Detection of Nitrofuran Metabolites in Shrimp

A. SCOPE

This method is used for total residues screening of 3-amino-2-oxazolidinone (AOZ), 5-methyl-morpholino-3-amino-2-oxazolidinone (AMOZ), semicarbazide (SC), and 1-aminohydantoin (AH), (which are metabolites of furazolidone, furaltadone, nitrofurazone, and nitrofirantion, respectively) in shrimp.

The method involves overnight acid hydrolysis and simultaneous derivatization with 2-nitrobenzaldehyde (2-NBA), pH adjustment, liquid-liquid extraction, and detection of 2-NBA derivatives of AOZ, AMOZ, SC, and AH by LC-MS-MS (liquid chromatography tandem mass spectrometry).

B. APPARATUS

1. Adjustable pipettors (Eppendorf)
2. Balance (AND HM-202)
3. Balance (Mettler PM 4800)
4. Water bath with shaker with a homemade platform (New Brunswick Scientific, Innova 3100)
5. Centrifuge (SORVALL, RC 5C Plus)
6. pH meter (ORION model 401 Aplus)
7. Vortex mixer (Vortex-Genie 2 mixer)
8. Solid phase extraction manifolder 24 port (Supelco/Sigma-Aldrich)
9. Evaporator: TurboVap with a 15 ml test tube rack (Zymark, Hopkinton, MA)
10. Disposable 50 ml centrifuge tubes (VWR, 21008-240)
11. Disposable 20 ml reservoirs (part No.12131011, Varian)
12. Disposable frits (part No.12131023, Varian)
13. Disposable 15 ml tube (part No. 3131-340, LabCon)
14. Disposable 3 ml syringe (part No. 309595, B.D.)
15. Disposable 2 µm syringe filter (GelmanLab, No. 2003-09)
16. 1.8 ml autosampler vial (Finnigan, No. A4954-010)

C. LC-MS-MS EQUIPMENT

D. CHEMICALS and SOLVENTS

1. Methanol (MeOH) (High purity solvent, Burdick & Jackson)
2. Ethyl Acetate (EtOAc) (High purity solvent, Burdick & Jackson)
3. Hydrochloric Acid (HCl) (Concentrated, Fisher Scientific)
4. Formic acid (class IIIA, 90%, Fisher Scientific)
5. Sodium Chloride (NaCl,)(Certified A.C.S, Fisher Scientific)
6. Dipotassium hydrogen phosphate trihydrate (K₂HPO₄) (Certified A.C.S, Fisher Scientific)
7. Solid and dry pellet sodium hydroxide (NaOH) (Mallinckrodf)
8. 2-Nitrobenzaldehyde (Sigma-Aldrich)
9. Distilled and deionized water generated in house
10. NBA-SC, NBA-AH, NBA-AMOZ, and NBA-AOZ (2-NBA derivatives of SC, AH, AMOZ, and AOZ, respectively; 10 mg analytical standards, identity confirmed by NMR, HPLC purity > 99% (Sigma-Aldrich cat. # 33868, 33869, 33870, and 33871, respectively)

E. SAFETY

Safety procedures for handling, storage, and disposal of chemical solvent and their wastes should be strictly followed. The safety precautions for running instruments and apparatus should be strictly followed. Protective clothing, gloves, and safety glasses should be worn at all times when handling solvents or chemicals, and when performing procedure. Fume hood should be used when handling hazardous solvents and chemicals. The material safety data sheet (MSDS) for chemicals used should be followed.

F. REAGENTS

1. 0.125M HCl
   Add 5.2 ml of conc. HCl to approximately 200 ml of water in a 500 ml volumetric flask. Add water to mark and mix by inverting.

2. 0.1M K₂HPO₄
   Dissolve 17.4 g K₂HPO₄ in less than 1 L of water. Transfer the solution into 1L volumetric flask and add water to the mark.

3. 0.8M NaOH
   Dissolve 3.2 g NaOH in less than 100 ml of water. Transfer the solution in 100 ml volumetric flask and add water to the mark.

4. 0.125M NaOH
   Dilute 10 ml of 0.8 M NaOH into 630 ml of water.

5. 0.1% formic acid
   Add 1 ml of formic acid into less than 1 L of water in a 1 L water volumetric flask and add water to the mark.

6. 50 mM 2-NBA
   Dissolve 0.07555g 2-NBA in 10 ml MeOH.

7. 50/50 v/v water/MeOH
   Add 50 ml MeOH into 50 ml of water.

G. STANDARD SOLUTIONS

1. Dissolve 0.43, 0.56, 0.46, and 0.33 mg of NBA-AH, NBA-SC, NBA-AOZ, and NBA-AMOZ, respectively, in 100 ml MeOH. The resulting solutions (stock mixtures) contain 4.3 ng/µl, 5.6 ng/ml, 4.6 ng/µl, and 3.3 ng/µl of NBA-AH, NBA-SC, NBA-AOZ, and NBA-AMOZ, respectively. These concentrations correspond to 2 ng/µl of the underivatized metabolites (AH, SC, AOZ, and AMOZ).
2. Dilute the stock mixture 500 times to prepare solution D500: 10 µl of stock solution plus 5 ml of 50/50 (v/v) MeOH/water (this concentration corresponds to 4 pg/µl of the underivatized metabolites).
3. Prepare solution D1000: 2 ml of D500 solution plus 2 ml of 50/50 (v/v) MeOH/water (this concentration corresponds to 2 pg/µl of the underivatized metabolites).
4. Prepare solution D2000: 2 ml of D1000 solution plus 2 ml of 50/50 (v/v) MeOH/water (this concentration corresponds to 1 pg/µl of the underivatized metabolites).
5. Prepare solution D4000: 2 ml of D2000 solution plus 2 ml of 50/50 (v/v) MeOH/water (this concentration corresponds to 0.5 pg/µl of the underivatized metabolites).
6. Prepare solution D8000: 2 ml of D4000 solution plus 2 ml of 50/50 (v/v) MeOH/water (this concentration corresponds to 0.25 pg/µl of the underivatized metabolites).

H. SAMPLE PREPARATION PROCEDURES

1. Weigh 2.0 (± 0.1) g homogenized shrimp sample into the bottom of a 50 ml labeled centrifuge tube (VWR 21008-240). Record the weight of the sample.
2. Add 10 ml of 0.125 M HCl and 400 µl of freshly prepared 50 mM 2-NBA in MeOH to each sample. Vortex-mix the tube with a cap on at setting 70 for 15 seconds.
3. Place samples overnight (16 hours) into a water bath at 37°C with shaker set at 80 rpm.
4. Cool samples to room temperature. Add 1 ml of 0.1M K₂HPO₄ and 1 ml of 0.8 M NaOH. Vortex-mix at setting 70 for 15 seconds.
5. Determine the pH of the sample with a pH meter. Adjust pH to 7.3 ± 0.2 with 0.125 M HCl or NaOH. Record the final pH. Wash the electrode with water. Collect washing water. The final volume of the sample should be no more than 20 ml. Record the final volume.
6. Centrifuge samples at 4°C and 3000 rpm for 5 min.
7. Clean inside and outside of metal tubing on the filtration manifold with water. Attach 20 ml reservoirs (part No.12131011, Varian) to the manifold. Insert a frit (part No. 12131023, Varian) on the bottom of the reservoir. Place the frit rough face up. Put a new labeled 50 ml centrifuge tube under each reservoir.
8. Decant the supernatant into the reservoir. Keep shrimp pellet for washing in next step. Apply vacuum and collect the filtrate.
9. Add 3 ml of water into the shrimp pellet. Vortex-mix at setting 70 for 15 seconds. Centrifuge at 4°C and 3000 rpm for 5 min.
10. Decant the second supernatant into the same reservoir. Apply vacuum and collect the second filtrate in the same 50 ml tube.
11. Add water to adjust the total volume of the filtrate to 20 ml. Add approximately 0.5 g of NaCl and 12 ml of EtOAc to the filtrate. Close the tube with a cap and vortex-mix at setting 70 for 15 seconds.
12. Centrifuge samples at 4°C and 3000 rpm for 5 min.
13. Pipet top layer of EtOAc into a new labeled 15 ml tube. Add 2 ml of water into EtOAc extract. Pipet out the bottom (water) layer and discard it.
14. Evaporate EtOAc extract to dryness using Zymark evaporator at 40°C.
15. Add 1ml of 50/50 (v/v) MeOH/water into each tube with dried sample. Vortex-mix at setting 70 for 15 seconds.
16. Transfer the final solution into a 3 ml syringe (B.D. 309595) with a 2 µm syringe filter (GelmanLab, 2003-09). Filter the solution into a labeled 1.8 ml autosampler vial (Finnigan, No. A3954-010) for LC/MS/MS analysis.

I. LC-MS-MS ANALYSIS PROCEDURES

J. CONFIRMATION CRITERIA

1. Retention time of the analytes must agree within 5% of that of standard.
2. The two ion ratios of the analytes must agree within 10% of that of standard.

Comments or questions on method procedures may be directed to:

Sensui Wang, Chemist
FDA/ORA/NERL/HFR-NE580
158-15 Liberty Ave.
Jamaica, NY 11433
718-340-7176
sensui.wang@fda.hhs.gov

or

Albert Thompson, Chemist
FDA/ORA/NERL/HFR-NE580
158-15 Liberty Ave.
Jamaica, NY 11433
718-340-7172
albert.thompson@fda.hhs.gov

Press inquiries MUST be directed to:

FDA Office of Public Affairs
Tel: 301-827-6242
Fax: 301-827-1683