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?