4.10.03 (l) Standard food grinder.

AOAC Official Method 996.13 (m) Membrane filter.—0.4 µm porosity.

Ethoxyquin in Feeds
C. Reagents
Liquid Chromatographic Method
First Action 1996 (a) Water.—LC grade.
Final Action 1997 (b) Acetonitrile.—LC grade.
(Applicable for determination of 0.5–300 µg/g ethoxyquin in dry (c) Ammonium acetate.—0.01M. Dissolve 0.231 g ammonium
extruded pet food or meat meal.) acetate (ACS grade) in 300 mL H2O. Prepare fresh solution daily.
(d) LC mobile phase.—Acetonitrile and 0.01M ammonium
See Table 996.13 for the results of the interlaboratory study
acetate (70 + 30, v/v). Prepare fresh LC mobile phase daily. Before
supporting acceptance of the method.
use, filter and degas 30 min.
A. Principle (e) Ethoxyquin.—Liquid; approximately 93.5% purity.
Ethoxyquin is extracted with acetonitrile. Extract is analyzed by D. Preparation of Standard Solutions
isocratic liquid chromatography with fluorescence detection.
(a) Ethoxyquin standard stock solution.—400 µg/mL. Weigh the
B. Apparatus
equivalent of 0.1000 g liquid ethoxyquin into 250 mL amber
(a) Liquid chromatograph.—Generating 1500 ± 200 psi; with volumetric flask and dilute to volume with acetonitrile. (Note:
peak area integrator (manual or computer), isocratic LC pump, and Amount of ethoxyquin needed for preparation of stock solution is
column heater. Operating conditions: injection volume, 20 µL; flow based on purity of liquid, e.g., for purity of 93.5%, amount of liquid
rate, 1.3 mL/min; temperature, 35°C; fluorescence detector output, ethoxyquin = 0.100/0.935 = 0.1070 g.)
analog to digital conversion; detector settings: excitation, 360 nm; (b ) Ethoxyqui n standard solutions.—(1)
emission, 432 nm. Ethoxyquin retention time, 6 min. Pump LC
4.0 µg/mL.—Transfer 5.0 mL ethoxyquin standard stock solution
mobile phase through column 30–60 min at flow rate 1.3 mL/min
into 50 mL amber vol umetric flask, di lute to vol ume with
until system is equilibrated.
acetonitrile, and mix well. Transfer 5.0 mL to 50 mL amber
(b) LC column.—250 × 4.6 mm id, C18 octadecylsilane, 5 µm volumetric flask and dilute to volume with acetonitrile. (2)
spherical, 100 Å pore size.
1.0 µg/mL.—Transfer 25 mL 4.0 µg/mL ethoxyquin standard
(c) Autosampler.—Optional; delivering 20 µL. solution into 100 mL amber volumetric flask and di lute to vol ume
(d) Amber autosampler vials. with acetonitrile. (3) 0.4 µg/mL.—Transfer 10.0 mL 4.0 µg/mL
(e) Centrifuge or microcentrifuge.—Maintaining 3000 × g. ethoxyquin standard solution into 100 mL amber volumetric flask
(f) Microcentrifuge tubes.—2 mL. and dilute to volume with acetonitrile. (4) 0.10 µg/mL.—Transfer
(g) Amber volumetric flasks.—50, 100, and 250 mL. 5.0 mL 1.0 µg/mL ethoxyquin standard solution to 50 mL amber
(h) Analytical balance.—Readability 0.1 mg. vol u met ric flask and di lute to vol ume with acetonitrile.
(i) Repipetor.—Delivering 1.0–5.0 mL ± 1%. (5) 0.06 µg/mL.—Trans fer 6.0 mL 1.0 µg/mL ethoxyquin
(j) Vortex mixer. standard solution to 100 mL amber volumetric flask and di lute to
(k) Volumetric pipets.—5, 6, 10, and 25 mL. volume with acetonitrile. (6) 0.04 µg/mL.—Transfer 10.0 mL

Table 996.13. Interlaboratory study results for determination of ethoxyquin in feeds by liquid chromatography
a b
Sample Spike, ppm Mean, ppm Recovery, % No. of labs sr sR RSDr RSDR r R HorRat
c d
Poultry meal — 0.25 — 8 0.08 0.12 32 46 0.23 0.33 2.35
c
Dog food — 71.1 — 11 3.2 7.5 4.5 11 8.9 21 1.31
e
Poultry meal — 289 — 11 13 4.5 36 0.66
e
Cat food — 0.41 — 8 0.21 50 0.58 2.75
e
Dog food — 15.7 — 11 2.9 18 8.0 1.71
e
Fish meal — 0.52 — 9 0.15 28 0.42 1.60
e
Dog food 3.0 1.80 60.0 11 0.52 29 1.4 1.99
e
Cat food — 41.5 — 11 6.4 16 18 1.76
e
Dog food 30.0 24.8 82.7 11 2.4 9.5 6.6 0.97
e
Cat food — 0.42 — 10 0.23 55 0.64 3.04
e
Fish meal — 83.0 — 10 4.3 5.2 12 0.64
a
r = 2.8 × sr.
b
R = 2.8 × sR.
c
Repeatability (r) values were determined by comparing duplicate values of 1 sample to duplicate values of the other as blind duplicates.
d
Naturally contaminated samples.
e
Statistics were determined using average value obtained from duplicate values obtained from separate weighings of the same test sample.

 2005 AOAC INTERNATIONAL

0.4 µg/mL ethoxyquin stan dard so lu tion to 100 mL amber Signal-to-noise ratio of 0.01 µg/mL ethoxyquin standard solution
vol u met ric flask and di lute to vol ume with acetonitrile. should be ≥3. Five replicate injections of 0.1 µg/mL ethoxyquin
(7) 0.02 µg/mL.—Trans fer 2.0 mL 1.0 µg/mL ethoxyquin standard solution should give relative standard deviation (RSD)
standard solution to 100 mL amber volumetric flask and di lute to ≤2.0%. Otherwise, repeat calibration steps and check precision with
volume with acetonitrile. (8) 0.01 µg/mL.—Transfer 25 mL 0.1 µg/mL ethoxyquin standard solution. If RSD is still >2.0%,
0.04 µg/mL ethoxyquin standard solution to 100 mL amber check equipment for potential problems.
volumetric flask and dilute to volume with acetonitrile. Calculate ethoxyquin concentration using manual or computer
Ethoxyquin standard solutions are stable 1 day. It is critical that integration.
exposure to light is minimized.
G. LC Determination
Depending on analyte concentrations, certain concentrations of
ethoxyquin calibrating standard solutions may be eliminated. Be fore an a lyz ing test so lu tions, run ethoxyquin stan dard
Use separate calibration curves for matrixes with concentrations solutions to establish linearity. Use autosampler or manual injection.
above and below 0.1 µg ethoxyquin/mL. Run standard solution every 10 tests and at the end of batch to ensure
minimum drift of calibration.
E. Extraction
Use separate calibration curves for matrixes with concentrations
Grind laboratory sample to particle size of 0.85 mm or smaller. above and below 0.1 µg ethoxyquin/mL.
Store test samples sealed with minimized exposure to light and heat.
Dilute solutions containing >4.0 µg/mL with acetonitrile (i.e., add
Analyze test samples within 30 days of receipt.
200 µL supernatant to 1.8 mL acetonitrile and use 10 as dilution
Weigh (in du pli cate) 0.06–0.1 g test por tion into 2 mL
factor, F, in calculations.)
microcentrifuge tube. Using calibrated repipetor or volumetric
pipet, add 1.5 mL acetonitrile to each tube and cap securely. If larger H. Calculations
test portion is analyzed, proportionately increase volume of Calculate concentration of ethoxyquin, µg/g or ppm, in test
acetonitrile to maintain the ratio of 0.5 g test portion to 7.5 mL sample from calibration curve (using linear regression with line
solvent. forced through zero intercept) as follows:
Thoroughly mix 1 min on Vortex mixer and let stand 20 min.
Remix 1 min on Vortex mixer and then centrifuge 1 min at 3000 × g. C × 15
. ×F
Ethoxyquin, µg/g or ppm =
Transfer acetonitrile supernatant layer to amber autosampler vial W
and proceed with LC analysis.
F. System Suitability where C = ethoxyquin concentration from LC calibration curve,
Calibrate LC system with ethoxyquin calibrating standard µg/mL; 1.5 = volume of acetonitrile added to test solution, mL; F =
solutions. dilution factor; W = weight of test portion, g.
LC system must meet the following criteria: Ethoxyquin should Average duplicate values and report the result.
elute at ca 6 min. Total run time should not exceed 8–9 min. Slight Reference: J. AOAC Int. 80, 725(1997).
chromatographic adjustments may be needed to ensure proper
retention time. CAS-91-53-2 (ethoxyquin) 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline

 2005 AOAC INTERNATIONAL