CN104897767A - Method for rapidly detecting chlorpyrifos in honey by using neutral desorption-extractive electrospray ionization mass spectrometry - Google Patents

Abstract

The invention belongs to the technical field of detection, and discloses a method for directly detecting chlorpyrifos in honey by neutral desorption-electrospray extraction ionization mass spectrometry, including (1) preparation of standard-added honey; (2) neutral desorption-electrospray extraction ionization mass spectrometry detection of chlorpyrifos extraction (3) Select the tertiary characteristic fragment ion m/z296 of m/z352 for quantitative analysis to obtain the amount of chlorpyrifos in honey. The invention does not need to pretreat the sample to be tested, realizes the rapid detection and identification of chlorpyrifos in honey, and improves the defects of the existing analysis technology such as complex operation steps and time-consuming.

Description

Rapid detection of chlorpyrifos in honey by neutral desorption-electrospray extraction ionization mass spectrometry

technical field

The invention belongs to the technical field of detection, in particular to a detection method for chlorpyrifos in honey.

Background technique

Chlorpyrifos is a widely used high-efficiency broad-spectrum organophosphorus pesticide. It can not only kill mites, but also be used as a hygienic insecticide. It is mainly used for the control of diseases and insect pests of various crops such as vegetables, fruit trees, and cotton, but it poses a greater threat to honey plants. . Toxicological studies have shown that chlorpyrifos can significantly inhibit the activity of cholinesterase, produce toxicity to the central nervous system of mice, and have certain teratogenic effects. The analysis and research of organophosphorus pesticide residues in honey showed that the phenomenon of excessive residues of chlorpyrifos in honey was common. China is the largest beekeeping country in the world, as well as a major producer and exporter of bee products. With the improvement of people's living standards and the rapid development of import and export trade, chlorpyrifos residues in honey have become the primary factor affecting my country's honey export, seriously endangering national food safety and economic development.

At present, commonly used detection methods for chlorpyrifos include enzyme-linked immunoassay, high performance liquid chromatography, liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry. These methods generally require a complicated sample pretreatment process, and have the defects of cumbersome operation steps and time-consuming, which bring a lot of inconvenience to the detection of chlorpyrifos in honey.

Contents of the invention

The purpose of the invention is to provide a new method for direct detection of chlorpyrifos residues in honey. The invention directly and rapidly detects chlorpyrifos in honey without sample pretreatment. The invention adopts the three-stage serial series fragments of chlorpyrifos to carry out quantitative analysis, avoids false positives and improves detection accuracy.

The detection method of the present invention is by means of conventional LTQ-XL linear ion trap mass spectrometer and neutral desorption-electrospray extraction ionization (neutral desorption-extractive electrospray ionization mass spectrometry, ND-EESI) source to carry out the detection of honey samples containing chlorpyrifos .

Technical scheme of the present invention is as follows:

A method for directly detecting chlorpyrifos in honey by neutral desorption-electrospray extraction ionization mass spectrometry, comprising the following steps:

(1) preparation of standard-added honey: take the sample honey to be tested and set aside;

Preparation of spiked honey: Weigh several portions of chlorpyrifos-free honey with the same weight as the sample honey to be tested, add chlorpyrifos solution with gradient concentration respectively, stir evenly, and prepare spiked honey for later use;

Seal the sample honey and the spiked honey with plastic wrap in a 65°C water bath for 5 minutes; wait for the honey to cool to room temperature before testing;

(2) Neutral desorption—electrospray extraction ionization mass spectrometry detection: set ND-EESI-MS to positive ion detection mode, mass spectrometry detection scanning range is m/z 50-400; methanol aqueous solution is used as neutral desorption reagent; ionization voltage 3.5kV; the temperature of the ion transfer tube is 300°C; the atomizing gas is nitrogen, the pressure is 1.0MPa; the extractant formic acid:methanol:water volume ratio is 1:2:2, and the flow rate is 4μL/min;

In the collision-induced dissociation experiment, the m/z 352 width of the chlorpyrifos parent ion was set to 1.2Da, the collision energy was 30%, and the collision duration was 30ms; the secondary m/z 324 ion width was set to 1.4Da, and the collision energy was 15%. The collision duration is 30ms, and the tertiary m/z 296 is used as the quantitative ion;

Other parameters are automatically optimized by the LTQ-MS system; the angle between the spray solvent channel and the horizontal plane is 30°, and the distance between the solvent spray port and the mass spectrometer port is 0.5 cm; The included angle is maintained at 60°, and the distance between the sample spray port and the mass spectrometer inlet is 0.5 cm;

(3) Judgment of whether chlorpyrifos is contained: if there is an ion signal peak of m/z 296 in the tertiary fragment of m/z 352, it means that there is chlorpyrifos in the sample;

The tertiary characteristic fragment ion m/z 296 of m/z 352 was selected for quantitative analysis, the concentration of chlorpyrifos in the spiked honey was taken as the abscissa, and the absolute signal intensity of m/z 296 was used as the ordinate to draw a standard curve. According to the standard curve, The concentration of chlorpyrifos in the sample honey to be tested was obtained.

The beneficial effects of the present invention are: (1) the detection limit is lower, and the detection limit for chlorpyrifos in honey is 1.64 ng/mL. (2) The three-level signal is used as the quantitative signal to avoid false positives. (3) The method of the present invention adopts formic acid:methanol:water (1:2:2) to spray for neutral desorption, the desorption efficiency is high, and the intensity of the target signal is improved. (4) The in situ analysis of honey is realized without pretreatment of samples. (5) Make it possible to realize the direct and rapid application of honey pollutant detection technology, and lay the foundation for the sustainable development of the honey industry.

Description of drawings

Fig. 1 is a diagram of the ND-EESI experimental device of the method of the present invention.

Fig. 2 is the MS ⁿ spectrogram of chlorpyrifos (100ng/mL) of the present invention, among the figure, (a) is the primary mass spectrogram of chlorpyrifos methanol solution, (b) is the tandem mass spectrogram of chlorpyrifos methanol solution, (c) is the spiked honey Chlorpyrifos MS spectrum, (d) is the tandem mass spectrum of chlorpyrifos spiked honey.

Fig. 3 is the change trend figure of m/z 296CID signal strength with electrospray extractant in the spray reagent optimization experiment of the present invention, among the figure (1) is different organic solvents as extractant, (2) is the formic acid methanol solution of different concentrations as extraction Agent, (3) A-D are respectively 20% formic acid+80% methanol; 20% formic acid+40% methanol+40% acetone; 20% formic acid+40% methanol+40% ethyl acetate; 20% formic acid+40% methanol+ 40% water.

Fig. 4 is the m/z 296 signal intensity figure under different neutral desorbents of the present invention.

Fig. 5 is the optimization diagram of the conditions affecting chlorpyrifos mass spectrometry behavior in the present invention, among the figure, (a) is the optimization diagram of electrospray voltage, (b) is the optimization diagram of extractant flow rate, (c) is the optimization of atomization gas pressure Figure, (d) is the optimization diagram of the desorption pressure, (e) is the optimization diagram of the ion transfer tube temperature.

Figure 6 is the working curve of different concentrations of spiked honey.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings.

The mass spectrometer used in the example of the present invention is the LTQ-XL linear ion trap mass spectrometer of the U.S. Finnigan company, and the data processing system is the XCalibur data processing system of the U.S. Finnigan company.

The neutral desorption device and the EESI ion source are independently developed by the Jiangxi Key Laboratory of Mass Spectrometry Science and Instruments; the precision electronic balance (METTLERTOLEDO). Methanol (chromatographically pure, SKCHEM works with LS); Chlorpyrifos standard substance (purity is 99%, Aladdin reagent company); The mass spectrometer that the example described in the present invention uses is the LTQ-XL type linear ion trap mass spectrometer of U.S. Finnigan company , the data processing system is the Xcalibur data processing system of the American Finnigan Company.

Example 1

This experiment uses the ND-EESI-MS method shown in Fig. 1 to carry out experiment to chlorpyrifos honey solution (100ng/mL),

(1) Chlorpyrifos standard solution: Accurately weigh an appropriate amount of chlorpyrifos standard product, dissolve it in methanol, make a chlorpyrifos standard solution with a concentration of 2 mg/mL, and store it at -3°C in the dark. Take 100 μL 2mg/mL chlorpyrifos standard solution, dilute to 4 mL with ultrapure water, the concentration is 50 μg/mL.

(2) Adding standard honey chlorpyrifos solution: place the measuring cylinder on an analytical balance, peel and return to zero, accurately measure 9.9 mL of honey with the measuring cylinder, and weigh it, which is 13.96232 g. Then put each 10mL Erlenmeyer flask into an analytical balance, peel and return to zero, and weigh 13.96g of honey respectively. After weighing, seal it with plastic wrap and bathe in water at 65°C for 5 minutes, measure 100 μL of 50 μg/mL dilution solution in each Erlenmeyer flask filled with honey, and stir evenly. After the honey was cooled to room temperature, mass spectrometry was performed.

The results are shown in Figure 2. When chlorpyrifos is analyzed in ND-EESI ion source, positive ion mode, m/z352 chlorpyrifos [C ₉ H ₁₁ Cl ₃ NO ₃ PS] ⁺ tandem mass spectrometry, the fragment ion has m/z324, m/ z 296, m/z 278. They lost 1 (-C ₂ H ₅ ), 2 (-C ₂ H ₅ ), 2 (-C ₂ H ₅ ) and a part of H ₂ O at m/z 352, respectively. Then CID was performed on m/z 324 and m/z 296 to further confirm that the above fragments were consistent with the characteristic fragments in the literature. In order to ensure the reliability of the experiment, MS/MS mass spectrometry analysis of 100ng/mL chlorpyrifos standard solution was carried out by using EESI-MS. The results showed that the ND-EESI-MS ⁿ spectrum of chlorpyrifos was completely consistent with its EESI-MS ⁿ spectrum. Therefore, if the signal peak m/z352 can be detected in the actual sample, and the main characteristic ions m/z 324 and m/z296 are observed in the MS/MS spectrum, it can be judged that the sample contains chlorpyrifos.

In order to exclude false positives during detection, the three-level characteristic fragment ion peak m/z296 was used as the target signal. In order to improve its signal intensity, the spray reagent was optimized in this experiment, and 100ng/mL chlorpyrifos spiked honey was used as the experimental object. Taking the response intensity of the tertiary mass spectrometry signal m/z296 as the standard, the optimization experiments of water, methanol, ethanol, ethyl acetate, propanol, butanol, benzene, p-xylene, acetonitrile and acetone are carried out, as shown in Figure 3(1) shown. After a lot of exploration, methanol with different volume fractions of formic acid was used as the extraction agent to measure the signal intensity of the tertiary characteristic ion m/z296 of chlorpyrifos, and it was found that adding acid had a very good effect. Methanol solution is used as a spray reagent, and the third-order characteristic fragment m/z296 of chlorpyrifos in honey has the strongest signal intensity, but the maximum acid concentration allowed by the mass spectrometer is 20%, so the acid concentration cannot be increased. To explore better spray reagents, use 20% formic acid + 80% methanol; 20% formic acid + 40% methanol + 40% acetone; 20% formic acid + 40% methanol + 40% ethyl acetate; 20% formic acid + 40% methanol +40% water is used as the electrospray reagent, and the results show that 20% formic acid+40% methanol+40% water has the best effect, as shown in Figure 3 (3). Therefore, the extractant in this experiment is a mixed solution of 20% formic acid+40% methanol+40% water.

In this experiment, a neutral desorbent was explored. Using 20% formic acid + 40% methanol + 40% water as the electrospray extraction agent, 90% methanol + 10% water, 80% methanol + 20% water, 60% methanol + 40% water, 40% methanol + 60% water, 20% methanol + 80% water, and 100% water were used as desorbents to detect the signal intensity of chlorpyrifos triple-tandem characteristic mass spectrum fragment m/z 296, and the results are shown in Figure 4.

In this experiment, the ion source conditions were explored. According to the mass spectrometry behavior of the chlorpyrifos standard solution, in the positive ion mode, the mass spectrum response signal intensity of the chlorpyrifos three-stage tandem characteristic mass spectrum fragment m/z 296 was selected to represent the detection efficiency of the chlorpyrifos molecule. . As shown in Figure 5(a), when the voltage is too small (less than 1.5kV), the signal strength is low. As the voltage increases, the target signal strength increases rapidly. When the voltage is too large (greater than 3.5kV), the signal strength On the contrary, it decreased to some extent and reached the optimum at 3.5kV; as shown in Figure 5(b), the extraction efficiency of the electrospray solvent flow rate was at the maximum at 4μL/min; as shown in Figure 5(c), the experiment showed that with As the atomization gas pressure increases, the target signal intensity also increases. When the air pressure exceeds a certain value (greater than 1.0MPa), most ions will be annihilated at the edge of the mass spectrometer, and the target signal intensity will also drop significantly. As shown in Figure 5(d), the signal is the strongest when the desorption pressure is 1.2MPa, and the desorption efficiency is the highest; as shown in Figure 5(e), the target signal intensity increases rapidly from 100°C to 300°C However, when the temperature is higher than 300°C, the signal intensity gradually decreases. Therefore, the temperature of the ion transfer tube is selected as 300°C for the experiment.

The chlorpyrifos spiked experiment was carried out according to the above experimental method to determine the linear range and detection limit of the method. Due to the real-time detection in the honey sample, in order to eliminate the false positive of the signal, the experiment selected the tertiary characteristic fragment ion m/z 296 of m/z 352 for quantitative analysis, and deducted the corresponding blank background at the same time. Standard samples of each concentration were measured 6 times, and a curve was drawn between the average absolute signal intensity of m/z 296 and the corresponding chlorpyrifos concentration, as shown in FIG. 6 . Experiments show that within the range of chlorpyrifos concentration from 100 to 1000ng/mL, the ion strength has a good linear relationship with the concentration, and the linear regression equation is y=2.444x-63.76, R ² =0.997. According to LOD=c3σ/S, S/N=3, c is the concentration of the standard, σ is the standard deviation, and S is the average value of the net corresponding signal intensity), the detection limit of this method is calculated to be 1.64ng to the spiked honey /mL.

Example 2

A method for directly detecting chlorpyrifos in honey by neutral desorption-electrospray extraction ionization mass spectrometry, comprising the following steps:

(1) preparation of standard-added honey: take the sample honey to be tested and set aside;

Preparation of spiked honey: Weigh several portions of chlorpyrifos-free honey with the same weight as the sample honey to be tested, add chlorpyrifos solution with gradient concentration respectively, stir evenly, and prepare spiked honey for later use;

Seal the sample honey and the spiked honey with plastic wrap in a 65°C water bath for 5 minutes; wait for the honey to cool to room temperature before testing;

(2) Neutral desorption—electrospray extraction ionization mass spectrometry detection: set ND-EESI-MS to positive ion detection mode, mass spectrometry detection scanning range is m/z 50-400; methanol aqueous solution is used as neutral desorption reagent; ionization voltage 3.5kV; the temperature of the ion transfer tube is 300°C; the atomizing gas is nitrogen, the pressure is 1.0MPa; the extractant formic acid:methanol:water volume ratio is 1:2:2, and the flow rate is 4μL/min;

In the collision-induced dissociation experiment, the m/z 352 width of the chlorpyrifos parent ion was set to 1.2Da, the collision energy was 30%, and the collision duration was 30ms; the secondary m/z 324 ion width was set to 1.4Da, and the collision energy was 15%. The collision duration is 30ms, and the tertiary m/z 296 is used as the quantitative ion;

Other parameters are automatically optimized by the LTQ-MS system; the angle between the spray solvent channel and the horizontal plane is 30°, and the distance between the solvent spray port and the mass spectrometer port is 0.5 cm; The included angle is maintained at 60°, and the distance between the sample spray port and the mass spectrometer inlet is 0.5 cm;

(3) Judgment of whether chlorpyrifos is contained: If there is an ion signal peak of m/z 296 in the tertiary fragment of m/z 352, it means that there is chlorpyrifos in the sample.

The tertiary characteristic fragment ion m/z 296 of m/z 352 was selected for quantitative analysis, the concentration of chlorpyrifos in the spiked honey was taken as the abscissa, and the absolute signal intensity of m/z 296 was used as the ordinate to draw a standard curve. According to the standard curve, The concentration of chlorpyrifos in the sample honey to be tested was obtained.

Example 3

(1) Weigh 13.96g of commercially available honey into a 10mL Erlenmeyer flask, seal with plastic wrap and bathe in water at 65°C for 5 minutes; wait for the honey to cool down to room temperature, then test;

(2) Neutral desorption-electrospray extraction ionization mass spectrometry detection: set the ND-EESI-MS to positive ion detection mode, and the mass spectrometry detection scanning range is m/z 50-400; the methanol:water volume ratio is 1:1 as the medium Desorption spray reagent; ionization voltage 3.5kV; ion transfer tube temperature 300°C; atomizing gas nitrogen, pressure 1.0MPa; extractant formic acid: methanol: water volume ratio 1:2:2, flow rate 4μL/min;

In the collision-induced dissociation experiment, the m/z 352 width of the chlorpyrifos parent ion was set to 1.2Da, the collision energy was 30%, and the collision duration was 30ms; the secondary m/z 324 ion width was set to 1.4Da, and the collision energy was 15%. The collision duration is 30ms, and the tertiary m/z 296 is used as the quantitative ion;

Other parameters are automatically optimized by the LTQ-MS system; the angle between the spray solvent channel and the horizontal plane is 30°, and the distance between the solvent spray port and the mass spectrometer port is 0.5 cm; The included angle is maintained at 60°, and the distance between the sample spray port and the mass spectrometer inlet is 0.5 cm;

(3) Judgment of whether chlorpyrifos is contained: If there is an ion signal peak of m/z 296 in the tertiary fragment of m/z 352, it means that there is chlorpyrifos.

Determination of the content of chlorpyrifos in the sample: There is an ion signal peak of m/z 296 in the tertiary fragment of m/z 352 in the honey sample collected by this device, and the signal intensity data is obtained, and the data is substituted into the formula: y=2.444x-63.76 , to calculate the content of chlorpyrifos in the sample (concentration unit is ng/mL).

The 50 and 500ng/mL chlorpyrifos honey solutions were measured in parallel 6 times, and calculated by the standard curve, the recoveries were 82.02% and 109.28%, respectively, and the precision (RSD) were 4.02% and 4.34%, respectively.

Claims (2)

1. a neutral desorption-electrospray extraction ionization mass spectrometry directly detects the method for chlorpyrifos in honey, it is characterized in that comprising the steps: (1) preparation of standard-added honey: take the sample honey to be tested and set aside; Preparation of spiked honey: Weigh several portions of chlorpyrifos-free honey with the same weight as the sample honey to be tested, add chlorpyrifos solution with gradient concentration respectively, stir evenly, and prepare spiked honey for later use; Seal the sample honey and the spiked honey with plastic wrap in a 65°C water bath for 5 minutes; wait for the honey to cool to room temperature before testing; (2) Neutral desorption—electrospray extraction ionization mass spectrometry detection: set ND-EESI-MS to positive ion detection mode, mass spectrometry detection scanning range is m/z 50-400; methanol aqueous solution is used as neutral desorption reagent; formic acid Methanol aqueous solution is the electrospray extraction reagent; the ionization voltage is 3.5kV; the temperature of the ion transfer tube is 300°C; the atomizing gas is nitrogen, and the pressure is 1.0MPa; the flow rate of the electrospray extraction reagent is 4 μL/min; In the collision-induced dissociation experiment, the m/z 352 width of the chlorpyrifos parent ion was set to 1.2Da, the collision energy was 30%, and the collision duration was 30ms; the secondary m/z 324 ion width was set to 1.4Da, and the collision energy was 15%. The collision duration is 30ms, and the tertiary m/z 296 is used as the quantitative ion; Other parameters are automatically optimized by the LTQ-MS system; the angle between the spray solvent channel and the horizontal plane is 30°, and the distance between the solvent spray port and the mass spectrometer port is 0.5 cm; The included angle is maintained at 60°, and the distance between the sample spray port and the mass spectrometer inlet is 0.5 cm; (3) Judgment of whether chlorpyrifos is contained: if there is an ion signal peak of m/z 296 in the tertiary fragment of m/z 352, it means that there is chlorpyrifos in the sample; The tertiary characteristic fragment ion m/z 296 of m/z 352 was selected for quantitative analysis, the concentration of chlorpyrifos in the spiked honey was taken as the abscissa, and the absolute signal intensity of m/z 296 was taken as the ordinate to draw a standard curve. The signal intensity of the tertiary characteristic fragment ion m/z 296 of /z 352 was substituted into the standard curve formula to obtain the concentration of chlorpyrifos in the honey to be tested. 2. The method according to claim 1, characterized in that methanol: water volume ratio of 1:1 is the neutral desorption reagent; formic acid: methanol: water volume ratio of 1:2:2 is the electrospray extraction reagent.