CN111398501A - Method for determining content of sex pheromone in tobacco beetle sex pheromone lure core - Google Patents

Abstract

The invention belongs to the field of detection and analysis, and in particular relates to a method for determining the content of sex pheromone in tobacco beetle sexual pheromone lure cores, comprising the following steps: (1) immersing the tobacco beetle sexual pheromone lure cores with a solvent to obtain a mixture; wherein , the solvent is ethanol containing sodium borohydride, and the mass of sodium borohydride accounts for 0.05% to 1% of the mass of the solvent; (2) passing the mixture through a headspace sampler with a trap to a gas chromatograph-triple The sample is injected into the quadrupole mass spectrometer, and then detected, and the detection result is obtained; it also discloses the specific operating conditions of the headspace sampler with a trap. The method of the invention has good repeatability, high sensitivity, low detection limit and high accuracy for determining the content of the tobacco beetle sex pheromone in the tobacco beetle sex pheromone lure core and the tobacco beetle sex pheromone trap.

Description

Method for determining the content of sex pheromone in tobacco beetle sex pheromone lure

technical field

The invention belongs to the field of detection and analysis, and in particular relates to a method for determining the content of sex pheromone in a sex pheromone lure of tobacco beetle.

Background technique

Tobacco beetles belong to the family Coleoptera, and are one of the main pests in tobacco warehouses in my country. Tobacco beetles are widely distributed, reproduce in large numbers, and cause serious harm. They not only eat tobacco leaves to form holes, thereby affecting the filamentation rate of tobacco leaves, but also discharge a large amount of feces and leave insect carcasses, which lead to the deterioration of tobacco taste and quality. Tobacco beetles have caused serious losses to the tobacco industry. According to rough estimates, the direct economic loss caused by tobacco beetles in the country is more than 1 billion yuan each year.

At present, tobacco beetle control mainly uses tobacco beetle sex pheromone traps to monitor the distribution and density of tobacco beetles, and kill tobacco beetles or intervene to inhibit the reproduction of tobacco beetles; usually, it can also be combined with phosphine fumigation to improve cleanliness and hygiene for auxiliary prevention.

The main component in the tobacco beetle pheromone trap is the tobacco beetle pheromone lure. Usually, tobacco beetle sexual pheromone lure core contains tobacco beetle sex pheromone, carrier, synergist, killer (sterile agent), synergist, diluent, slow-release agent and other ingredients. Plant-derived attractants, synthetic attractants, etc. are also added. The main component of tobacco beetle sex pheromone is 4,6-dimethyl-7-hydroxy-3-nonanone (6-Serricornin), wherein C ₇ is the configuration of S, namely (7S)-(one)-4 , 6-dimethyl-7-hydroxy-3-nonanone had stronger sex-inducing activity. Tobacco beetle sex pheromone content and content stability in tobacco beetle sex pheromone lures have an important impact on the effectiveness of the trap, so it is necessary to accurately know the tobacco beetle sex pheromone content in tobacco beetle sex pheromone lures. However, the components in tobacco beetle sex pheromone lure are complex and diverse, and general detection methods (such as GC-MS) cannot accurately determine the content of tobacco beetle sex pheromone in the lure. Therefore, there is an urgent need for a method for accurately detecting the content of the tobacco beetle sex pheromone in the tobacco beetle sex pheromone lure.

SUMMARY OF THE INVENTION

The invention provides a method for determining the content of sex pheromone in tobacco beetle sex pheromone lure core. Using mass spectrometry and certain detection conditions, the content of sex pheromone in the lure core can be accurately determined. The method has good repeatability, high sensitivity, low detection limit and high accuracy. On this basis, the present invention also provides a method for determining the content of sex pheromone in a tobacco beetle sex pheromone trap.

A first aspect of the present invention relates to a method for measuring the (tobacco beetle) sex pheromone content in the sex pheromone lure of tobacco beetle, comprising the steps of:

(1) using a solvent to immerse the sexual pheromone lure of the tobacco beetle to obtain a mixture; wherein, the solvent is ethanol containing sodium borohydride, and the mass of sodium borohydride accounts for 0.05% to 1% of the mass of the solvent, such as 0.06%, 0.07% %, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 1%;

(2) injecting the mixture into a gas chromatography-triple quadrupole mass spectrometer through a headspace sampler with a trap, and then detecting to obtain a detection result;

Among them, the operating conditions of the headspace sampler with trap include:

The heating equilibrium temperature is 70°C to 80°C (eg 72°C, 75°C, 78°C), the heating equilibrium time is 20 to 40 minutes (eg 25 minutes, 30 minutes), and the injection needle temperature is 90°C to 100°C (eg 93°C, 95°C, 98°C), transfer line temperature is 105°C to 125°C (eg 110°C, 115°C, 120°C), trap low temperature is -55°C to -25°C (eg -50°C, -45°C) °C, -40 °C, -35 °C, -32 °C, -30 °C), trap high temperature is 160 °C ~ 200 °C (for example, 165 °C, 170 °C, 175 °C, 180 °C, 185 °C, 190 °C, 195 °C ℃), the desorption time is 0.1 to 2 minutes (such as 0.5 minutes, 1 minute, 1.5 minutes), the desorption pressure is 25 to 45Psi (such as 30Psi, 35Psi, 40Psi), and the bottle pressure is 30 to 50Psi (such as 35Psi, 40Psi, 45Psi) ), the column pressure is 20~50Psi (such as 30Psi, 35Psi, 40Psi, 45Psi), the trap retention time is 1~10 minutes (such as 2, 5, 7, 8 minutes), and the dry purge time is 0.5~8 minutes (eg 1, 2, 3, 5, 7 minutes).

In some embodiments of the first aspect of the invention, the headspace sampler with trap is splitless during operation.

In some embodiments of the first aspect of the present invention, the (tobacco beetle) sex pheromone is 4,6-dimethyl-7-hydroxy-3-nonanone, preferably (7S)-(one)-4, 6-Dimethyl-7-hydroxy-3-nonanone.

In some embodiments of the first aspect of the present invention, in step (1), the mass of sodium borohydride accounts for 0.05% to 0.4% of the mass of the solvent.

In some embodiments of the first aspect of the present invention, in step (1), the volume of the solvent is just enough to submerge the tobacco beetle sexual pheromone lure.

In some embodiments of the first aspect of the present invention, the solvent is anhydrous ethanol containing sodium borohydride, which can also be understood as a mixture of sodium borohydride and anhydrous ethanol.

In some embodiments of the first aspect of the present invention, in step (2), the operating conditions of the gas chromatography include one or more of the following A to G:

A. The chromatographic column is HP-5MS (UI) column;

Preferably, the HP-5MS (UI) column is a capillary column;

B. The size of the chromatographic column is 60m×0.25mm, 0.25μm;

C. The carrier gas is helium;

D. The inlet temperature is 80℃～120℃, such as 85℃, 90℃, 95℃, 100℃, 105℃, 110℃, 115℃;

E. Adopt constant voltage mode;

F. The heating program of the chromatographic column is: 30℃～50℃ (eg 35℃, 40℃, 45℃) for 1～5 minutes (eg 2, 3, 4 minutes), at 5～12℃/min (eg 6 minutes) °C/min, 8°C/min, 10°C/min) at a rate of 110°C to 130°C (eg 115°C, 120°C, 125°C), at 110°C to 130°C (eg 115°C, 120°C, 125°C) ℃) for 1 to 5 minutes (such as 2, 3, 4 minutes), and then increase to 150 ℃ to 175 °C at a rate of 1 to 6 ℃/min (such as 2 ℃/min, 4 ℃/min, 5 ℃/min) °C (eg 155°C, 160°C, 170°C), then at a rate of 40-60°C/min (eg 45°C/min, 50°C/min, 55°C/min) to 250°C-270°C (eg 255°C/min) ℃, 260℃, 265℃), keep at 250℃～270℃ (such as 255℃, 260℃, 265℃) for 1 to 10 minutes (such as 2, 3, 4, 6, 8 minutes) and finish;

G. The temperature of the auxiliary heater is 250°C to 290°C, for example, 260°C, 270°C, and 280°C.

In some embodiments of the first aspect of the present invention, in step (2), the operating conditions of the triple quadrupole mass spectrometer include one or more of the following a to d:

a. The temperature of the ion source is 240℃～260℃, such as 245℃, 250℃, 255℃;

b. The temperature of the quadrupole is 140℃～160℃, such as 145℃, 150℃, 155℃;

c. The ionization energy is 60～80eV, such as 65eV, 70eV, 75eV;

d. The scanning range is 29～350amu.

In some embodiments of the first aspect of the present invention, the qualifying ion of (7S)-(mono)-4,6-dimethyl-7-hydroxy-3-nonanone is 55.

In some embodiments of the first aspect of the present invention, the quantitative ion of (7S)-(mono)-4,6-dimethyl-7-hydroxy-3-nonanone is 86.

In some embodiments of the first aspect of the present invention, in step (2), the detection result is obtained by an external standard quantitative analysis method.

A second aspect of the present invention relates to a method for measuring (tobacco beetle) sex pheromone content in a tobacco beetle sex pheromone trap, comprising the steps of:

Measure the (tobacco beetle) sex pheromone content in the tobacco beetle sex pheromone lure according to the method described in the first aspect of the present invention;

According to the (tobacco beetle) sex pheromone content in the tobacco beetle sex pheromone trap and the number of lures in the tobacco beetle sex pheromone trap, the sex pheromone content in the tobacco beetle sex pheromone trap was calculated.

In some embodiments of the second aspect of the present invention, the (tobacco beetle) sex pheromone is 4,6-dimethyl-7-hydroxy-3-nonanone, preferably (7S)-(one)-4, 6-Dimethyl-7-hydroxy-3-nonanone.

In the present invention, the tobacco beetle pheromone lure core or the tobacco beetle pheromone trap are conventional commodities in the art and can be obtained commercially.

The beneficial effects obtained by the present invention:

The method of the present invention is used to determine the sex pheromone (for example (7S)-(one)-4,6-dimethyl-7-hydroxy-3) in the tobacco beetle sex pheromone trap and the tobacco beetle sex pheromone trap. -nonanone) content has good repeatability, high sensitivity, low detection limit and high accuracy.

Description of drawings

In order to make the content of the present invention easier to understand clearly, the present invention will be described in further detail below according to specific embodiments of the present invention and in conjunction with the accompanying drawings, wherein

Fig. 1 is the chromatogram of the sample obtained in step (2) of Example 1.

Detailed ways

The embodiments of the present invention will be clearly and completely described below with reference to the examples. Obviously, the described examples are only a part of the examples of the present invention, but not all of the examples. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

(1) Preparation of a series of standard solutions:

In a 10mL volumetric flask, add accurately weighed (7S)-(mono)-4,6-dimethyl-7-hydroxy-3-nonanone standard 25mg (accurate to 0.1mg), add 0.1% (mass) Concentration) of sodium borohydride in ethanol to constant volume to obtain (7S)-(one)-4,6-dimethyl-7-hydroxy-3-nonanone concentration 1-grade standard solution of 2500 μg/mL. Pipette 5mL, 2mL, 1mL, and 0.2mL of the 1st grade standard solution into a 10mL volumetric flask, dilute to volume with ethanol containing 0.1% (mass concentration) sodium borohydride, and prepare a 2nd to 5th grade standard solution.

(2) Pretreatment:

Put a freshly opened tobacco beetle pheromone lure core 1 into a 20mL headspace bottle, add 1ml of ethanol containing 0.1% (mass concentration) sodium borohydride to immerse the lure core, obtain a sample, and seal it for later use.

(3) Detection:

The series of standard solutions and the samples obtained in step (2) were passed through a headspace sampler with trap (PerkinElmer TurboMatrix HST-40 headspace sampler) to a gas chromatograph-triple quadrupole mass spectrometer (Agilent 7890B GC). The chromatograph and Agilent 5977A MS were used in combination) to inject samples for detection; wherein,

Operating conditions for the headspace sampler with traps include:

Heating equilibrium temperature is 75℃; heating equilibrium time is 30min; injection needle temperature is 95℃; transfer line temperature is 115℃; trap low temperature is -40℃; trap high temperature (desorption temperature) is 180℃; desorption The time was 0.5min; the desorption pressure was 35Psi; the bottle pressure was 40Psi; the column pressure was 35Psi; the trap retention time was 5.0min; the dry purge time was 3.0min;

Operating conditions for gas chromatography include:

The chromatographic column is a capillary column HP-5MS(UI) column (60m×0.25mm, 0.25μm); the carrier gas is helium; the inlet temperature is 100°C; Heat up to 120°C at a rate of 8°C/min, hold at 120°C for 2 minutes, then heat up to 160°C at a rate of 2°C/min, then heat up to 260°C at a rate of 50°C/min, and hold at 260°C for 4 minutes to end; The heater temperature is 270℃;

Operating conditions for mass spectrometry include:

The temperature of the ion source is 250℃; the temperature of the quadrupole is 150℃; the ionization energy is 70eV; the scanning range is 29～350amu; The qualifier ion for the ketone is 55 and the quantifier ion is 86.

The chromatogram of the sample in step (2) is shown in Figure 1 (1 represents the compound peak of (7S)-(mono)-4,6-dimethyl-7-hydroxy-3-nonanone).

(4) Data processing:

The concentration-quantitative ion peak area standard working curve Y=0.6237x+0.0021 (R ²⁼ 0.997) was drawn according to the concentration of the series of standard solutions and the measured quantitative ion peak area. Combined with the quantitative ion peak area of the sample, the content of the active ingredient (7S)-(one)-4,6-dimethyl-7-hydroxy-3-nonanone in tobacco beetle sexual pheromone lure 1 was calculated to be 595.11 μg /indivual.

Example 2

The content of (7S)-(mono)-4,6-dimethyl-7-hydroxy-3-nonanone in tobacco beetle sexual pheromone lure core 2-13 was determined according to the method in Example 1, wherein the lure Cores 2-6 were just opened, and the rest were lure cores that were left for a period of time after opening. The results are shown in Table 1.

Table 1 Measurement results

Content of (7S)-(one)-4,6-dimethyl-7-hydroxy-3-nonanone (μg/piece) lure 2 550.50 lure 3 554.64 lure 4 515.39 lure 5 635.96 lure 6 302.46 lure 7 54.48 lure 8 39.43 lure 9 36.62 lure 10 36.29 lure 11 36.19 lure core 12 36.08 lure 13 36.45

It can be seen from Table 1 that the content of sex pheromone in the just-opened lure is relatively high; after a period of opening, the content of sex pheromone in the lure is significantly reduced, which seriously affects the effectiveness of the lure. Therefore, it is necessary to measure and monitor the sex pheromone content of the lure.

Repeatability, recovery, limit of detection and limit of quantification of the method of Example 3

The fifth-grade standard solution in Example 1 was repeatedly injected and tested ten times according to the method in Example 1, and three times and ten times the deviation were used as the detection limit and the quantification limit, respectively, as shown in Table 2.

Add 500 μg (7S)-(mono)-4,6-dimethyl-7-hydroxy-3-nonanone standard on the basis of lure 1 in Example 1 to obtain a spiked sample, and then add the spiked sample according to Steps (2)-(4) in Example 1 were carried out for pretreatment, detection and data processing, the recovery rate was calculated, and the experiment was repeated six times in parallel. The results are shown in Table 2.

Table 2 Repeatability, recovery rate, detection limit and quantification limit results of the method of the present invention

It can be seen from Table 2 that the relative standard deviation of the detection results of the method of the present invention is less than 5%, the detection limit and the limit of quantification are both low, and the recovery rate is within the usual acceptable range of 80% to 120%, indicating that the method of the present invention is repeated. Good performance, high sensitivity and high accuracy.

Comparative Example 1

Detection method: direct injection into gas chromatography-triple quadrupole mass spectrometer (Agilent 7890B gas chromatograph combined with Agilent 5977A MS) for detection (without headspace sampler with trap); The operating conditions and other operations of gas chromatography and triple quadrupole mass spectrometry are the same as those in Example 1.

Take the 1st to 5th grade standard solutions in Example 1 and detect according to the above-mentioned "detection method", and draw a standard working curve according to the concentration of the series of standard solutions and the measured quantitative ion peak area. The fifth-grade standard solution was repeatedly detected ten times, and three times and ten times the deviation were taken as the detection limit and the quantification limit, respectively, as shown in Table 3.

Add 1 mL of ethanol containing 0.1% (mass concentration) sodium borohydride to the spiked sample in Example 3 to immerse the sample, and then perform detection according to the above "detection method", perform data processing in combination with the above standard working curve, and calculate the recovery The experiment was repeated six times in parallel, and the results are shown in Table 3.

Table 3 Results of repeatability, recovery, limit of detection and limit of quantification of the method of Comparative Example 1

It can be seen from Tables 2 to 3 that the detection limit and quantification limit of the method of Comparative Example 1 are respectively more than three times that of the method of the present invention, the relative standard deviation of the method of Comparative Example 1 is greater than that of the method of the present invention, and the relative standard deviation of the method of Comparative Example 1 is greater than that of the method of the present invention. The recovery rate exceeds the generally acceptable range of 80% to 120%, which shows that, compared with the method of Comparative Example 1, the method of the present invention has better repeatability, lower detection limit, higher sensitivity and higher accuracy .

Comparative Example 2

(1) Preparation of a series of standard solutions:

In a 10mL volumetric flask, add accurately weighed (7S)-(mono)-4,6-dimethyl-7-hydroxy-3-nonanone standard 25mg (accurate to 0.1mg), add absolute ethanol to volume , to obtain (7S)-(one)-4,6-dimethyl-7-hydroxy-3-nonanone, a grade 1 standard solution with a concentration of 2500 μg/mL. Pipette 5mL, 2mL, 1mL, and 0.2mL of the 1st grade standard solution into a 10mL volumetric flask, dilute to volume with absolute ethanol, and prepare the 2nd to 5th grade standard solution.

(2) Pretreatment:

Put a freshly opened tobacco beetle pheromone lure core 1 into a 20 mL headspace bottle, add 1 ml of absolute ethanol to immerse the lure core, obtain a sample to be tested, and seal it for later use.

Put a freshly opened tobacco beetle pheromone lure core 1 into a 20mL headspace vial, and then add 500μg (7S)-(one)-4,6-dimethyl-7-hydroxy-3-nonanone standard , and then 1 mL of absolute ethanol was added to it to immerse all the substances to obtain a spiked sample, which was sealed for later use.

(3) Detection: Take the 1st to 5th grade standard solutions, the samples to be tested and the spiked samples for detection according to step (3) in Example 1.

Draw a standard working curve according to the concentration of a series of standard solutions and the measured quantitative ion peak area. The fifth-grade standard solution was repeatedly detected ten times, and three times and ten times the deviation were taken as the detection limit and the quantification limit, respectively, as shown in Table 4. Data processing was carried out according to the quantitative ion peak area and standard working curve measured by the sample to be tested and the spiked sample, and the recovery rate was calculated, and the experiment was repeated six times in parallel. The results are shown in Table 4.

Table 4 Results of repeatability, recovery, limit of detection and limit of quantification of the method of Comparative Example 2

As can be seen from Table 2 and Table 4, the relative standard deviation of the method of Comparative Example 2 is more than three times that of the method of the present invention, the detection limit and the limit of quantification of the method of Comparative Example 2 are significantly higher than those of the method of the present invention, and the recovery of the method of Comparative Example 2 is significantly higher than that of the method of the present invention. The rate exceeds the generally acceptable range of 80% to 120%, which shows that, compared with the method of Comparative Example 2, the method of the present invention has better repeatability, lower detection limit, higher sensitivity and higher accuracy.

Obviously, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation manner. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. And the obvious changes or changes derived from this are still within the protection scope of the present invention.

Claims (10)

1. A method for determining the content of sex pheromone in a sex pheromone lure of tobacco beetles comprises the following steps:

(1) immersing the tobacco beetle sex pheromone lure core by using a solvent to obtain a mixture; wherein the solvent is ethanol containing sodium borohydride, and the mass of the sodium borohydride accounts for 0.05-1% of the mass of the solvent;

(2) introducing the mixture into a gas chromatography-triple quadrupole mass spectrometer through a headspace sampler with a trap, and then detecting to obtain a detection result;

wherein the operating conditions of the trap-equipped headspace sampler comprise:

the heating balance temperature is 70-80 ℃, the heating balance time is 20-40 minutes, the temperature of a sample injection needle is 90-100 ℃, the temperature of a transmission line is 105-125 ℃, the low temperature of a trap is-55-25 ℃, the high temperature of the trap is 160-200 ℃, the desorption time is 0.1-2 minutes, the desorption pressure is 25-45 Psi, the bottle pressure is 30-50 Psi, the pressure of a chromatographic column is 20-50 Psi, the holding time of the trap is 1-10 minutes, and the dry purging time is 0.5-8 minutes.

2. The method of claim 1, wherein the sex pheromone is 4, 6-dimethyl-7-hydroxy-3-nonanone.

3. The method of claim 1, wherein the sex pheromone is (7S) - (mono) -4, 6-dimethyl-7-hydroxy-3-nonanone.

4. The method according to claim 1, wherein in the step (1), the mass of the sodium borohydride accounts for 0.05-0.4% of the mass of the solvent.

5. The method of claim 1, wherein in step (2), the trapped-band headspace sampler is not shunted during operation.

6. The method of claim 1, wherein in step (2), the operating conditions of the gas chromatograph comprise one or more of the following A to G:

A. the chromatographic column is HP-5MS (UI) column;

B. the specification of the chromatographic column is 60m × 0.25mm,0.25 μm;

C. the carrier gas is helium;

D. the temperature of a sample inlet is 80-120 ℃;

E. a constant pressure mode is adopted;

F. the temperature rising program of the chromatographic column is as follows: keeping the temperature at 30-50 ℃ for 1-5 minutes, raising the temperature to 110-130 ℃ at the speed of 5-12 ℃/min, keeping the temperature at 110-130 ℃ for 1-5 minutes, raising the temperature to 150-175 ℃ at the speed of 1-6 ℃/min, raising the temperature to 250-270 ℃ at the speed of 40-60 ℃/min, keeping the temperature at 250-270 ℃ for 1-10 minutes, and finishing;

G. the temperature of the auxiliary heater is 250-290 ℃.

7. The method of any one of claims 1 to 6, wherein in step (2), the operating conditions of triple quadrupole mass spectrometry comprise one or more of the following a to d:

a. the temperature of the ion source is 240-260 ℃;

b. the temperature of the quadrupole rods is 140-160 ℃;

c. the ionization energy is 60-80 eV;

d. the scanning range is 29-350 amu.

8. The method of claim 1, wherein in step (1), the volume of solvent is just sufficient to submerge the tobacco beetle sex pheromone attractant core.

9. The method according to claim 1, wherein in the step (2), the detection result is obtained by an external standard quantitative analysis method.

10. A method of determining the sex pheromone content in a tobacco beetle sex pheromone trap comprising the steps of:

measuring the sex pheromone content in the sex pheromone attractant core of the tobacco beetles according to the method of any one of claims 1 to 9;

and calculating the content of the sex pheromone in the tobacco beetle sex pheromone trap according to the content of the sex pheromone in the tobacco beetle sex pheromone trap and the number of the trap cores in the tobacco beetle sex pheromone trap.

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