CN102628844A - Content determining method for trichlorfon in dried fish - Google Patents

Abstract

The invention belongs to the technical field of content determination of trichlorfon, and provides a content determining method for trichlorfon in dried fish. The method comprises the following specific steps of: (1) preparing a standard curve; (2) preparing a sample solution, to be specific, (1) pretreating; (2) extracting; (3) concentrating; and (4) purifying; and (3) detecting the sample with a gas chromatography-mass spectrometry method. Compared with the prior art, the method has the advantages that: during preparation of the sample solution, a purifying step in a pretreatment process is mainly increased, so that the interference of greasy substances in the sample on determination is greatly lowered, and the probability of a false positive result is lowered; and trichlorfon is detected by using the gas chromatography-mass spectrometry method, so that determination is accurate, and the detection sensitivity is high.

Description

The content assaying method of metrifonate in the drying fish

Technical field

The invention belongs to the assay field of metrifonate, be applicable to the detection of metrifonate residual quantity in the drying fish.

Background technology

Metrifonate is the pesticide of efficient, low toxicity and low-residual.Cladocera, oar angle class, freshwater mussel hook Jie larva and kyllinga brevifolia etc. to the parasitic fluke in fish inside and outside, nematode, spiny-headed worm and harm fry, fish-egg all have good killing action.But because metrifonate under weak basic condition, can form the bigger DDVP of residual hazard property, when pH value was 8～10, metrifonate is transformed into DDVP only needed half an hour.Therefore, not only to take the poisonous effect of fishes and shrimps into account, and also very important to the safety of people, animal.

In recent years, China once repeatedly found to have illegal retailer to use metrifonate processing cured fish, had brought great potential safety hazard for people's healthy and life security.To this phenomenon, we have set up the assay method of metrifonate in the drying fish.This method can accurately detect the metrifonate in the drying fish, can technical support be provided for the food and drink food safety risk supervision.

To the otherness of sample, every profession and trade also there are differences for the examination criteria of metrifonate, and the method that relates to comprises: methods such as liquid phase chromatography, vapor-phase chromatography, liquid chromatograph mass spectrography method.Main prior art is following 2 at present: 1, the mensuration of metrifonate residual quantity in No. 783 bulletin-3-2006 aquatic products of the Ministry of Agriculture; The assay method of No. 783 bulletin-3-2006 of the Ministry of Agriculture is to extract the metrifonate in the dried fish with acetonitrile, behind zinc acetate degrease and chloroform extraction, directly uses gas Chromatographic Determination.Impurity is a lot of in the sample solution after the processing, occurs a lot of assorted peaks in the gas chromatogram, and mensuration is had interference.And still remain with a large amount of grease constituents in the sample solution, not volatile, be accumulated in injection port, can pollute sampling systems such as bushing pipe, be prone to mensuration is caused cross pollution.2, the assay method of SNT 1920-2007 is only to the mensuration of metrifonate in fresh meat, casing and the honey, extract metrifonate through methylene chloride after, be concentrated into driedly, behind the acetonitrile dissolved residue, add a small amount of cyclohexane degrease.Exist more impurity to disturb equally, and LC-MS appearance operating cost is high, the required a large amount of organic solvents of moving phase are prone to environment is polluted.

Summary of the invention

Deficiency to prior art; The present invention aims to provide the content assaying method of metrifonate in the drying fish; Can detect the content of metrifonate in the dried fish accurately and rapidly, and reduce the interference of oil substances to measuring in the sample, reduce the probability that false positive results occurs.

For realizing above-mentioned purpose, technical scheme of the present invention is:

The content assaying method of metrifonate in a kind of drying fish may further comprise the steps:

1) preparation of typical curve;

2) preparation of need testing solution:

1. pre-service: get dried fish and rub mixing, get pretreated sample, subsequent use;

2. extract: a, get pretreated sample, add zinc acetate earlier, add the acetonitrile solution homogeneous again, centrifugal, supernatant is transferred in the separating funnel; Residue repeating step a is 1-2 time in b, the centrifuge tube, merges supernatant in same separating funnel, adds aqueous sodium persulfate solution, the concussion mixing; C, in above-mentioned supernatant, add methenyl choloride, after the shaken, standing demix; D, 2-3 c step of repetition merge lower floor's liquid, get lower floor's methenyl choloride liquid;

3. concentrate: lower floor's methenyl choloride liquid is flow in the concentrated bottle through the anhydrous sodium sulfate post, be concentrated into dried;

4. purify: add the acetonitrile dissolution residual substance, add the saturated normal hexane of acetonitrile, vortex vibration 2-3 minute is put refrigerator and cooled and is frozen preservation 2-3 hour, and low-temperature centrifugation 2-3 minute, get supernatant, promptly get need testing solution;

3) with the gas chromatography mass spectrometry method need testing solution is detected.

Wherein,

Said step 2) preparation of need testing solution is preferably:

1. pre-service: get dried fish, rub, mixing gets pretreated sample, and is subsequent use;

2. extract: a, get pretreated sample 5g, put in the 50ml centrifuge tube, add zinc acetate 0.5g, add acetonitrile solution 18-29mL again, homogeneous appearance homogeneous 1min, the centrifugal 3min of 4000r/min, supernatant are transferred in the 250ml separating funnel; Residue repeats above operation a once in b, the centrifuge tube, merges extract in same 250ml separating funnel; C, add 20g/L aqueous sodium persulfate solution 100mL, the concussion mixing adds the 30mL methenyl choloride, shaken 3min, standing demix; D, 2-3 c step of repetition merge lower floor's liquid, get lower floor's methenyl choloride liquid;

3. concentrate: lower floor's methenyl choloride liquid is flow to 250mL through the anhydrous sodium sulfate post concentrate in the bottle, be concentrated into dried with rotary evaporator;

4. purify: quantitatively add 1mL acetonitrile dissolution residual substance, add the saturated normal hexane of 2mL acetonitrile, vortex vibration 2 minutes is put refrigerator and cooled and is frozen preservation 2 hours, and 10000r/min low-temperature centrifugation 3 minutes is got supernatant, promptly gets need testing solution.

Wherein,

Said step 3) is said to be preferably the testing conditions that need testing solution detects with the gas chromatography mass spectrometry method:

Chromatographic column: DB-5MS capillary column; Injector temperature: 200 ℃-220 ℃; Interface temperature: 250 ℃-260 ℃; Ion source temperature: 220 ℃-230 ℃; The quadrupole rod temperature: 145 ℃-155 ℃, electron energy 65eV-75eV; Carrier gas: N ₂, flow velocity is 0.8ml/min-1.5ml/min, pulse is split sampling not; Sample size: 1 μ l-1.5 μ l;

Heating schedule is: from 40 ℃-50 ℃, kept 2-3 minute, rise to 165 ℃-175 ℃ with 8 ℃/min-15 ℃/min, kept 1-3 minute, rise to 198 ℃-205 ℃, kept 2-4 minute with 15 ℃/min-25 ℃/min again.

Said testing conditions is more preferably: chromatographic column: DB-5MS capillary column; Injector temperature: 200 ℃; Interface temperature: 260 ℃; Ion source temperature: 230 ℃; The quadrupole rod temperature: 150 ℃, electron energy 70eV; Carrier gas: N ₂, flow velocity is 1.0ml/min, pulse is split sampling not; Sample size: 1 μ l;

Heating schedule is: from 50 ℃, kept 2 minutes, rise to 170 ℃ with 10 ℃/min, kept 1 minute, rise to 200 ℃ with 20 ℃/min again, kept 3 minutes.

Do further explanation and explanation in the face of the present invention down:

Principle: metrifonate extracts through methenyl choloride in the sample, and extract concentrates, after the degreasing, purification, and with vapor-phase chromatography-GC-MS mensuration, the external standard peak area method is quantitative, and the daughter ion abundance ratio is qualitative.

Dried fish after pre-service, extraction, concentrating, because of the polarity and the grease constituents of methenyl choloride approaching, so still there are a large amount of grease constituents in the need testing solution.After purifying step, because of normal hexane polarity is a lot of a little less than than acetonitrile, the grease constituents can get into the normal hexane layer, reaches purification effects.After need testing solution removed grease class impurity, a lot of less (see figure 2)s of the assorted summit of chromatogram had avoided assorted peak that mensuration is caused interference.And the grease constituents is not volatile, if do not remove, then can be accumulated in injection port, pollutes sampling systems such as bushing pipe, and mensuration is caused cross pollution.

Compared with prior art, advantage of the present invention is:

1, increases purifying step in the pre-treatment process, increased the purifying step in the pre-treatment process, greatly reduced the interference of oil substances to measuring in the sample; Reduced the probability that false positive results occurs; And adopt the gas chromatography mass spectrometry method to detect, qualitative accurate, detection sensitivity is high.

2, adopted the gas chromatography mass spectrometry method to detect the method for metrifonate, this is not have in the existing standard system.

Description of drawings

Fig. 1 is the typical curve of embodiment 1;

The gas chromatography-mass spectrography figure of Fig. 2, metrifonate, wherein 1 is dimethylphosphite; The 2nd, DDVP.

Embodiment

For a better understanding of the present invention, below in conjunction with embodiment the present invention is done detailed description further.

Embodiment 1:

1 reagent and material

1.1 liquid reagent: acetonitrile (chromatographically pure), methenyl choloride (chromatographically pure), ethyl acetate (chromatographically pure), normal hexane (the residual level of farming)

1.2 solid reagent: anhydrous sodium sulfate (analyzing pure), zinc acetate (analyzing pure)

1.3 purified water

1.4 experiment apparatus: sample introduction bottle, centrifuge tube (50ml), accurate adjustable liquid-transfering gun (100～1000 μ l, 10～100 μ l), volumetric flask, concentrated bottle (250ml), a separating funnel (250ml)

1.5 standard items: metrifonate purity is all more than 98.0%

2 instruments and analysis condition:

2.1Agilent 7890N/5975C gas chromatography appearance

2.2 Rotary Evaporators

2.3 homogeneous appearance

2.4 vortex vortex mixer

2.5 hydro-extractor (5000r/min)

2.6 balance: precision 10mg/0.01mg, do the test sample weighing with and do the standard items weighing and use

3 specimen preparations and preservation

3.1 the preparation of standard solution

3.1.1 standard items storing solution: get the about 10mg of metrifonate standard items, precision is weighed, and puts in the 20ml volumetric flask, uses ethyl acetate to be mixed with the standard reserving solution that concentration is 500 μ g/ml.Be diluted to the standard operation liquid of debita spissitudo as required again with ethyl acetate.

3.1.2 typical curve: get blank sample, take a sample 9 parts every part of 5g; The standard operation liquid 0.01,0.02,0.05,0.1,0.2,0.5, the 1.0ml that add debita spissitudo respectively; Press the method for need testing solution preparation,, process series standard solution from " adding the 0.5g zinc acetate " beginning; Inject gas chromatograph-mass spectrometer, measure.Typical curve is as shown in Figure 1.

3.2 the preparation of need testing solution

1. pre-service: get dried fish, rub, mixing, subsequent use;

2. extract: a, get pretreated sample 5g, put in the 50ml centrifuge tube, add zinc acetate 0.5g, add acetonitrile solution 18-29mL again, homogeneous appearance homogeneous 1min, the centrifugal 3min of 4000r/min, supernatant are transferred in the 250ml separating funnel; Residue repeats above operation a once in b, the centrifuge tube, merges extract in same 250ml separating funnel; C, add 20g/L aqueous sodium persulfate solution 100mL, the concussion mixing adds the 30mL methenyl choloride, shaken 3min, standing demix; D, 2-3 c step of repetition merge lower floor's liquid, get lower floor's methenyl choloride liquid;

3. concentrate: lower floor's methenyl choloride liquid is flow to 250mL through the anhydrous sodium sulfate post concentrate in the bottle, be concentrated into dried with rotary evaporator;

4. purify: quantitatively add 1mL acetonitrile dissolution residual substance, add the saturated normal hexane of 2mL acetonitrile, vortex vibration 2 minutes is put refrigerator and cooled and is frozen preservation 2 hours, and 10000r/min low-temperature centrifugation 3 minutes is got supernatant, promptly gets need testing solution.

3.3 the preparation of application of sample sample solution

During the preparation of each need testing solution, the application of sample of making a collection of sample of should accompanying reclaims, and adds above-mentioned standard operation liquid 0.1ml; Press the method for need testing solution preparation,, process the application of sample sample solution from " adding the 0.5g zinc acetate " beginning; Inject gas chromatograph-mass spectrometer, measure.The recovery scope of each agricultural chemicals should be in 70%～120%.Do not reach requirement like the recovery, this sample that extracts should all be reformed, and (purpose of retinue recovery is to monitor in real time the operation of this experiment to make experimental result more true and reliable.If the recovery is lower than 70%, then lose in the sample pretreatment process probably; If the recovery is higher than 120%, then sample substrate has interference or misoperation to measuring the result).

3.4 interference test: make the blank assay of total reagent table,,, process blank solution from " adding the 0.5g zinc acetate " beginning by the method for above-mentioned need testing solution preparation.Blank should be noiseless.

4 tests

Use instrument and chromatographic condition Agilent 7890N/5975C gas chromatography appearance; Mass spectrometer is a quadrupole mass spectrometer, selects ion monitoring (the monitoring ion sees table 1 for details, and the characteristic ion of each pesticide composition monitoring sees table 2 for details).DB-5MS capillary column (column length 30m, internal diameter 0.25mm, film thickness 0.25 μ m); Carrier gas: high-purity helium, constant current, flow velocity is 1.0ml/min; Pulse is split sampling not, sample size 1 μ l.200 ℃ of injector temperatures, 260 ℃ of interface temperature, ion source temperature is 230 ℃, the quadrupole rod temperature is 150 ℃, electron energy 70eV.

Heating schedule: from 50 ℃, kept 2 minutes, rise to 170 ℃, kept 1 minute, rise to 200 ℃ with 20 ℃/min again, kept 3 minutes with 10 ℃/min.

Table 1 monitoring ion

Retention time (min) scope	The characteristic ion of monitoring
		?0～3.5	/ (solvent delay)
?3.5～19.5	79、80、95、109、185、220

The characteristic ion of each pesticide composition monitoring of table 2

5 results

5.1GC-MS qualitative: the qualitative of each target compound carries out with retention time with the pairing GC-MS chromatographic peak relative abundance of three pairs/four pairs ions (characteristic ion right/quota ion to); Require the retention time of target pesticide composition in the sample to be tested consistent with the retention time of target pesticide composition in the typical curve solution; The GC-MS chromatographic peak relative abundance that the three couples/four pair ion pair of target pesticide composition is answered in the tested sample simultaneously is than consistent, and the deviation of permission is seen table 3:

The maximum allowable offset of relative abundance of ions during table 3 qualitative determination

Relative abundance of ions	＞50％	＞20% to 50%	＞10% to 20%	≤10％
					The relative deviation that allows	±10％	±15％	±20％	±50％

5.2 metrifonate is prone to resolve into dimethylphosphite and trichloroacetaldehyde, Mass Spectrometer Method also possibly be transformed into DDVP.So with reference to " mensuration of metrifonate residual quantity in No. 783 bulletin-3-2006 aquatic products of the Ministry of Agriculture ", metrifonate carries out quantitatively in dimethylphosphite content.

Quantitatively: this standard adopts external standard method quantitative.Preparation typical curve before each the mensuration, sample introduction is measured, and obtains the working curve of dimethylphosphite concentration and peak area.

5.2.1 the sample peak area is in the interior content that calculates metrifonate in the sample by formula (1) of working curve scope:

$X=\frac{C_S\×V}{m}...\left(1\right)$

In the formula:

X---the residual content of metrifonate (mg/kg) in the sample;

C _S---the concentration (mg/kg) of dimethylphosphite in the last machine sample solution that calculates by regression curve;

V---the final constant volume of need testing solution (ml);

M---sampling amount (g)

5.2.2 the sample peak area is lower than the working curve scope is calculated metrifonate in the sample by formula (2) content:

$X=\frac{C_S\×V\×A}{m\×A_S}...\left(2\right)$

In the formula:

X---the residual content of metrifonate (mg/kg) in the sample;

C _S---mix the concentration (mg/kg) of dimethylphosphite typical curve minimum point in the contrast solution;

V---the final constant volume of need testing solution (ml);

A---the peak area of dimethylphosphite component in the sample

A _S---the peak area of dimethylphosphite component in the contrast solution

M---sampling amount (g)

5.2.3 the sample peak area is higher than the working curve scope, should increase the standard solution addition by the method for typical curve preparation, enlarges the scope of typical curve again, enlarges standard curve range.

5.3 the result who the product on the market is inspected by random samples by method of the present invention:

179 batches of drying fishes to the market random sampling are tested, and wherein 1 batch detects metrifonate, and content is 0.34mg/kg.The method recovery all in 70%～120% scope, detects and is limited to 0.10mg/kg.

6 positive tests (carrying out in case of necessity)

6.1 positive test purpose

When the experimenter is with suspicion to test process and test macro, or to important test sample (if can force rate to, relate to public inspection when judicial), or during methodological study, need verify result's accuracy, with warranty test result's reliability.

6.2 the preparation of typical curve solution

With 5.1.

6.3 the preparation of positive need testing solution:

Method preparation by 5.3 application of samples (each target pesticide composition of adding is by 100%, 200%, 1000% adding that detects lower bound) sample solution preparation.

6.4GC-MS measure:

Get typical curve solution, positive need testing solution by the analysis of method of inspection sample introduction.Recovery scope should be 70%～120%.

6.5 result

With 5.

Claims (4)

1. the content assaying method of metrifonate in the drying fish is characterized in that, may further comprise the steps:

1) preparation of typical curve;

2) preparation of need testing solution:

1. pre-service: get dried fish and rub mixing, get pretreated sample, subsequent use;

2. extract: a, get pretreated sample, add zinc acetate earlier, add the acetonitrile solution homogeneous again, centrifugal, supernatant is transferred in the separating funnel; Residue repeating step a is 1-2 time in b, the centrifuge tube, merges supernatant in same separating funnel, adds aqueous sodium persulfate solution, the concussion mixing; C, in above-mentioned supernatant, add methenyl choloride, after the shaken, standing demix; D, 2-3 c step of repetition merge lower floor's liquid, get lower floor's methenyl choloride liquid;

3. concentrate: lower floor's methenyl choloride liquid is flow in the concentrated bottle through the anhydrous sodium sulfate post, be concentrated into dried;

4. purify: add the acetonitrile dissolution residual substance, add the saturated normal hexane of acetonitrile, vortex vibration 2-3 minute is put refrigerator and cooled and is frozen preservation 2-3 hour, and low-temperature centrifugation 2-3 minute, get supernatant, promptly get need testing solution;

3) with the gas chromatography mass spectrometry method need testing solution is detected.

2. according to the content assaying method of metrifonate in the said a kind of drying fish of claim 1, it is characterized in that said step 2) being prepared as of need testing solution:

1. pre-service: get dried fish, rub, mixing gets pretreated sample, and is subsequent use;

2. extract: a, get pretreated sample 5g, put in the 50ml centrifuge tube, add zinc acetate 0.5g, add acetonitrile solution 18-29mL again, homogeneous appearance homogeneous 1min, the centrifugal 3min of 4000r/min, supernatant are transferred in the 250ml separating funnel; Residue repeats above operation a once in b, the centrifuge tube, merges extract in same 250ml separating funnel; C, add 20g/L aqueous sodium persulfate solution 100mL, the concussion mixing adds the 30mL methenyl choloride, shaken 3min, standing demix; D, 2-3 c step of repetition merge lower floor's liquid, get lower floor's methenyl choloride liquid;

3. concentrate: lower floor's methenyl choloride liquid is flow to 250mL through the anhydrous sodium sulfate post concentrate in the bottle, be concentrated into dried with rotary evaporator;

4. purify: quantitatively add 1mL acetonitrile dissolution residual substance, add the saturated normal hexane of 2mL acetonitrile, vortex vibration 2 minutes is put refrigerator and cooled and is frozen preservation 2 hours, and 10000r/min low-temperature centrifugation 3 minutes is got supernatant, promptly gets need testing solution.

3. according to the content assaying method of metrifonate in claim 1 or the 2 said a kind of drying fishes, it is characterized in that the said use gas chromatography mass spectrometry of said step 3) method to the testing conditions that need testing solution detects is:

Chromatographic column: DB-5MS capillary column; Injector temperature: 200 ℃-220 ℃; Interface temperature: 250 ℃-260 ℃; Ion source temperature: 220 ℃-230 ℃; The quadrupole rod temperature: 145 ℃-155 ℃, electron energy 65eV-75eV; Carrier gas: N ₂, flow velocity is 0.8ml/min-1.5ml/min, pulse is split sampling not; Sample size: 1 μ l-1.5 μ l;

Heating schedule is: from 40 ℃-50 ℃, kept 2-3 minute, rise to 165 ℃-175 ℃ with 8 ℃/min-15 ℃/min, kept 1-3 minute, rise to 198 ℃-205 ℃, kept 2-4 minute with 15 ℃/min-25 ℃/min again.

4. according to the content assaying method of metrifonate in the said a kind of drying fish of claim 3, it is characterized in that said testing conditions is: chromatographic column: the DB-5MS capillary column; Injector temperature: 200 ℃; Interface temperature: 260 ℃; Ion source temperature: 230 ℃; The quadrupole rod temperature: 150 ℃, electron energy 70eV; Carrier gas: N ₂, flow velocity is 1.0ml/min, pulse is split sampling not; Sample size: 1 μ l;

Heating schedule is: from 50 ℃, kept 2 minutes, rise to 170 ℃ with 10 ℃/min, kept 1 minute, rise to 200 ℃ with 20 ℃/min again, kept 3 minutes.

Images