The People's Republic of China Entry-Exit Inspection and Quarantine Industry Standard
sn/t 5442-2022
Determination of prothioconazole and its metabolite residues in foods of plant origin for export
Liquid chromatographyMass spectrometryMass spectrometry
determination of prothioconazole and its metabolite residues
in foodstuffs of plant origin for export-lc-ms/ms method
Foreword
This document is in accordance with GB T 11-2020 "Guidelines for Standardization Work Part 1: Structure and Drafting Rules of Standardization Documents".
gb/t 20001.4-2015 "Rules for the Preparation of Standards Part 4: Standards for Test Methods" and SN T 0001-2016 "Basic Provisions for the Preparation of Standards for Physical and Chemical Determination Methods of Exported Food and Cosmetics" were drafted.
Please note that some of the contents of the document may refer to patents. The issuer of this document assumes no responsibility for identifying these patents.
This document is submitted and centralized by the General Administration of Customs of the People's Republic of China.
This document was drafted by: Hefei Customs Technology Center of the People's Republic of China, Yinchuan Customs Technology Center of the People's Republic of China, Hangzhou Customs Technology Center of the People's Republic of China, and Civil Aviation University of China.
The main drafters of this document: Song Wei, Han Fang, Wang Fanghuan, Chen Da, Lou Chengjie, Lu Yaning, Liu Yuxin, Zhou Dianbing, Ding Lei, Xu Manman, Wang Yu, Jia Xueying, Hou Jianbo, Zheng Ping.
Scope
This document specifies the liquid chromatography-mass spectrometry mass spectrometry method for the detection of prothioconazole and its metabolite dethiproconazole residues in foods of plant origin for export.
This document is applicable to the qualitative identification and quantitative determination of prothioconazole and its metabolite desthioconazole residues in wheat, barley, oats, rye, corn, red beans, rapeseed, soybeans, peanut kernels, sunflower seeds, sugar beets, lettuce, tomatoes, cucumbers, potatoes, pears, grapes, strawberries and other plant-derived foods.
Normative references
The content of the following documents constitutes an essential provision of this document by means of normative references in the text. Among them, the reference document with the date is noted, and only the version corresponding to that date applies to this document;For undated references, the most recent version of the document (including all change orders) applies to this document.
GB 2763 National Food Safety Standard Maximum residue limits for pesticides in food.
GB T6682 Analytical Laboratory Water Specifications and Test Methods.
Terms and Definitions
There are no terms and definitions that need to be defined in this document.
Methodological principle
The residues of prothioconazole and its metabolite dethiproconazole in the sample were 01% formic acid acetonitrile solution extraction, matrix dispersion solid phase extraction and purification, liquid chromatography mass spectrometry mass spectrometry determination, external standard method quantification.
Reagents and materials
Unless otherwise stated, the reagents used are analytically pure, and the water is grade 1 water according to GB T 6682.
5.1 Acetonitrile: chromatographically pure.
5.2 Acetic acid: chromatographically pure.
5.3 formic acid: chromatographically pure.
5.4 Ammonium acetate: chromatographically pure.
5.5 n-hexane: chromatographically pure.
5.6. Ascorbic acid.
5.7 sodium chloride.
5.8 Anhydrous magnesium sulfate: burned at 650 for 4 h, stored in a dryer, cooled and set aside.
5.9 1% acetonitrile formate solution: take 10 ml formic acid (5.)3) to a 100 ml volumetric flask and use acetonitrile (51) Set the volume to the scale, mix well, and set aside.
5. 10 1.5% ascorbic acid solution: take 15 g of ascorbic acid (56), dilute with water and set to 1 000 ml.
5.11 20% acetonitrile solution: take 20 ml of acetonitrile (5.)1) Dilute with water and set to 100 ml.
5.12 Prothioconazole: C14H15Cl2N3OS, CAS No. 178928-70-6;Prothioconazole-desthio: C14H15Cl2N3O, Casno 120983-64-4。The purity is greater than or equal to 98%.
5.13. Preparation of standard stock solution: accurately weigh the appropriate amount of prothioconazole and desthioproconazole standards (accurate to 0.).1mg), respectively, dissolved with acetonitrile, prepared to a concentration of 10 mg ml standard stock solution, 0 4 Store in the dark, can be used for 6 months.
5.14 Intermediate mixed standard solutions: Pipette 100 ml of prothioconazole standard stock solution and 100 ml of desthiprothioconazole standard stock solution (5.)13), transfer into a 100 ml brown volumetric flask and set the volume with acetonitrile, the solution concentration is 10 g ml. 0 4 Refrigerate and protect from light, can be used for 3 months.
5.15. Standard working solution: As needed, draw an appropriate amount of standard intermediate solution (514), formulated into 125μg/l、2.Mix standard solutions of 5 g l, 5 g l, 25 g l, 50 g l, prepare before use.
Octadecyl bonded silica gel (c18m
n-Propylethylenediamine (psam~60m
5.18 Membrane: 022 m, organic faculty.
Instruments and equipment
6.1 Liquid Chromatography-Mass Harmonic Mass Spectrometer: Power Distribution Spray Ion Source (ESI).
6.2. Analytical balance: inductance is 001 g and 0000 1 g。
6.3 pulverizers.
6.4. Tissue masher.
6.6 sample sieve: pore size is 2 mm.
6.6 homogenizer: 15000 r min.
6.7. Vortex shaker.
6.8 High-speed refrigerated centrifuge: the rotational speed is not less than 10 000 r min.
Analyze the steps
7.1. Sample preparation and preservation.
7.1.1 Wheat, barley, oats, rye, corn, red beans, rapeseed, soybeans, peanut kernels, sunflower seeds.
Take out 500 g of representative samples, crush them all with a pulverizer, mix them thoroughly, evenly divide them into 2 parts as samples, pack them into clean containers, seal them and mark them, and store them at a temperature below -18 in the dark.
7.1.2 Beets, lettuce, tomatoes, cucumbers, grapes, strawberries, pears, potatoes.
Sampling is performed in accordance with the provisions of GB 2763. For samples with small individuals, all samples are processed after sampling;For essentially homogeneous samples with large individuals, they can be split or cut into small pieces on the axis of symmetry or on the surface of symmetryFor samples that are elongated, flattened, or have different component content in each part, small pieces can be cut or cut into small sections at different parts for processingAfter taking the sample, it will be minced, mixed thoroughly, sampled by quartering or directly put into a tissue masher to be crushed into a homogenate, evenly divided into 2 parts as a sample, packed into a clean container, sealed and marked, and stored at a temperature below -18.
7.2 extraction.
7.2.1Wheat, barley, oats, rye, soybeans, corn, red beans, potatoes.
Weigh about 5 g of specimen (accurate to 0.).01 g), place in a 50 ml brown centrifuge tube with stopper, and quickly add 10 ml of ascorbic acid solution (510), vortex for 1 min to completely wet the sample, and then add 10 ml of 1% acetonitrile formate solution (59) Ultrasonic extraction for 10 min after vortex shakingAdd 5 g of sodium chloride (5.) to the centrifuge tube7) Vortex shaking for 1 min;8 000 r min 4 centrifugation for 5 min, take 10 ml of supernatant to be purified.
7.2.2 Beets, lettuce, tomatoes, cucumbers, grapes, pears, strawberries.
Weigh about 5 g of specimen (accurate to 0.).01 g), place in a 50 ml brown centrifuge tube with stopper, and quickly add 10 ml of ascorbic acid solution (510) Vortex for 1 minAdd 10 ml of 1% acetonitrile formate solution (5.)9) Vortex and shake for 2 min, after which 5 g of sodium chloride (57) Vortex shaking for 1 min;8 000 r min 4 centrifugation for 5 min, take 10 ml of supernatant to be purified.
7.2.3 Rapeseed, peanut kernels, sunflower seeds.
Weigh about 5 g of specimen (accurate to 0.).01 g), place in a 50 ml brown centrifuge tube with stopper, and quickly add 10 ml of ascorbic acid solution (510), vortex for 1 min to completely wet the sample, and then add 10 ml of 1% acetonitrile formate solution (59) Ultrasonic extraction for 10 min after vortex shakingAdd 5 g of sodium chloride (5.) to the centrifuge tube7) Vortex shaking for 1 min;Centrifuge at 8000 r min4 C for 5 min, take 20 ml of supernatant, then add acetonitrile-saturated n-hexane solution to remove the oil several times, take 10ml of the lower layer of Yiyan solution to be purified.
7.3. Purification.
7.3.1Wheat, barley, oats, rye, corn, red beans, soybeans, beets, lettuce, tomatoes, cucumbers, grapes, strawberries, potatoes, pears.
Will 10 ml of supernatant into the containment50 mg c1850 mg psaand150 mgAnhydrous magnesium sulfate (Centrifuge tubes in 2 ml, vortex for 30 s, 10,000 rmin, 4 centrifuge for 5 min;Take 05 ml of supernatant, add 05 ml of 20% acetonitrile solution (5.)11) Diluted through an organic filter membrane to a brown vial for HPLC-MS MS detection.
7.3.2 Rapeseed, peanut kernels, sunflower seeds.
Will 10 ml of supernatant was placed in a state filled with 150 mg C mg PSA (517) and 150 mg anhydrous magnesium sulfate (58) in a 2 ml centrifuge tube;Vortex for 30 s, 10 000 r min 4 and centrifuge for 5 minTake 05 ml of supernatant, add 05 ml of 20% acetonitrile solution (5.)11) After dilution, pass through the organic filter membrane (if there is turbidity, it can be centrifuged at 10 000 r min4 for 5 min, and then take the supernatant through the organic filter membrane) to the brown sample bottle for HPLC-MS MS detection.
7.4 Assay.
7.4.1 Liquid chromatography reference conditions.
7.4.1.1 LC column: C18 column, 50 mm20 mm (inner diameter), 18 m, or equivalent.
7.4.1.2 Mobile phase: A is pure water, B is acetonitrile.
7.4.1.3 column temperature: 35 1.
7.4. 1.4 Injection volume: 100μl。
7.4.1.5 Flow rate: 04 ml/min。
7.4.1.6 Mobile phase and gradient elution conditions: see Table 1.
7.4.2 Mass spectrometry reference conditions.
7.4.2.1 Ion source: Electrospray ion source (ESI).
7.4.2.2. Scanning mode: positive and negative switching mode.
7.4.2.3. Detection method: multiple reaction monitoring mode (MRM).
7.4.2.4. Atomized gas, dry gas, sheath gas and collision gas are all high-purity nitrogen and other suitable gasesBefore use, the flow rate of each gas should be adjusted to make the mass spectrometry sensitivity meet the detection requirements. Voltage values such as spray voltage, fragmentation voltage, and collision energy should be optimized to the best sensitivity, and the reference mass spectrometry parameters are shown in Appendix A.
7.4.3. Qualitative determination.
Samples and standard working solutions were determined according to liquid chromatography-mass spectrometry mass spectrometry conditions if the retention time of the detected mass chromatographic peaks differed from the standard retention time by no more than 25%, the relative abundance of the qualitative ion pair (expressed as a percentage of intensity relative to the abundance of the strongest ion) is the relative abundance of the standard working solution of the same concentration, and the allowable deviation of the relative abundance does not exceed the range specified in Table 2, then the corresponding analyte can be judged in the sample.
7.4.4. Quantitative determination.
Under the same experimental conditions, a series of standard working solutions (515) Injection detection from low to high concentration, and the peak area of quantitative product ions was plotted to obtain the standard curve. The response values of prothioconazole and its metabolites in the sample to be tested should be within the linear range of the standard curve, and if the linear range is exceeded, it should be diluted to the corresponding range before injection analysis. External standard method is used for quantification. The multiple reaction monitoring (MRM) chromatograms of prothioconazole and its metabolite dethiproconazole are shown in Appendix B under the above chromatographic conditions, and the peak time is 2., respectively9min and 32min。
7.4.5. Blank test.
Except for the addition of no sample, the above determination steps are followed.
7.4.6 Calculation and presentation of results.
The amount of analyte in the sample is calculated by chromatographic data processing software or according to equation (1), and the blank value is deducted from the calculation result.
where: x - the amount of analyte residue in the sample, in milligrams per kilogram (mg kg);
c——The concentration of the measured component solution from the standard working curve in micrograms per milliliter (g ml);
v——The final volume of the sample solution, in milliliters (ml);
m - the equivalent mass of the sample solution, in grams (g).
Limit of quantification and ** rate
8.1 Limit of quantification (LOQ).
The limits of quantification of prothioconazole and desthiprothioconazole in this method were 0005mg/kg。
8.2 ** rate.
The average ** rate data of different addition levels of prothioconazole and its metabolites in wheat, barley, oats, rye, corn, red beans, rapeseed, soybeans, peanut kernels, sunflower seeds, sugar beets, lettuce, tomatoes, cucumbers, potatoes, pears, grapes, and strawberries are shown in Appendix C.
*From: