CN113466356A - Sample pretreatment and detection method for determining pesticide residue content in cow milk - Google Patents

Abstract

The invention discloses a sample pretreatment and detection method for determining pesticide residue content in cow's milk, which comprises the steps of effectively extracting acetonitrile formic acid extract in the sample pretreatment process, filtering by a solid phase extraction column, evaporating to dryness, dissolving by methanol water, putting the cow's milk into a high performance liquid chromatograph and connecting a mass spectrometer in series, and determining the content of chlormequat chloride, methamidophos, cyromazine, aminopyralid, chloropyrimidic acid and imazamox pesticide residue in cow's milk. According to the invention, the problem of difficulty in extracting specific pesticide residue components in cow milk sample detection is effectively solved by adding the acetonitrile formic acid extract liquid with a reasonable proportion, the precision and the recovery rate are obviously improved, and the method is suitable for detecting the pesticide residue content in cow milk.

Description

Sample pretreatment and detection method for determining pesticide residue content in cow milk

Technical Field

The invention belongs to the technical field of pesticide residue detection methods, and relates to determination of pesticide residue content in cow milk, in particular to a sample pretreatment and detection method for determination of pesticide residue content in cow milk.

Background

Pesticide residues are gradually accumulated in human food along with the food nutrition supply and demand sequence of the food chain, more than 90 percent of pesticide components entering the human body are accumulated from the food chain, and in addition, the pesticide residues can be ingested by the human body through the atmosphere and drinking water. With the milk products being recorded into daily recipes of all ages, trace but long-term harm of residual micro-dose pesticide ingredients in milk to human bodies cannot be ignored, pesticide residue pollution in the milk products is controlled, pesticide ingredients are accurately detected, and the safety of the milk products is ensured, so that the milk products are receiving increasing attention.

At present, methods for measuring the content of pesticide residues in milk are few, and methods for measuring 511 pesticides and 493 pesticides in milk are respectively specified in GB/T23210-2008 and GB/T23211-2008, but the detection methods have the problems of complicated sample pretreatment operation, large reagent consumption and the like.

For methamidophos (methamidophos), chlormequat chloride (chlormequat), cyromazine (cyromazine), imazapyr (imazapyr), aminopyralid (aminopyralid) and aminocyclopyrachlor (aminocyclopyrachlor) which are all easily soluble in water and have strong polarity, the existing detection method removes water in a sample and is not beneficial to keeping a compound in an extraction solvent; through quantitative analysis and verification of pesticide residue content in cow milk, the recovery rate of residue content measurement results is low, target substance loss is high, and the accuracy of detection results is influenced by the method in national standards at present. Therefore, it is necessary to study a simple and fast sample pretreatment and detection method with high recovery rate to determine the residual amount of the 6 pesticides in cow milk.

Disclosure of Invention

The invention aims to provide a sample pretreatment and detection method for determining pesticide residue content in cow milk, which adopts the sample pretreatment processes of extraction, elution, evaporation to dryness and filtration and adopts a high performance liquid chromatography tandem mass spectrometer for detection so as to achieve the purposes of improving the recovery rate, reducing the loss of a target object, being simple and rapid and improving the accuracy of a detection result.

In order to achieve the purpose, the invention adopts the technical scheme that:

a sample pretreatment and detection method for determining pesticide residue content in cow milk comprises the following steps in sequence:

s1, sample pretreatment:

adding acetonitrile formic acid extract into raw milk to be detected, uniformly mixing by vortex, centrifuging, and purifying supernatant by a solid phase extraction column to obtain effluent liquid and a solid phase extraction column to be eluted;

taking acetonitrile formic acid extract to pass through a to-be-eluted solid-phase extraction column to obtain eluent;

mixing the effluent and the eluent, drying, dissolving with methanol water solution, and filtering with a filter membrane to obtain supernatant;

wherein: the volume ratio of acetonitrile to formic acid in the acetonitrile-formic acid extraction liquid is 450-550: 1;

and taking another 1.00mL (accurate to 0.01mL) of standard pesticide-free raw milk to replace the 'raw milk to be detected' to repeat the sample pretreatment process to obtain a blank extracting solution for preparing the standard working solution.

S2, sample analysis:

and (3) taking the upper liquid, and respectively measuring the contents of chlormequat chloride, methamidophos, cyromazine, aminopyralid, chloropropimidic acid and imazapyr in a high performance liquid chromatograph tandem mass spectrometer to obtain detection results.

Wherein, the measurement process adopts blank extract to dilute chlormequat chloride, methamidophos, cyromazine, aminopyralid, chloropyrimidinic acid and imazapyr standard solution respectively for preparing standard working solution.

By way of limitation, in step S1, the volume fraction of the aqueous methanol solution is 50-80%.

As another limitation, in step S1, the volume ratio of the raw milk to be tested to the acetonitrile formic acid extract is 1: 3.5-4.

As a third limitation, in step S1,

the temperature of the centrifugation is 0-4 ℃, the rotating speed is 7000-12000r/min, and the time is 5-15 min;

the solid phase extraction column is an Oasis HLB solid phase extraction column activated by acetonitrile aqueous solution with the volume fraction of 75-80%.

As a fourth definition, the drying is a evaporation to dryness at a temperature of 38-42 ℃;

the filter membrane is an organic microporous filter membrane, and the aperture of the filter membrane is 0.22 mu m.

As a fifth limitation, in step S2, the parameters measured by the hplc tandem mass spectrometer are:

I. parameters of the high performance liquid chromatograph:

the chromatographic column is a C18 chromatographic column with the diameter of 1.7 mu m and the diameter of 2.1 multiplied by 100 mm;

the mobile phase A is methanol;

the mobile phase B is 1L of aqueous solution containing 1mL of formic acid and 5mmol of ammonium formate;

the column temperature is 34-37 ℃; the sample injection amount is 2 mu L; the flow rate is 0.3 mL/min;

parameters of the mass spectrometer:

the ion source is an electrospray ion source;

the detection mode is multi-reaction selective ion detection;

the atomizing gas, the gas curtain gas and the auxiliary heating gas are all high-purity nitrogen; the collision gas is high-purity argon;

the capillary voltage is 2 kV;

the back blowing of the taper hole is 150L/h;

the temperature of the desolventizing gas is 450 ℃;

the desolventizing agent flow rate is 900L/h.

Wherein, the instrument and the parameter can be adjusted correspondingly according to different models of instruments.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:

(1) according to the method, raw milk is added with acetonitrile formic acid extract, an Oasis HLB solid-phase extraction column activated by acetonitrile aqueous solution is used for extraction and purification, methanol aqueous solution is used as a solvent in a sample to be detected, the acetonitrile and formic acid are reasonable in proportion, and the Oasis HLB solid-phase extraction column, the acetonitrile formic acid extract and the methanol aqueous solvent are matched with each other, so that the pretreatment effect of the sample is improved; in the conventional method, only raw milk is taken and added with extraction reagents such as acetonitrile, methanol and the like, and a clear supernatant cannot be obtained after centrifugation, so that the purification is difficult, the extraction effect is poor, and a pure methanol redissolved sample has poor redissolved effect and low recovery rate;

(2) the extraction efficiency is enhanced by using the acidified extraction solvent, the 6 compounds related by the invention are all easy to dissolve in water and have strong polarity, raw milk contains rich protein, acetonitrile is used as the extraction solvent, which is beneficial to precipitating the protein and is beneficial to obtaining supernatant by centrifugation, and a large amount of fat-soluble substances cannot be extracted together;

(3) according to the method, the pretreated sample is quantitatively determined by adopting an external standard method, the standard working solution is prepared from the blank extracting solution, the influence of other components in the sample to be detected on the accuracy of the detection result is reduced, the recovery rate of the detection method is obviously improved, the residual contents of chlormequat chloride, methamidophos, cyromazine, aminopyralid, chloropyrimidinic acid and imazethapyr can be simultaneously determined in the cow milk, the steps are simplified, and the cost is saved; filtering with organic microporous filter membrane to remove fine particulate matter in organic solution, and protecting chromatographic column and instrument;

the method is simple, convenient and quick, has high result accuracy, can carry out high-throughput detection, can be applied to industrial production, and is suitable for measuring the content of pesticide residues in cow milk.

The invention is described in further detail below with reference to the figures and the embodiments.

Drawings

FIG. 1 is an MRM chromatogram of chlormequat chloride in example 1 of the present invention;

FIG. 2 is an MRM chromatogram of methamidophos according to example 1 of the present invention;

FIG. 3 is an MRM chromatogram of cyromazine according to example 1 of the present invention;

FIG. 4 is an MRM chromatogram of aminopyralid in example 1 of the present invention;

FIG. 5 is an MRM chromatogram of chloropropanoic acid from example 1 of the present invention;

FIG. 6 is an MRM chromatogram of imazapyr in example 1 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the described embodiments are only for illustrating the present invention and do not limit the present invention.

The experimental conditions in the following examples, if not indicated, are generally according to conventional conditions or according to the conditions recommended by the reagents company; reagents, consumables and the like used in the following examples can be obtained commercially without special instructions, analytically pure reagents meeting national standards are used during analysis, experimental water is pure water, and the used methods are conventional process methods in the field without special instructions.

Example 1 sample pretreatment and detection method for determining pesticide residue content in cow milk

The reagents used in this example were: acetonitrile (CH)₃CN), methanol (CH)₃OH), both chromatographically pure, purchased from merck chemical technology (shanghai) ltd; formic acid (HCOOH), mass spec grade, purchased from shijiazhuang modern instrumentation and chemical company, ltd;

the pesticide standard substance is as follows: cycocel, methamidophos, cyromazine, aminopyralid, chloropyrimidinic acid and imazapic acid are 6 standard substances which are all purchased from Dr Ehrenstorfer company in Germany;

the instrument materials are as follows: oasis HLB solid phase extraction column; high performance liquid chromatography tandem mass spectrometer TQ-XS, Vorters science and technology, Inc.; TGL-20M centrifuge, shanghai luxiang instrument centrifuge instruments ltd; RE311 rotary evaporator, yamata.

The embodiment comprises the following steps which are carried out in sequence:

s1, sample pretreatment:

putting 1.00mL (accurate to 0.01mL) of raw milk to be detected into a 50mL centrifuge tube, adding 4mL of acetonitrile formic acid extract (the volume ratio of acetonitrile to formic acid is 450: 1), carrying out vortex oscillation for 1min, mixing uniformly, and centrifuging at the low temperature of 8000r/min for 5min at the temperature of 4 ℃ to obtain supernatant;

activating an Oasis HLB solid-phase extraction column by using acetonitrile aqueous solution with the volume fraction of 80%, taking supernatant to flow through the column at the flow rate of 1 s/drop, collecting effluent liquid, taking 4mL of acetonitrile formic acid extract liquid to elute the used Oasis HLB solid-phase extraction column, and collecting eluent;

mixing the effluent with the eluate, rotary evaporating at 40 deg.C to dryness, adding 1mL 50% methanol water solution, dissolving, filtering with 0.22 μm organic microporous membrane, and collecting filtrate as upper machine liquid for on-machine detection.

And taking another 1.00mL (accurate to 0.01mL) of standard pesticide-free raw milk to replace the 'raw milk to be detected' to repeat the sample pretreatment process to obtain a blank extracting solution for preparing the standard working solution.

S2, sample analysis:

respectively taking chlormequat chloride, methamidophos, cyromazine, aminopyralid, chloropyrimidinic acid and imazapic acid standard substances, and using methanol as a solvent to prepare chlormequat chloride, methamidophos, cyromazine, aminopyralid, chloropyrimidinic acid and imazapic acid standard solutions with respective concentrations of 1 mg/mL;

respectively taking 100 mu L of the 6 standard solutions, uniformly mixing, and using methanol as a solvent to fix the volume to 6 mixed standard solutions with pesticide concentrations of 10 mu g/mL;

and diluting the mixed standard solution step by using the blank extracting solution to prepare standard working solutions with different concentration gradients (the specific concentrations are shown in a table 4).

Loading the standard working solution to a high performance liquid chromatograph tandem mass spectrometer for determination to obtain chromatograms and retention times corresponding to different pesticides;

and (4) measuring the upper machine liquid in the step S1 by using a high performance liquid chromatograph tandem mass spectrometer with 2 mu L of the upper machine liquid to obtain a detection result.

Wherein the MRM chromatogram of chlormequat chloride is shown in figure 1; the MRM chromatogram of methamidophos is shown in FIG. 2; the MRM chromatogram of cyromazine is shown in FIG. 3; the MRM chromatogram of aminopyralid is shown in FIG. 4; the MRM chromatogram of chloropropionic pyrimidinic acid is shown in FIG. 5; the MRM chromatogram of imazalil is shown in FIG. 6;

wherein the determination conditions are as follows:

(i) the instrument parameters are as follows: the parameters of the HPLC tandem mass spectrometer are as follows:

liquid chromatography conditions:

(1) a chromatographic column: c18, (100 mm. times.2.1 mm, 1.7 μm);

(2) mobile phase: a is methanol, B is 1L of an aqueous solution containing 1mL of formic acid and 5mmol of ammonium formate, and the gradient elution procedure is shown in Table 1;

TABLE 1 mobile phase and gradient elution procedure

(3) Column temperature: 35 ℃;

(4) sample introduction amount: 2.0 mu L; the flow rate is 0.3 mL/min;

(ii) Mass Spectrometry Condition

(6) An ion source: an electrospray ion source;

(7) the detection mode is as follows: detecting multiple reaction selection ions; the scanning mode is ES⁺

(8) The atomizing gas, the gas curtain gas and the auxiliary heating gas are all high-purity nitrogen, and the collision gas is high-purity argon;

(9) capillary voltage: 2.00 (kV);

(10) back blowing of the taper hole: 150 (L/h);

(11) desolventizing gas temperature: 450 (. degree. C.);

(12) desolventizing agent gas flow: 900 (L/h).

(13) Mass spectrometer detection parameter conditions: the monitoring ion pairs, the quantitative ion pairs, the declustering voltage and the collision energy of each pesticide component are shown in the table 2:

TABLE 2 Mass spectrometer test parameter conditions

(ii) And (3) verifying the detection result:

(a) and (3) qualitative determination and verification:

measuring the organic liquid and the standard working liquid according to the conditions, and measuring that the retention time of the chromatographic peak of the measured object in the organic liquid is consistent with the retention time deviation corresponding to the standard working liquid, wherein the allowable deviation is less than +/-2%; and in the sample spectrogram after background subtraction, the maximum allowable relative deviation of the relative abundance of each qualitative ion compared with the standard solution spectrogram obtained under the same condition with the similar concentration does not exceed the range specified in table 3, and then the corresponding detected object exists in the sample.

TABLE 3 maximum permissible deviation of relative ion abundance in qualitative confirmation

Relative abundance (basal peak)	＞50％	More than 20% to 50%	More than 10% to 20%	≤10％
					Allowable relative deviation	±20％	±25％	±30％	±50％

And selecting a mixed standard working solution instrument with similar concentration for high performance liquid chromatography-mass spectrometry according to the content of the substance to be detected in the organic solvent. The response value of various pesticides in the organic liquid is in the linear range of instrument detection. And (4) carrying out equal-volume sample injection measurement on the standard working solution and the measurement machine loading solution.

And (3) qualitative determination results:

by comparing the retention time of various pesticides in the organic solution and the standard working solution, the retention time is basically consistent, the deviation is less than or equal to 2.5 percent, and the maximum allowable deviation of relative ion abundance meets the requirement in qualitative confirmation;

the measurement result of the organic liquid: the chlormequat chloride retention time is 0.87 min; the retention time of the methamidophos is 2.09 min; the retention time of cyromazine is 2.14 min; the retention time of the aminopyralid is 1.87 min; the retention time of the chloropropionic pyrimidinic acid is 2.34 min; the imazapic retention time was 3.33 min.

(b) And (3) quantitative determination and verification:

the method comprises the steps of adopting external standard quantitative determination, preparing standard working solution through blank extracting solution, effectively reducing influence of a matrix on a detection result, carrying out liquid chromatography analysis on the standard working solution, drawing a standard working curve by taking a peak area as a vertical coordinate and taking the concentration of the standard working solution as a horizontal coordinate, and determining a regression equation and a correlation coefficient (shown in table 4). And (4) operating the upper machine liquid on the machine, measuring the peak area, and calculating to obtain the content of the corresponding pesticide component (the result is shown in table 5).

TABLE 4 Peak area, standard curve and regression equation of each pesticide component to be tested

TABLE 5 pesticide ingredient contents to be measured

Example 2-6 sample pretreatment and detection method for determination of pesticide residue content in cow milk

Examples 2 to 6 are sample pretreatment and detection methods for determining the content of pesticide residue in cow milk, and the steps are basically the same as those in example 1, except for the differences in process parameters and detection results, which are specifically shown in tables 6 and 7:

table 6 examples 2-6 process parameters

TABLE 7 results of tests of examples 2 to 6

The detection results of the above embodiments show that the sample pretreatment and detection method for determining the pesticide residue content in cow milk has appropriate retention time, the obtained regression equation has effective R value and good detection effect, and can be used for effectively detecting six pesticide components at the same time.

Example 7 Effect test of sample pretreatment and detection method for measuring residual content of pesticide in cow milk

This example is a sample pretreatment and effect experiment of the detection method for determining the content of pesticide residues in cow milk, and is used to verify the determination effect of the detection method of the present invention.

The recovery rate of the detection method of the present invention was calculated by measuring the content (measured value) of the pesticide residue by the method in example 1 for each of the standard working liquids (the concentration is the theoretical value), and the results are shown in table 8:

table 8 results of effect test

The results show that the recovery rates of all groups are stable, and the sample pretreatment and detection method for determining the content of the pesticide residues in the cow milk has a good detection effect, effectively solves the problem of poor extraction effect of the pesticide residues in the cow milk, can improve the recovery rate, has stable results, and has good detection results for pesticide residues with different concentrations.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms, and any person skilled in the art may use the above-mentioned technical content as a teaching to make changes or modifications to the equivalent embodiments with equivalent changes, but all those simple changes, equivalent changes and modifications made to the above-mentioned embodiments without departing from the technical spirit of the present invention, and still all those embodiments are within the scope of the present invention as claimed in the claims.

Claims (6)

1. A sample pretreatment and detection method for determining pesticide residue content in cow milk is characterized by comprising the following steps of:

s1, sample pretreatment:

adding acetonitrile formic acid extract into raw milk to be detected, uniformly mixing by vortex, centrifuging, and purifying supernatant by a solid phase extraction column to obtain effluent liquid and a solid phase extraction column to be eluted;

taking acetonitrile formic acid extract to pass through a to-be-eluted solid-phase extraction column to obtain eluent;

mixing the effluent and the eluent, drying, dissolving with methanol water solution, and filtering with a filter membrane to obtain supernatant;

wherein: the volume ratio of acetonitrile to formic acid in the acetonitrile-formic acid extraction liquid is 450-550: 1;

s2, sample analysis:

and (3) taking the upper liquid, and respectively measuring the contents of chlormequat chloride, methamidophos, cyromazine, aminopyralid, chloropropimidic acid and imazapyr in a high performance liquid chromatograph tandem mass spectrometer to obtain detection results.

2. The method for pretreating and detecting the sample for detecting the content of the pesticide residues in the cow' S milk according to claim 1, wherein in the step S1, the volume fraction of the methanol aqueous solution is 50-80%.

3. The sample pretreatment and detection method for detecting the content of pesticide residues in cow 'S milk according to claim 1, wherein in step S1, the volume ratio of the raw cow' S milk to be detected to the acetonitrile formic acid extract is 1: 3.5-4.

4. The method for pretreating and detecting a sample for measuring the content of residual pesticide in cow' S milk according to claim 1, 2 or 3, wherein in step S1,

the temperature of the centrifugation is 0-4 ℃, the rotating speed is 7000-12000r/min, and the time is 5-15 min;

the solid phase extraction column is an Oasis HLB solid phase extraction column activated by acetonitrile aqueous solution with the volume fraction of 75-80%.

5. The method for pretreating and detecting a sample for measuring the content of residual pesticide in cow' S milk according to claim 1, 2 or 3, wherein in step S1,

the drying is carried out by evaporating at 38-42 ℃;

the filter membrane is an organic microporous filter membrane, and the aperture of the filter membrane is 0.22 mu m.

6. The method for pretreating and detecting the sample for detecting the content of the pesticide residues in the cow' S milk according to claim 1, 2 or 3, wherein in step S2, the parameters measured by the HPLC-tandem mass spectrometer are as follows:

I. parameters of the high performance liquid chromatograph:

the chromatographic column is a C18 chromatographic column with the diameter of 1.7 mu m and the diameter of 2.1 multiplied by 100 mm;

the mobile phase A is methanol;

the mobile phase B is an aqueous solution containing 1mL of formic acid and 5mmol of ammonium formate;

the column temperature is 34-37 ℃; the sample injection amount is 2 mu L; the flow rate is 0.3 mL/min;

parameters of the mass spectrometer:

the ion source is an electrospray ion source;

the detection mode is multi-reaction selective ion detection;

the atomizing gas, the gas curtain gas and the auxiliary heating gas are all high-purity nitrogen; the collision gas is high-purity argon;

the capillary voltage is 2 kV;

the back blowing of the taper hole is 150L/h;

the temperature of the desolventizing gas is 450 ℃;

the desolventizing agent flow rate is 900L/h.

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