GB 31657.2-2021National Food Safety Standard Determination of Multiple Residues of Quinolones in Bee Products Liquid Chromatography-Tandem Mass Spectrometry (English PDF)

1 Scope

This document specifies the sample preparation and liquid chromatography-tandem mass spectrometry detection methods for the detection of quinolone residues in honey and royal jelly: This document applies to oxalic acid, nalidixic acid, flumequine, cinofloxacin, difloxacin, sarafloxacin, ofloxacin, enrofloxacin, lomefloxacin, and pefluxacin in honey and royal jelly single or Determination of multiple drug residues:

2 Normative reference documents

The contents of the following documents constitute essential provisions of this document through normative references in the text: Among them, for dated reference documents, only the version corresponding to the date applies to this document; for undated reference documents, the latest version (including all amendments) applies to this document:

GB/T 6682 Specifications and test methods for water used in analytical laboratories

3 Terms and Definitions

There are no terms or definitions to be defined in this document:

4 Principles

The remaining quinolones in the sample were extracted with phosphate buffer, purified with HLB solid phase extraction column, measured by liquid chromatography-tandem mass spectrometry, calibrated by matrix matching standard curve, and quantified by external standard method:

5 Reagents and Materials

Unless otherwise specified, all reagents are of analytical grade and the water is first-grade water that complies with GB/T 6682:

5.1 Reagents

5.1.1 Acetonitrile (CH3CN): chromatographically pure:

5.1.2 Methanol (CH3OH): chromatographically pure:

5.1.3 Formic acid (CHOOH): chromatographically pure:

5.1.4 Ethyl acetate (C4H8O2):

5.1.5 Disodium hydrogen phosphate (Na2HPO4·12H2O):

5.1.6 Sodium dihydrogen phosphate (NaH2PO4·2H2O):

5.1.7 Phosphoric acid (H3PO4):

5.2 Solution preparation

5.2.1 Phosphate buffer: Take 7:16 g of disodium hydrogen phosphate and 3:12 g of sodium dihydrogen phosphate, add 800 mL of water to dissolve, and adjust the pH to 3:0±0:05, dilute with water to 1000mL:

5:2:2 5% formic acid in methanol solution: Take 5mL of formic acid, add 95mL of methanol, and mix:

5:2:30.1% formic acid solution: Take 0:5 mL of formic acid, add water to dilute to 500 mL, and mix well:

5:2:4 20% acetonitrile formic acid solution: Take 20 mL of acetonitrile, add 80 mL of 0.1% formic acid solution, and mix well:

5.3 Standard products Oxalinic acid, flumequine, cinofloxacin, difloxacin, sarafloxacin, ofloxacin, enrofloxacin, lomefloxacin, pefloxacin, norfloxacin, Ciprofloxacin, nalidixic acid, enoxacin, danofloxacin, marbofloxacin, flerofloxacin, gatifloxacin, orbifloxacin, pipemidic acid and sparfloxacin, the contents are all >=95% ,See Appendix A for specific information:

5.4 Preparation of standard solution

5.4.1 Standard stock solution (1 mg/mL): Take oxalinic acid, flumequine, cinofloxacin, diloxacin, sarafloxacin, ofloxacin, enrofloxacin, lomefloxacin, and Floxacin, norfloxacin, ciprofloxacin, nalidixic acid, enoxacin, danofloxacin, marbofloxacin, fleroxacin, gatifloxacin, orbifloxacin, piperonic acid, and difloxacin Pafloxacin reference substance is 10 mg each, weighed accurately, dissolved in 0:5 mL ammonia water, diluted with methanol to a 10 mL volumetric flask, and prepared into a standard stock solution with a concentration of 1 mg/mL: Store at -18°C away from light, valid for 6 months:

5.4.2 Mixed standard working solution (10 µg/mL): Accurately measure 1 mL of each of the above standard stock solutions in a 100 mL volumetric flask, dilute to the mark with methanol, and prepare a mixed standard working solution with a concentration of 10 µg/mL: Store at 4°C away from light, valid for 1 month:

5.5 Materials

5.5.1 HLB solid phase extraction column::200 mg/6 mL, or equivalent:

5.5.2 Microporous nylon filter membrane: 0:22µm:

5.5.3 Glass fiber filter paper: 1:6µm:

6 Instruments and equipment

6.1 Liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source:

6.2 Analytical balance: sensitive to 0:00001g and 0:01g:

6.3 Vortex mixer:

6.4 Vortex oscillator:

6.5 High-speed refrigerated centrifuge: the speed can reach:20000 r/min:

6.6 Ultrasonic cleaning machine:

6.7 Solid phase extraction device:

6.8 Nitrogen blower,

7 Preparation and preservation of samples

7.1 Preparation of samples Take an appropriate amount of fresh or thawed blank or test bee products and mix them evenly: For honey samples with crystallization, place them in a water bath not exceeding 60°C under closed conditions to decrystallize them: After the samples are completely melted, stir well and cool to room temperature: The prepared sample is placed in a sample bottle, sealed, and marked:

a) Take the homogenized test sample as the test material;

b) Take the homogenized blank sample as the blank sample;

c) Take the homogenized blank sample, add the standard working solution of appropriate concentration, and add the sample as a blank:

7.2 Storage of samples Honey samples were stored at room temperature away from light; royal jelly samples were stored at -18°C:

8 Measurement steps

8.1 Extraction

8.1.1 Honey

Take 2g of the sample (accurate to ±0:02g), put it in a 50mL polypropylene centrifuge tube, add 20mL of phosphate buffer, vortex for 1 min, shake for 10 min, centrifuge at 10000 r/min for 5 min, take the supernatant and set aside: :

1) The HLB solid-phase extraction cartridges listed here are for reference only and are not for commercial purposes: Standard users are encouraged to try solid-phase extraction cartridges from different manufacturers or models:

8.1.2 Royal jelly

Take 2g of the sample (accurate to ±0:02g), put it in a 50mL polypropylene centrifuge tube, add 10mL of phosphate buffer, vortex for 1 min, shake for 10 min, centrifuge at 10000 r/min for 10 min at 4°C, and transfer Transfer the supernatant to another centrifuge tube: Add 10 mL of phosphate buffer to the residue, repeat the extraction, combine the two supernatants, filter with glass fiber filter paper, and set aside:

8.2 Purification

The HLB solid-phase extraction column was activated with 10 mL of methanol and 10 mL of water respectively: Pass the backup solution through the column, rinse with 5 mL of water, and drain: Elute with 4 mL of 5% formic acid methanol and ethyl acetate in sequence, drain, and collect the eluate: Blow dry with nitrogen at 40°C: Dissolve the residue with 1:00 mL of 20% acetonitrile formic acid aqueous solution, sonicate for 1 min, and vortex to mix (the royal jelly sample solution needs to be centrifuged at 20,000 r/min for 10 min at 4°C): The supernatant is passed through a microporous nylon filter for the liquid phase: Chromatography-tandem mass spectrometry:

8.3 Preparation of matrix matching standard curve Accurately measure an appropriate amount of the mixed standard working solution, dilute it with 20% acetonitrile formic acid aqueous solution, and prepare a series of standard solutions with concentrations of 1ng/mL, 2 ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 50ng/mL: Take 1:00mL of each and add it to the blank sample processed by the extraction and purification steps, dissolve the residue (the royal jelly matrix matching standard solution needs to be centrifuged at:20000 r/min for 10 min at 4°C), and the supernatant is passed through microporous nylon After filtering, it was analyzed by liquid chromatography-tandem mass spectrometry: Using the characteristic ion mass chromatographic peak area of each drug as the ordinate and the concentration of the standard solution as the abscissa, draw a matrix matching standard curve: Find the regression equation and correlation coefficient:

8.4 Determination

8.4.1 Liquid Chromatography Reference Conditions

a) Chromatographic column: Cis column (50mm×2:1mm, 1:7µm), or equivalent;

b) Column temperature: 35°C;

c Injection volume: 10L;

d) Flow rate: 0:3mL/min;

e) Mobile phase: A is 0.1% formic acid aqueous solution, B is methanol, and the gradient elution conditions are shown in Table 1:

8.4.2 Mass spectrometry reference conditions

a) Ion source: electrospray ion source;

b) Scanning method: positive ion scanning;

c) Detection method: multiple reaction monitoring;

d) Capillary voltage: 1:0kV;

e) Source temperature: 150°C;

f) Atomization temperature: 500°C;

g) Cone air flow rate: 50 L/h;

h) Atomizing gas flow rate: 1000 L/h;

i) The reference values of test drug retention time, qualitative ion transition, quantitative ion transition, cone voltage and collision energy are shown in Table 2:

Under the same test conditions, the retention time of the quinolone peak in the sample solution is the same as the retention time of the corresponding peak in the matrix-matched standard solution:

The relative deviation is within ±2.5%, and the detected relative ion abundance should be consistent with the relative abundance of the matrix-matched standard solution of equivalent concentration: The allowable deviation should comply with the requirements of Table 3:

8.4.3.2 Quantitative determination Take the sample solution and the corresponding matrix-matched standard solution, perform single-point or multi-point calibration, and quantify the peak area of characteristic ion mass chromatography, and calculate it according to the external standard method: The response values of quinolones in the matrix-matched standard working solution and sample solution should be within the linear range of instrument detection: Under the above chromatography-mass spectrometry conditions, the characteristic ion mass chromatogram of the matrix-matched standard solution is shown in Appendix B:

8.5 Blank test

Take a blank sample and perform parallel operations using the same measurement steps except that no standard solution is added:

9 Result calculation and presentation

The residual amount of quinolones in the sample is calculated according to the standard curve or formula (1):

10 Sensitivity, accuracy and precision of detection methods

10.1 Sensitivity

The detection limit of this method is 0:5µg/kg, and the quantification limit is 1:0µg/kg:

10.2 Accuracy

This method has a recovery rate of 60% to 120% at a concentration of 1 µg/kg to 5 µg/kg:

10.3 Precision

The intra-batch relative standard deviation of this method is <=20%, and the inter-batch relative standard deviation is <=20%: