49.10.02
AOAC Official Method 991.26 Refrigerate when not in use. Warm to room temperature before use.
Domoic Acid in Mussels
Liquid Chromatographic Method
First Action 1991
Final Action 1999
(Applicable to determination at ca 20 mg domoic acid/g tissue.)
Results of the interlaboratory study supporting acceptance of the
method:
sr = 3.5–3.6; sR = 1.8–23.9; RSDr = 1.9–4.8%; RSDR = 7.5–19.4%
A. Principle
Domoic acid is extracted from homogenized mussel tissue by
boiling for 5 min with 0.1M HCl. Mixture is cooled and centrifuged,
and an aliquot of supernate is diluted, filtered, and analyzed by
isocratic LC with UV detection at 242 nm with mobile phase of
CH3CN–H2O acidified to pH ca 2.5.
B. Apparatus
Equivalent apparatus may be substituted.
(a) Liquid chromatograph.—With in jec tion valve, solvent
delivery system, recording integrator, and variable wavelength UV
detector. Operating conditions: flow rate 0.5–1.5 mL/min (domoic
acid should elute between 5 and 15 min); injection volume 20 mL;
detector wavelength 242 nm.
(b) LC column.—Stainless steel, 15 cm ´ 4.6 mm id, packed with
reversed-phase C18, 5 mm material. Column brand does not affect
analysis if CH3CN concentration is adjusted in mobile phase as in
C(c).
(c) Membrane filters.—Disposable, plastic-sealed mini-filters
with Luer-Lok hub, 0.45 mm (3 cm diameter), attached to 5 mL glass
or disposable plastic syringe. [Millex HV (Millipore Corp.) meets
these specifications.]
(d) Centrifuge.—High speed, with timer. Capable of 3000 rpm
using 100 mL centrifuge tubes. (Sorvall Superspeed SS-3 meets
these specifications.)
C. Reagents
(a) Acetonitrile (CH3CN).—LC grade.
(b) Hydrochloric acid (HCl).—0.1M.
(c) Mobile phase.—Add 2 mL 8.5% aqueous H3PO4 to 873 mL
deionized water. Mix and check to ensure pH is ca 2.5. Add 125 mL
CH3CN, mix, and degas. Perform preliminary analysis of domoic acid
standard and adjust CH3CN concentration as necessary to give domoic
acid retention time ca 8 min (k¢, ca 6) under method conditions.
(d) Domoic acid standard solution.—1.09 ng/mL. Aqueous
solution of domoic acid (available from National Research Council
of Canada, ARL, Halifax, Nova Scotia B3H 3Z1, Canada).
D. Preparation of Test Solution
Extract and analyze test portion on same day. Refrigerate test
samples and extracts when not being handled. (Note: Domoic acid in
acidic extracts slowly decomposes if left at room temperature.)
Prepare homogenate as in 959.08D(a) (see 35.1.37). Accurately
weigh ca 50 g homogenate in clean 250 mL beaker. Add 50 mL 0.1M
HCl and stir thoroughly. Quickly heat mixture to boiling (within
10 min) and continue gentle boiling, with stirring, for exactly 5 min.
Immediately transfer hot beaker and contents to ice bath and let cool
to room temperature (ca 10 min).
Transfer cooled mixture to 100 mL graduated cylinder (with
stopper) or volumetric flask and dilute to 100 mL with 0.1M HCl.
Stir with glass rod, or stopper and shake to homogeneity. Transfer
portion of at least 50 mL homogeneous mixture to 100 mL
centrifuge tube. Centrifuge 5 min at 3000 rpm. Use volumetric pipet
to transfer 1.0–5.0 mL clear supernate to 50 mL volumetric flask
(vol ume trans ferred de pends on an tic i pated domoic acid
concentration). Dilute to volume with water and mix well.
Filter 1–2 mL aliquot of diluted supernate through disposable
filter attached to clean, dry 5 mL glass or disposable plastic syringe
and collect filtrate in 4 mL vial for LC determination.
E. Determination
Inject replicate 20 mL portions of domoic acid standard solution
into LC system until peaks (measured as height or area) for 3
consecutive injections do not vary by more than 3%. Ensure
baseline resolution of L-tryptophan from domoic acid; adjust mobile
phase composition accordingly.
Make alternate, duplicate injections of test solution from D and
standards.
Determine recoveries of domoic acid at 20 mg/g level.
F. Calculations
Calculate results as follows:
Domoic acid, mg/g = (R/R¢) ´ (W¢/W)
where R and R¢ are average peak heights or areas of test solution and
standard, respectively, and W and W¢ are weights injected of test
portion, mg, and standard, ng, respectively.
Reference: JAOAC 74, 68(1991).
ã 2005 AOAC INTERNATIONAL