6000r/min are centrifuged 5min, take supernatant and cross neutral alumina column in 50mL pear shape bottles.15mL is added in residue
Ethyl acetate is repeated to extract 1 time, and 6000r/min centrifugation 5min, supernatant is incorporated in 50mL pear shape bottles, in 40 DEG C of rotations of water-bath
It is evaporated near dry, addition acetonitrile:1mmol/L ammonium acetate solutions contain 0.1% formic acid=50:501mL constant volume solution, is adding 2mL just
Hexane degreasing, crosses 0.2 μm of microfiltration membranes, treats machine.The rate of recovery and relative standard deviation are shown in Table 3.Result of the test shows：With/without hole
The recovery of standard addition of sparrow malachite green, quinolone, sulfamido in aquatic products can reach detection and require, illustrate that this method has higher
Accuracy.Meanwhile, relative standard deviation RSD is below 15%, shows result in confidential interval, and this method has higher
Reliability.
TIANZHU XINGNAO Capsul and relative standard deviation (n=6) of the malachite green of table 3 in blank sample
Tab.3 Recoverries and relative standard deviations ofMG and LMG in
Blank samples (n=6)
The quinolone of table 4 and TIANZHU XINGNAO Capsul and relative standard deviation (n=6) of the sulfamido in blank sample
Tab.4 Recoverriesandrelative standarddeviations ofquinolone and
Sulfas inblank samples (n=6)
Embodiment 5, sensitivity, the degree of accuracy and precision
Under the sample volume and constant volume that method specifies, sensitivity obtains the method with/without hole by 3 times of signal-to-noise ratio computations
The μ g/kg of sparrow malachite green detection limit 0.1, quinolone, sulfamido detection are limited to 0.5 μ g/kg.The degree of 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 inventive method 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 it is positive to be more than 1.0 μ g/kg by Ministry of Agriculture's regulation malachite green vestigial, and actual qualification rate is 97%；Quinoline
Promise ketone recall rate is 46%, and wherein Enrofloxacin accounts for the 90% of detection, and 100 μ are more than by Ministry of Agriculture's regulation enrofloxacin residual
G/kg or Ciprofloxacin are positive sample more than 100 μ g/kg with Norfloxacin total residual, and actual qualification rate is 98%；Sulfanilamide (SN)
Class recall rate is 2%, is positive more than 100 μ g/kg by Ministry of Agriculture's regulation sulfamido residual total amount, and actual qualification rate is
100%, above testing result is consistent with adoption of existing standard testing result, it was demonstrated that the inventive method has practical application.
Claims (2)
1. a kind of to determine aquatic products Malachite Green, quinolones, the method for sulfa drugs, the method is using liquid matter-matter
The detection that combined instrument is carried out to aquatic products, it is characterised in that as follows including method and step：
Step one, sample preparation
Detected aquatic products are taken after edible muscle parts sample is cut into the block of no more than 0.5cm × 0.5cm × 0.5cm, is put
Blended in meat grinder, the sample for preparing is preserved under the conditions of being placed in -16 DEG C to -20 DEG C；
Step 2, extracts
(1) weigh the above-mentioned 5.00 ± 0.01g of sample for preparing to be placed in 50mL centrifuge tubes, add 20 μ L to be 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, add 10g without
Aqueous sodium persulfate, is vortexed and mixes, and adds 15mL, VAcetonitrile：VConcentrated hydrochloric acid=1000:4 Acidifying acetonitrile, is homogenized 1min, ultrasonic extraction 10min,
6000r/min is centrifuged 5min, takes supernatant and crosses neutral alumina column in 50mL pear shape bottles；
(2) add 15mL ethyl acetate in the residue of aforesaid operations to repeat to extract 1 time, 6000r/min centrifugation 5min, supernatant
Liquid is incorporated in above-mentioned 50mL pear shape bottles；
(3) by above-mentioned 50mL pear shape bottles in 40 DEG C of rotary evaporations of water-bath near dry, add 1mL constant volume solution, add 2mL just oneself
Alkane degreasing, crosses 0.2 μm of microfiltration membranes, must treat press proof product；
Step 3, determines chromatographic condition
Chromatographic column：Shim-pack XR-ODS (75mm × 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, design parameter is shown in Table 1,
The eluent gradient elution program of table 1
Step 4, determines Mass Spectrometry Conditions
(1) ionization mode：Electric spray ion source (ESI), positive ion mode；
(2) ion source temperature：600℃；
(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, taper hole voltage and collision energy are shown in Table
2；
The target compound Mass Spectrometry Conditions of table 2
Step 5, the drafting of standard working curve
(1) measure with/without color malachite green 100ng/mL standard working solutions 0.05,0.1,0.2,0.5,1.0,2.0mL, then measure
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
It is 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 20.0ng/mL to spend, 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 measures the 1.0 μ deuterated sulphurs of g/mL internal standards
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, result of the test, it is determined that with/without color malachite green mass concentration in the range of 0.1~100ng/mL
Be in good linear relationship with peak area, sulfamido and quinolones mass concentration in the range of 0.005~1 μ g/mL with peak
Area is in good linear relationship, linearly dependent coefficient r2It is all higher than 0.999；
Step 6, the determination of sensitivity, the degree of accuracy and precision
(1) sensitivity：Under the sample volume and constant volume of our law regulation, obtained by 3 times of signal-to-noise ratio computations, with/without peacock
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) degree of accuracy：Its lower limit of quantitation (LOQ) is obtained with signal to noise ratio S/N ＞ 10,0.25 μ g/ are quantitatively limited to 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. according to claim 1 to determine aquatic products Malachite Green, quinolones, the method for sulfa drugs, it is special
Levy and be：Constant volume solution is in (3) step of the step 2：Acetonitrile:1mmol/L ammonium acetate solutions contain 0.1% formic acid=50:
50。
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| CN115078589A (en) * | 2022-07-15 | 2022-09-20 | 贵州省烟草科学研究院 | Chromatographic analysis method for amine additive in biodegradable mulch film |
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