Comparison of Simultaneous Nitrofuran (NF) and ... of Simultaneous Nitrofuran (NF) and...
Transcript of Comparison of Simultaneous Nitrofuran (NF) and ... of Simultaneous Nitrofuran (NF) and...
Comparison of Simultaneous
Nitrofuran (NF) and Chloramphenicol
(CAP) Analytical Methods for AOAC
Validation Study
Haejung An
Pacific Regional Lab-Southwest, FDA, Irvine, CA
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
NF Parent Drug, Metabolites and
Their Derivatives
Furazolidone AOZ NBA-AOZ
Ref: McCracken et al., Food Add.
Contamin. 2001, 18(11): 954-959. Ref: Cooper et al, Food Add.
Contamin. 2005, 22(5): 406-414.
Depletion of Furazolidone and AOZ
Chicken Pork
Ref: McCracken and Kennedy. Food Add. Contamin. 1997, 14(11): 507-513.
Distribution of Free and Bound NF
Metabolites in Therapeutically
Treated Porcine Muscle
Release of Bound NF Metabolites
and 2-NBA Derivatization
6
2-NBA with 0.125 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. 1991. Toxic. in Vitro 5(1):31-38
Verdon et al., 2007. Anal. Chim. Acta 586:336-347
Treatment
16-Hr Incubation 1-Hr Sonication
Samples
AOZ Conc
(ppb)
Recovery
(%)
AOZ Conc
(ppb)
Recovery
(%)
Matrix BL (#659397-12) 0.006 0.002
Matrix Std 1 ppb A
(#659397-12) 1.000 99.4 1.000 99.8
Matrix Std 1 ppb B
(#659397-12) 1.014 100.8 0.995 99.3
Incurred Sample (#665227-Lot34-11)
1.292 0.311
Incurred Sample Spike NF 1 ppb (#665227-Lot34-11)
2.358 106.7 1.389 107.8
Free vs. Bound NF Metabolites Impact on AOZ Quantitation in Shrimp Muscle
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
Details of Three NF/CAP Methods Steps El-Demerdash et al. 2015 Veach et al. 2015
Hydrolysis and
Derivatization
0.125 M HCl and 2-NBA and 16
Hr Incubation at 37°C with
Shaking
0.125 M HCl and 2-NBA and
microwave digest, 5 min ramp to
95°C and 1 min hold
Neutralization/
Extr Recovery
0.1 M K2HPO4 and 0.8 M NaOH
to pH 6.25-7.25
Centrifuge and Collect SPN (re-
extr with water)
1 M K2HPO4 to pH 7.3
Centrifuge and collect SPN and re-
centrifuge on PVDF 50mL filtration
tube.
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.
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
Steps An et al. 2015
Hydrolysis and
Derivatization
0.125 M HCl and 2-NBA with
16 Hr Incubation with Shaking
Neutralization/
Extr
1 M K2HPO4 (pH 7.3)
Acetonitrile
Fat Removal -
Extraction Liquid-Liquid Extraction:
• Add ACN (Done on previous
step)
• Add Salt for Phase Separation
LC Prep Evaporate and Chill and
Microfuge
LC-MS/MS
Detection
Single Injection
(+/-)
Details of Three NF/CAP Methods
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 - - 30 min
Reconstitute Filter Filter Microfuge
Detection Single Injection
(+/-)
Double Injection
NF (+) APCI and CAP
(-) ESI
Single Injection
(+/-)
Time Table and Work Flow of Methods 3 Samples Excluding Sample Homogenization
Neutral/
Filtration
Evap/
Recon
LCMS
Detection
5
min Auto SPE: ?? 1 Hr 8x2 Hr
16 Hr, Overnight
1 Hr
16 Hr, Overnight Manual SPE: ?? 1 Hr
8 Hr
1 Hr
Spike
3 Hr
3 Hr
3 Hr 30
min 30 min 2 Hr
8 Hr
Overnight??
El-Demerdash et al., 2015
Veach et al., 2015
An et al., 2015
30
min
Hexane Clean up
Hydrolysis Extraction Color
Codes
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%
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
NF: 0.5 ppb
CAP: 0.3 ppb
NF: 0.037-0.067 ppb
CAP: 0.003 ppb
NF: 0.1 ppb
CAP: 0.03 ppb
Range (ppb) NF: 0.5-5 ppb (4 pt)
CAP: 0.3-10 ppb (4 pt)
NF: 0.25-2 (5 pt)
CAP: 0.05-0.4 (5 pt)
NF: 0.25-5 (5 pt)
CAP: 0.075-1.5 (5 pt)
Linearity (R2) >0.99 >0.995 >0.995
Spike Levels
(ppb)
NF: 0.75, 2, 4
CAP: 0.45, 1.2, 2.4
NF: 0.25-2 & 10 (1)
CAP: 0.05-0.4 & 2 (1)
NF: 0.5-2 ppb
CAP: 0.15-0.6 ppb
Reps (n) for
ea spike level
6 9 (7 for lowest and 1 at
highest)
27
(3 levels, 9 studies)
%Recovery
(Accuracy)
NF/CAP:
93-107%
NF: 95.1-105%
CAP: 91.3-106%
NF/CAP:
98-109%
%RSD
(Precision)
1.2-8.2% NF: <8.3%
CAP: <4%
<18%
IRC Confirm.
For Spikes
Not Reported Not Reported All at or above
regulatory levels
Incurred
Sample Test
No No Yes
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
THANK YOU! ANY COMMENTS?