CN104730191B - A kind of LC-MS/MS assay method of fluorine ether bacterium amide residual quantity - Google Patents

Abstract

The invention discloses a LC-MS/MS method for determining the residual amount of fluoxacillin. The method is mainly used for measuring the residual amount of fluoxybactam in food and agricultural products with complex substrates such as grains and animal-derived foods. Use acetonitrile or an acetonitrile solution containing 1% acetic acid to homogeneously extract the residual flurocystamide in the sample. After purification by C ₁₈ /PSA solid phase extraction column, liquid chromatography tandem mass spectrometry (LC‑MS/MS) is used for detection. The blank matrix solution of the pesticide to be tested was used to establish a calibration standard curve, and the external standard method was used for quantification. The average recovery rate of this method is 83.1%-89.5%, the average relative standard deviation (RSD) is 4.0%-6.3%, and the detection limit is lower than 0.12 μg/kg. Good repeatability, accurate qualitative and quantitative advantages. It can meet the "uniform standard" technical requirements of 0.01 mg/kg residue limit, and provide strong technical support for ensuring the food safety of our people and the healthy development of foreign export trade.

Description

A kind of LC-MS/MS determination method of fluoxetin residue

technical field

The invention relates to a LC-MS/MS method for determining the residual amount of fluoxetin, more specifically, qualitative and quantitative determination of grains, pork, beef, mutton, The invention relates to a method for the content of fluoxetyram residues in animal and plant-derived foods with complex matrices such as chicken and other animal muscles and products, and belongs to the technical field of determination of pesticide residues.

Background technique

Bisamide insecticides are popular insecticide products in the world in recent years. They can be widely used in the pest control of rice, vegetables, cotton and other crops. They have the advantages of low toxicity, environmental safety and high activity, including chlorantraniliprole , cyantraniliprole, flubendiamide and other varieties, fluroxystamide (LH-2010A) is a new product developed by Shenyang Research Institute of Chemical Industry, a subsidiary of Sinochem Group, and operated by Sinochem Agrochemical Co., Ltd., the first in my country with independent knowledge The bisamide insecticides with proprietary rights have been temporarily registered by the Ministry of Agriculture.

Fluoreosinamide (LH-2010A) belongs to the ryanodine receptor activator insecticide. It binds to the ryanodine receptor in the pest to open the calcium ion channel, so that the calcium ion stored in the cell is continuously released to the In the sarcoplasm, calcium ions combine with the matrix protein in the sarcoplasm, causing continuous muscle contraction. Insect body symptoms are convulsions, refusal to feed, and eventually death. Fluoroxystrobin is a low-toxicity, broad-spectrum insecticide, and has good activity against Lepidoptera pests. The control objects include rice leaf roller, rice stem borer, diamondback moth, beet armyworm, corn borer, sugarcane borer, small tortillaria, borer and so on.

With the registration, promotion and use of fluoxyroxybactam, research on environmental behaviors such as fluoxyroxybactam residue digestion dynamics and final residues is bound to increase. When the pesticides are not registered in the country and the corresponding residue limit standard is not formulated, the residue limit of food and agricultural products exported to the country, including animal-derived foods such as livestock and poultry meat, shall be subject to a "uniform standard" of 0.01mg/L.

So far, there have been no domestic and foreign reports on the detection method of fluoxetin residues in food and agricultural products. The use of LC-MS/MS to determine pesticide residues in food and agricultural products has the advantages of rapidity, simplicity, and high sensitivity. The matrix of food and agricultural products such as sexual food is relatively complex, and it is necessary to establish a sample pretreatment method with good purification effect to meet the detection requirements. The LC-MS/MS detection method is of great significance.

Contents of the invention

The purpose of the present invention is to provide a LC-MS/MS method for determining the residual amount of fluetherbactam, which is mainly used for the determination of the residual amount of fluetherbactam in agricultural products with complex substrates such as grains and animal-derived foods.

In order to achieve the above purpose, the technical solution adopted in the present invention is: a LC-MS/MS method for determining the residual amount of fluoxetin, comprising the following steps:

(1) extraction

Weigh the pulverized sample into a centrifuge tube with a stopper, add an appropriate amount of water for resuscitation, quantitatively add acetonitrile or an acetonitrile solution containing 1% acetic acid to homogenize or oscillate for ultrasonic extraction, then add one of sodium chloride or sodium acetate and anhydrous Magnesium sulfate, vortex vigorously for 1 min and centrifuge.

(2) purification

Pipette a certain volume of sample extract, concentrate to about 1mL, clean up with C ₁₈ /PSA solid-phase extraction column, elute with acetonitrile, collect the eluate, use acetonitrile to make up to volume, take the constant volume to pass through the membrane, and wait for liquid chromatography Tandem mass spectrometry (LC-MS/MS) detection;

(3) Preparation of standard working solution

Treat the blank sample of the same kind of matrix without fluoxyrhebstramide according to the above steps (1) and (2) to obtain the sample extraction and purification solution, and use the blank extraction and purification solution to prepare at least 3 concentrations of fluoxyrhebstramide series mixed standards working fluid;

(4) Determination by liquid chromatography tandem mass spectrometry (LC-MS/MS)

The standard working solution of each concentration gradient in step (3) is carried out LC-MS/MS measurement, carries out regression analysis to its corresponding concentration with the chromatographic peak area of standard working solution, obtains standard working curve; Under the same condition, step ( 2) Inject the purified sample liquid into LC-MS/MS for measurement, measure the chromatographic peak area of fluetheribactin in the sample liquid, and substitute it into the standard curve to obtain the content of fluetheribactin in the sample liquid. The mass of the representative sample is calculated to obtain the residual amount of fluoxybactam in the sample.

If the sample in step (1) is a sample with low water content such as grains and animal livers, it must be fully infiltrated with an appropriate amount of water before extraction.

In step (1), sodium chloride is added for salting out when acetonitrile is used for extraction, and sodium acetate is added for salting out when an acetonitrile solution containing 1% acetic acid is used for extraction.

In step (2), C ₁₈ /PSA solid phase extraction purification is carried out, and when acetonitrile is eluted, the elution volume is 6-8 mL.

The mobile phase of the liquid chromatography in step (4) is: aqueous solution containing 5 mmol/L ammonium acetate and acetonitrile, the flow rate is 0.2-0.4 mL/min, and the injection volume is 5 μL.

In the step (4), liquid chromatography uses the method of gradient elution, and the gradient elution program is:

In step (4), the chromatographic column packing of the liquid chromatography is C ₁₈ , and the column temperature is 30°C.

The mass spectrometry detection in step (4) is detected by electrospray mass spectrometry (ESI), the electrospray voltage is -3500 to -4500V, the atomization gas pressure is 275.9kPa, both the drying gas and the sheath gas are nitrogen, and the drying gas temperature is 300°C. The drying gas flow rate is 5.0L/min, the sheath gas temperature is 250°C, the sheath gas flow rate is 11.0L/min, and the nozzle voltage is 500V.

In the step (4), the mass spectrometry detection uses the multiple reaction monitoring (MRM) negative ion scanning mode; the parent ion of fluorethyramide is 414.4-415.4, and the product ions are 163.3-164.3 and 178.4-179.4, respectively.

In the step (4), detect the parent ion and the product ion pair of the pesticide in the filtrate, if its ion chromatogram peak retention time is consistent with the standard working solution; and the relative abundance and concentration of the two product ions of the target compound in the filtrate (sample) are equivalent When the ion relative abundance deviation of the blank matrix standard solution does not exceed 30%, it is judged that the pesticide exists in the sample; if the above two conditions cannot be satisfied simultaneously, it is judged that the pesticide does not exist.

The beneficial effects of the present invention are:

The present invention utilizes dispersive solid-phase extraction technology to establish a sample pretreatment method that is simple and fast and can effectively avoid matrix interference in the sample, and applies the pretreatment method combined with LC-MS/MS to fluoroethers in grains and animal-derived foods Qualitative confirmation and quantitative detection of bacteramide, the average recovery rate is 83.1%-89.5%, the average relative standard deviation (RSD) is 4.0%-6.3%, and the detection limit is lower than 0.12μg/kg. It is easy to operate, fast, accurate, The advantages of high sensitivity and good repeatability. It can meet the "uniform standard" technical requirements of 0.01mg/kg residue limit, and provide strong technical support for ensuring the food safety of our people and the healthy development of foreign export trade.

Description of drawings

Figure 1 is the LC-MS/MS multiple reaction monitoring chromatogram of the 5.0ng/mL fluorethasperamide standard solution added in the blank rice matrix.

Fig. 2 is the LC-MS/MS multiple reaction monitoring chromatogram of the rice blank sample without fluoxyroxystamide.

Fig. 3 is the standard working curve of fluoxybactam prepared by using the blank rice sample without fluoxyroxystat as the matrix.

detailed description

Now illustrate the present invention with the following implementation examples, but not limit the scope of the present invention.

Instruments and reagents used in the examples

T18Basic homogenizer (IKA, Germany); CR21GⅢ centrifuge (Hitachi, Japan); MS3 basic vortex mixer (IKA, Germany); TurboVap LV automatic sample concentrator (Caliper, USA); 1290 fast high performance liquid chromatography -6460 triple quadrupole mass spectrometer (Agilent, USA); C ₁₈ /PSA solid phase extraction column (6mL, 500mg/500mg) was purchased from Tianjin Bona Agel Technology Co., Ltd.

Reagents: acetonitrile (HPLC grade, Merke, Germany); acetic acid (HPLC grade, CNW, Germany); anhydrous magnesium sulfate, sodium chloride and sodium acetate were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd.

Standard substance: Purity 97.8%, purchased from Shandong Union Pesticide Industry Co., Ltd.

Example 1: Detection of Fluorapyramide Residues in Pork

(1) Sample pretreatment

extract

Weigh 5g of fully mixed pork sample into a 50mL centrifuge tube, add 5mL of water to mix, let stand for 30min, accurately add 20mL of acetonitrile, extract homogeneously for 2min, add 3g of anhydrous magnesium sulfate and 2g of sodium chloride, and vortex for 1min , 7000r/min centrifugal 5min. After centrifugation, take 8 mL of the acetonitrile extract at 40°C for rotary steaming or nitrogen blowing to about 1 mL for purification.

purify

Pre-wash the C ₁₈ /PSA solid-phase extraction column with 5 mL of acetonitrile. When the liquid level reaches the top of the adsorbent, transfer the above extraction solution into the column, wash the test tube with 2 mL of acetonitrile, and transfer the washing liquid into the SPE column. When reaching the top of the adsorbent, add 4mL of acetonitrile to the column for elution, all the eluate is received in the quantitative test tube, and the volume is adjusted to 10mL with acetonitrile, and the purified volume is passed through a 0.22μm filter membrane, and the LC-MS/ MS determination.

(2) Preparation of standard working solution

Accurately weigh an appropriate amount of standard product in a 25mL volumetric flask, dissolve it with acetonitrile, and dilute to get 1000.0μg/mL standard stock solution; pipette 1.0mL standard stock solution into a 100mL volumetric flask, and dilute to volume with acetonitrile to get 10.0μg/mL Standard intermediate solution: Weigh 10g pork blank sample, prepare blank matrix solution through the above pretreatment steps, dilute the standard intermediate solution with blank matrix solution to prepare 0.5, 1, 2, 5, 10, 20, 50ng/mL series standard working solution, the standard working solution was analyzed by LC-MS/MS, and the corresponding concentration of the obtained peak area was used for regression analysis to obtain the standard working curve.

(3) Determination by liquid chromatography tandem mass spectrometry (LC-MS/MS)

The standard working solutions with different concentration gradients were respectively injected into LC-MS/MS, and the quantitative analysis of the content of fluoxetin was carried out by the external standard method, that is, the chromatographic peak area of the standard working solution was used to perform regression analysis on the corresponding concentrations to obtain the standard curve; Under the same conditions, the sample extract was injected into LC-MS/MS for determination, and the chromatographic peak area of fluetheribactin in the sample liquid was measured, which was substituted into the standard curve to obtain the content of fluetheribactin in the sample liquid, and then according to the The mass of the representative sample is calculated to obtain the residual amount of fluoxybactam in the sample.

Wherein the chromatographic conditions are:

Chromatographic column: Agilent, Eclipse plus C ₁₈ , 2.1mm×100mm, particle size 3.5μm;

Mobile phase: aqueous solution and acetonitrile containing 5mmol/L ammonium acetate;

Flow rate: 0.3mL/min;

Injection volume: 5 μL;

Column temperature: 30°C;

The gradient elution program is shown in Table 1.

Table 1: Gradient elution program of Example 1

time (min) Aqueous solution of 5mmol/L ammonium acetate (%) Acetonitrile (%) 0 95 5 0.5 95 5 2.0 5 95 5.0 5 95 5.2 95 5 8.0 95 5

Among them, the mass spectrometer parameters are:

Scanning mode: multiple reaction ion monitoring (MRM) negative ion scanning;

Electrospray voltage: -4000V; nozzle voltage 500V;

Atomizing gas pressure: 275.9kPa;

Drying gas: nitrogen, 300°C, flow rate 5.0L/min;

Sheath gas: nitrogen, 250°C, flow rate 11.0L/min;

MRM detection parameters are shown in Table 2.

Table 2 MRM detection parameters of Example 1

^* is the quantitative transition.

Qualitative identification: for the parent ion and product ion pair of pesticides, under the same conditions, if the ion chromatographic peak in the sample is consistent with the blank matrix standard working solution (within ± 2.5%); When the relative abundance of a product ion deviates no more than 30% from the relative abundance of a standard solution with an equivalent concentration, it is judged that the pesticide exists in the sample; if the above two conditions cannot be satisfied simultaneously, it is judged that the pesticide does not exist.

Using the chromatographic peak area of the standard working solution to perform regression analysis on its corresponding concentration, the standard working curve is shown in Table 3.

Table 3 Standard curve of fluorysamide in pork blank matrix

name retention time (min) regression equation correlation coefficient Fluorobactamide LH-2010A 3.05 Y=1299.9X-204.22 0.9998

Spike recovery and repeatability:

Add 10, 20 and 200 μg/kg three concentration levels of Fluorysyramide standard solution to the pork without Fluorysyramide, and measure the residual amount according to the above-mentioned treatment steps after adding the pesticide for 30 minutes. The measured concentration was compared with the theoretical addition concentration of the pesticide to obtain the recovery rate of the addition of the pesticide. Each addition level was measured 6 times in parallel to obtain the relative standard deviation. The measurement results are shown in Table 4. As can be seen from Table 4, on 3 standard addition levels, the average recovery rate of fluorethyrhebstramide is 85.3%～89.5%, and the average relative standard deviation (RSD) is 4.1%～6.3%, illustrates the recovery of the inventive method. High rate and good repeatability.

Table 4 The recovery rate and repeatability of fluoxystrobin (n=6)

The detection limit:

Inject different concentrations of Fluorysin amide matrix standard working solution into LC-MS/MS, and calculate with the signal-to-noise ratio of 3 times the chromatographic peak of the lowest concentration matrix standard solution and the concentration multiple of the sample processing process (the concentration multiple of pork is 0.2 times) The limit of detection, the limit of detection of fluroxystrobin is 0.12μg/kg.

Example 2: Detection of Fluorapyramide Residues in Rice

Sample pretreatment

extract

Weigh 5g of rice sample (ground into flour) that has been thoroughly mixed into a 50mL centrifuge tube, add 20mL of water to resuscitate for 30min, then accurately add 20mL of acetonitrile solution containing 1% acetic acid, shake and extract for 20min, ultrasonically extract for 5min, add 3g of anhydrous Magnesium sulfate and 2g sodium acetate, after vortexing for 1min, centrifuge at 7000r/min for 5min. After centrifugation, take 8mL of the extract solution at 40°C by rotary steaming or blowing with nitrogen until it is nearly dry, add 1mL of acetonitrile and vortex, and wait for purification.

purify

Pre-wash the C ₁₈ /PSA solid-phase extraction column with 5 mL of acetonitrile. When the liquid level reaches the top of the adsorbent, transfer the above extraction solution into the column, wash the test tube with 2 mL of acetonitrile, and transfer the washing liquid into the SPE column. When reaching the top of the adsorbent, add 4mL of acetonitrile to the column for elution, all the eluate is received in a quantitative test tube, dilute to 10mL with acetonitrile, pass through a 0.22μm filter membrane, and wait for LC-MS/MS determination.

The preparation of the standard working solution, liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination and qualitative identification procedures, chromatographic and mass spectrometry conditions were consistent with the determination of fluoxetin in pork samples above.

Linear relationship:

Regression analysis was carried out on the corresponding concentration of the chromatographic peak area of the standard working solution, and the standard working curve was obtained as Y=1645.7X-469.68, and the correlation coefficient was 0.9998.

Spike recovery and repeatability:

Add 10, 20 and 200 μg/kg standard solutions of fluoxyrhebstramide at three concentration levels to the rice without fluoxyroxystat, and measure the residual amount according to the above-mentioned treatment steps after adding the pesticide for 30 minutes. The measured concentration and the pesticide theory The addition concentration was compared to obtain the recovery rate of pesticide addition. Each addition level was measured 6 times in parallel to obtain its relative standard deviation. The measurement results are shown in Table 5. As can be seen from Table 5, on the 3 standard addition levels, the average recovery rate of fluorethyrhebstramide is 83.1%～86.7%, and the average relative standard deviation (RSD) is 4.0%～5.5%. High rate and good repeatability.

Table 5 The recovery rate and repeatability of fluoxystrobin (n=6)

The detection limit:

Inject different concentrations of Fluorethyroxystamide matrix standard working solutions into LC-MS/MS, and calculate with the signal-to-noise ratio of 3 times the chromatographic peak of the lowest concentration matrix standard solution and the concentration multiple of the sample processing process (the concentration multiple of rice is 0.2 times) The limit of detection, the limit of detection of fluroxystrobin is 0.086μg/kg.

The above embodiments are only descriptions of preferred implementations of the present invention, and are not intended to limit the scope of the present invention. On the premise of not departing from the design spirit of the present invention, various modifications and modifications made to the technical solutions of the present invention by ordinary engineering techniques in the field can be made. Improvements should all fall within the scope of protection determined by the claims of the present invention.

Claims (4)

1. A LC-MS/MS method for determining the residual amount of Fluorethyroxystamide, characterized in that, the method comprises the following steps: (1) extraction Weigh the mixed sample into a stoppered centrifuge tube, add appropriate amount of water, add acetonitrile or acetonitrile solution containing 1% acetic acid to homogenize or oscillate for ultrasonic extraction, then add one of sodium chloride or sodium acetate and anhydrous sulfuric acid Magnesium, vortex vigorously for 1 min and then centrifuge; (2) purification Pipette a certain volume of sample extract, concentrate to about 1mL, clean up with C ₁₈ /PSA solid-phase extraction column, elute with acetonitrile, collect the eluate, use acetonitrile to make up to volume, take the constant volume to pass through the membrane, and wait for liquid chromatography Tandem mass spectrometry (LC-MS/MS) detection; (3) Preparation of standard working solution Treat the blank sample of the same kind of matrix without fluoxyrhebstramide according to the above steps (1) and (2) to obtain the sample extraction and purification solution, and use the blank extraction and purification solution to prepare at least 5 concentrations of the fluoxyrhebstramide series standard work liquid; (4) Determination and result calculation The liquid chromatography analysis condition of LC-MS/MS in the step (4) is: packing is the chromatographic column of C ₁₈ , and column temperature is 30 ℃; Injection volume is 5 μ L; Mobile phase is: the aqueous solution containing 5mmol/L ammonium acetate and acetonitrile, gradient elution; the gradient elution program is: The mass spectrometry conditions are: electrospray mass spectrometry detection; electrospray voltage is -3500 to -4500V; atomization gas pressure is 275.9kPa; drying gas and sheath gas are both nitrogen; drying gas temperature is 300°C; drying gas flow rate is 5.0L/ min; the temperature of the sheath gas is 250°C, the flow rate of the sheath gas is 11.0L/min, the nozzle voltage is 500V, and the multiple reaction monitoring (MRM) negative ion scanning mode; 164.3 and 178.4~179.4; The standard working solution of each concentration gradient in step (3) is carried out LC-MS/MS measurement, carries out regression analysis to its corresponding concentration with the chromatographic peak area of standard working solution, obtains matrix standard working curve; (2) The purified sample solution in (2) is injected into LC-MS/MS for determination, and the chromatographic peak area of fluetherbactin in the sample liquid is measured, and then substituted into the matrix standard working curve to obtain the content of fluetheribactin in the sample liquid, and then according to The mass of the sample represented by the sample liquid is calculated to obtain the residual amount of fluoxetin in the sample. 2. The LC-MS/MS assay method of a kind of fluoxetin residue according to claim 1, characterized in that, if the samples in step (1) are grains and animal liver samples, an appropriate amount must be added before extraction Fully soaked with water. 3. The LC-MS/MS assay method of a kind of fluoxetin amide residual amount according to claim 1, is characterized in that, when adopting acetonitrile to extract in step (1), needs to add sodium chloride salting-out, adopts containing 1 When extracting with acetonitrile solution of % acetic acid, sodium acetate should be added for salting out. 4. the LC-MS/MS assay method of a kind of fluoxetin residual amount according to claim 1, is characterized in that, in step (2), carry out C ₁₈ /PSA solid-phase extraction column purification, when acetonitrile elutes , the elution volume is 6-8 mL.