CN108519454B

CN108519454B - Pretreatment method for measuring various pesticide residues in tea and detection method thereof

Info

Publication number: CN108519454B
Application number: CN201810335822.3A
Authority: CN
Inventors: 李少华; 陈达炜; 陈荣冰; 张渤; 王飞权
Original assignee: Wuyi University
Current assignee: Wuyi University
Priority date: 2018-04-16
Filing date: 2018-04-16
Publication date: 2021-01-19
Anticipated expiration: 2038-04-16
Also published as: CN108519454A

Abstract

The invention discloses a pretreatment method for measuring various pesticide residues in tea and a detection method thereof, wherein the pretreatment method comprises the following steps: adding water into tea powder for pre-soaking, adding acetonitrile for ultrasonic extraction, and filtering; adding polyvinylpyrrolidone and sodium chloride into the extracting solution, mixing by vortex, and centrifuging; taking the supernatant fluid I, adding a strong cation exchange filler PCX, mixing in a vortex mode, and centrifuging; eluting the precipitate with acetonitrile and 5% ammonia water acetonitrile in sequence, and collecting the eluent of 5% ammonia water acetonitrile; taking the supernatant II, adding PCX, mixing in a vortex mode, centrifuging to obtain a supernatant III, and repeating the step for 1-2 times; and mixing the eluent of 5% ammonia water acetonitrile and the supernatant III, and filtering by an organic filter membrane to obtain the compound. The method can ensure that the adsorption clearance rate of tea polyphenols reaches more than 94 percent and the adsorption clearance rate of caffeine reaches more than 85 percent, greatly improves the pretreatment effect of the sample, and has the advantages of simple operation, short time consumption, less solvent consumption, low cost and the like.

Description

Pretreatment method for measuring various pesticide residues in tea and detection method thereof

Technical Field

The invention belongs to the field of food safety detection, and particularly relates to a pretreatment method for determining various pesticide residues in tea and a detection method thereof.

Background

China is a large country for tea production and consumption, tea is always popular with common people as a healthy beverage, but in recent years, the six kinds of tea pesticide residues are reported to exceed standards, so that the worry of people about tea safety is caused, and the development of the tea industry is not facilitated. In order to meet the increasingly stringent tea pesticide residue standard, the mass spectrometry technology is widely applied, and especially the high-resolution mass spectrometry has received great attention in recent years. However, the tea polyphenol content in the tea is up to 20-40%, and the caffeine content is also 2-5%, so that the accurate quantification of the high-resolution mass spectrum on the pesticide residue in the tea is seriously influenced, and the ion source is polluted, and the chromatographic column, the ion source and the detector are greatly damaged.

The existing method for analyzing the pesticide residue in the tea leaves mostly focuses on a single type of pesticide or different treatment modes are used for different types of tea samples so as to overcome the influence of matrix effect, and the method is complex. CN201310056873 discloses a tea solid phase extraction column and a sample pretreatment method for detecting tea pesticide residues, which have more operation steps and higher cost and reagent consumption. CN201410226018 discloses a pretreatment and detection method for determining various nicotine pesticide residues in tea, which comprises removing tea polyphenols by pvpp, then adsorbing by using various mixed materials such as PSA/GCB/C18, and finally drying and redissolving, wherein the operation steps are complicated, the application range is only used for 8 kinds of nicotine pesticides, and the application range is relatively limited.

Therefore, there is a need to develop a pretreatment method for tea leaves with high pesticide residue content, which is simple in operation, short in time consumption, low in cost and high in efficiency and is suitable for six kinds of tea.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a pretreatment method for measuring various pesticide residues in tea, which is simple to operate, can effectively remove interfering matrixes such as polyphenols, caffeine and the like in the tea, basically does not influence the recovery rate of pesticides, greatly improves the pretreatment effect of samples, and is suitable for the pretreatment of six major tea leaves with high pesticide residues.

The invention is realized by the following technical scheme:

a pretreatment method for measuring various pesticide residues in tea comprises the following steps:

(1) crushing a tea sample to obtain tea powder;

(2) adding water into the tea powder obtained in the step (1) for pre-soaking, then adding acetonitrile into a soaking solution for ultrasonic extraction, and filtering to obtain an extracting solution;

(3) adding polyvinylpyrrolidone and sodium chloride into the extracting solution obtained in the step (2), mixing by vortex, and centrifuging to obtain supernatant I;

(4) taking the supernatant I obtained in the step (3), adding a strong cation exchange filler PCX, mixing in a vortex mode, and centrifuging to obtain a precipitate and a supernatant II;

(5) eluting the precipitate obtained in the step (4) with acetonitrile and 5% ammonia acetonitrile in sequence, and collecting the eluent of the 5% ammonia acetonitrile;

(6) taking the supernatant II obtained in the step (4), adding a strong cation exchange filler PCX, mixing in a vortex mode, centrifuging to obtain a supernatant III, and repeating the step for 1-2 times;

(7) and (5) combining the eluent of 5% ammonia water acetonitrile in the step (5) and the supernatant III in the step (6), and filtering through an organic filter membrane to obtain a sample to be detected.

Among them, the strong cation exchange packing PCX, which is commonly used as an adsorption packing, has a good adsorption effect on alkaloids substances and can be obtained commercially.

Because the tea contains a large amount of matrix components such as tea polyphenol (20-40%) and caffeine (2-5%), and the liquid phase-high resolution mass spectrometry is sensitive to matrix interference, before the detection and analysis of instruments, a proper sample pretreatment method is usually adopted to avoid the interference of the matrix on the analysis of target components so as to improve the detection accuracy. Based on the above, through research, two materials are preferably used as the adsorption filler, wherein PVP mainly adsorbs polyphenols, amino acids and pigment substances, and PCX mainly adsorbs alkaloids substances. The invention utilizes the dispersed solid phase extraction technology, firstly PVP is added to directly remove tea polyphenol, amino acids, pigment substances and the like, the adsorption clearance rate of the tea polyphenol substances can reach more than 94 percent, then a small amount of PCX is used for extracting alkaline pesticides which can be almost completely adsorbed, the adsorption precipitation is carried out, then an elution solvent is used for desorbing the alkaline pesticides for later use, the adsorption clearance rate of the adsorption precipitation to the caffeine is less than 10 percent, then a large amount of PCX is used for removing the caffeine substances from the supernatant after adsorption, and the adsorption clearance rate of the adsorption clearance rate to the caffeine substances can reach more than 85 percent. By the method, the tea polyphenol, caffeine and other substrates in the tea can be basically removed, the residual pesticide is basically reserved, the pretreatment effect of the sample is greatly improved, and the accuracy of subsequent detection is guaranteed.

Preferably, in the step (2), the mass ratio of the tea powder to the water is 1: 1-5, and the pre-soaking time is 10-60 min.

Preferably, in the step (2), the ratio of the volume of the acetonitrile to the mass of the tea powder is in the range of 5-20 ml: 1g, and the ultrasonic extraction time is 10-30 min.

The present invention compares the influence of several adsorbing materials on the adsorption effect of tea polyphenol through experiments, and the specific method is as follows: adding 1.0g of tea powder into 2mL of water for pre-soaking for 30min, adding 8mL of acetonitrile into the soaking solution, performing ultrasonic extraction for 15min, and filtering to obtain an extracting solution; adding each adsorbent filler shown in Table 1 and 2g sodium chloride into the extractive solution, mixing by vortex for 1min, centrifuging at 5000rpm for 3min to obtain supernatant I, and testing the adsorption clearance of tea polyphenols, with the results shown in Table 1:

table 1:

PVPP/1g	PSA/1g	PAX/1g	PCX/1g	PVP/1g
					94％	69％	62％	8％	97％

from the results in table 1, it can be seen that the adsorption effect of PVP on tea polyphenol is the best at the same addition amount, and therefore, polyvinylpyrrolidone (PVP) was selected as the adsorbent of the present invention.

The invention further optimizes the addition of polyvinylpyrrolidone (PVP), and the specific results are shown in Table 2:

table 2:

PVP/1g	PVP/0.75g	PVP/0.5g	PVP/0.25g
				97％	95％	94％	83％

from the results in table 2, it can be seen that when the mass ratio of polyvinylpyrrolidone (PVP) to tea powder is 0.25 to 1: 1, the adsorption effect on tea polyphenol can reach more than 80%, when polyvinylpyrrolidone (PVP) is added to a certain amount, the adsorption is saturated, and preferably, the mass ratio of the polyvinylpyrrolidone (PVP) to the tea powder is 0.5-1: 1.

preferably, in the step (3), the mass ratio of the sodium chloride to the tea powder is 1-3: 1.

the invention selects PCX, and the aim is to adsorb alkaloid substances such as caffeine and the like and reduce matrix interference.

The invention compares the influence of several exchange materials (COOH, SCX and PCX) on the adsorption effect of caffeine and theophylline through experiments, and the specific operation method comprises the following steps: adding 1.0g of tea powder into 2mL of water for pre-soaking for 30min, adding 8mL of acetonitrile into the soaking solution, performing ultrasonic extraction for 15min, and filtering to obtain an extracting solution; adding PVP and 2g sodium chloride into the extract, carrying out vortex mixing for 1min, carrying out centrifugation at 5000rpm for 3min to obtain a supernatant I, adding various exchange fillers shown in the table 3 into 2ml of the supernatant I, carrying out vortex mixing for 30s, carrying out centrifugation at 5000rpm for 3min to obtain a precipitate and a supernatant II, testing the adsorption rate of caffeine and theophylline, and obtaining the results shown in the table 3:

table 3:

	PCX/100mg	SCX/100mg	COOH/100mg
				caffeine	65％	29％	11％
Theophylline	65％	42％	20％

As can be seen from the results in Table 3, the adsorption effect of PCX on tea alkaloid is optimal, therefore, PCX is screened as the tea alkaloid adsorbing material of the invention.

The invention further optimizes the addition of PCX, 2ml of supernatant II (containing 0.2g of tea powder) is added with the PCX amount filler shown in the table 4, vortex mixing is carried out for 30s, and the adsorption rate of caffeine and theophylline is tested, and the results are shown in the table 4:

TABLE 4

As can be seen from the results of table 4, when the mass ratio of PCX to tea powder was 75 mg: 0.2g, and when the step is repeated for 1 time, the adsorption of the tea alkaloid is complete, preferably, in the step (6), the mass ratio of the strong cation exchange filler PCX to the tea powder is 250-500 mg: 1g, preferably 375-500 mg: 1g of the total weight of the composition.

Besides adsorbing tea alkaloid, PCX also adsorbs other alkaline substances, such as alkaline pesticides and the like, so that pesticide loss is caused, and as can be seen from Table 4, when a small amount of PCX is added, the adsorption on tea caffeine and theophylline is small (less than 10%), and the alkaline pesticides are almost completely adsorbed, so that, preferably, in the step (4), the mass ratio of the strong cation exchange filler PCX to the tea powder is 50-100 mg: 1g of the total weight of the composition.

Preferably, in the steps (3), (4) and (6), the vortex mixing time is 30 s-2 min, the centrifugal rotation speed is 3000-6000 rpm, and the centrifugal time is 1-5 min.

The invention also provides a detection method for measuring various pesticide residues in tea, which comprises the following steps:

a. pretreating the tea leaves by using the pretreatment method to obtain a sample to be detected;

b. and detecting the sample to be detected by adopting a liquid chromatogram-mass spectrometer.

Preferably, in step b, the detection conditions are:

liquid phase conditions: a chromatographic column: a C18 column; column temperature: 40 ℃; flow rate: 0.4 mL/min; sample introduction amount: 10 mu L of the solution; mobile phase: the phase A is methanol; phase B is water and contains 0.1% of formic acid and 5mmoL/L of ammonium formate; the gradient elution conditions were: and (3) 0-4 min, wherein the volume concentration of the phase A is as follows: 2% -20%; 4-5.5 min, volume concentration of phase A: 20 to 40 percent; 5.5-10.5min, 40% -100% of phase A volume concentration; 10.5-12.9min, 100%, volume concentration of phase A;

mass spectrum conditions: an ion source: an electrospray ion source; scanning mode: a positive ion mode; the spraying voltage is 3.8 Kv; the capillary temperature was 325 ℃; the temperature of the heater is 350 ℃; the sheath gas is 30arb; auxiliary gas 10 arb; the scanning mode is as follows: FS/DIA mode; the collection range is as follows: m/z 150-750; the resolution ratio adopts MS Full Scan 70000 FWHM; quantification: m/z 175,225,275,325,375,425,475,525,575,625,675,725.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention utilizes the dispersive solid phase extraction technology, can lead the adsorption clearance rate of tea polyphenols to reach more than 94 percent, and the adsorption clearance rate of theine and theophylline substances to reach more than 85 percent, greatly improves the pretreatment effect of samples, and has the advantages of simple operation, short time consumption, less solvent consumption, low cost and the like.

(2) The pretreatment method provided by the invention is combined with liquid chromatography-mass spectrometry to detect the pesticide in the tea, so that the sensitivity and accuracy of detection are greatly improved, and the method is suitable for detecting the multiple pesticide residues in the six teas.

Detailed Description

The present invention is further illustrated by the following specific embodiments, which are not intended to limit the scope of the invention.

The polyvinylpyrrolidone PVP and the strong cation exchange filler PCX adopted in the embodiment of the invention are both from commercial sources.

Example 1:

(1) crushing a tea sample to obtain tea powder;

(2) adding 1.0g of the tea powder obtained in the step (1) into 2mL of water for pre-soaking for 30min, then adding 8mL of acetonitrile into the soaking solution for ultrasonic extraction for 15min, and filtering to obtain an extracting solution;

(3) adding 0.5g of polyvinylpyrrolidone (PVP) and 2g of sodium chloride into the extracting solution obtained in the step (2), mixing for 1min in a vortex mode, and centrifuging for 3min at 5000rpm to obtain supernatant I;

(4) taking 2ml of the supernatant I obtained in the step (3), adding 20mg of PCX, mixing by vortex for 30s, and centrifuging at 5000rpm for 3min to obtain a precipitate and a supernatant II;

(5) eluting the precipitate obtained in the step (4) with 2.0ml of acetonitrile and 2.0ml of 5% ammonia acetonitrile in sequence, and collecting the eluent of the 5% ammonia acetonitrile;

(6) taking the supernatant II obtained in the step (4), adding 75mg of PCX, carrying out vortex mixing for 30s, centrifuging at 5000rpm for 3min to obtain a supernatant III, and repeating the step 1 time;

(7) and (4) combining the eluent of 5% ammonia water acetonitrile in the step (5) and the supernatant III in the step (6), and filtering through a 0.22 mu m organic filter membrane to obtain a sample to be detected.

Example 2:

(1) crushing a tea sample to obtain tea powder;

(2) adding 1.0g of the tea powder obtained in the step (1) into 3mL of water for pre-soaking for 40min, then adding 7mL of acetonitrile into the soaking solution, carrying out ultrasonic extraction for 20min, and filtering to obtain an extracting solution;

(3) adding 0.8g of polyvinylpyrrolidone (PVP) and 1.5g of sodium chloride into the extracting solution obtained in the step (2), mixing for 40s in a vortex mode, and centrifuging for 5min at 5000rpm to obtain supernatant I;

(4) taking 2ml of the supernatant I obtained in the step (3), adding 10mg of PCX, mixing by vortex for 20s, and centrifuging at 5000rpm for 5min to obtain a precipitate and a supernatant II;

(6) taking the supernatant II obtained in the step (4), adding 100mg of PCX, carrying out vortex mixing for 20s, centrifuging at 5000rpm for 5min to obtain a supernatant III, and repeating the step 1 time;

Example 3:

(1) crushing a tea sample to obtain tea powder;

(2) adding 1.0g of the tea powder obtained in the step (1) into 5mL of water for pre-soaking for 20min, then adding 5mL of acetonitrile into the soaking solution, carrying out ultrasonic extraction for 10min, and filtering to obtain an extracting solution;

(3) adding 1.0g of polyvinylpyrrolidone (PVP) and 1.0g of sodium chloride into the extracting solution obtained in the step (2), mixing for 1min in a vortex mode, and centrifuging for 3min at 5000rpm to obtain supernatant I;

(4) taking 2ml of the supernatant I obtained in the step (3), adding 15mg of PCX, mixing by vortex for 15s, and centrifuging at 5000rpm for 3min to obtain a precipitate and a supernatant II;

(6) taking the supernatant II obtained in the step (4), adding 90mg of PCX, carrying out vortex mixing for 40s, centrifuging at 5000rpm for 3min to obtain a supernatant III, and repeating the step 1 time;

Example 4:

a detection method for determining various pesticide residues in tea leaves is characterized in that a sample to be detected obtained by the pretreatment method in the embodiment 1 is detected by a liquid chromatography-mass spectrometer, wherein the detection conditions are as follows:

The invention examines parameters such as linearity, detection Limit (LOD), quantification Limit (LOQ), recovery rate and the like of 167 pesticides, and the results are shown in Table 5:

as can be seen from Table 5, 167 species of agricultural cropsThe drug has good linear relation in the range of 0.01-50 mu g/L, R²More than 0.9920, detection limit and quantification limit are respectively in the range of 0.2-10 mug/kg and 1.0-25 mug/kg, and recovery rate is 70-120%, which shows that the method has good reproducibility.

TABLE 5 methodological validation

Example 5:

in order to further verify the accuracy of the method, the pesticide residue evaluation sample FV-T02 in the tea leaves of the European Union pesticide residue comparison laboratory organization is detected by the invention, the pretreatment method and the detection method are the same as those in the example 1, and the results are shown in Table 6:

TABLE 6 detection values and relative mean deviations of Standard pesticide residue tea samples FV-T02 in European Union pesticide residue reference laboratory

As can be seen from the results in Table 6, the detection value of the European Union pesticide residue reference laboratory standard pesticide residue tea sample is in the range of 0.022-0.707 mg/Kg, and the relative average deviation is in the range of 4.4-16.7% by adopting the method, which indicates that the method is accurate and reliable.

Claims

1. A pretreatment method for measuring various pesticide residues in tea is characterized by comprising the following steps:

(1) crushing a tea sample to obtain tea powder;

(3) adding polyvinylpyrrolidone and sodium chloride into the extracting solution obtained in the step (2), mixing by vortex, and centrifuging to obtain supernatant I; the mass ratio of the polyvinylpyrrolidone to the tea powder is 0.5-1: 1; the mass ratio of the sodium chloride to the tea powder is 1-3: 1;

(4) taking the supernatant I obtained in the step (3), adding a strong cation exchange filler PCX, mixing in a vortex mode, and centrifuging to obtain a precipitate and a supernatant II; the mass ratio of the strong cation exchange filler PCX to the tea powder is 50-100 mg: 1g of a compound;

(6) taking the supernatant II obtained in the step (4), adding a strong cation exchange filler PCX, mixing in a vortex mode, centrifuging to obtain a supernatant III, and repeating the step for 1-2 times; the mass ratio of the strong cation exchange filler PCX to the tea powder is 250-500 mg: 1g of a compound;

2. The pretreatment method for measuring various pesticide residues in tea leaves according to claim 1, wherein in the step (2), the mass ratio of the tea leaf powder to water is 1: 1-5, and the pre-soaking time is 10-60 min.

3. The pretreatment method for measuring various pesticide residues in tea leaves according to claim 1, wherein in the step (2), the ratio of the volume of the acetonitrile to the mass of the tea leaves powder is in the range of 5 to 20 ml: 1g, and the ultrasonic extraction time is 10-30 min.

4. The pretreatment method for measuring various pesticide residues in tea leaves according to claim 1, wherein in the step (6), the mass ratio of the strong cation exchange filler PCX to the tea leaf powder is 375-500 mg: 1g of the total weight of the composition.

5. The pretreatment method for measuring various pesticide residues in tea leaves according to claim 1, wherein in the steps (3), (4) and (6), the vortex mixing time is 30 s-2 min, the centrifugal rotation speed is 3000-6000 rpm, and the centrifugal time is 1-5 min.

6. A detection method for measuring various pesticide residues in tea leaves is characterized by comprising the following steps:

a. pretreating tea leaves by using the pretreatment method of any one of claims 1 to 5 to obtain a sample to be tested;

b. and detecting the sample to be detected by adopting a liquid chromatogram-high resolution mass spectrometer.

7. The detection method for detecting various pesticide residues in tea leaves as claimed in claim 6, wherein in the step b, the detection conditions are as follows:

liquid phase conditions: a chromatographic column: a C18 column; column temperature: 40 ℃; flow rate: 0.4 mL/min; sample introduction amount: 10 mu L of the solution; mobile phase: the phase A is methanol; phase B is water and contains 0.1% of formic acid and 5mmoL/L of ammonium formate; the gradient elution conditions were: and (3) 0-4 min, wherein the volume concentration of the phase A is as follows: 2% → 20%; 4-5.5 min, volume concentration of phase A: 20% → 40%; 5.5-10.5min, volume concentration of phase A: 40% → 100%; 10.5-12.9min, volume concentration of phase A: 100 percent;

mass spectrum conditions: an ion source: an electrospray ion source; scanning mode: a positive ion mode; the spraying voltage is 3.8 Kv; the capillary temperature was 325 ℃; the temperature of the heater is 350 ℃; 30arb of sheath gas, 10arb of auxiliary gas; the scanning mode is as follows: FS/DIA mode; the collection range is as follows: m/z 150-750; the resolution ratio adopts MS Full Scan 70000 FWHM; quantification: m/z 175,225,275,325,375,425,475,525,575,625,675, 725.