Comparison of Simultaneous Nitrofuran (NF) and Chloramphenicol (CAP) Analytical Methods for AOAC Validation Study
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1 Comparison of Simultaneous Nitrofuran (NF) and Chloramphenicol (CAP) Analytical Methods for AOAC Validation Study Haejung An Pacific Regional Lab-Southwest, FDA, Irvine, CA
2 Three NF/CAP Methods Published in JAOAC Special Issue El-Demerdash et al. FDA Kansas Lab Veach et al., FDA Arkansas Lab An et al., FDA, PRLSW
3 NF Parent Drug, Metabolites and Their Derivatives Furazolidone AOZ NBA-AOZ
4 Depletion of Furazolidone and AOZ Chicken Pork Ref: McCracken et al., Food Add. Contamin. 2001, 18(11): Ref: Cooper et al, Food Add. Contamin. 2005, 22(5):
5 Distribution of Free and Bound NF Metabolites in Therapeutically Treated Porcine Muscle Ref: McCracken and Kennedy. Food Add. Contamin. 1997, 14(11):
6 Release of Bound NF Metabolites 6 and 2-NBA Derivatization 2-NBA with M HCl 16 Hr incubation with agitation at 37 C Originally developed by Hoogenboom et al., 1991 Center for Veterinary Medicine, FDA Northeast Regional Laboratory, FDA Pacific Regional Lab Southwest, FDA 4 Hr incubation at 55 C (Verdon) 2 Hr incubation with sonication at 37 C (In-House) 6 Min microwave digest, ramp to and at 95 C (Veach) Hoogenboom et al Toxic. in Vitro 5(1):31-38 Verdon et al., Anal. Chim. Acta 586:
7 Free vs. Bound NF Metabolites Impact on AOZ Quantitation in Shrimp Muscle Treatment 16-Hr Incubation 1-Hr Sonication Samples AOZ Conc (ppb) Recovery (%) AOZ Conc (ppb) Recovery (%) Matrix BL (# ) Matrix Std 1 ppb A (# ) Matrix Std 1 ppb B (# ) Incurred Sample (# Lot34-11) Incurred Sample Spike NF 1 ppb (# Lot34-11)
8 Three Parts to NF Analysis Hydrolysis of covalently bound NF metabolites 1. Need mild acid hydrolysis overnight to release them 2. Need to derivatize the released metabolites to stabilize them and to increase MW for the detection in mass spec using 2-nitrobenzaldehyde (NBA) Extraction of released metabolites 1. SPE or liquid-liquid extraction 2. Choice of organic solvents, EA and ACN Detection using LC-MS/MS 1. (+) for NF and (-) for CAP 2. APCI/ESI for NF and ESI for CAP
9 Details of Three NF/CAP Methods Steps El-Demerdash et al Veach et al Hydrolysis and Derivatization Neutralization/ Extr Recovery M HCl and 2-NBA and 16 Hr Incubation at 37 C with Shaking 0.1 M K 2 HPO 4 and 0.8 M NaOH to ph Centrifuge and Collect SPN (reextr with water) Fat Removal Hexane _ Extraction Manual SPE: (Oasis HLB 3 ml, 60 mg) Load samples Condition with EA, methanol, and water. Wash with 30% MeOH and hexane. Dry cartridge. Elute with EA. Wash with water and recover EA M HCl and 2-NBA and microwave digest, 5 min ramp to 95 C and 1 min hold 1 M K 2 HPO 4 to ph 7.3 Centrifuge and collect SPN and recentrifuge on PVDF 50mL filtration tube. Automated SPE: (Gilson GX 274-ASPEC, Oasis HLB 3 ml, 60 mg) Precondition with methanol and water. Load SPN. Wash with water and 30% MeOH Dry cartridge. Elute with EA. LC Prep Evaporate and Filter Evaporate and Filter LC-MS/MS Detection Single Injection (+/-) Double Injection APCI (+) for NF and ESI (-) for CAP
10 Details of Three NF/CAP Methods Steps An et al Hydrolysis and Derivatization Neutralization/ Extr M HCl and 2-NBA with 16 Hr Incubation with Shaking 1 M K 2 HPO 4 (ph 7.3) Acetonitrile Fat Removal - Extraction LC Prep LC-MS/MS Detection Liquid-Liquid Extraction: Add ACN (Done on previous step) Add Salt for Phase Separation Evaporate and Chill and Microfuge Single Injection (+/-)
11 Comparison of NF/CAP Extraction Methods Steps El-Demerdash et al. Veach et al. An et al. Hydrolysis and Derivatization 16 Hr Mild Agitation 5 min using Microwave 16 Hr Mild Agitation Neutralization Yes Yes Yes Filtration Yes Yes - Fat Removal Yes/Hexane - - Extraction Manual SPE Automated SPE Liquid-Liquid Extr Evaporation Ethyl Acetate Ethyl Acetate Acetonitrile Chilling min Reconstitute Filter Filter Microfuge Detection Single Injection (+/-) Double Injection NF (+) APCI and CAP (-) ESI Single Injection (+/-)
12 Time Table and Work Flow of Methods 3 Samples Excluding Sample Homogenization Color Codes Spike Hydrolysis Neutral/ Filtration Hexane Clean up Extraction Evap/ Recon LCMS Detection El-Demerdash et al., Hr Hr, Overnight 1 Hr Manual SPE:?? 1 Hr min 8 Hr Veach et al., Hr 5 min 1 Hr Overnight?? Auto SPE:?? 1 Hr 8x2 Hr An et al., Hr 16 Hr, Overnight 30 min 30 min 2 Hr 8 Hr
13 Method Validation Criteria According to the Office of Foods Target levels: NF at 1 ng/g (ppb) CAP at 0.3 ng/g (ppb) Three matrices with duplicate (3x2 experiments) Three spike levels in triplicate (min of 18 reps/level) LOD: Four fold lower than target levels Linearity: R2 >0.995 Accuracy: +/-20% from the target levels Precision: <20% RSD CCV and ICV: <10%
14 Method Validation and Performance Target: NF 1 ppb and CAP 0.3 ppb, Shrimp Parameters El-Demerdash et al. Veach et al. An et al. LOD Range (ppb) NF: 0.5 ppb CAP: 0.3 ppb NF: ppb (4 pt) CAP: ppb (4 pt) NF: ppb CAP: ppb NF: (5 pt) CAP: (5 pt) Linearity (R2) >0.99 >0.995 >0.995 NF: 0.1 ppb CAP: 0.03 ppb NF: (5 pt) CAP: (5 pt) Spike Levels (ppb) Reps (n) for ea spike level %Recovery (Accuracy) %RSD (Precision) IRC Confirm. For Spikes Incurred Sample Test NF: 0.75, 2, 4 CAP: 0.45, 1.2, 2.4 NF: & 10 (1) CAP: & 2 (1) 6 9 (7 for lowest and 1 at highest) NF/CAP: % NF: % CAP: % % NF: <8.3% CAP: <4% NF: ppb CAP: ppb 27 (3 levels, 9 studies) NF/CAP: % <18% Not Reported Not Reported All at or above regulatory levels No No Yes
15 Decisions Need To Be Made Hydrolysis method to ensure the complete release of bound NF metabolites Overnight Rapid microwave digest Extraction method depending on the labs need SPE Automated SPE Liquid-Liquid extraction LCMS detection depending on the labs capabilities Polarity switching Dual injection using APCI for NF and ESI for CAP
16 THANK YOU! ANY COMMENTS?
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