Liquid Chromatography Tandem Mass Spectrometry for the Determination of 19 Veterinary Drug Residues in Organic Fertilizers (Draft for Solicitation of Comments)

This standard is compiled in accordance with the rules given in GB/T 1.

Appendix A of this standard is an informative appendix

This standard was proposed by the China Petroleum and Chemical Industry Federation

This standard is under the jurisdiction of the National Standardization Technical Committee for Fertilizers and Soil Conditioners (SAC/TC105)

Drafting organizations of this standard: Shanghai Customs Industrial Products and Raw Material Testing Technology Center, Shanghai Academy of Agricultural Sciences, Shanghai Institute of Chemical Technology Testing Co.

1 Scope

This standard specifies the method for the determination of 19 veterinary drug residues in organic fertilizers by liquid chromatography-tandem mass spectrometry

This standard applies to amantadine (AMT), sulfapyridine (SPD), sulfapyridazine (SDZ), sulfamethoxazole (SMX), sulfathiazole (STZ), and sulfonamides in commercial organic fertilizers with poultry manure as the main raw material.

Detection limit: STZ, SMM, SMT, SCP are all 5µg/Kg; the rest are all 20µg/Kg

2 Normative references

The following documents are indispensable for the application of this document

GB/T 1.

GB/T 6682 Analyze laboratory water specifications and test methods

3 Principle

The residual veterinary drug in the sample is extracted with McIlvain-EDTA solution and acetonitrile/acetic acid (9/1, v/v) solution, purified by HLB solid phase extraction column, and determined by high performance liquid chromatography tandem mass spectrometer, external standard or Quantification by internal standard method

4 Reagents and materials

Unless otherwise specified, only reagents confirmed to meet the requirements of residual detection and water that meet the requirements of GB/T 6682 are used in the analysis

4.

4.

4.

4.
4 Formic acid (HCOOH): chromatographically pure
.

4.
5 Citric acid (C5H8O7·H2O)
.

4.
6 Disodium hydrogen phosphate (Na2HPO4·12H2O)
.

4.
7 Disodium ethylenediaminetetraacetic acid (C10H14N2O8Na2·2H2O)
.

4.
8 Acetic acid (CH3COOH)
.

4.
9 0.
1 mol/L citric acid solution: Weigh 21.
01 g of citric acid (4.
5), dissolve in water, and dilute to 1 000 mL
.

4.
10 Disodium hydrogen phosphate solution: 0.
2 mol/L
.
Weigh 71.
63 g of disodium hydrogen phosphate (4.
6), dissolve in water, and dilute to 1000 mL
.

4.
11 Mcllvaine buffer solution: mix 1 000 mL of 0.
1 mol/L citric acid solution (4.
9) with 625 mL of 0.
2 mol/L disodium hydrogen phosphate solution (4.
10), if necessary, use sodium hydroxide or hydrochloric acid to adjust pH=4.
0 ± 0.
05
.

4.
12 Na2EDTA-Mcllvaine buffer solution: 0.
02 mol/L
.
Weigh 60.
5 g of disodium edetate (4.
7) into 1625 mL of Mcllvaine buffer solution (4.
11) to dissolve it, then dilute it with pure water 5 times and mix
.

4.
13 Acetonitrile + acetic acid (9+1): Measure 10 mL of acetic acid (4.
8) and mix with 90 mL of acetonitrile (4.
3)
.

4.
14 0.
1% formic acid water: accurately pipette 1 mL of formic acid (4.
4) into a 1,000 mL volumetric flask, dilute with pure water and dilute to 1,000 mL
.

4.
15 HLB solid phase extraction cartridge: Waters Oasis PRiME HLB 60 mg, 3 mL solid phase extraction cartridge
.

4.
16 Mixed standard stock solution (100 μg/mL)

Weigh 19 appropriate amounts of veterinary drug standard substances (4.
1) and dissolve them in methanol to prepare a standard stock solution of 100 μg/mL.
Store them in a refrigerator at -18°C.
The validity period is 3 months
.

4.
17 Mixed standard working solution (1μg/mL)

Accurately draw an appropriate amount of the mixed standard stock solution (4.
16), dilute with methanol to prepare a 1μg/mL mixed standard working solution, store it at 4 ℃, and have a validity period of 1 week
.

4.
18 Deuterated isotope internal standard stock solution (100 μg/mL)

Weigh appropriate amounts of deuterated norfloxacin, deuterated ciprofloxacin, and deuterated enrofloxacin standard materials, and dissolve them in methanol to prepare a standard stock solution of 100 μg/mL, and store in a refrigerator at -18 ℃.
The expiry date 3 months
.

4.
19 Mixed internal standard working solution (5μg/mL and 1 μg/mL)

Accurately draw an appropriate amount of deuterated isotope internal standard stock solution (4.
18), dilute with methanol to prepare internal standard working solutions of 5 μg/mL and 1 μg/mL, store at 4 ℃, valid for 1 week
.

4.
20 Compound solution configuration: accurately suck 6.
6 mL of phosphoric acid and 7.
4 mL of triethylamine and mix with 1,000 mL of water, and mix 85 mL of the mixed solution with 15 mL of acetonitrile

5 Apparatus and equipment

5.
1 High performance liquid chromatography tandem mass spectrometer
.

5.
2 Balance: Sensitivity 0.
1 mg, 0.
00001 g
.

5.
3 Centrifuge: the speed is not less than 4 000 r/min
.

5.
4 Oscillator
.

5.
5 Filter membrane: 0.
22μm
.
(Hydrophilic PTFE membrane or equivalent)

5.
6 pH meter: measurement accuracy is 0.
02
.

6 Sample preparation

Prepare laboratory samples in accordance with GB/T 8571
.
The sample is ground through a 1 mm test sieve during sample preparation.
If the sample is difficult to grind, it can be ground to pass the 2 mm test sieve
.
The sample is stored at -18°C and protected from light
.

7 Analysis steps

7.
1 Extraction

Weigh (2.
0 ± 0.
02) g of the sample in a 50 mL centrifuge tube, add 100 μL of 5μg/mL internal standard working solution (4.
19) and mix well, then add 5 mL of Na2EDTA-Mcllvaline buffer (4.
12), and mix well Then add 20 mL of acetonitrile + acetic acid (4.
13), shake on a shaker for 20 minutes, centrifuge at 4 000 r/min for 10 minutes, and use the supernatant for use
.

7.
2 Purification

Take Waters Oasis PRiME HLB 60 mg, 3 mL solid phase extraction column, pass 2 mL of the supernatant through the solid phase extraction column, maintain a flow rate of about one drop per second, accurately measure 1 mL of the effluent through a 0.
22 μm filter membrane, and wait for loading Machine testing
.

7.
3 Preparation of matrix matching standard curve

7.
3.
1 Preparation of external standard matrix matching standard solution

Take a blank organic fertilizer sample, follow steps 7.
1 and 7.
2, add a mixed standard working solution (4.
17) to the eluent, and configure a series of standard curves with concentrations of 0.
010, 0.
020, 0.
050, 0.
100, 0.
150 and 0.
200 μg/mL to work The solution was measured by liquid chromatography-tandem mass spectrometer (conditions refer to Table 1, Table 2)
.
Using the peak area of ​​the quantitative ion as the ordinate and the mass concentration of the standard solution as the abscissa, draw the matrix matching standard curve
.

7.
3.
2 Preparation of internal standard matrix matching standard curve

Take a blank organic fertilizer sample and process it according to steps 7.
1 and 7.
2.
Add the mixed standard working solution (4.
17) and mixed internal standard working solution (4.
19) to the eluent to make the concentration 0.
010, 0.
020, 0.
050, 0.
100, 0.
150 And 0.
200 μg/mL series of matrix-matching standard solutions (internal standard concentration of 0.
010 μg/mL), measured by liquid chromatography-tandem mass spectrometer
.
Ofloxacin and norfloxacin use deuterated norfloxacin as internal standard; ciprofloxacin uses deuterated ciprofloxacin as internal standard; enrofloxacin uses deuterated enrofloxacin as internal standard
.
The ratio of the peak area of ​​the quantitative ion of norfloxacin, ciprofloxacin, enrofloxacin, and ofloxacin to the peak area of ​​the quantitative ion of the corresponding internal standard is the ordinate, and the mass concentration of the standard solution is the abscissa , Draw the internal standard matrix matching standard curve
.

7.
4 Determination

7.
4.
1 Chromatographic reference conditions

a) Chromatographic column: C18 column, Thermo Hypersil GOLD aQ, 100 mm × 2.
1 mm (id), 1.
9 μm, or equivalent chromatographic column;

b) Chromatographic column temperature: 30 ℃; c) Mobile phase: Phase A is methanol; Phase B is 0.
1% formic acid aqueous solution (4.
14);

d) Flow rate: 0.
4 mL/min; e) Gradient elution: See Table 1 for gradient elution conditions
.
f) Injection volume: 2 μL;

7.
4.
2 Reference conditions for mass spectrometry

a) Ion source: Electrospray ion source (ESI);

b) Scan mode: positive ion scan;

c) Detection method: select reaction monitoring;

d) Spray voltage: 3800 V;

e) Ion transfer tube temperature: 350 ℃;

f) Nitrogen is used as sheath gas and auxiliary gas, sheath gas pressure is 40 arb, auxiliary gas pressure is 10 arb;

g) Argon is used as the collision gas, and the collision pressure is 1.
5 mTorr;

h) The precursor ions, quantitative/qualitative ions of the test substance and the corresponding collision energy and cone voltage are shown in Table 2
.

7.
5 Qualitative and quantitative methods

7.
5.
1 Qualitative method

Under the same experimental conditions, the deviation between the retention time of the analyte in the sample and the corresponding retention time in the matrix matching standard solution is within ±2.
5%, and the relative abundance of the target compound of the analyte in the sample is consistent with the matrix matching standard If the relative abundance deviation of the corresponding ion in the solution does not exceed the range specified in Table 3, it can be determined that the corresponding analyte exists in the sample
.

7.
5.
2 Quantitative method

1.
Take the sample solution and matrix matching standard solution for multi-point calibration, external standard method and internal standard method calculation
.
Norfloxacin, ciprofloxacin, enrofloxacin, and ofloxacin (four types in total) were quantified by internal standard method; the rest (15 types) amantadine, sulfapyridine, sulfapyridazine, and sulfamethoxazole , Sulfathiazole, Sulfamethazine, Sulfamethoxazole, Sulfamethizole, Sulfamethazine, Sulfa-6-methoxypyrimidine, Sulfamethoxypyridazine, Sulfamethoxazole, Sulfachloropyridine Sulfamethoxine, sulfadimethoxine, and sulfadimethoxine were quantified by external standard method
.
The response value of each veterinary drug in the sample solution and matrix matching standard solution should be within the linear range detected by the instrument.
If the range is exceeded, it needs to be diluted and injected for determination.
If it is below the range, it needs to be extracted and purified according to steps 7.
1 and 7.
2.
mL effluent, dried by nitrogen in a 40℃ water bath, dissolved in 1 mL reconstituted solution (4.
20), passed through a 0.
22μm filter membrane, and waited for the machine to test
.

7.
6 Blank test

Except that no sample is added, proceed as described above (7.
1 ~ 7.
4)
.

8 Result calculation

The residual amount of each veterinary drug in the sample is calculated as the mass fraction ω, and the value is expressed in milligrams per kilogram (mg/kg).
The internal standard method is calculated according to formula (1), and the external standard method is calculated according to formula (2)
.

Where:

ω Internal standard --- The residual amount of the analyte in the sample measured by the internal standard method, in milligrams per kilogram (mg/kg);

ω External standard———The residual amount of the analyte in the sample measured by the external standard method, in milligrams per kilogram (mg/kg)

Ci ——— The concentration of quinolones in the sample preparation solution, in milligrams per liter (mg/L)

ρ—The mass concentration of the test substance in the standard working solution of the matrix, in micrograms per milliliter (µg/mL);

A——The chromatographic peak area of ​​the test substance in the sample solution;

As——The chromatographic peak area of ​​the analyte in the matrix standard working solution;

V——The final volume of the sample solution, the unit is milliliters (mL);

m——The mass of the sample represented by the sample solution, in grams (g)
.

Note: The calculation result should be deducted from the blank value.
The calculation result is expressed as the arithmetic average of two independent measurement results obtained under repeatability conditions, and two significant digits are retained
.

8 Precision

Under repeatability conditions, the absolute difference between two independent test results obtained shall not exceed the repeatability limit (r), see Appendix D
.

In the reproduction conditions, the absolute difference between two independent test results must not exceed the reproducibility limit (R), see Appendix D
.

Appendix A (informative appendix)

Total ion chromatogram of standard solution and blank sample addition and recovery

A.
1 Total ion current diagram of 19 veterinary drugs and 3 internal standard mixed standard solutions

See Figure A.
1

A.
2 Selected ion chromatogram of 19 veterinary drugs and three internal standard mixed standard solutions

See Figure A.
2

A.
3 Selected ion chromatogram of blank organic fertilizer sample added and recovered

See Figure A.
3

Appendix B Nitrogen Blowing Concentration Operation

B.
1 Reconstituted solution configuration: accurately draw 6.
6 mL of phosphoric acid and 7.
4 mL of triethylamine and mix with 1,000 mL of water, and mix 85 mL of the mixed solution with 15 mL of acetonitrile
.

B.
2 Nitrogen blowing and concentration operation: After extraction and purification according to steps 7.
1 and 7.
2, accurately measure 2.
5 mL of the effluent, dry it in a 40℃ water bath with nitrogen, and dissolve it with 1 mL of reconstituted solution, pass it through a 0.
22 µm filter membrane, and wait for machine testing
.