Search

CN-121540835-B - Method for measuring 14 mycotoxins in peanuts by high performance liquid chromatography tandem mass spectrometry

CN121540835BCN 121540835 BCN121540835 BCN 121540835BCN-121540835-B

Abstract

The invention discloses a method for measuring 14 mycotoxins in peanuts based on a high performance liquid chromatography-tandem mass spectrometry, and belongs to the technical field of food detection. The method comprises the steps of solution preparation, preparation of a sample solution, and high performance liquid chromatography-tandem mass spectrometry detection, wherein acetonitrile-water-acetic acid mixed solution is used as an extracting solution, and a peanut sample is subjected to grinding and crushing, low-temperature ultrasonic extraction, centrifugation, concentration, re-dissolution and filtration by a filter membrane, and then is separated by a GL SCIENCES InertSustain AQ C chromatographic column, and is detected by a triple quadrupole mass spectrometer in a multistage reaction monitoring mode. The method realizes synchronous determination of 14 mycotoxins, has the advantages of simple pretreatment, high detection efficiency, good sensitivity and high accuracy, and can be used for accurately detecting mycotoxins in peanuts.

Inventors

  • YANG TINGTING
  • SUN TIAN
  • LI HUI
  • LIU ZEYU

Assignees

  • 南京维百瑞检测技术有限公司

Dates

Publication Date
20260508
Application Date
20260119

Claims (7)

  1. 1. A method for measuring 14 mycotoxins in peanuts by using high performance liquid chromatography tandem mass spectrometry, which is characterized by comprising the following steps of: Step S1, preparing a solution, namely respectively preparing a mobile phase, an extracting solution, a standard stock solution, a standard working solution and an internal standard solution, wherein the mobile phase consists of an organic phase and an inorganic phase, the organic phase is chromatographic acetonitrile, the inorganic phase is formic acid aqueous solution with the volume percentage concentration of 0.1%, the extracting solution is a mixed solution formed by mixing acetonitrile, water and acetic acid according to the volume ratio of 35:64.5:0.5, and the internal standard solution is a stable isotope internal standard mixed solution with the volume ratio of 100ng/mL and is used for matrix effect correction; S2, preparing a sample solution, namely selecting a peanut sample, removing impurities, crushing the peanut sample by a high-speed crusher, sieving the peanut sample by a 20-mesh sieve, placing the crushed peanut sample in a sealed bag for normal temperature preservation to ensure uniformity of the sample, weighing the crushed peanut sample, placing the crushed peanut sample in a 50mL centrifuge tube, adding 20mL of extracting solution, performing ultrasonic extraction for 30min at normal temperature, shaking the crushed peanut sample once every 5min to ensure that the peanut sample is fully contacted with the extracting solution to promote toxin dissolution, adding 100mg of N-propylethylenediamine, 50mg of C18 adsorbent and 30mg of graphitized carbon black into the centrifuge tube after the extraction is completed, performing vortex shaking for 2min, then placing the peanut sample into the centrifuge tube, performing centrifugal 5min at a rotating speed of 5000rpm, taking supernatant, transferring the supernatant into a nitrogen blowing tube, performing nitrogen blowing-drying or freeze-drying treatment on the supernatant, adding 1mL of the extracting solution and 10 mu L of mixed internal standard solution, performing vortex shaking for 1min re-dissolution, and uniformly mixing the obtained mixture by stirring, and then passing through a polytetrafluoroethylene membrane of 0.22 mu m to collect filtrate to serve as the sample solution to be detected; S3, instrument detection, namely detecting by adopting an ultra-high performance liquid chromatography and triple quadrupole mass spectrometer, and realizing effective separation and high-sensitivity detection of 14 toxins through cooperative optimization of chromatography-mass spectrometry parameters, wherein the chromatographic conditions of the detection are that a chromatographic column is GL SCIENCES InertSustain AQ C, the column temperature is 30 ℃, the sample injection volume is 2 mu L, the flow rate is 0.4mL/min, a gradient elution program is designed aiming at the retention characteristics of 14 toxins, wherein an organic phase is kept for 0-2min, so that DON and PAT with stronger polarity are eluted first, an organic phase is linearly lifted from 20% to 60% for realizing separation of the toxins with medium polarity for 2-8min, the organic phase is lifted to 90%, the elution polarity is weaker AFB1, AFB2 and T-2, and the organic phase is kept for 12-15min, so as to ensure complete elution of the toxins with strong hydrophobicity, the mass spectrometry conditions are that the ion source is an electrospray ion source, the detection is switched between 0-2min and 20%, the detection of AFB1, FB2 and FB2, and FB2 are respectively, and the air pressure is monitored in a mode of 20 psi-550 psi and 50 psi-20 psi, and the air pressure is optimized for detecting the specific air pressure of the toxins; And S4, analyzing results, namely, according to the detection result of the standard working solution, drawing a matrix matching standard curve by taking the ratio of the target toxin peak area to the internal standard peak area as an ordinate and the target toxin concentration as an abscissa, quantitatively analyzing 14 mycotoxins in the sample solution by adopting an internal standard method, and qualitatively confirming the ratio of the retention time to the characteristic ion pair to ensure the accuracy of the detection result.
  2. 2. The method for measuring 14 mycotoxins in peanuts by using the high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein the preparation method of the formic acid aqueous solution in the step S1 comprises the following steps of sequentially adding 900mL of ultrapure water and 1mL of formic acid into a 1L volumetric flask, and then fixing the volume to a scale by using the ultrapure water.
  3. 3. The method for measuring 14 mycotoxins in peanuts by using the high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein the preparation method of the extracting solution in the step S1 comprises the steps of sequentially adding 400mL of ultrapure water, 350mL of acetonitrile and 5mL of acetic acid into a 1L volumetric flask, and then fixing the volume to a scale by using the ultrapure water.
  4. 4. The method for determining 14 mycotoxins in peanuts by using the high performance liquid chromatography tandem mass spectrometry according to claim 1 is characterized in that the preparation method of the standard stock solution in the step S1 comprises the steps of respectively preparing 1mg/mL single standard stock solutions of AFB1, AFB2, AFG1, AFG2 and ZEN, CIT, OTA by using chromatographic methanol as a solvent, respectively preparing 1mg/mL single standard stock solutions of FB1, FB2 and FB3 by using an acetonitrile aqueous solution with a volume percentage concentration of 50% as a solvent, and respectively preparing 1mg/mL single standard stock solutions of T-2 and ST, DON, PAT by using chromatographic acetonitrile as a solvent.
  5. 5. The method for determining 14 mycotoxins in peanuts by using the high performance liquid chromatography tandem mass spectrometry according to claim 1, wherein the preparation method of the standard working solution in the step S1 comprises the steps of mixing the single standard stock solutions, taking the extracting solution as a diluent, adding the stable isotope internal standard mixed solution to enable the final concentration of each internal standard to be 1ng/mL, and gradually diluting to prepare the mixed standard working solution with the concentration of 14 toxins of 0.1, 1, 5, 10, 20, 50, 75 and 100ng/mL for preparation.
  6. 6. The method for determining 14 mycotoxins in peanuts by using the high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein the preparation method of the internal standard solution in the step S1 comprises the steps of weighing 1mg of each AFB1- 13 C 17 、OTA- 13 C 20 、ZEN- 13 C 18 standard substance, dissolving the standard substance to 100mL by using chromatographic methanol to obtain 10 mug/mL of single standard internal standard stock solution, and respectively weighing 1mL of single standard internal standard stock solution, and dissolving the standard substance to 100mL by using chromatographic methanol to obtain mixed internal standard solution with the concentration of 100ng/mL of each AFB1- 13 C 17 、OTA- 13 C 20 、ZEN- 13 C 18 .
  7. 7. The method for determining 14 mycotoxins in peanuts by high performance liquid chromatography tandem mass spectrometry according to claim 1, wherein the power of ultrasonic extraction in step S2 is 300W and the frequency is 40kHz.

Description

Method for measuring 14 mycotoxins in peanuts by high performance liquid chromatography tandem mass spectrometry Technical Field The invention relates to the technical field of food detection, in particular to a method for measuring 14 mycotoxins in peanuts by using a high performance liquid chromatography-tandem mass spectrometry. Background Peanut is used as important oil crop and economic crop in China, is not only the core raw material for producing edible oil, but also the important source of high-quality protein, dietary fiber and various mineral matters in daily diet, and occupies a key position in national diet structure and agricultural economic system. However, the rich nature of oil and protein makes peanuts extremely vulnerable to contamination by fungi such as aspergillus, penicillium, fusarium and the like in the whole industrial chain links of planting, harvesting, storage, processing and the like. The fungi are propagated in a large quantity under the proper temperature and humidity condition, and can be metabolized to generate various mycotoxins with strong toxicity, thereby forming a serious threat to food safety. Mycotoxins are natural toxic secondary metabolites, have various harm such as carcinogenicity, teratogenicity, mutagenicity, hepatorenal toxicity and the like, have stable chemical properties, and are difficult to thoroughly destroy by common processing and cooking means. Among them, aflatoxin B1, ochratoxin a, fumonisins, etc. have been listed as food pollutants with important control by the united nations Food and Agricultural Organization (FAO) and the World Health Organization (WHO), and the residual amount thereof in foods is strictly limited. If the peanuts and peanut products are polluted by mycotoxins, the quality and market circulation of the products can be affected, and long-term potential harm is caused to the health of consumers through accumulation of food chains, so that the establishment of an accurate and comprehensive mycotoxin detection method is a key link for guaranteeing the safety of peanut foods. Currently, methods for detecting mycotoxins in foods mainly comprise thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), high Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS/MS) and the like. The method has the advantages of complex operation and low sensitivity, which are gradually eliminated, the ELISA method has the advantages of rapidness and high flux, but has insufficient specificity and accuracy, is easy to generate false positive results, is only suitable for preliminary screening, and the HPLC method has the characteristics of good separation effect and accurate quantification, has wider application in conventional detection, but has poorer selectivity and limited detection capability for trace toxins in synchronous detection of multi-component mycotoxins. Therefore, the mycotoxin synchronous detection method with comprehensive detection indexes, simple pretreatment and excellent detection performance is developed, and has very important practical significance and application value for perfecting a peanut food safety detection system, preventing food safety risks and guaranteeing consumer health. Disclosure of Invention The invention aims to overcome the defects of the prior art, and provides a detection method based on a high performance liquid chromatography tandem mass spectrometry, which realizes synchronous, rapid and accurate detection of 14 mycotoxins and improves detection efficiency and detection coverage. In order to achieve the aim, the technical scheme adopted by the invention is that the method for measuring 14 mycotoxins in peanuts by using high performance liquid chromatography tandem mass spectrometry comprises the following steps of: Step S1, preparing a solution, namely respectively preparing a mobile phase, an extracting solution, a standard stock solution, a standard working solution and an internal standard solution, wherein the mobile phase consists of an organic phase and an inorganic phase, the organic phase is chromatographic acetonitrile, the inorganic phase is formic acid aqueous solution with the volume percentage concentration of 0.1%, the extracting solution is a mixed solution formed by mixing acetonitrile, water and acetic acid according to the volume ratio of 35:64.5:0.5, and the internal standard solution is a stable isotope internal standard mixed solution with the volume ratio of 100ng/mL and is used for matrix effect correction; S2, preparing a sample solution, namely selecting a peanut sample, removing impurities, crushing the peanut sample by a high-speed crusher, sieving the peanut sample by a 20-mesh sieve, placing the crushed peanut sample in a sealed bag for normal temperature preservation to ensure uniformity of the sample, weighing the crushed peanut sample, placing the crushed peanut sample in a 50mL centrifuge tube, adding 20mL of extracting solution,