CN115047112A - Detection method for determining mycotoxin in cereal grains by QuEChERS pretreatment combined with LC-MS/MS - Google Patents

Abstract

The invention belongs to the field of food safety detection, and discloses a detection method for determining mycotoxin in cereal grains by combining pretreatment of a QuEChERS sample with LC-MS/MS (liquid chromatography-mass spectrometry), in particular to a detection method for detecting the mycotoxin in the cereal grains by using a liquid chromatography-mass spectrometer after the cereal grains are extracted and purified, which has strong anti-interference capability on a complex matrix, high sensitivity, good accuracy and repeatability and more convenient pretreatment.

Description

Detection method for determining mycotoxin in cereal grains by QuEChERS pretreatment combined with LC-MS/MS

Technical Field

The invention belongs to the field of food safety detection, and relates to a detection method for determining mycotoxin in cereal grains by QuEChERS pretreatment in combination with LC-MS/MS.

Background

Mycotoxin is a toxic secondary metabolite produced by filamentous fungi under appropriate conditions, widely exists in food, pollutes food in a large amount, has strong carcinogenicity, and can cause various food poisoning, acute and chronic diseases. The mycotoxin pollutes the food inevitably, fumonisins and T-2 toxins are easy to generate in warm and humid south, aflatoxins are easy to generate in warm and dry north peanuts and corns, the problem of mycotoxin pollution is very important to China, and the limit indexes of the mycotoxins in various foods are limited in the national food safety standard GB 2761 + 2017. In order to ensure the food quality safety of China and avoid unnecessary economic loss, it is important to monitor and investigate the mycotoxin pollution type, level and risk quickly and timely. Due to the variety and the existence of synergistic pollution of mycotoxins, a simple, rapid, sensitive and accurate method for simultaneously determining various mycotoxins in grains is urgently needed to ensure that the pollution condition of the mycotoxins in the grains is comprehensively and rapidly known. The prior pretreatment detection method of mycotoxin mainly comprises an enzyme-linked immunosorbent assay, an immunoaffinity column method and the like. The enzyme-linked immunosorbent assay based on the antigen-antibody interaction has high selectivity, but cannot accurately quantify, and simultaneously, because the pretreatment is simple, effective purification is not realized, the enzyme-linked immunosorbent assay is easily interfered by a matrix, and false positive is generated. The immunoaffinity column pretreatment purification method has good purification effect due to specific adsorption, but on one hand, the operation is complex, on the other hand, the cost is high, and the immunoaffinity column pretreatment purification method is not suitable for large-scale detection. The method uses QuEChERS as a pretreatment purification method, and has the advantages of simple operation, good repeatability and high recovery rate. By adopting the LC-MS/MS method, the molecular structure information and the corresponding retention time, accuracy, selectivity and sensitivity of the target compound can be detected, the analysis of the low-concentration mycotoxin can be quickly completed within 15min, the simultaneous detection of multiple components can be met, and the method is well suitable for the analysis and detection of multiple mycotoxins in cereal and grain.

Disclosure of Invention

(1) Problems to be solved

The invention aims to provide a detection method for determining mycotoxin in cereals by combining QuEChERS pretreatment with LC-MS/MS.

The 12 mycotoxins of the present invention comprise: deoxynivalenol (DON), 15-acetylated deoxynivalenol (15-ADON), 3-acetylated deoxynivalenol (3-AcDON), aflatoxin G2 (AFG2), HT-2 toxin (HT-2), aflatoxin B2(AFB2), aflatoxin G1(AFG1), AFG1 aflatoxin B1(AFB1), T2-toxin (T2), Zearalenone (ZEN), varicolor (ST), ochratoxin A (OTA)

The invention is realized by adopting the following technical scheme

1) Weighing 5g of homogenized sample, putting the sample into a 50mL centrifuge tube, adding 10mL of water, and shaking and uniformly mixing for 20 min;

2) then 10mL of 2% acetonitrile formate is added, and vortex extraction is carried out for 5 min;

3) adding 4g MgSO4 and 1g NaCl, and quickly shaking for 1 min;

4) centrifuging 50ml of centrifuge tube in a high-speed centrifuge at a rotation speed of more than or equal to 3000r/min for 5 min;

5) transferring 1ml of centrifuged supernatant into a small bottle filled with QuEChERS purified salt bag (150mg MgSO4,50mg PSA 50mg C18), shaking for 30s, and centrifuging for 5min in a high-speed centrifuge at the rotating speed of more than or equal to 3000 r/min;

6) and (5) transferring 500ul of the purified solution in the step (5), diluting the purified solution by 4 times by using pure water, and feeding the diluted solution to a liquid chromatography mass spectrometer for analysis after passing through a filter membrane with the diameter of 0.2 mu m.

Analyzing conditions of the liquid chromatographic mass spectrometer:

a chromatographic column of Accucore aQ,100 multiplied by 2.1mm,2.6 mu m; the mobile phase consists of a mobile phase A and a mobile phase B, wherein the ratio of the mobile phase A: 0.1% formic acid water mobile phase B0.1% formic acid acetonitrile; column flow rate: 0.3 mL/min: column temperature: 35 ℃; sample introduction amount: the 10 μ L gradient elution conditions were as follows:

time min	Mobile phase A%	Mobile phase B%	Flow rate ml/min
				0.0	95	5	0.3
0.7	95	5	0.3
				2.0	50	50	0.3
4.0	5	95	0.3
				6.0	5	95	0.3
6.5	95	5	0.3
				8.0	95	5	0.3

Mass spectrum conditions:

an ion source: an electrospray ion source;

mass spectrum scanning mode: multiple reaction monitoring mode (MRM);

taper hole voltage: 3.0 kV;

heating gas temperature: 500 ℃;

ion source temperature: 150 ℃;

removing the solvent gas: 800L/H.

The reaction monitoring ion pair information was selected as follows:

the invention has the beneficial effects that:

the method adopts QuEChERS sample pretreatment and LC-MS/MS combined to determine 12 kinds of mycotoxins in the cereal grains, has the advantages of simple pretreatment and simple and convenient operation, adopts LC-MS/MS detection and dual verification of qualitative ions and quantitative ions, is accurate in qualitative and quantitative determination and is not easy to generate false positive, and can be used for detecting the mycotoxins in the cereal grains such as corn, wheat and the like.

Drawings

The following detailed description of embodiments of the invention is provided in conjunction with the accompanying drawings:

FIG. 1 is a diagram of the selective ion flow of deoxynivalenol;

FIG. 2 is a diagram showing the selective ion flow of 15-acetylated deoxynivalenol;

FIG. 3 is a diagram showing the selective ion flow of 3-acetylated deoxynivalenol;

FIG. 4 is a selected ion flow diagram of aflatoxin G2;

FIG. 5 is a selected ion flow graph of HT-2 toxin;

FIG. 6 is a selected ion flow diagram of aflatoxin B2;

FIG. 7 is a selected ion flow diagram of aflatoxin G1;

FIG. 8 is a selected ion flow diagram of aflatoxin B1;

FIG. 9 is a selected ion flow diagram of the T2-toxin;

FIG. 10 is a selected ion flow diagram of zearalenone;

FIG. 11 is a selected ion flow diagram of a variegated aspergillin;

FIG. 12 is a selected ion flowsheet of ochratoxin A;

FIG. 13 is a chromatogram of total ion flux of 12 mycotoxin standard solutions in ESI + ionization mode;

FIG. 14 is a total ion flow chromatogram for the detection of 12 mycotoxins in a commercial maize sample under ESI + ionization mode.

Detailed Description

Example 1

A detection method for determining mycotoxin in cereal grains by QuEChERS pretreatment and LC-MS/MS combination comprises the following steps,

1) 5g of the homogenized sample is weighed into a 50mL centrifuge tube, 10mL of water is added, and the mixture is shaken and mixed for 20 min.

2) Adding 10mL of 2% acetonitrile formate, and performing vortex extraction for 5min

3) Adding 4g MgSO4 and 1g NaCl, and quickly shaking for 1min

4) Centrifuging 50ml centrifuge tube in high speed centrifuge at rotation speed of 3000r/min or more for 5min

5) Transferring 1ml of centrifuged supernatant into a small bottle containing QuEChERS purified salt bag (150mg MgSO4,50mg PSA,50mg C18), shaking for 30s, and centrifuging at a rotation speed of not less than 3000r/min in a high-speed centrifuge for 5min

6) And (5) transferring 500ul of the purified solution in the step (5), diluting the purified solution by 4 times by using pure water, and feeding the diluted solution to a liquid chromatography mass spectrometer for analysis after passing through a filter membrane with the diameter of 0.2 mu m.

Analyzing conditions of the liquid chromatographic mass spectrometer:

a chromatographic column of Accucore aQ,100 multiplied by 2.1mm,2.6 mu m; the mobile phase consists of a mobile phase A and a mobile phase B, wherein the ratio of the mobile phase A: 0.1% formic acid water mobile phase B0.1% formic acid acetonitrile; column flow rate: column temperature 0.3 mL/min: 35 ℃; sample introduction amount: the 10 μ L gradient elution conditions were as follows:

time min	Mobile phase A%	Mobile phase B%	Flow rate ml/min
				0.0	95	5	0.3
0.7	95	5	0.3
				2.0	50	50	0.3
4.0	5	95	0.3
				6.0	5	95	0.3
6.5	95	5	0.3
				8.0	95	5	0.3

Mass spectrum conditions:

an ion source: an electrospray ion source;

mass spectrum scanning mode: multiple reaction monitoring mode (MRM);

taper hole voltage: 3.0 kV;

heating gas temperature: 500 ℃;

ion source temperature: 150 ℃;

removing the solvent gas: 800L/H.

The reaction-monitoring ion pair information is selected in the following table:

QuEChERS pretreatment optimization:

the sample pretreatment compares two purification modes (adding GCB filler and not adding GCB filler), and a labeling experiment is respectively carried out, so that the purification mode containing the GCB filler has strong adsorbability on aflatoxin B1, aflatoxin G2, variegated aspergillocin, zearalenone, aflatoxin G1, aflatoxin B2 and ochratoxin A, and is not suitable for multi-component purification. And finally, the clean detection purification effect and the recovery rate of the filler without GCB are better.

Standard curve, linear relationship and detection limit:

and (5) performing linear regression on the mass concentration (X, ug/ml) by using the peak area (Y) corresponding to each concentration point in the standard curve, and determining the detection limit by using the signal-to-noise ratio of 3 times.

The result shows that the correlation coefficient of the method is 0.9911-0.9982, and the mass concentration and the peak area have a good linear relation. The detection limit is between 0.0025 and 0.0506 ug/kg.

Example 2 recovery experiment

(1) Addition of standard solution

Taking a negative corn sample, pretreating according to the method of the invention, adding 12 kinds of mycotoxin mixed standard solutions with horizontal concentration, and measuring the pretreated supernatant by using an ultra performance liquid chromatography-tandem mass spectrometer. The results of 6 horizontal runs (n-6) were performed as shown in the following table. The average recovery rate of 12 mycotoxins is 70.2-98.2%, and the relative standard deviation is 5.6-12.6%.

(2) Sample pretreatment

1) Weighing 5g of homogenized sample, putting the sample into a 50mL centrifuge tube, adding 10mL of water, and shaking and uniformly mixing for 20 min;

2) then adding 10mL of 2% acetonitrile formate, and performing vortex extraction for 5 min;

3) adding 4g MgSO4 and 1g NaCl, and quickly shaking for 1 min;

4) centrifuging 50ml of centrifuge tube in a high-speed centrifuge at a rotation speed of more than or equal to 3000r/min for 5 min;

5) transferring 1ml of centrifuged supernatant into a small bottle filled with QuEChERS purified salt bag (150mg MgSO4,50mg PSA,50mg C18), shaking for 30s, and centrifuging for 5min in a high-speed centrifuge at the rotating speed of more than or equal to 3000 r/min;

6) and (5) transferring 500ul of the purified solution in the step (5), diluting the purified solution by 4 times by using pure water, and feeding the diluted solution to a liquid chromatography mass spectrometer for analysis after passing through a filter membrane with the diameter of 0.2 mu m.

(3) Analytical conditions for liquid chromatography mass spectrometer reference example 1

The results show that: the average recovery rate of 12 mycotoxins is 70.2-98.2%, and the relative standard deviation is 5.6-12.6%.

Example 3

For randomly purchased corns in the market, the invention is used for carrying out experiments

(1) Sample pretreatment

1) Weighing 5g of homogenized sample, putting the sample into a 50mL centrifuge tube, adding 10mL of water, and shaking and uniformly mixing for 20 min;

2) then adding 10mL of 2% acetonitrile formate, and performing vortex extraction for 5 min;

3) adding 4g MgSO4 and 1g NaCl, and quickly shaking for 1 min;

4) centrifuging 50ml of centrifuge tube in a high-speed centrifuge at a rotation speed of more than or equal to 3000r/min for 5 min;

5) transferring 1ml of centrifuged supernatant into a small bottle filled with QuEChERS purified salt bag (150mg MgSO4,50mg PSA,50mg C18), shaking for 30s, and centrifuging for 5min in a high-speed centrifuge at the rotating speed of more than or equal to 3000 r/min;

6) and (5) transferring 500ul of the purified solution in the step (5), diluting the purified solution by 4 times by using pure water, and feeding the diluted solution to a liquid chromatography mass spectrometer for analysis after passing through a filter membrane with the diameter of 0.2 mu m.

(2) The liquid chromatography mass spectrometer analysis conditions refer to example 1.

(3) The results show that: no 12 mycotoxins were detected in maize.

Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims (1)

1. A detection method for determining mycotoxin in cereal grains by QuEChERS pretreatment and LC-MS/MS combined is characterized by comprising the following steps: the method comprises the following steps:

1) weighing 5g of homogenized sample, putting the sample into a 50mL centrifuge tube, adding 10mL of water, and shaking and uniformly mixing for 20 min;

2) then 10mL of 2% acetonitrile formate is added, and vortex extraction is carried out for 5 min;

3) adding 4g MgSO4 and 1g NaCl, and quickly shaking for 1 min;

4) centrifuging 50ml of centrifuge tube in a high-speed centrifuge at a rotation speed of more than or equal to 3000r/min for 5 min;

5) transferring 1ml of centrifuged supernatant into a small bottle filled with QuEChERS purified salt bag (150mg MgSO4,50mg PSA 50mg C18), shaking for 30s, and centrifuging for 5min in a high-speed centrifuge at the rotating speed of more than or equal to 3000 r/min;

6) in the step 5, 500ul of the purified solution is diluted by 4 times by pure water, and the purified solution is subjected to liquid chromatography mass spectrometer analysis after passing through a filter membrane of 0.2 mu m;

analyzing conditions of the liquid chromatographic mass spectrometer:

a chromatographic column of Accucore aQ,100 multiplied by 2.1mm,2.6 mu m; the mobile phase consists of a mobile phase A and a mobile phase B, wherein the ratio of the mobile phase A: 0.1% formic acid water mobile phase B is 0.1% formic acid acetonitrile; column flow rate: 0.3 mL/min: column temperature: 35 ℃; sample introduction amount: the 10 μ L gradient elution conditions were as follows:

time min Mobile phase A% Mobile phase B% Flow rate ml/min 0.0 95 5 0.3 0.7 95 5 0.3 2.0 50 50 0.3 4.0 5 95 0.3 6.0 5 95 0.3 6.5 95 5 0.3 8.0 95 5 0.3

Mass spectrum conditions:

an ion source: an electrospray ion source;

mass spectrum scanning mode: multiple reaction monitoring mode (MRM);

taper hole voltage: 3.0 kV;

heating gas temperature: 500 ℃;

ion source temperature: 150 ℃;

removing the solvent gas: 800L/H;

the reaction monitoring ion pair information was selected as follows:

Images