GB 31658.20-2022National food safety standards-Determination of residues of amidoalcohol drugs and their metabolites in animal foods-liquid chromatography-tandem mass spectrometry (English PDF)

5.1 Reagents

5.1.1 Methanol (CH3OH). chromatographically pure.

5.1.2 Acetonitrile (CH3CN). chromatographically pure.

5.1.3 Ammonia (NH3.H2O).

5.1.4 Ethyl acetate (C4H8O2).

5.1.5 Anhydrous sodium sulfate (Na2SO4).

5.1.6 Sodium Chloride (NaCl).

5.1.7 Ammonium formate (HCOONH4).

5.1.8 n-Hexane (C6H14).

5.2 Solution preparation

5.2.1 2% ammoniated ethyl acetate solution. Take 20mL ammonia water and dilute it to 1000mL with ethyl acetate.

5.2.2 4% sodium chloride solution. Take 4g of sodium chloride, dissolve it in water and dilute to 100mL.

5.2.3 4% sodium chloride saturated n-hexane. Take an appropriate amount of 4% sodium chloride solution, add excess n-hexane, mix, let stand to separate layers, take the upper layer of n-hexane

Hexane.

5.2.4 20% methanol solution. Take 20mL of methanol and dilute with water to 100mL.

5.2.5 10mmol/L ammonium formate solution. Take 0.63g of ammonium formate, dissolve it in water and dilute to 1000mL.

5.3 Standards

Chloramphenicol, thiamphenicol, florfenicol, florfenicol amine, the content of which was >=99%, chloramphenicol-D5, thiamphenicol-D3, florfenicol-D3, fluorfenicol

Benicolamide-D3 internal standard, the contents were all >=98%, see Appendix A for details.

5.4 Preparation of standard solution

5.4.1 Standard stock solution. Take an appropriate amount of each standard substance of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine (equivalent to about

10mg), accurately weighed, respectively add appropriate amount of methanol to dissolve and dilute to a volume of 100mL volumetric flask, and prepare a concentration of 100µg/mL

Standard stock solution. Store below -18°C, valid for 12 months.

5.4.2 Internal standard stock solution. take appropriate amount of internal standard of chloramphenicol-D5, thiamphenicol-D3, florfenicol-D3, florfenicol amine-D3 (equivalent to each active

(about 1 mg of active ingredients), accurately weighed, add appropriate amount of methanol to dissolve and dilute to volume into a 10mL volumetric flask, and prepare the concentration of

5.5 Materials

Nylon microporous membrane. 0.22 µm.

6.1 Liquid chromatography-tandem mass spectrometer. with electrospray ionization source (ESI).

6.2 Balance. Sensitivity 0.00001g and 0.01g.

6.3 Homogenizer.

6.4 Vortex mixer.

6.5 Multi-tube vortex shaker.

6.6 High-speed refrigerated centrifuge. the speed can reach 8000r/min.

6.7 Nitrogen blowing instrument.

7.1 Preparation of samples

7.1.1 Muscle, liver, kidney and adipose tissue

Take an appropriate amount of fresh or thawed blank or test tissue, mince it, and homogenize it.

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

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.

7.1.2 Milk

Take an appropriate amount of fresh or thawed blank or test milk or goat milk, and mix well.

a) Take the uniformly mixed test sample as the test sample;

b) Take the uniformly mixed 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.

7.1.3 Eggs

Take an appropriate amount of fresh or refrigerated blank or test eggs, shell them, and homogenize them.

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

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

Store below -18°C.

8.1 Extraction

Take 2 g of the test material (accurate to +/-0.05 g), put it in a 50 mL centrifuge tube, add 100 µL of mixed internal standard working solution, vortex to mix, and add

2% ammoniated ethyl acetate solution 10mL (milk, goat milk samples need to add anhydrous sodium sulfate 3g), vortex 30s, vortex oscillation 10min,

Centrifuge at 8000r/min for 5min. Transfer the supernatant to another 50mL centrifuge tube, add 10mL of 2% ammoniated ethyl acetate solution to the residue, repeat

Extract once. Combine the two extracts, blow dry at 50°C with nitrogen, and wait for purification.

8.2 Purification

Take the residue to be purified, add 3mL of 4% sodium chloride solution, vortex to dissolve, add 5mL of n-hexane saturated with 4% sodium chloride, vortex for 30s,

Centrifuge at 8000r/min for 5min, discard the upper n-hexane layer, repeat degreasing with 4% sodium chloride-saturated n-hexane, add 2% ammoniated acetic acid

Ethyl acetate solution 5mL, vortex 5min, 8000r/min centrifuge 5min, take the upper organic phase. Use 2% ammoniated ethyl acetate solution 5mL

Repeat the extraction once, combine the organic phases, blow dry with nitrogen at 50°C, add 1.0 mL of 20% methanol solution, vortex for 30 s, pass through a 0.22 µm filter membrane, and supply the liquid phase.

Chromatography-tandem mass spectrometry.

8.3 Preparation of standard curve

Precisely measure the appropriate amount of mixed standard intermediate solution and mixed internal standard working solution, dilute with 20% methanol solution, and prepare the concentration of chloramphenicol as

0.2 µg/L, 0.5 µg/L, 1 µg/L, 2 µg/L, 5 µg/L, 10 µg/L, the concentrations of thiamphenicol, florfenicol and florfenicol amine were respectively

1 µg/L, 2.5 µg/L, 5 µg/L, 10 µg/L, 25 µg/L, 50 µg/L, chloramphenicol-D5 concentration was 1 µg/L, thiamphenicol-D3, florfenib

A series of standard solutions with concentrations of 5 µg/L and 5 µg/L of florfenicol amine-D3 and florfenicol amine-D3 were prepared immediately before use for determination by liquid chromatography-tandem mass spectrometer.

Draw the standard curve with the peak area ratio as the ordinate and the concentration as the abscissa, and find the regression equation and correlation coefficient.

8.4 Determination

8.4.1 Reference conditions for liquid chromatography

a) Chromatographic column. C18 chromatographic column (100mmx2.1mm, 1.7µm), or equivalent;

b) Column temperature. 30°C;

c) Injection volume. 5 µL;

d) Flow rate. 0.3mL/min;

e) Mobile phase. A is 10 mmol/L ammonium formate solution; B is acetonitrile; the gradient elution program is shown in Table 1.

8.4.2 Reference conditions for mass spectrometry

a) Ion source. electrospray ion source;

b) Scanning mode. positive ion scanning/negative ion scanning;

c) Detection method. multiple reaction ion monitoring (MRM);

d) Desolvation gas, cone gas, and collision gas are all high-purity nitrogen or other suitable gases;

e) Parameters such as spray voltage and collision energy should be optimized to the optimum sensitivity;

f) Refer to Table 2 for reference values of the ion source of the analyte, qualitative ion pair, quantitative ion pair, cone voltage and collision energy.

8.4.3 Assay

8.4.3.1 Qualitative determination

Under the same test conditions, the retention time of amide alcohol drugs and their metabolites in the test solution was the same as that of amide alcohol drugs in the standard working solution.

The relative deviation of the retention time of its metabolites and its metabolites is within +/-2.5%, and the detected relative ion abundance should be the same as the concentration of the calibration standard.

The relative ion abundance of the solution is consistent. The allowable deviation should meet the requirements of Table 3.

8.4.3.2 Quantitative determination

Take the sample solution and the corresponding standard solution for single-point or multi-point calibration, and use the internal standard method to quantify the chromatographic peak area ratio. The standard solution and sample

The peak area ratios of amidoalcohol drugs and their metabolites in the solution to their corresponding internal standards should be within the linear range of instrument detection. For residues exceeding

In the linear range of the instrument, the amount of internal standard working solution should be increased according to the concentration of the drug during extraction, so that the sample solution can be diluted and the amide alcohol drug

The concentration of its metabolites is within the range of the curve, and the corresponding internal standard concentration is consistent with the standard working solution. The characteristic ion mass chromatogram of the standard solution is shown in

Appendix B.

8.5 Blank test

Take the blank sample, except that no drug is added, the exact same determination steps are used for determination.

10.1 Sensitivity

The detection limit of chloramphenicol in this method is 0.1 µg/kg, and the limit of quantification is 0.2 µg/kg; the detection of thiamphenicol, florfenicol and florfenicol amine

The limit of concentration was 0.5 µg/kg, and the limit of quantitation was 1 µg/kg.

10.2 Accuracy

In this method, chloramphenicol was added at a concentration level of 0.2 µg/kg~1 µg/kg, and thiamphenicol was added at a concentration level of 1 µg/kg~100 µg/kg.

The recoveries of florfenicol and florfenicol amine were both 70%-120% at the concentration levels of 1 µg/kg-6000 µg/kg.

10.3 Precision

In this method, the relative standard deviation within the batch was <=15%, and the relative standard deviation between batches was <=20%.