SN/T 5119-2019Determination of neomycin residues in edible animal for import and export - ELISA and LC-MS/MS (English PDF)

4.1 Water. secondary water specified in GB/T 6682-2008

4.2 Carbodiimide. Analytical grade

4.3 Ovalbumin

4.4 Bovine serum albumin

4.5 Dialysis Bag

4.6 Coating buffer

4.7 Washing buffer

4.8 Blocking buffer

4.9 Dilution buffer

4.10 Sample Extraction Solution

4.11 Diluent of horseradish peroxidase labeled conjugate

4.12 Substrate solution

4.13 Developer solution

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4.14 Stop solution

4.15 Antibodies

4.16 HRP conjugate

4.17 Ovalbumin-coated neomycin polystyrene micro 96-well reaction plate

4.18 Standard. neomycin sulfate, purity 90%.

4.19 Standard stock solution. Weigh the standard accurately, convert it to 10.0 mg of the target compound, dissolve it with an appropriate amount of water, and make up to volume with water.

10mL, mix well. Store below -18 ° C in the dark, with a validity of 12 months.

4.20 Standard intermediate solution. Dilute the standard stock solution with water to 10 mg/L, and store it in the dark below 4 ° C. The validity period is 6 months.

4.21 Standard working solution. Dilute the standard intermediate solution to a working concentration of 1.5 µg/L, 3 µg/L, 6 µg/L, 12 µg/L, 36 µg/L with dilution buffer.

Note. 4 For the preparation of various liquids involved in reagents and materials, see Appendix A.

6.1 Sample preparation and storage

6.1.1 Sample preparation

10 ml of animal blood was collected in anticoagulant test tubes containing sodium heparin, and 50 ml of fresh urine was placed in clean containers.

Two aliquots were sealed as samples and marked. During sample preparation, samples should be protected from contamination or residues.

Variety.

6.1.2 Sample preservation

The sample can be stored at 4 ° C for 3-4 days; if it is stored for a long time, the sample should be placed below -18 ° C.

6.2 Extraction

Take 50 µL of sample, add 1.2 mL of sample extract, mix well, centrifuge at 4000 × g for 5 min, and use the supernatant for detection.

6.3 Enzyme-linked immunoassay

6.3.1 According to the number of samples to be tested and the standard samples (2 parallel samples), determine the amount of microwells used.

6.3.2 Add 100 µL of ovalbumin-conjugated neomycin antigen-coated plate to each well overnight at 4 ° C. 6.3.3 Pour the liquid in the microwells vertically, add 250 µL of washing buffer, and shake it gently. Then repeat the washing of the microwells three times, and vigorously wipe off the remaining liquid on the wall on the filter paper.

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6.3.4 Add 250 µL 2% bovine serum albumin to each well, and block at 37 ° C for 2 hours. 6.3.5 The operation is the same as 6.3.3.

6.3.6 Add 50 µL of standard solution or test sample solution to each microwell. 6.3.7 Add 50 µL horseradish enzyme label and 50 µL antibody to each microwell, and mix well. 6.3.8 Incubate for 30 min at room temperature (20-25) ° C, protected from light.

6.3.9 The operation is the same as 6.3.3.

6.3.10 Add 50 µL of substrate solution and 50 µL of developer solution to each microwell, mix thoroughly and incubate for 15 min at room temperature (20-25) ° C in the dark.

6.3.11 Add 100 µL of stop solution to stop the reaction. After thorough mixing, measure the absorbance at 450 nm within 30 min.

6.4 Blank test

Except that no sample is added, it is carried out according to the measurement procedure of 6.3.

6.5 Quality control test

For each measurement, a quality control sample with a blank sample and a drug mixed standard should be prepared, and the addition concentration should be the detection of the corresponding product.

Low limit.

7.1 Drawing of standard curve

Six concentrations of standard solutions (0ng/mL, 1.5ng/mL, 3ng/mL, 6ng/mL, 12ng/mL, 36ng/mL) and the solution to be tested were measured to determine the corresponding absorbance values in accordance with the limit method. Take the absorbance A0 value of 0 concentration as the denominator, the absorbance A value of other standard concentrations is the ratio of the numerator, and then multiply by 100 to obtain the percentage of absorbance. Take the absorbance percentage as the ordinate, the corresponding 5 the standard concentration is the abscissa, and the standard curve is plotted on the semi-logarithmic scale. The calibration model is linear in the range of 1.5ng/mL to 36ng/mL.

Sexual.

7.2 Calculation of percent absorbance

Calculate the percent absorbance value according to formula (1).

Percent absorbance value = B sample B0 × 100% (1) B sample --- the average absorbance value of the sample well;

B0 --- Average absorbance value of zero standard.

7.3 Calculation of the content of the target substance in the test solution

Substitute the sample's percent absorbance value into the standard curve to obtain the corresponding drug concentration c (µg/L) from the standard curve. Calculate the residual amount of neomycin in plasma and urine according to formula (2).

X = c · f (2)

In the formula.

X --- the amount of drug residue in the sample, in micrograms/liter (µg/L); c --- the content of the drug in the sample, obtained from the standard curve, in micrograms/liter (µg/L); f --- sample dilution factor;

The calculation results retain three significant digits. Positive results need to be confirmed by the LC-MS/MS method.

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8.1 Sensitivity

The detection limit of this method in blood and urine samples from pigs, cattle, and sheep was 40ng/mL.

8.2 Recovery rate

The recovery rate of this method at the concentration level of 1.5ng/mL-36ng/mL is 80% -95%.

8.3 Precision

For quantitative analysis, at least two parallel samples should be taken for each sample for analysis. Results of two independent determinations obtained under repeatable conditions the absolute difference of the results must not exceed 20% of the arithmetic mean.

Note. Equivalent commercial ELISA kits can be used for testing.

Second method liquid chromatography tandem mass spectrometry

11.1 Extraction

Prepare the sample according to 6.1.1, take 3g to 50mL centrifuge tube, add 12mL of extraction solution 1 (9.9), mix and shake for 15min, Centrifuge at 10,000 r/min for 10 min. The supernatant was transferred to another new 50 mL centrifuge tube. 10 mL of n-hexane was added, and the mixture was shaken for 10 min.

Centrifuge at 8000 r/min for 5 min, discard the upper layer of liquid, and accurately transfer the lower layer of the extraction solution to 10 mL into a new 50 mL centrifuge tube.

Solution 2 (9.10) adjusted pH = 4.0-4.5, and waited for column purification.

11.2 Purification

Purify the SP column, pre-column with 5mL methanol and 5mL water, adjust the pH of the extraction solution and add it to the reservoir through the C18 column at a flow rate of 3mL/min or less. After the liquid completely flows out, rinse the column with 5mL water. , Discard all effluent, blow dry with positive pressure, and finally use 5 Methanol was used to elute. The eluate was collected into a 15mL polytetrafluoroethylene round bottom centrifuge tube, dried at 50 ° C in a water bath with nitrogen, and 1.0mL of mobile phase was allowed to dissolve.

Pass 0.22 µm filter membrane to the injection vial for determination by high performance liquid chromatography-tandem mass spectrometer.

11.3 Determination

11.3.1 Liquid chromatography conditions

11.3.1.1 Chromatographic column. ObeliSCR2.1 × 150mm, 5µm Lot. S02-002C. 11.3.3.1.2 Mobile phase. A organic phase. acetonitrile; B aqueous phase. 1% formic acid aqueous solution (containing 10mM ammonium formate) gradient elution conditions, see Appendix B.

11.3.3.1.3 Flow rate. 0.5mL/min.

11.3.3.1.4 Column temperature. 30 ° C.

11.3.3.1.5 Injection volume. 30 µL.

11.3.2 Mass spectrometry conditions

11.3.3.2.1 Ionization mode. Electrospray ionization positive ion mode (ESI +).

11.3.2.2.2 Mass spectrometry scanning method. See Appendix C for multiple reaction monitoring (MRM) spectra.

11.3.3.2.3 Resolution. Unit resolution.

11.3.3.2.4 For other mass spectrometry conditions, see Appendix D.

11.3.3 Qualitative determination

The measured component selects one parent ion and two or more product ions. Under the same experimental conditions, the retention time of the test substance in the sample is within ± 5% of the retention time of the base standard solution; and each group in the sample Matrix standard method for determining the relative abundance and concentration of qualifier ions Compare the relative abundance of the corresponding qualifier ions in the solution, and the deviation does not exceed the range specified in Table 1.

The object to be tested.

Table 1 Maximum allowable deviation of relative ion abundance during qualitative confirmation

Relative ion abundance /% > 50 > 20 ~ 50 > 10 ~ 20 <=10 Allowable relative deviation /% ± 20 ± 25 ± 30 ± 50

11.3.4 Quantitative determination

Under the optimal conditions of the instrument, the matrix standard working solution was injected, with the peak area as the ordinate and the matrix standard working solution concentration as the horizontal sitting.

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Standard draw the standard working curve, and use the standard curve to quantify.

11.4 Blank test

Except for not adding samples, all operations are carried out according to the above operation steps.

13.1 Lower limit of determination

The lower limit of determination of neomycin by this method is 100 µg/kg.

13.2 Recovery and precision

The recovery rate of neomycin in urine and blood was 71.0% -103.0%.

Non-commercial statement. The instrument models used in the text of this standard are for reference only and do not involve commercial purposes. Users of the standard are encouraged.

Try using a different manufacturer or model of instrument.

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Appendix A

(Informative appendix) Preparation of ELISA reagents A. 11mol/L sodium hydroxide solution. Weigh 40.00g sodium hydroxide, make up to volume with a 1L volumetric flask, and dissolve.

A. 20.1mol/L sodium dihydrogen phosphate buffer solution (pH = 6.0). Take 15.6g of sodium dihydrogen phosphate (NaH2PO4 · 2H2O), add an appropriate amount of water to make After 1000mL, the pH was adjusted to 6.0 with phosphoric acid.

A. 3 0.05mol/L carbonate coating buffer (pH 9.6). Weigh NaHCO31.46g, Na2CO30.97g, add water to volume to 500mL. After dissolving, store in 4 °C freezer.

A. 4 Dilution buffer 0.05% Tween20-PBS. 1,000mL, Tween-20 (Tween-20) 0.5mL, mix well.

A. 5 Blocking buffer 2% BSA solution. 1,000 PBS, add 20g bovine serum albumin, mix and dissolve.

A. 6 Washing buffer. 0.01-0.05mol/LpH7.5 sodium phosphate buffer, add 0.05% Tween-20.

A. 7 Horseradish peroxidase-labeled conjugate diluent. Contains 0.01mol/L-0.05mol/LpH7.5 sodium phosphate buffer, add 0.05% Tween-20, 2% bovine serum albumin.

A. 8 Substrate liquid. hydrogen peroxide with a concentration of 0.3%.

A. 9 developer solution. 0.2 g/L tetramethylbenzidine solution was prepared with pH 5.0 sodium acetate-citrate buffer.

A. 10 Stop solution. 2mol/L sulfuric acid solution.

A. 11 Explanation of three reagents. 4.9 sample extraction solution was provided by US REAGEN, 4.14 antibody was sheep-derived neomycin polyclonal antibody

4.15 HRP conjugate is donkey anti-sheep HRP secondary antibody.

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Appendix B

(Informative appendix) Gradient elution conditions for liquid chromatography Step running time (min) Flow rate (mL/min) Mobile phase A (%) Mobile phase B (%) 0 0 0 5 5 70.0 30.0 1 3.500 0.5 10.0 90.0 2 6.500 0.5 10.0 90.0 3 6.10 0.5 5.0 95.0 4 7.500 0.5 5.0 95.0 5 9.500 0.5 70.0 30.0 6 20.0 0.5 70.0 30.0 SN/T 5111-2019???? ????, ????,?

Appendix C

(Informative appendix) Standard multiple reaction detection (MRM) chromatogram SN/T 5111-2019???? ????, ????,?

Appendix D

(Informative appendix) Reference MS conditions D. 1 Impact gas pressure CAD. 4.0Pa D. 2 Curtain gas pressure CU. 20.00 Pa D. 3 Atomizing gas pressure GS1. 50.00 Pa D. 4 Auxiliary air pressure GS2. 50.00 Pa D. 5 Electrospray voltage IS. 550,000.00V D. 6 Ion source temperature TEM. 550,000 ° C D. 7 de-clustering voltage EP. 10.000V D. 8 CXF at the exit of the collision cell. 12.00V D. 9 See Table D for other mass spectrometry parameters. 1.

Table D. 1 Main reference mass spectrometry parameters

Compound name Qualitative ion pair m/z Quantitative ion pair declustering voltage/V Collision energy/V neomycin 615.4/293.3 615.4/323.3 615.4/293.3 3 110 2 32 SN/T 5111-2019???? ????, ????,?