GB 31656.12-2021National food safety standard - Determination of Penicillin multi-residues in aquatic products by liquid chromatography-tandem mass spectrometry method (English PDF)

1 Scope

This document specifies the sample preparation and liquid chromatography-tandem mass spectrometry determination methods for the detection of penicillin drug residues in aquatic products:

This document applies to amoxicillin, ampicillin, penicillin G, penicillin V, and oxacillin in the edible tissues of aquatic products such as fish, shrimp, turtle, and sea cucumber:

Determination of single or multiple drug residues of lin, cloxacillin, dicloxacillin, nafcillin, piperacillin, azlocillin and methicillin:

2 Normative reference documents

The contents of the following documents constitute essential provisions of this document through normative citations in the text: Among them, the cited documents with dates are:

Only the version corresponding to the date applies to this document; for undated referenced documents, the latest version (including all amendments) applies to this document:

document:

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

GB/T 30891-2014 Sampling specifications for aquatic products

3 Terms and definitions

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

4 Principles

The remaining penicillins in the sample were extracted with acetonitrile aqueous solution, purified with a solid phase extraction column, and measured by liquid chromatography-tandem mass spectrometry, using the internal standard method:

Quantitative:

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 Acetonitrile (CH3CN): chromatographically pure:

5:1:2 Formic acid (HCOOH): chromatographically pure:

5:2 Solution preparation 5:2:1 30% acetonitrile aqueous solution: Take 30mL of acetonitrile and 70mL of water, mix:

5:2:2 80% acetonitrile aqueous solution: Take 400mL of acetonitrile and 100mL of water, mix:

5:2:3 0:05% formic acid aqueous solution: Take 250 uL of formic acid and dilute to 500 mL with water:

5:2:4 0:05% formic acid acetonitrile solution: Take 250 uL of formic acid, add acetonitrile and dilute to 500 mL:

5:3 Standard products 5:3:1 Penicillins: amoxicillin, ampicillin, penicillin G, penicillin V, oxacillin, cloxacillin, dicloxacillin, nafcil Lin, piperacillin, azlocillin and methicillin, the content is >=95%, please see Appendix A for details:

5:3:2 Internal standards: amoxicillin Gd4, ampicillin Gd5, penicillin Gd7, nafcillin Gd6, the contents are all >=95%, see Appendix A for details:

5:4 Preparation of standard solution 5:4:1 Penicillin standard stock solution (0:10 mg/mL): Take about 10 mg of penicillin drug standard, weigh it accurately, and use 30% acetonitrile water Dissolve and dilute the solution to a volume of 100 mL, and prepare a standard stock solution with a concentration of 0:10 mg/mL: Store in the dark at -18°C to be effective:

Issue 5d:

5:4:2 Internal standard stock solution (0:10 mg/mL): Take about 10 mg of the internal standard standard, weigh it accurately, dissolve it in 30% acetonitrile aqueous solution and dilute it to the final concentration:

GB 31656:12-2021 The volume is increased to 100 mL, and the internal standard stock solution is prepared with a concentration of 0:10 mg/mL: Store in the dark at -18°C and has a validity period of 5 days:

5:4:3 Mix standard intermediate solution (1:0ug/mL): Precisely measure an appropriate amount of penicillin standard stock solution and dilute it with water to prepare amoxicillin, ammonia and ammonia:

Benzillin, Penicillin G, Penicillin V, Oxacillin, Cloxacillin, Dicloxacillin, Nafcillin, Piperacillin, Azlocillin and Methicillin Concentrate A mixed standard solution with a concentration of 1:0 ug/mL: Store in the dark at -4°C and have a validity period of 5 days:

5． d5, penicillin G-d7, and nafcillin-d6 are all internal standard working solutions with a concentration of 1:0 ug/mL: Store in the dark at -4°C and have a validity period of 5 days:

5:5 Materials 5:5:1 Pass-through reversed-phase mixed hydrophilic-lipophilic solid-phase extraction column::200mg/6mL, or equivalent:

5:5:5: Ultrafiltration tube: 10kD, 0:05mL:

6 Instruments and equipment

6:1 Liquid chromatography-tandem quadrupole mass spectrometer: equipped with electrospray ion source:

6:2 Analytical balance: sensitivity 0:01g and 0:00001g:

6:3 High-speed refrigerated centrifuge:

6:4 Solid phase extraction device:

6: Ultrasonic cleaning instrument:

6: Tissue homogenizer:

6: Vortex mixer:

6: 8 Nitrogen blowing instrument:

7: Preparation and preservation of samples 7:1 Preparation of samples Prepare samples according to the requirements of Appendix B of GB/T 30891-2014:

a) Take a homogeneous test sample as a test material;

b) Take a homogeneous blank sample as a blank sample;

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

7:2 Preservation of samples Store below -18 degrees C and conduct analysis and testing within 3 months:

8 Measurement steps 8:1 Extraction Take 2:5g of the sample (accurate to +/-0:02g), add 100uL of internal standard working solution, let it stand for 10min, add 5mL of 80% acetonitrile aqueous solution, and vortex Mix for 1 minute, ultrasonic for 10 minutes, centrifuge at 10,000 r/min for 5 minutes at 4°C, take the supernatant, add 4 mL of 80% acetonitrile aqueous solution to the residue, and repeat the extraction:

Take once, combine the supernatants, dilute to 10:0 mL with 80% acetonitrile aqueous solution, and set aside:

8:2 Purification Take about 1 mL of 80% acetonitrile aqueous solution to rinse the solid phase extraction column, discard the effluent, and pass 2:0 mL of the reserve solution through the column, keeping the flow rate at 1 drop/s:

Collect the effluent, blow it with nitrogen at 35°C to less than 0:5 mL, add water to adjust the volume to 0:5 mL, centrifuge with an ultrafiltration tube at 12000 r/min for 10 min, and take the filter liquid for determination by liquid chromatography-tandem mass spectrometry:

8:3 Preparation of standard curve Precisely measure appropriate amounts of the mixed standard intermediate solution and internal standard working solution, and prepare penicillin drugs with water concentrations of 2 ug/L, 5 ug/L, Mixed standard working solutions of 10 ug/L, 25 ug/L, 50 ug/L, 150 ug/L and 300 ug/L, in which the concentrations of penicillin internal standard solutions are all 40 ug/L: Take the measured characteristic ion mass chromatography peak external standard and internal standard peak area ratio as the ordinate, and the corresponding standard solution concentration as the abscissa, Draw a standard curve and find the regression equation and correlation coefficient:

8: Measurement 8:4:1 Liquid Chromatography Reference Conditions a) Chromatographic column: C18 chromatographic column (2:1mmx100mm, 2:6um), or one with equivalent performance;

b) Column temperature: 35 degrees C;

c) Flow rate: 0:4mL/min;

d) Injection volume: 10uL;

e) Mobile phase: A is 0:05% formic acid aqueous solution, B is 0:05% formic acid acetonitrile solution: Gradient elution conditions are shown in Table 1:

8:4:2 Mass Spectrometry Reference Conditions a) Ionization mode: electrospray ion source (ESI), multiple reaction monitoring (MRM), positive ion mode;

b) Spray voltage: 5:5kV;

c) Curtain air pressure: 0:24MPa;

d) Collision gas pressure: 0:02MPa;

e) Ion source temperature: 500 degrees C;

f) Collision chamber inlet voltage: 10V;

g) Collision chamber outlet voltage: 12V;

h) Dwell time: 50ms;

i) Ion source Gas1:0:34MPa;

j) Ion source Gas2:0:34MPa;

k) Multiple reaction monitoring precursor ions, product ions, declustering voltage and collision energy are shown in Table 2:

8:5 Measurement method 8:5:1 Qualitative determination Under the same test conditions, the retention time of penicillins in the sample solution and the retention time of penicillins in the standard working solution ratio, the deviation is within +/-2:5%, and the detected relative ion abundance should be consistent with the relative abundance of the standard solution with equivalent concentration: The deviation is allowed The difference should meet the requirements of Table 3:

8:5:2 Quantitative determination Inject equal volumes of sample solution and mixed standard working solution for measurement, perform single-point or multi-point calibration, and quantify by chromatographic peak area, according to the internal standard method Calculation, amoxicillin uses amoxicillin Gd4 as the internal standard, ampicillin uses ampicillin Gd5 as the internal standard, penicillin G, penicillin V, oxacillin Penicillin G-d7 was used as the internal standard for penicillin, cloxacillin and dicloxacillin, and nafcillin-Gd6 was used for nafcillin, piperacillin, azlocillin and methicillin:

is the internal standard: The response values of penicillins in the standard solution and sample solution should be within the linear range of instrument detection: In the above liquid phase color Under the conditions of spectrum-mass spectrometry, the characteristic ion mass chromatogram in the standard solution is shown in Appendix B:

8:6 Blank test Take a blank sample and perform parallel operations using the same measurement steps except that no standard solution is added:

9 Calculation and presentation of results The residual amount of penicillins in the sample is calculated according to the standard curve or formula (1):

10 Sensitivity, accuracy and precision of detection methods 10:1 Sensitivity Detection limit of this method: ampicillin, penicillin G, penicillin V, oxacillin, cloxacillin, dicloxacillin, nafcillin, piperacillin Lin, azlocillin, and methicillin are all 2 ug/kg, and amoxicillin is 10 ug/kg:

Quantitative limit of this method: ampicillin, penicillin G, penicillin V, oxacillin, cloxacillin, dicloxacillin, nafcillin, piperacillin Lin, azlocillin, and methicillin are all 5 ug/kg, and amoxicillin is 25 ug/kg:

10:2 Accuracy In this method, ampicillin, penicillin G, penicillin V, oxacillin, cloxacillin, dicloxacillin, nafcillin, piperacillin, alosil The recovery rates of methicillin and methicillin at 5 ug/kg~300 ug/kg and amoxicillin at 25 ug/kg~300 ug/kg were the same:

It is 70%~120%:

10:3 Precision The intra-batch relative standard deviation of this method is <=15%, and the inter-batch relative standard deviation is <=15%: