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35. Fish and Other Marine Products
John M. Tennyson and R. Steve Winters,
,Nationa/ Marine #Fisheries Service
35.1 .Ol
AOAC Official Method 937.07
Fish and Marine Products
Treatment and Preparation of Sample
Procedure
First Action 1937
Final ,Action 1996
To prevent loss of H,O during preparation and subsequent han-
dling, use test portions as large as practicable. Keep ground mate-
nal m container with air-tight cover. Begin all determinations as
soon as practicable. If any delay occurs, chill test portions to inhibit
decomposition. In general, prepare test portion of fish as it is usu-
ally prepared by consumer, by including skin and discarding
bones, but subject to overall rule of edibility, e.g., inedible catfilsh
skin is discarded; softened canned salmon bones are included, sad-
dines are examined whole. Instructions may be modified in accor-
dance with purpose of specific examination. Prepare test portion
for analysis as follows:
(a) Fresh fish.-Clean, scale, and eviscerate fish. In case Iof
small fish 6 in. (515 cm), use 5-10 fish. In case of large fish, from
each of 23 fish cut 3 cross-sectional slices 1 in. (2.5 cm) thick,
1 slice fromjust back of pectoral fins, 1 slice halfway between first
slice and vent, and 1 slice just back of vent. Remove bone. For in-
termediate-size fish, remove and discard heads, scales, tails, fins,
guts, and Inedible bones; fillet fish to obtain all flesh and skin from
head to tail and from top ofback to belly on both sides. For detenm-
natlon of fat and fat-soluble components, skin must be Included,
since many fish store large amounts of fat directly beneath skin.
Pass test portion sample rapidly through meat chopper 3 times.
Remove unground material fr’om chopper after each gnnding and
rmx thoroughly with ground material. Meat chopper should have
holes as small as practicable (1.5-3 mm L&-x in.] diameter) and
should not leak around handle end As alternative for soft fish,
high-speedblender may be us’ed. Blend several minutes, stopping
blender frequently to scrape down sides of cup.
(b) Canned fish, shellfish,. and other canned marine prod-
ucts.-Place entire contents of can (meat and liquid) in blender and
blend until homogeneous or grind 3 times through meat chopper. For
large cans, drain meat 2 min on No. 8-12 sieve and collect all liqmd.
Determine weight of meat and volume of liquid. Recombine test
portion of each in proportionate amounts. Blend test recombmed
portlons m blender (or grind) until homogeneous.
(c) Canned marine products packed in oil, sauce, broth, or wa-
ter.-Dram 2 min on No. 8 sieve. Prepare solid test portion as m (b).
Liquid maly be analyzed separately, if desired, or reincorporated
with solids. H,O 1susually discarded.
(d) Fish packed in salt or brine.-Drain and discard brine and
rinse off adhenng salt crystals with saturated NaCl solution. Dram
again 2 min and proceed as m (a).
(e) Dried smoked or dried saltfish.-Proceed as in (a).
(f) FrozenJish.--Let thaw at room temperature, and &scard dram-
mg. (I) Fillet-Use enbre piece. (2) Wholefish.-Proceed as m (a).
Chapter Editors
(g) Shellfish other than oysters, clams, andscallops.-If test sam-
ple is received in shell, wash as in (h) and separate edible test portions
in usual way. Prepare edible test portion for analysis as in (a).
(h) Shell oysters, shell clams, and scallops.-Wash shells in po-
table H,O to remove all loose silt and dirt, and drain well. Shuck
enough oysters or clams into clean dry container to yield 2500 mL
(1 pt) drained meats. Transfer shellfish meats to skimmer, 953.11A
(see 35.1.07), pick out pieces of shell, drain 2 min on skimmer, and
proceed as in (i) or (j).
(i) Shucked clams or scallops.-Prepare as in (b).
(j) Shucked oysters -Blend meats, including liquid, 1-2 min in
high-speed blender.
(k) Breadedfish, raw or cooked.-Do not remove breading or
skin. Proceed as in (a).
References: JAOAC 20,70(1937); 21,85(1938); 35,218(1952);
36,608(1953); 38, 194(1955); 59,312(1976).
Revised: March 1997
35.1.02
AOAC Official Method 963.18
Net Contents
of Frozen Seafoods
Drained Weight
Procedure
First Action 1963
Codex-Adopted-AOAC Method*
Set scale, 963.26A (see 42.3.01), on firm support and level. Ad-
Just 0 load indicator or rest point and check sensitivity.
(a) Glazed seafoods.-Remove package from low temperature
storage, open immediately, and place contents under gentle spray of
cold H,O. Agitate carefully so product IS not broken. Spray until all
Ice glaze that can be seen or felt 1sremoved. Transfer product to cir-
cular No. 8 sieve, 8 in. (20 cm) diameter for 0.9 kg (2 lb) and 12 in.
(30 cm) for >0.9 kg (2 lb). Without shifting product, incline sieve at
angle of 17-20°C to facilitate drainage and drain exactly 2 min (stop
watch). Immediately transfer product to tared pan(B) and weigh (A).
Weight product = A - B.
(b) Unglazed seafoods.-See 963.26B (see 42.3.01).
Reference: JAOAC46,31(1963).
Revised: March 1997
* Adopted as a Ccdex Defining Method (Type I) of water spraying and slev-
ing in net contents of products covered by glaze m quick frozen fillets of
ocean perch and gutted salmon.
Adopted as a Codex Defimng Method (Type I) of gravlmetry of net con-
tents of productscoveredby glaze in quick frozen blocks of fish, minced
fish and mixtures of fillets and minced fish flesh
Adopted as a Codex Defining Method (Type I) of weighmg of net contents
of products covered by glaze in quick frozen fish sticks (fish fingers) and
fish portions-breaded or in batter
0 2002 AOAC INTERNATIONAL
FISH AND OTHER MARINE PRODUCTS
Chapter 35, p. 2
35.1.03
AOAC Official Method 996.15
Fish Flesh Content (FFC)
in Frozen Coated Fish Products
First Action 1996
(Applicable to the determination of the FFC in frozen coated fish
products.)
(Note: The coated fish products industry uses the term “portions” in
the name of some of its products. The term “test portion” may be
rmsunderstood as only applicable to these products. Therefore, in
this method the term “test unit” is understood to apply to whatever
form IS being examined.)
Caurion: Use protective gloves when immersing and holding test
sample in water bath set at >43”C.
See Table 996.15 for the results of the interlaboratory study sup-
porting the acceptance of the method.
A. Principle
Method uses combination of (1) heat and H,O to breakdown adhe-
sive properties of coating (batter and/or breading) and (2) hands to
assrst in determinmg when coating’s ability to adhere to flesh’s
frozen surfaces is diminished and can be easily removed.
B. Apparatus
(a) Water baths-Pr~maty (17-49”(I) and secondary (17-30”(Z).
(b) Thermomererx-Two; immersion type, accurately measur-
ing to * 1°C.
(c) Thermometer holders.-Two; with clips.
(d) Balance.-Accurately weighing to 0.1 g.
(e) Stop watch.-Reading seconds.
(f) Paper towels.
(g) Spatula.4 m. (ca 10 cm) blade with rounded tip.
(h) Nur pick.
C. Preparation of Test Sample
Maintain integrity of frozen test samples by storing in freezer un-
til ready to remove batter and/or breading. Take mto account all ap-
plied coating when weighing coated test samples.
D. Determination
Set primary H,O bath temperature between 17-49”C. Set second-
ary H,O bath temperature between 17-30°C.
Weigh and record weight of each test umt while it is hard frozen.
Using hands, immerse and hold test umt in primary Ha0 bath until coat-
mg becomes soft and can be removed easily from still-frozen flesh.
Remove test unit from H,O bath and blot lightly with enough pa-
per towel to absorb excess H20. Complete blotting in <7 s. Scrape
and remove coatmg from flesh with spatula. If coating is difficult to
remove using hands, redip and hold partially debattered or
debreaded test unit m secondary Ha0 bath until coating becomes soft
and can be removed easily from still-frozen flesh.
Remove test unit from H,O bath and blot lightly with enough pa-
per towel to absorb excess H,O. Complete blotting in 17 s. Scrape
and remove coating from flesh with spatula. When necessary, repeat
redipping procedure and use nut pick to remove coating from any
voids (holes, spaces, or depressions) until all coating has been re-
moved from still-frozen flesh. Reweigh and record weight of
debattered and/or debreaded test unit.
-.~~--.~
0 2000 AOAC INTERNATIONAL
AOAC OFFICIALMETHODSOF ANALYSIS(2000)
(Nore: Several preliminary trials may be necessary to determme
optimum HZ0 bath temperatures, dip times, and number of dips re-
quired for debattering and/or debreadmg test units. The correct dip
time IS the mimmum time of immersion in Ha0 baths required before
coating on test unit can be scraped off easily, provided that coated
test unn is still solidly frozen.)
As a guide, no more than 1 initial dip (17-49”C) and 2 redtps
(17-30”(I) for a maximum of 2.5, 0.5, and 0.5 mm, respectrvely,
should be necessary.
E. Calculations
Calculate content of fish flesh, %, in test sample as follows:
Flesh, % = (IV, / Wb) x 100
where W, = weight of debattered and/or debreaded test unit; W, =
weight of coated test unit.
Reference: J. AOAC Inr. 80, 1235(1997).
Revised: March 1998
35.1.04
AOAC Official Method 976.16
Cooking Seafood Products
Procedure
First Action 1976
For fish blocks or other unbreaded test samples, cut 3 test portions,
each 4 x 3 x 0.5 in. (ca 10 x 7.5 x 1.2 cm) from test sample.
Cooking procedure is based on heating product to internal temper-
ature 160°F (70°C). Cooking times vary according to size of product
and equipment used. To determine cooking time, cook extra test por-
tion same way using temperature measuring device with probe of
known length to determine internal temperature. Cooking equip-
ment, Including cooking oil for deep fat frying, shall be free from
substances that interfere with sensory evaluation of cooked product.
Methods of heating product include, but are not limited to, baking,
bake-in-foil, broiling, boll-in-bag, shallow pan frying, deep fat frying,
oven frying, grilling, poachmg, steaming, and microwave heating.
References: JAOAC59,225(1976); 66,813(1983).
351.05
AOAC Official Method 973.24
Detection of Frozen
and Thawed Shucked Oysters
Disc Electrophoresis Method
First Action 1973
Final Action 1979
A. Principle
Latent forms of malic enzyme (ME) activity, solubthzed by freez-
ing and thawing, are differentiated from normally soluble form by
their drffermg rates of electrophoretic migration. Medium used to
develop satesof methyl activtty on gels 1sderived from reaction mix-
ture used for spectrophotometrtc assay of nicotinamide adenine
dinucleotide phosphate (NADP) reduction by ME, together with
tetrazolium dye and adjuvant. Sharply discontinuous (disc) buffer
system durmg polyacrylamrde gel electrophoresis and carefully
timed incubation period for htstochemical staining are essential.
Gels and solutions must be kept cold dunng electrophoresis. Full
work-day 1srequired for test. Do nor use commercially prepared gel
AOAC OFFICIAL METHODS OF ANALYSIS (2000) FISH AND OTHER MARINE PRODUCTS
Chapter35 p. 3

Table 996.15 Interlaboratory study results for determination of fish flesh content In frozen coated fish products (based on
intra-laboratory collaborative study results of the modified method)
Mean % fish flesh Mean%
Product APFFa DPFFb recovery' Sr SR RSDr,% RSDR, % rd Fle
Fillet blocks, raw breaded slicks
B/IO 36.1410 :33.5629 92.939 2.25190 2.27882 6.70948 6.78969 6.30532 6.38070
All4 49.2264 47.4115 98.303 2.05307 2.09314 4.33031 4.41482 5.74860 5.86079
IV01 62.3667 61.5532 98.724 2.47642 2.85438 4.02321 4.63726 6.93398 7.99226
Fillet blocks, raw breaded portions -
AJo 30.7542 28.6364 93.089 1.23469 1.46086 4.31160 5.10138 3.45713 4.09041
AJo3 56.4791 56.0997 99.336 1.34103 1.66026 2.39044 2.95948 3.75488 4.84873
c/o1 65.0645 65.7579 101.076 1.86375 2.16725 2.83426 3.29581 5.2185 6.06830
B/O1 77.0154 77.0468 100.044 0.80335 1.08992 1.04268 1.41462 2.24938 3.05178
Fillet blocks, raw breaded formed port ons
Afo4 36.5627 33.7388 92.250 2.08035 2.11721 6.16603 6.27529 5.82498 5.92819
Al05 52.5984 51.5287 97.993 2.45717 2.91705 4.76854 5.66101 6.88008 8.16774
52.4812)' (51.5292) (98.227) (3.27043) (3.47031) (6.34675) (6.73466) (9.15720) (9.71687)
c/o2 68.1410 67.3977 98.912 2.44424 3.12584 3.62659 4.63790 6.84387 8.75235
c/o3 85.3599 86.1709 100.955 1.23485 1.38000 1.43302 1.60147 3.45758 3.86400
(85.3518) (85.7242) (100.438) (1.43482) (2.09194) (1.67376) (2.44031) (4.01750) (5.857431
Fillet blocks, bafter-dipped sticks -
B/O2 43.6435 46.3990 106.402 3.01790 3.33241 6.50423 7.18208 8.45012 9.33075
D/O3 47.1455 45.1178 95.778 2.14514 2.96251 4.75453 6.56617 8.00839 8.29503
B/O3 56.9040 56.1931 98.743 2.19080 2.41134 3.89870 4.29116 6.13424 8.75175-
Fillet blocks, bafter-dipped portions -
0104 33.9510 33.2692 97.871 2.78348 3.97661 8.38653 11.95280 7.79374 11.13451
D/O4 50.9414 48.1860 94.550 1.93707 2.74004 4.01997 5.68840 5.42380 7.67211
AJO 61.6366 57.9171 93.973 1.92648 3.09530 3.32627 5.34435 5.39414 8.66684
(61.6322) (57.9880) (94.075) (2.46179) (3.30784) (4.24550) (5.70450) (6.89301) (9.26195)
Fillet blocks, precooked sticks -
Cl04 39.5959 34.1145 86.183 2.11368 2.41707 6.19582 7.08517 5.91830 6.76780
B/11 51.9958 48.4411 93.181 2.68598 3.00339 5.54483 6.20008 7.52074 8.40949
Al07 65.1124 60.6381 93.170 2.22276 3.48511 3.66562 5.74739 6.22373 9.75831 -
Fillet blocks, precooked portions -
AJO 40.9826 39.1195 95.584 1.24408 1.24408 3.18020 3.18020 3.48342 3.48342
B/O6 49.5313 48.1593 97.243 2.27357 2.45565 4.72093 5.09902 6.36600 6.87582
c/o5 68.0569 61.0507 92.419 1.34918 1.88435 2.20993 3.08854 3.77770 5.27618
Fillet blocks, fully cooked portions -
C/O6 76.2462 54.5157 71.746 4.22557 4.62680 7.75111 8.48710 11.83160 12.95504
Minced blocks, batter-dipped portions -
B/O7 30.3455 30.6260 100.988 2.1851.5 2.79689 7.13496 9.13241 6.11842 7.83129
D/05 47.8147 46.6756 97.682 2.91981 3.07373 6.25553 6.58531 8.17547 8.60644
B/O8 60.8697 58.5523 96.201 1.22854 1.89274 2.09818 3.23256 3.43991 5.29967 -
Minced blocks, precooked sticks -
Cl08 31.3085 24.7942 79.214 1.35011 1.72982 5.44527 6.97673 3.78031 4.84350
8109 47.7750 48.5133 97.368 2.18135 2.87328 4.68974 6.17734 6.10778 8.04518
D/06 53.5220 52.2995 97.758 1.90382 2.57832 3.84022 4.92991 5.33070 7.21930
Minced blocks,prewoked portons
Cl07 32.4689 30.0538 92.850 1.47933 1.86630 4.92228 6.20988 4.14212 5.22564
D/O7 59.8867 60.8030 101.533 1.9421!5 2.38702 3.19417 3.92582 5.43802 6.68366
FISH AND OTHER MARINE PRODUCTS AOAC OFFICIAL METHODSOF ANALYSIS(2000)
Chapter 35, p. 4

Table 996.16 (continued)

Mean % fish flesh Mean %
Product APFFa DPFF* recove$ Sr SR RSD,, % RSDR, % rd Re
Natural fillets, raw breaded fillets
Al10 38.0024 34.6633 91.195 2.31547 2.41498 6.67991 6.96698 6.48332 6.76194
A/11 49.2663 43.5693 88.451 2.93463 3.05227 6.73555 7.00555 8.21696 8.54636
A/12 65.4867 59.3448 90.607 3.17842 3.48347 5.35586 5.86989 8.89958 9.75372
All3 81.0088 79.9051 98.645 2.65963 2.65963 3.32848 3.32848 7.44696 7.44696
* APFF = actual percent fish flesh = weight of test sample before battering and/or breading and/or flying divided by weight of test sample after battering and/or
breading and/or frying and multiplied by 100. APFF was considered the control (unknown to collaborators)
b DPFF = determined percent fish flesh = weight of test sample after scraping divided by weight of test sample before scraping and multiplied by 100 DPFF was
considered the test (results from collaborators).
’ Mean percent recovery = the mean of the percent recoveries [DPFF/APFF) x IOO].
d r=28xs,
* R=2.8xs,.
’ Values in parentheses were calculated including outliers

solutions; pH controI is critical. All glassware must be meticulously X5521, or equivalent) and 1.8375 g BIS
cleaned, with final soak 26 h in Hz0 before rinsing and drying. Give (N,N’-methylenebisacrylamide, Eastman Kodak Co. No. 8383, or
glass columns final rinse with Kodak Photo-F10 solution (I+ 200) to equivalent) in H,O and dilute to 250 mL. (4) SoEution B.-O. 14%
ease removal of electrophoresed gels. (w/v) ammonium persulfate in H,O. (Caution: Acrylamide mono-
mers are neurotoxins. Wear impervious gloves, avoid pipetting solu-
tions by mouth, and when dry, handle only in effective fume removal
(a) Articles used in making gel columns.-(l) Glass col- device. Close medical supervision is advisable for persons exposed
umns.-62 mm long x 5 mm id soft glass tubing, with unpolished to this material.)
ends. (2) Disposable syringes.-5 mL without needle, used with Solutions A-l and A-2 are stable ca 6 months stored in brown bot-
0.76mm id, 1.22 mmod polypropylene tubing, for loading gels. (Sy- tles in refrigerator; Solutions A and B, only 1 week in refrigerator.
ringes may be washed and reused.) (3) Water-layer applica- Prepare running gel by mixing equal volumes Solutions A and B just
ror.-2 mL syringe, without plunger, used with 25 gage 1 in. before use.
(2.54 cm) hypodermic needle bent slightly for ease of application. (b) Stacker and sample gels.-(Large pore, photopolymerized.)
(4) Disposable syringes.-2.5 mL, with needle, for removing (1) Solution C.-Prepare by mixing Solutions C-l, C-2, and C-3
unpolymerized gel solutions during rinse. (5) Removing (I + 2 + 1). (2)Solution C-I.-Mix 14.95 g Tris, 1.15 mLTEMED,
tooZ.-Grind off flanges of 1 mL glass syringe; no plunger. Attach and ca 120 mL 1N HCl to pH 5.1, and dilute with H,O to 250 mL.
22 gage 5 cm (2 in.) hypodermic needle to one end and short length (3) Solution C-2.-Dissolve 50.0 g acrylamide and 12.5 g BIS in
of rubber tubing to other. Keep another short length of rubber tubing H,O and dilute to 500 mL. (4) Solution C-3.4.0 mg riboflavin in
attached to faucet adapter; add yZ plastic tubing-connector to each 100 mL H,O. (5) Solution 0.40% (w/v) sucrose in H,O.
piece of rubber tubing so that removing tool can be easily attached. All solutions are stable ca 6 months stored in brown bottles in re-
(b) Apparatus for photopolymerization of gels.-Convenient frigerator. Prepare stacker gel by mixing equal volumes Solutions C
set-up for photopolymerizing large pore gels is open-ended and D within 1 h of use; store at room temperature away from light.
wooden box with removable bottom panel. Glue row of small rub- Prepare gels by mixing 0.04 mL centrifuged tissue fluid with 1 mL
ber-base caps to bottom panel; (use those supplied with apparatus stacker gel just before use.
or obtain rubber cushions for file tray feet from office supply (c) Electrode bufler (IOX).-Dissolve 57.6 g glycine and 12.0 g
house). Mount fluorescent light beneath top of box, positioned so Tris in H,O and dilute to 2 L. Dilute 1 + 9 just before each run; pH
that row of rubber caps will be directly under light when bottom should be 8.3. Add few drops ca 0.1% (w/v) bromophenol blue aque-
panel is in place. Tops of glass columns, placed in rubber caps, ous solution to cathode buffer to light blue tint. Discard both cathode
should be ca 2.5 cm from light. and anode buffers after each run.
(c) Electrophoresis apparatus.-Disc electrophoresis bath as-
D. Preparation of Histochemical Reagent
sembly with regulated power source capable of maintaining con-
stunt current over O-50 milliamp range. For incubating 12 columns, prepare mixture of 20.0 mg NADP
(nicotinamide adenine dinucleotide phosphate,
C. Preparation of Electrophoretic Reagents ICN-Pharmaceuticals, Life Sciences Group), 15.0 mL 0.144M Tris
(a) Running gel.-(Small pore, chemical polymerized.) (I) Solu- buffer (17.44 g/L; store in refrigerator; pH 8.0), 9.0 mL 0.095M po-
tion A.-Prepare by mixing equal volumes Solutions A-l and A-2. tassium malate (127 mg L-malic acid neutralized with KOH and di-
(2) Solution A-I.-Mix 91.5 g Tris [tris(hydroxymethyl) luted to 10 mL), 2.4 rnL 0.06M KCN (dissolve 0.40 g in H,O, add
aminomethane], 0.575 mL TEMED (N,N,N’,NL-tetramethyl- 1M HCl to pH 8.0 [ca 6 mL] in hood, and dilute to 100 mL; refriger-
ethylenediamine, Eastman KodakCo. No. 8178, or equivalent), and ate in glass-stoppered Erlenmeyer) (to block electron flux to respira-
ca 116 mL 1M HCl to pH 9.0, and dilute with H,O to 250 mL. (3) So- tory chain), and ca 1.0 g polyvinylpyrrolidone (Type K30 [MW
lution A-2.-Dissolve 70.0 g acrylamide (Eastman Kodak Co. No. 40 000], GAF Chemicals Corp., 1361 Alps Rd, Wayne, NJ 07470,

0 2000 AOAC INTERNATIONAL

AOAC OFFICIAL METHODS OF ANALYSIS(2000)
USA) (to help solubilize dye). Mixture may be prepared several h in
advance and stored m refrigerator. Also prepare control medium
with H,O replacing potassium malate, if considered necessary.
Prepare 2.4 mg phenazine methosulfate/mL H,O. This solution is
stable several weeks in refrigerator in dark.
Within h of use, prepare solution of 15.4 mg
p-nitrobluetetrazolium chloride (NBT.Cl) in 7.0 mL 0.144M Tris
buffer (1.74 g/l00 mL). (Double amount if using control medium.)
Immediately before incubation of gels, add 0.4 mL phenazine
methosulfate solution and the 7.0 mLNTB.Cl solution to main solution.
E. Preparation of Centrifuged Tissue Fluid
Centrifuge 4 or 5 whole oyster meat test samples 20 min at
20 000 g m refrigerated centrifuge. (Never use minced or homoge-
nized test samples. Mechanical disruption of tissue produces results
similar to freezing.) Withdraw clear supematant fluid (CTP) with
disposable syringe and refrigerate until ready to prepare gels. Begin
centrifuging early in morning, before setting up running gel, so that
CTF will be ready in ample time.
F. Electrophoresis
(a) Preparation ofgeZ columns.-Remove solutions for prepar-
ing gels from refrigerator and warm slightly to lo-15°C
(50-60’F). It is not necessary to bring them to room temperature,
but the colder the solutions, the slower the polymerization. Place
glass columns in vertical position in small rubber base caps glued
to bottom panel of photopolymerization box. (Caution: Protect
eyes from direct rays of UV light.) If run is for 12 columns, set up
14 or 15, to have replacements available for any defective columns.
Slowly pour equal volumes Solution A and ammonium persulfate
successively into small beaker and swirl gently (10 mL each i;s
enough for I6 columns). Avoid getting air bubbles in mixture. This
running gel will polymerize ca 45 min after mixing.
Using 5 mL disposable syringe, 973.24B(a)(2), gently draw up run-
ning gel solution; wipe off tip of syringe and insert 7.5-10 cm length of
small polypropylene tubing. Push gel solution through tubing to re-
move any air; then fill columns to within 15 mm of top, refilling syringe
and repeating as needed.
When running gel is in all columns, they must be H,O-layered,
both to keep gel from contact with air and to establish flat inter
face between running gel and subsequently added stacker gel.
Add drop of 0.1% (w/v) aqueous bromophenol blue tracking dye
to small flask of H,O, enough to tint H,O pale blue; add ca 2 mL.
to 2 mL syringe, (a)(3). Perform HzO-layering carefully; even
small drop falling suddenly can violently disturb (“bomb”) gel,
and bombed gels must be discarded. Steady each column with one
hand, holding H,O-layering syringe in other. Wipe tip of needle
(finger of hand steadying column is simplest), to keep any sizable
drop from formmg as it is brought to column. Place needle
quickly and gently against inne,r side of column, sliding it rapidly
down to gel surface. If gel is “‘bombed,” remove disturbed gel
with disposable syringe, (a)(4), and refill column with fresh run-
ning gel mixture.
When H,O-layering is completed, set timer for 40 min polymeriza-
tion period. Mix stacker gel solution at this point, pouring equal vol-
umes Solution C and 40% (w/v) sucrose solution carefully into small
beaker. Allow 5 or 6 mL for stacker gels and 2 mL each for sample gels,
Swirl mixture and store at room temperature away from light.
When running gels are polymerized, invert columns and gently re-
move H,O layer. Use cotton swabs to draw off any large remaining
drops, being careful not to disturb gel surface or to leave fibers on glass.
FISH AND OTHER MARINE PRODUCTS
Chapter 35, p. 5
Set columnsupright again and add small amount stacker gel to each to
rinse out last of H,O, using another 5 mL disposable syringe with short
length of polypropylene tubing attached.Remove rinse mixture with dis-
posable syringe, (a)(+ Add stacker gel to each column, to ca 6 mm
above running gel surface, and HrO-layer as before.
Push columns to within 7.5-10 cm of fluorescent light in box, turn
on light, let gels stand 10 min, and then position columns directly under
light for final 10 min. Light should be ca 2.5 cm from top of columns.
Retrieve CTF from centrifuge and refrigerate during this step.
When stacker gels are photopolymerized, turn off light, remove
HZ0 layer from columns as before, and prepare sample gels. Add
0.08 mL CTF of each specimen to 2 mL stacker gel in 5 mL beaker, or
any amount in proportion. Mix well; add to columns first as rinse, then
as 6 mm sample gel, as in stacker gel procedure (use individual 5 mL
disposable syringes and tubing), and H,O-layer. Photopolymerize
as for stacker gels.
(b) Separation.-Set up electrophoresis in cold room and chill
electrode buffers before use. (Alternatively, place bath assembly in
refrigerator, or circulate ice-H,0 through cooling coils immersed in
baths.) Prepare histochemical medium during run, reserving
tetrazolium dye and adjuvant until just before gel incubation; keep
solutions refrigerated until use.
As soon as sample gels are polymerized, set up electrophoresis in
cold room. Place columns in upper bath container with sample gel on
top, toward cathode. (Caution: Cover for electrophoresis chamber
should have switch to disconnect current to electrodes when
chamber is open. Shield contacts and electrodes against body
contact.) Add 1 L diluted electrode buffer to lower (anode) bath,
and bead ends of columns with buffer before setting upper (cath-
ode) bath (from which columns extend) in place above lower
bath. Add 1 L diluted electrode buffer, tinted pale blue with
bromophenol blue, to upper bath, first pipetting small amounts
into tops of columns, to avoid creating air bubbles. Bromophenol
blue migrates more rapidly than proteins, which it will overtake
and pass to form visible moving front.
Use constant current and do not stop electrophoresis during run.
Set at 1 mA/column for first 0.5 h and then at 2 mA/column for 2 h.
Continue until tracking dye has left columns.
G. Incubation of Gels
(a) Removal of gel columns.-When electrophoresis is finished,
remove glass columns from bath assembly, discard buffers, and
remove gels from glass columns. Connect rubber tubing on removal
tool to suitable faucet adapter and use steady flow of Hz0 while
gently reaming gels from glass columns with needle. Loosen anode
end of gel column first, then cathode end; gels will slip out easily un-
der H,O pressure. Discard sample and stacker gels andplacerunning
gels, cathode end up, in 10 x 75 mm test tubes.
(b) Staining of geEs.-Add histochemical reagent immediately,
cork tubes, and invert several times; then place in dark 25-30 min
(but not >30 min) at room temperature. Rinse gels immediately and
thoroughly with tap H,O; then store in H,O. No counterstaining is
necessary.
H. Interpretation
Use densitometer or Polaroid camera for permanent records, as
formazan bands on gels will diffuse with time. Unfrozen oysters
show, at most, single band, often faint or missing. Frozen and
thawed oysters produce broad band at same place with or without 1
or 2 accessory bands and a thin cathodal band.
0 2000 AOAC INTERNATIONAL
FISH AND OTHER MARINE PRODUCTS AOAC OFFICIAL METHODSOF ANALYSIS(2000)
Chapter 35, p. 6

Freeze and thaw test portion of questionable lot and test against 35.1.06
test portion of reference laboratory sample. If patterns are alike, lot AOAC Official Method 967.13
has been frozen and thawed; if different, lot has not been frozen and Drained Weight
thawed. of Frozen Shrimp and Crabmeat
First Action 1967
References: JAOAC 53,1237(1970);56,541(1973).
Final Action 1971

35.1.06 (Applicable to Alaska king and snow crabmeat.)

AOAC Official Method 937.06
Volume of Shucked
Oysters, Clams, or Scallops
First Action 1937
Final Action
Fluff entire contents of commercial container, or container in
which test sample is recetved (13.8 L; 1 gal.), by pouring into stari-
dardmeasuring vessel through distance of 1 ft. 230 cm, then pouring
back mto contamer from same height, and again pouring into mea-
suring vessel. Use metal funnel (stainless steel preferable) 8-10 in.
(20-25 cm) diameter at top, with stem 3 in. (7.6 cm) diameter and ca
7.6 cm long, to facilitate pouring from one vessel to another. Mea-
sures are straight-side, cylindrical, made of metal (stainless steel
preferable), holding exactly 1 gallon or 1 quart, respectively, and
havmg smooth nms. Plane of nm must be level when measure is
standing on level surface. Diameter of top of gallon measure is
4.25-5.25 in. (10.8-13.3 cm), and that of quart measure is
3.25-3.5 in. (8.3-8.9 cm). Calibrate with standard glass measures,
and for estimating volumes less than level full, use graduated me-
chanic’s depth gage to measure distance from rim to surface of con-
tents. Tabulate depth gage readings against volumes or % shortages
as desired for each measuring vessel. Measure head space with depth
gage and determine volume. For $1 pint containers, calibrated glass
cylmders may be used.
References: JAOAC 20,70(1937); 21,85(1938); 35,218(1952);
36,608(1953);38,194(1955);59,312(1976).
35.1.07
AOAC Official Method 953.11
Drained Liquid
from Shucked Oysters
First Action 1953
Final Action
A. Apparatus
Skimmer or strainer-.-Flat-bottom metal pan or tray with ca 2 in.
(5 cm) sides, with area of 21900 cm2 (300 square inches) for each
gal. of oysters to be poured on tray, and with perforations 0.25 in.
(0.6 cm) diameter and 1.25 in. (3.2 cm) apart in square pattern, or
perforations of equtvalent area and distribution. Support skimmer
over slightly larger solid tray so that liquid drains into solid tray.
B. Determination
Weigh tared contamer with shellfish meats, transfer contents to
skimmer, and quickly distribute meats evenly over draining surface
with mimmum of handling. Drain 2 min, return meats to container,
and reweigh. Calculate loss of weight as % drained liquid. Make de-
terminattons at 7 + 1“C (45 + 2°F). If further analysis is desired, pro-
ceed as m 937.07(j) (see 35.1.01).
References. CFR Title 21,161.130(c)(2)(i).
JAOAC 36,947(1953); 38, 194(1955).
A. Apparatus
(a) Container.-Wire mesh basket large enough to hold contents
of one package and with openings small enough to retain all pieces.
Expanded metal test-tube basket or equivalent, fully lined with stan-
dard 16 mesh per linear inch insect screen is satisfactory.
(b) Balance.-Sensitive to 0.25 g or 0.01 oz.
(c) Sieves.-U.S.No. 8,8 in. (20cm) and 12in. (30cm)diameter.
B. Determination
Place contents of individual package in wire mesh basket and im-
merse m 215 L (4 gal.) container of fresh Ha0 at 26 + 3°C
(80 + 5°F) so that top of basket extends above H,O level. Introduce
H,O of same temperature at bottom of container at flow rate of
4-l 1 L (l-3 gal.)lmin. As soon as product thaws, as determined by
loss of rigidity, transfer all material to 12 in. (30 cm) (for package
450 g [ 1 lb]) or 8 in. (20 cm) (for package 51 lb) No. 8 sieve, distrib-
uting evenly. Without shifting material on sieve, incline sieve to ca
30” from horizontal to facilitate drainage. Two mm from time placed
on sieve, transfer product to previously weighed pan, and weigh.
Weight so found minus weight of pan is drained weight of product.
References: JAOAC 50,275(1967); 52,692(1969); 53,9(1970);
56,886(1973).
35.1.09
AOAC Official Method 970.60
Drained Weight
of Frozen Crabmeat
First Action 1970
Final Action 1971
Alaska King Crab Marketing
and Control Board-AOAC Method
(Applicable to Alaska king and snow crabmeat.)
A. Apparatus
(a) &lance.-Sensitive to 1 g or 0.01 lb.
(b) Thermometer.-Accurate in 0-30°C (30-80°F) range.
(c) Plastic bowls.-Marked at 48 oz (1440 mL), 64 oz
(1920 mL), or 1 gal. (3840 mL) level for 6 oz, 8 oz, or 1 lb pack-
ages, respectively.
B. Determination
Weigh bare block free of all wrappings and record weight. Place
block in bowl containing amount fresh potable water at 27°C (80°F)
equal to 8xdeclared weight. Leave block in Hz0 until all ice is melted.
Turn block over several times during thawing. Point at which thawing
is complete can be determined by probing block apart.
Pour entire thawed test portion onto tared 8 in. (20 cm) No. 8
sieve. Incline screen to aid drainage, drain exactly 2 min, and
weigh. Subtract tare weight of sieve for thawed drained weight of
test portion.

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AOAC OFFICIAL METHODS OF ANALYSIS (2000) FISHAND OTHER MARINE PRODUCTS
Chapter 35, p. 7

thawed drained weight x 100 35.1 .12

Drained weight, % = -
declared net weight AOAC Official Method 968.09
Minced Fish Flesh
References: JAOAC 53,9(1970); 56,886(1973).
in Mixed Fillet-Minced Cod Blocks
Physical Separation Method
First Actlon 1966
Final Action 1996
35.1 .lO
AOAC Official Method 976.17
Shrimp in Shrimp Cocktail Codex-Adopted-AOAC Method*
Drained Weight Method
First Action 1976 A. Principle
Final Action 1979 Mixed fillet-minced cod block is air-thawed, drained, weighed,
A, Preparation of Test Sample and immersed in cold H,O bath. Fillets are separated from mince by
Thaw unopened jars in 16 * 5°C H,O bath until product is de- hand and placed on perforated tray to drain. HZ0 from bath is poured
frosted to temperature of 6 f 5°C. Keep jar lids above H,O level. Al- through sieve, and any fillet pieces on sieve are removed and added to
ternatively, place frozen jars in refrigerator until contents have drain tray. Fillets are drained 15 min and weighed. Amount of minced
thawed. fish is calculated from drained weight of block and drained weight of
B. Determination fillets.
Empty thawed contents of jar onto No. 8 sieve. Wash jar and lid B. Apparatus
with H,O and pour washings onto sieve until jar is clean. Rinse
shrimp on sieve with gentle stream or spray of cold tap H,O. Use (a) Trays.-Al or galvanized. (1) Shallow; large enough to hold
rubber spatula to remove adhering material. Cover sieve with metal 1 fish block for thawing. (2) Perforated, with holes X in. diameter
cover or moisture barrier film, incline at 17-20” angle, and let drain drilled to cover entire bottom of tray, or made from U.S. Standard
No. 8 metal wire cloth.
exactly 2 min. Transfer shrimp to container previously tared with
cover and weigh to 0.1 g. (b) Container.-Tub of sufficient size to hold fish block plus H,O.
(c) Scale.-Sensitive to 0.25 oz (7 g).
weight shrimp x 100 (d) Sieve.-U.S. No. 8.
Shrimp, % =
declared weight total contents C. Determination
Thaw test sample (entire fish block) on preweighed tray at ambi-
Reference: JAOAC 59,644(1976). ent (room) temperature (usually takes overnight). Perform determi-
nation within 8 h after block is completely thawed.
Drain any exuded fluid (thaw-drip) and determine weight of
35.1 .ll drained fish block (weight = W,>.
AOAC Official Method 977.12 Immerse drained fish flesh in tub of cold, lO-21°C (SO-7O”F),
Seafood in Seafood Cocktail H,O. Use ratio of 2-3 parts H,O to 1 part fish by weight. Separate fil-
Drained Weight Method lets by hand and wash minced fish flesh from fillets in tub. Place
Procedure washed fillets on upper section of preweighed perforated tray. In-
cline tray at angle of 2040” to facilitate draining.
(a) For seafood other than crabmeat.-Proceed as in 976.17A
Pour H,O containing minced fish and any small pieces of fillet
and B (see 35.1. lo), using rubber spatula gently to remove sauce
from irregular surfaces without unduly fragmenting seafood. from tub through sieve. Remove any fillet pieces from sieve, place
(b) For rrabmeat.-Prepare test sample as in 976.17A (see them on drain tray with fillets, and let fillets drain 15 min. Remove
35.1 .lO). Empty thawed contents of jar onto No. 8 sieve nested on any excess HZ0 from lower section of tray and weigh drained fillets
top of No. 20 sieve. Wash jar and lid with H,O until jar is clean and (weight = W,).
pour washings onto sieves. Rinse crabmeat on No. 8 sieve with gen-
tle stream or spray of cold tap H,O. Use rubber spatula to remove ad- D. Calculation
hering material. Cover sieves with metal cover or moisture barrier film,
incline at 17-20” angle, and let drain exactly 2 min. Separate sieves. In-
vert, dropping No. 8 sieve onto nonabsorbent surface (such as wax pa- Minced fish, % = w,-w,lC)o
per). Transfer crabmeat to container previously tared with cover. W,
Transfer crabmeat from No. 20 sieve to same container and weigh to
M.l g. Reference: JAOAC 71,38(1988).

weight crabmeat x 100 Revised: March I997

Crabmeat, % =
declared weight total contents
* Adopted as a Codex Defining Method (Type I)for physical separation of
proportion of fillet and minced fish in quick frozen blocks of fish fillet,
Reference: JAOAC 60,963(1977). minced fish flesh and mixtures of fillets and minced fish flesh.

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FISH AND OTHER MARINE f910ouCTS AOAC OFFICIAL METHODS OF ANALYSIS (2000)
Chapter 35, p. 8

35.1.13 35.1.18
AOAC Official Method 952.08 AOAC Official Method 973.25
Solids (Total) Ammonia in Crabmeat
in Seafood Calorimetric Method
Gravimetric Method First Action 1973
First Action 1952 Final Action 1963
Final Action 1961
A. Reagents
A. For All Marine Products Except Raw Oysters
Weigh 2 g sand or glass fiber of type used in preparing Gooches. (Use NH,-free Hz0 throughout; ordinary distilled Hz0 is suitable.)
Place cut fibers and glass stirring rod ca 8 cm long with flat end into (a) Bromine solution.-Dilute 10 mL NaOH solution (1 + l),
flat-bottom metal weighing dish, ca 9 cm diameter, with cover. Dry 936.16B(b) (see A.1.12), to ca 100 mL with H,O, add 1.0 mL Br,,
dish, sand or glass fiber, and rod in oven 1 h at lOO”C, cool, and shake, and dilute to 200 mL with H,O. Prepare fresh daily.
weigh. Quickly weigh into dish, to nearest mg, 9.5-10.5 g prepared (b) Thymolsolution.-10% (w/v) in alcohol. Prepare fresh daily.
test sample. Add 20 mL Ha0 and mix test portion thoroughly with (c) Dilute sodium hydroxide solution--Dilute 25 mL NaOH so-
sand or glass fiber. Support end of rod on edge of dish and evaporate lution (1 + l), 936.16B(b) (see A. 1.12), to 100 mL with H,O.
just to dryness on steam bath, stirring once while still moist. Drop (d) Ammonia standard solution.40 pglmL. Dissolve 0.314 g
rod into dish and heat 4 h in oven at lOO”C, or in preheated NH&l, previously dried 1 h at 100°C in H,O, and dilute to 100 mL.
forced-draft oven set for full draft, 1 h at 100°C. Cover dish, cool in Transfer 4.0 mL to 100 mL volumetric flask, and dilute to volume
desiccator, and weigh promptly. with H,O.

References: JAOAC 35,216(1952); 37,602(1954). B. Preparation of Samples

B. For Raw Oysters Only Remove shell, if necessary, and grind meat 3 times through food
Quickly weigh, to nearest mg, 9.5-10.5 g prepared test portion chopper, mixing after each grinding.
into weighed, flat-bottom metal dish ca 9 cm diameter and 2 cm high
C. Determination
with cover. Spread test portion evenly over bottom of dish. Then:
(a) Evaporate just to dryness on steam bath and dry 3 h in oven at Place 20 g prepared test portion in 500 mL glass-stoppered
lOO”C, or Erlenmeyer. Add 180 mL 2.5% (w/v) phosphotungstic acid solu-
(b) Insert directly into preheated forced-draft oven set at full draft tion, shake vigorously 2 min, and filter through Whatman No. 1, or
and dry 1.5 h at 100°C. equivalent, paper into 250 mL glass-stoppered Erlenmeyer. Plpet
Cover, cool in desiccator, and weigh promptly. 2 mL filtrate (equivalent to 0.2 g test portion) mto 125 mL separator.
Save remainder of filtrate. To another separator, add 2.0 mL 2.5%
References: JAOAC20,71(1937); 35,218(1952); 36,608(1953); (w/v) phosphotungstic acid solution as blank.
37,607(1954); 44,276(1961); 46,744(1963). To each separator add 8.0 mL HsO. Then, in immediate succes-
sion, add 1.0 mL dilute NaOH solution, A(c), swirl to mix, 2.0 mL
Revised: March 1996
thymol solution, A(b), swirl to mix, and 5.0 mL Br, solution, A(a),
in ca 30 small additions, swirling vigorously after each addition.
Shake vigorously 1 min. With series of samples or standards, com-
35.1.14 plete reagent additions in sequence on each separator before pro-
AOAC Official Method 938.08 ceeding to next. Let stand 220 min.
Ash of Seafood To each separator add 20.0 mL n-butanol and shake vigorously
First Action 1936 1 min. Let stand 20 min. Drain aqueous layer and pass n-butanol
Final Action through ca 30 g anhydrous Na,SO, in glass funnel plugged with
glass wool mto glass-stoppered Erlenmeyer. Measure A of solution
Dry test portion containing ca 2 g dry material and proceed as in at maximum, ca 680 nm, in 1 cm cell against blank as reference.
900.02A or B (see 44.1.05), using temperature 550°C. If material IfA is greater than that ofhighest NH, standard, quantitatively di-
contains large amount of fat, make preliminary ashing at low enough lute reserved filtrate with 2.5% (w/v) phosphotungstic acid solution
temperature to allow smoking off of fat without burning. so that 2.0 mL diluted solution will produce A below this level.
References: JAOAC21,85(1938); 23,589(1940). D. Preparation of Standard Curve
Pipet 0, 1,2, 3,4, and 5 mL standard NH, solution into 125 mL
separators. Add 2.0 mL 2.5% (w/v) phosphotungstic acid solutton to
35.1.15 each and dilute to 10.0 mL with HzO. Proceed as in C, beginning
AOAC Official Method 940.25 “Then, in immediate succession, add 1 0 mL dilute NaOH solu-
Nitrogen (Total) tion,. . .I’. Using 0 solution as reference, measure A of each standard
in Seafood at maximum as above. Prepare standard curve.
First Action 1940
Final Action Reference: JAOAC 56,598(1973).

See 955.04 (see 2.4.03). CAS-7664-41-7 (ammonta)

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AOAC OFFICIAL METHODS OF ANALYSIS (2000) FISH AND OTHER MARINE PRODUCTS
Chapter 35, p. 9

35.1.17 blended solution at 2000-3000 rpm until supemate is practically

AOAC Official Method 971.14 clear.
Trimethylamine Nitrogen
D. Determination
in Seafood
Calorimetric Method Pipet aliquot (preferably containing 0.01-0.03 mg TMA-N) into
First Action 1971 20 x 150 mm Pyrex glass-stoppered test tube and dilute to 4.0 mL
Final Action 1972 with H,O. For standards, use 1 .O, 2.0, and 3.0 mL working standard
solution, diluting to 4.0 mL with HzO; for blank, use 4.0 mL H,O.
(Do not use stopcock grease; mixture of sugar and glycerol ground Add 1 mL HCHO, A(e), 10 mL toluene from automatic pipet, and
together may be used if necessary. Do not wash tubes with soap or 3 mL K,CO, solution. Stopper tube and shake vigorously by hand ca
detergent; rinse with H,O and occasionally clean thoroughly with 40 times. Pipet off 7-9 mL toluene layer into small test tube contain-
HNO,.) ing ca 0.1 g anhydrous Na2S04. Avoid removing droplets of aque-
ous layer. Stopper tube and shake well to dry toluene. Pipet 5 mL
A. Reagents
toluene layer into dry calorimeter tube. Add 5 mL picric acid solu-
(a) Trichloroacetic acid solution.-7.5% (w/v) aqueous solu- tion and mix by swirling gently. Determine A at 410 nm against
tion. (Caution: See Appendix B, safety notes on di- and blank carried through determination. Color is stable. If original
trichloroacetic and trifluoroacetic acids.) aliquot contains >0.03 mg TMA-N, dilute extract with
(b) Toluene.-Dried over anhydrous N&SO,. To remove inter- trichloroacetic acid solution and repeat determination.
ferences, shake 500 mL toluene with 100 mL OSM H,S04, distil,
and dry with anhydrous NaaSO,. (Caution: See Appendix B, safety mg TMA-N/100 g test sample (based on 1 mL aliquot) =
notes on distillation and flammable solvents.) (ALA) x (mg TMA-N/mL standard solution)
(c) Picric acidsolutions.- Stock solution.-Dissolve 2 g pic- x mL standard solution used x 300
ric acid (Caution: See Appendix B, safety notes on picric acid) in
100 mL H,O-free toluene. (2) Working solution.-Dilute 1 mL
stock solution to 100 mL with H,O-free toluene. Use A of standard nearest to A of sample for calculation.
(d) Potassium carbonate solution.-Dissolve 100 g K&O, in
Reference: JAOAC 54,725(1971).
100 mL H,O.
(e) Formaldehyde.-20%. Shake 1 L commercial formalin
(40%) with 100 g MgCO, until nearly colorless and filter. Dilute
100 mL to 200 mL with H,O. (Caution: See Appendix B, safety 35.1 .18
notes on formaldehyde.) AOAC Official Method 937.09
(f) Indicator solution.-(l) Methyl red-methylene blue.-Mix Salt (Chlorine as Sodium Chloride) in Seafood
2 parts 0.2% (w/v) alcoholic methyl red solution with 1 part 0.2% Volumetric Method
(w/v) alcoholic methylene blue solution; or (2) Methyl First Action 1937
red-bromocresol green solution-Mix 1 part 0.2% (W/V) alcoholic Final Action
methyl red solution with 5 parts 0.2% (w/v) alcoholic bromocresol
A. Reagents
green solution.
(a) Silver nitrate standard solution.-O.lM. Prepare as in
(g) Trimethylamine (TMA) standard solutions.-(l) Stock solw
941.1SA (see A. 1.11) and standardize against O.lM NaCl containing
tion.-Add 0.682 g (CH,),N.HCl to 1 mL HCl (1 + 3) and dilute to 5.844 g of pure dry NaCVL.
100 mL with H,O. Check basic N content of 5 mL aliquots by addin,g (b) Ammonium thiocyanate standard soZution.-O.lM. Prepare
6 mL 10% (w/v) NaOH solution, distilling into 10 r&4% (w/v) bo- as in 941.18D(b) (see A.l.ll) and standardize against O.lM
ric acid in micro-Kjeldahl distillation apparatus, and titrating with AgNO,.
0. 1N HISO,, using indicator, (f), This solution is stable. (2) Working (c) Ferric indicator.-Saturated solution of FeNH,(S0,),.12H,O.
soZution.4.01 mg TMA-N/n&. Add 1 mL stock solution to 1 ml, B. Determination
HCI (1 + 3) and dilute to 100 mL with H,O. (a) Shellfish meats.-Weigh 10 g meats, liquid, or mixed meats
B. Apparatus and liquid, into 250 mL Erlenmeyer or beaker.
(a) Digestion rack.-With either gas or electric heaters which (b) Otherfish products.-Use suitable size test sample, depend-
ing on NaCl content.
will supply enough heat to 30 mL flask to cause 15 mL Hz0 at 25°C
Add known volume 0. 1M AgNOs solution, more than enough to
to come to rolling boil in 22 but <3 min. precipitate all Cl as AgCl, and then add 20 mL HNO,. Boil gently on
(b) Distillationapparatus.-one-piece or Parnas-wagnerdistil- hot plate or sand bath until all solids except AgCl dissolve (usually
lation apparatus recommended by Committee on Microchemical 15 min). Cool, add 50 mL Hz0 and 5 mL indicator, and titrate with
Apparatus, ACS.
O.lN NH,SCN solution until solution becomes permanent light
(c) DigestionjZasks.-Use 30 mLregular Kjeldahl or Soltys-type brown. Subtract mL O.lM H$CN used from mL O.lM AgNOs
flasks [Reference: Anal. Chem. 23,523(1951)]. For small samples, added and calculate difference as NaCl. With 10 g test sample each
10 mL Kjeldahl flasks may be used. mL O.lN AgNO, = 0.058% NaCl.
C. Preparation of Sample
References: JAOAC 20,410(1937); 23,589(1940).
Weigh 100 g minced or chopped, well-mixed sample. Add
200 mL 7 S% (w/v) trichloroacetic acid and blend. Centrifuge CAS-7647-14-5 (sodium chloride)

0 2000 AOAC INTERNATIONAL

FISH AND OTHER MARINE PRODUCTS AOAC OFFICIALMETHODSO F ANALYSIS(2000)
Chapter 35, p. 10

35.1 .19 (a) Test Samples with unknown or known high oil content.-Add
AOAC Official Method 976.16 ca 10 mL petroleum ether, and warm on steam bath or low tempera-
Salt (Chlorine as Sodium Chloride) in Seafood ture hot plate until oil is extracted. Decant and repeat until test sam-
Potentiometric Method ple is defatted. Proceed as in (b).
First Action 1976 (b) Test Samples with low oil content.-Add 5 mL HNO, (if total
Final Action 1964 Cl content is desired, add enough O.lM AgNO, to precipitate chlo-
rides [3.0 mL]) to each beaker. Digest on steam bath or low tempera-
Prepare test sample as in 937.07 (see 35.1.01) and proceed as in ture hot plate until test sample dissolves; evaporate to dryness. Add
971.27 (see 42.1.15). 5 mL HNO, and take to dryness. Repeat. Add 2 mL HNO, and warm
to dissolve. Proceed as in (c) or (d).
35.1.20 (c) For flame emission.-Transfer digest to 25 mL volumetric
AOAC Official Method 976.19 flask with hot H,O, wash down sides of beaker 3 times with hot H,O,
Salt (Chlorine as Sodium Chloride) in Seafood and add washings to flask. Cool, and dilute to volume with H,O. If
Indicating Strip Method particles are too finely dispersed to settle, centrifuge aliquot at
Procedure 2000 rpm.
(d) Forflame absorption.-Transfer digest to 100 mL volumet-
See 91.19, 15th Ed. ric flask and proceed as above. Dilute for direct readout as follows:
Place 1 mL aliquot in 25 mL volumetric flask and dilute to volume
35.1.21 with H,O for Na; place 2 mL aliquot in 10 mL volumetric flask and
AOAC Official Method 969.23 dilute to volume with Hz0 for K.
Sodium and Potassium in Seafood Prepare blank solution by diluting 2 mL HNO, to 100 mL with
Flame Photometric Method H,O.
First Action 1969
Final Action 1971 C. Dry Ashing
Prepare test sample as in 937.07 (see 35.1 .Ol).
A. Apparatus and Reagents Weigh 4 g test sample into crucible, and char on electric hot plate
(a) Glassware.-Borosilicate glassware and intact Vycor, Pt, or or over low flame. Place in cold furnace and bring to 525°C. Ash 2 h
Si crucible precleaned with dilute HNO, and rinsed in distilled Hz0 to white ash. Cool, and weigh if total ash is desired.
immediately before use. Add 15 mL dilute HNO, (1 + 4) to crucible, breaking up ash with
(b) Distilled wafer.-H,O, free from Na and K; either dou- stirring rod if necessary. Filter through acid-washed quantitative pa-
ble-distilled or deionized. Use for preparing standards and dilutions. per into 100 mL volumetric flask. Wash residue and paper 3 times
(c) Sodium standard solutions.-(l) Stock solution.-1 mg with H,O. Dilute to volume. Proceed as in (a) or (b).
Na/mL. Dry reagent grade NaCl2 h at 110°C; cool in desiccator. (a) Forflame emission.-Read directly.
Weigh 2.5421 g into 1 L volumetric flask and dilute to volume with (b) Forflame absorption.-Dilute for direct readout as follows:
HzO. (2) Working solutions for flame emission.--0.01, 0.03, and Place 1 mL aliquot in 100 mL volumetric flask and dilute to volume
0.05 mg NaImL. Pipet 1.3, and 5 mL Na stock solution into separate with Hz0 for Na; place 1 mL aliquot in 25 mL volumetric flask and
100 mL volumetric flasks; add 7 mL K stock solution and 2 mL dilute to volume with H,O for K.
HNO, to each flask; dilute to volume with H,O. (3) Working solu- Prepare blank solution by diluting 2 mL HNO, to 100 mL with
tions for flame absorption.-0.00003, 0.0001, 0.0003, and H,O.
0.0005 mg Na/mL. Pipet 1 mL Na stock solution into 100 mL volu-
metric flask and dilute to volume with H,O. Pipet 0.3, 1.0,3.0, and D. Determination
5.0 mL diluted stock solution into separate 100 mL volumetric flasks (Caution: See Appendix B, safety notes on AAS and flame pho-
and dilute to volume with H,O. tometers.)
(d) Potassium standard solutions.-(l) Stock solution-l mg Follow manufacturer’s directions for type of instrument avail-
WmL. Dry and cool reagent grade KC1 as in (c). Weigh 1.9068 g into able. Dilute test solution if necessary to bring T readings within
1 L volumetric flask and dilute to volume with H,O. (2) Working so- range of working standards. Read blank, standards, and test solu-
lutions forflame emission.-O.04,0.07, and 0.10 mg KImL. Pipet 4, tions at 589 nm for Na and 767 nm for K until results are reproduc-
7, and 10 mL stock solution into separate 100 mL volumetric flasks; ible; record percent T or percent absorptron for each.
add 3 mL Na stock solution to each flask; dilute to volume with H,O. E. Calculations
(3) Working solutions for flame absorption.-0.0001, 0.0005,
For flame emissionphotometersnot equipped withdirect readout:
0.0007, and 0.0010 mg WmL. Pipet 1 mL K stock solution into
100 mL volumetric flask and dilute to volume with H,O. Pipet 1, 5,
mgNaorWlOOg=
7, and 10 mL diluted stock solution into separate 100 mL volumetric
flasks and dilute to volume with H,O.
B. Wet Ashing
100 x F
([ (4 -4) 11
(E, + 4) x (c, - C, ) + C, /g test sampk

(Caution: See Appendix B, safety notes on distillation, wet oxida-

tion, and nitric acid.) where E, = (percent T of unknown) - (percent Tof blank); E, = (per-
Preparetest sample as in 937.07 (see 35.1.01). cent Tof standard of lower concentration than test sample) - (percent
Weigh 1 g test sample into 50 mL Pyrex beaker. Dry 2.5 h at T of blank); .!?z= (percent T of standard of higher concentration than
110°C. cool, and weigh if percent solids is to be determined. test sample) - (percent Tofblank); CI = mg Naor KImLin standardof

0 2000 AOAC INTERNATIONAL

 AOAC OFFICIAL METHODS OF ANALYSIS (2000) FISHAND OTHER MARINE PRODUCTS
Chapter 35, p. 11

lower concentration than test sample; C, = mg Naor WrnL in standard 35.1.24

of higher concentration than test sample; F = dilution factor. AOAC Official Method 964.12
For flame absorption photometers: Convert percent absorption to Fat (Crude) in Seafood
absorbance (A). Plot standard curve ofA against concentration. Read Rapid Modified Babcock Method
unknown concentrations. First Action 1964
Final Actlon 1996
mg Na or K/l00 g =
(concentration unknown x F x 100)/g test sample (Applicable to raw, canned, and frozen fish.)

Reference: JAOAC 52,55(1969). Caution: See Appendix B, safety notes on acetic acid and
CAS-7440-09-7 (potassium) perchloric acid.
CAS-7440-23-5 (sodium)
Weigh 9.0 g ground and mixed test sample into Paley-type Bab-
cock cheese bottle (Kimble Glass No. 508,20% size), stopper, and
35.1.22 add ca 30 mL reagent prepared by mixing equal volumes CH,COOH
AOAC Official Method 991.26 and 70-72% HC104. Place in H,O bath (2 L stainless steel beaker is
Domoic Acid in Mussels satisfactory) maintained at 92 YZ2”C, swirling occasionally until no
Liquid Chromatographlc Method lumps remain (usually ca 20 min). Remove from bath, add reagent
First Actlon 1991 until fat is well up in calibrated neck of bottle, centrifuge 2 min at ca
600 rpm, and read percent fat with dividers, using bottom of top me-
See 991.26 (see 49.10.02). niscus. If fat falls below calibration, add more reagent, centrifuge
Revised: June 2000 1 min, and read again.
With very fat fish, it may be necessary to use <9 g test sample. In
this case, correct reading by multiplying percent fat by factor 9/g test
35.1.23 sample.
AOAC Official Method 946.15 References: JAOAC40,343(1957); 42,261(1959); 45,259(1962);
Fat (Crude) in Seafood 46,746(1963); 47,708(1964).
Acid Hydrolysis Method

- First Action 1946

A. Preparation of Test Samp/e

Final Action

Prepare test sample according to type of pack as in 937.07 (see

Revised: March 1997

35.1.25
AOAC Official Method 946.16
351.01) and keep ground material in sealed jar. If jar has been Fat (Crude) in Fish Meal
chilled, let test sample come to room temperature and shake jar so
Acetone Extraction Method
that any separated liquid is absorbed by fish. Open jar and stir con- First Action 1948
tents with spatula, thoroughly scraping sides and lid so as to incorpo- Final Action 1976
rate any separated liquid or fat.
B. Determination Caution: See Appendix B, safety notes on monitoring equip-
Weigh 8 g well-mixed test sample into 50 mL beaker and add 2 mL ment, distillation, flammable solvents, and acetone.
HCl. Using stirring rod with extra large flat end, break up coagulated
Weigh 4-5 g test sample to nearest 0.01 g into Alundum or paper
lumps until mixture is homogeneous. Add additional 6 mL HCl, mix,
extraction thimble, cover with light layer of cotton, and extract with
cover with watch glass, and heat on steam bath 90 min, stirring occa-
acetone in continuous extractor 16 h. Distil off acetone until volume
sionally with rod. Cool solution and transfer to Mojonnier
in flask is 10-15 mL, transfer to 100 rnL tared beaker, washing flask
fat-extraction flask. Rinse beaker and rod with 7 mL alcohol, add to
free of all oil with fresh acetone, and evaporate with current of warm
extraction flask, and mix. Rinse beaker and rod with 25 mL ether,
air at edge of steam bath. When no H,O or acetone can be observed,
added in 3 portions; add rinsings to extraction flask, stopper with cork
place beaker in vacuum oven at 80°C and apply 24-25 in;
or stopper of synthetic rubber unaffected by usual fat solvents, and
81.3-85.3 kPa (610-640 mm) vacuum 1 h. Transfer to desiccator,
shake vigorously 1 min. Add 25 mL petroleum ether (bp <6O”C) to ex .
cool, and weigh.
traction flask and repeat vigorous shaking. Centrifuge Mojonnier
Transfer extracted meal residue from thimble to 150 mL beaker.
flask 20 min at ca 600 rpm and proceed as in 922.06 (fee 32.1.14), be-
Remove any remaining solvent by heating on warm surface and then
ginning “Draw off as much as possible of ether-fat solution .“.
add 60 mL 4M HCl. Digest 1 h at or near bp on hot plate, stirring oc-
Drying to constant weight takes ca 40 min for fish. Long heating
casionally with glass rod and adding H,O as needed to maintain vol-
periods may increase weight of fat. If centrifuge is not available, ex-
ume in beaker. (Complete removal of acetone is necessary before
traction can generally be made by letting Mojomner flask stand until
this digestion, otherwise vaporization of solvent will carry meal
upper liquid is practically clear, then swirling flask and again letting
particles over side of vessel onto hot plate.) Filter through 12.5 cm
stand until clear. If troublesome emulsion forms, let stand, pour ofJ
fluted paper. Wash residue on filter until acid-free, using methyl
as much of ether-fat solution as possible, add l-2 rnL alcohol to
red on portions of filtrate to follow progress of washing. Place filter
Mojonnier flask, swirl, and again let mixture separate.
and meal in 150 mL beaker and dry 1 h in air oven at SO-90°C.
Reference: JAOAC 31,334(1948). Transfer filter and contents to thimble and extract 16 h with ace-

0 2000 AOAC INTERNATIONAL

FISH AND OTHER MARINEPRODUCTS AOAC OFFICIAL METHODSOFANALYSIS (2000)
Chapter35,p. 12

tone. Remove solvent and weigh extract as above. Sum of weights Add 30 mL CHCl,, 20 mL methanol, and 7 mL H,O. Attach receiving
of extracts = total fat. chamber with screen inserted, using spnngs or rubber bands. Fasten
apparatus on stming plate with standard clamp and stir magnetically
Reference: JAOAC 31,98,606( 1948).
15 min. Remove apparatus and invert. Open stopcock of reaction
chamber (now on top) to equalize air pressure.
35.1.26 Place opening of receiving chamber (now on bottom) in 100 mL
AOAC Official Method 969.24 graduate containing 10 mL H,O, and open top stopcock. Close bot-
Fat (Crude) in Fish Meal tom stopcock. With top stopcock open, briefly apply vacuum from
Semimicro Method aspirator to remove solvents more thoroughly, and drain solvent into
First Action 1969 graduate. Invert apparatus to original position. Add 10 mL CHCl,
Final Actlon 1976 through top stopcock. Invert apparatus and drain solvent into same
100 mL graduate. Invert apparatus to original position and close bot-
A. Apparatus
tom stopcock. Slowly add 40 mL CHCl, through top stopcock so
Emaction apparatus.-See Figure 969.24. (I) Reaction charn- that sides of apparatus will be rinsed clean.
ber (Coming Glass Works, No. 9820 tube [38 x200 mm] sealed to Close top stopcock, place apparatus on stirrer, and extract 2 min.
standard taper 4.5/50 joint, No. 6560); (2) receiving chamber (stan- Repeat rinsing of apparatus as above, beginning “Remove apparatus
dard taper 45/50 joint, No. 6.580); (3) No. 100-150 stainless steel and invert” except use 2 rinses of 10 mL CHCl,. Collect extraction
screen. Add openings, Teflon stopcocks, and hooks as shown. Use solvent and rinses in same graduate.
Teflon ring to seal joint at screen. Opening in I lets atmosphere pres-
sure be exerted on liquid surface when apparatus is inverted to filter C. Determination
test sample. Flared opening in 2 IS used to remove filtrate and to add Let filtrate stand overnight to clarify; then record volume CHCI,
solvent for second extraction. Side outlet in 2 is used to apply rapid layer. Remove most of methanol layer by suction. Mix contents of grad-
short gusts of vacuum to remove maximum amount solvent from test uate well; remove remaining methanol layer and small amount CHCI,
samples or to aid in filtration. layer by suction. Pipet 25-50 mL CHCl, layer into 50 mL tared beaker,
and dry under N, in 50°C HZ0 bath. Place dried sample in vacuum des-
6. Extraction
iccator over P,O, or silica gel 90 min before weighing.
Close all stopcocks of extraction apparatus. Place Tef-
lon-coated magnetic stirring bar and ca 2-4 g accurately weighed
test sample, ground to pass No. 40 sieve, in reaction chamber. Weight lipid in test sample = w
V’

where W = weight lipid in aliquot taken, V = volume CHCl, layer,

and V’ = volume CHCl, aliquot taken.
h 22mm flare
References: JAOAC 52,688(1969); 54,1132(1971);
2 RECEIVINGCHAMBER 55,654( 1972).
PYREXJOINTNO 6580
f4mm BORE Revised: June 2000
r,

it \
TEFLONII GLASS
Bmm od VALVENO. 7282
35.1.27
AOAC Official Method 936.09
t Fatty Acids (Volatile) in Seafood
100-150 MESH 50mm
STAINLESSSTEELSCREEN

cf
I \ Column Chromatographic Method
=d
First Action 1936
Final Action 1960
38mm
’ GLASS HOOWS
,(EIOTH SIDES) A. Apparatus
(a) Steam distillation assembly.-See Figure 938.09. Assembly
consists of boiler flask (3 L) giving steam at constant rate so as to pro-
e/50 I
duce constant rate of distillation, distillation flask, condenser, and
PYREX JOINT
N O 6560 200 mL volumetric flasks as receivers. Standard distillation flask with
Q side arm (ca 9 mm od) attached near center of neck, and with steam inlet
230mm tube (ca 10 mm od), is satisfactory. Heating coil of steam generator IS
1 AEACllONCHAMBER made by winding 5 ft. (1.5 m) 28 gage Chrome1 wire (or equivalent)
PYREXNO. 9820 TUBE TEFLON-COATED around hollow pipe ca 0.25 in. (6 mm)) diameter and heating red hot to
(38 x 200mm) STIRRER
detemper wire. Leads into boiler flask are brass, Cu, or other nonferrous
$ 4mn lE TEFLON& GLASS metal ca 3/32in. (2 mm) diameter. Insulate and shield electric leads and
0 VALVENO 7282 contacts to avoid possible shorting and electric shocks.
:1 /
SCALE= l/5
Any similar distillation assembly may be used if it is of capacity to
TUBESEPARATED handle volumes specified in method and gives 57 + 2% recovery of ace-
tic acid on distillation.
Figure 969.24-Extraction apparatus. (b) Chrmnatogruphic tube.-Approximately 15 x 250 mm.

O2000AOAClNTERNATlONAL
AOAC OFFICIAL METHODSOF ANALYSIS(2000)
Figure 938.09-Steam distillation assembly.
(c) Source of air pressure or compressed N2 gas equipped with
pressure regulator.-If such source isnot available, following system
serves purpose: Fit 1 L side arm flask with 2-hole rubber stopper. Pass
glass manometer tube 70 cmlong through one hole in stopper so that it
reaches bottom of flask, and through other hole pass glass tube ca 8 cm
long, whose upper end is connected to top of chromatographic tube by
rubber tubing. Connect rubber hand-aspirator bulb to side arm of
flask. Fill flask with Hg to depth of 1.5 cm. (Height of Hg column in
manometer tube indicates pressure in system; 25 cm is equivalent to
ca 5 lb.) To maintain reservoir pressure when chromatographic tube is
disconnected, fit stopcock into line leading from flask to chromato-
graphic tube; and to prevent valve in hand aspirator from leaking, in-
sert second stopcock between side arm and bulb.
(d) Rubber bulb.-5 mL. (Type used on dropping bottles.)
(e) MicrofunneL-Bilchner-type, 2 mL capacity with coarse fiitted
disk; Corning No. 36060.
B. Reagents
(a) Butanol in chloroform.-1 % (v/v). Remove alcohol from USP
CHCl, by washing 3 times with p; volume H,O. Add 10 mL n-butanol
to 1 L washed CHCl, in separator, shake vigorously, add 25 mL H,O,
and shake again. Let stand until lower layer clears, and drain. Discard
upper aqueous layer. Store in contact with granular anhydrous Na$O,.
(b) Butanol in chloroform.-10% (v/v). To 900 mL USP CHCl,
(not previously washed) in separator add 100 mL n-butanol, shake vig-
orously, add 25 mL H,O, and shake again. Let CHCl, stand until clear,
and drain. Discard aqueous layer. Store in contact with granular anhy-
drous Na$O,.
(c) Alphamine red R indicator.-Dissolve 50 rn8
mono-ammonium salt 3-(4-anilino-1-naphthylazo)-2,7-naphthalene
disulfonic acid (TCI America, 9211 N. Harborgate St., Portland, OR
97203) in 25 mL H,O. (Red solution produced by one drop indicator
in 20 mL H,O must be changed to violet by one drop O.OlN HCl.) Pre..
pare fresh weekly.
(d) CresoI red indicator.-Dissolve 50 mL o-cresolsulfonphthalein
in 20 mL alcohol, add 1.3 mL 0. 1M NaOH, and dilute to 50 mL with
H,O. Use 2 drops for each 25 mL aqueous solution.
(e) Barium hydroxide standard solution.-O.01N. (Store in poly-
ethylene or paraffin-lined bottle and protect from CO2 of atmosphere
with soda-lime or Ascarite; dispense from 10 mL buret.) [O.OlN
FISH AND OTHER MARINE PRODUCTS
Chapter 35, p. 13
NaOH may be used instead of Ba(OH), except in titration of Solu-
tion B, E. NaOH solution must be used in titration of Solution A, E,
since sodium salts are required for chromatography.]
(f) Sodium acetate-sodium chloride solution.-Dissolve 12 g
NaCl and 25 g NaCH,C00.3H,O in Hz0 and dilute to 500 mL.
(g) Silicic acid.-Reagent grade “100-mesh” powdered, suitable
for chromatography (Mallinckrodt Chemical Works No. 2847, or
equivalent).
C. Standardization of Distillation Apparatus
Place apparatus, A(a), in laboratory so that it is free fromdrafts
and sudden changes in temperature. Make mark on boiler flask at
1.5 L level, fill to this mark with H,O, heat to boiling, and boil
several min before starting distillation. Transfer 150 mL H,O to
distillation flask, add 1 drop H,SO, (1 + l), connect condenser,
insert steam inlet tube into distillation flask, and bring contents of
flask to incipient boiling with burner. Connect steam inlet tube
with steam supply from boiler and steam distil. Regulate rate of
evolution of steam and height of small flame of burner under dis-
tillation flask so that volume of liquid in distillation flask is kept
constant at 150 mL and distillate collects at 200 mL/h (Period of
collection may vary 5 min for 200 mL distillate. Keep volume in
distillation flask at 150 10 mL. Stop boiling, if necessary, to per-
mit test of constancy of 150 mL volume by momentarily inter-
rupting steam supply. Few trials will show conditions necessary
to maintain constant volume in distillation flask and constant rate
of distillation.) Determine blank on 2 successive 200 mL portions
of distillate by titrating with O.OlN alkali (phenolphthalein) in
CO,-free atmosphere.
Transfer 50 mL ca O.lN CH,COOH (concentration must be accu-
rately known) to distillation flask; add 1 drop H,SO, (1 + 1; avoid
contact with neck of flask) and 100 mL H,O. Collect 200 mL distil-
late and titrate with O.lN alkali. Correct for titration blanks and com-
pute percent acid distilled. Distillation technique and apparatus are
satisfactory when recovery is 57 f 2%. Apparatus so adjusted gives
recoveries (2%) of formic, propionic, and butyric acids of 40.5, 81,
and 92%, respectively [JAOAC21,684,688( 1938)], on 200 mL dis-
tillate.
0. Preparation of Solution
Weigh 50 g comminuted material, 937.07 (see 35.1 .Ol), into tared
500 mL wide-mouth Erlenmeyer, add ca 150 mL H,O, stopper flask,
and shake vigorously ca 1 min to effect thorough suspension of ma-
terial. Add 25 mL 0.5M H,SO,, mix, precipitate proteins with 20%
(w/v) phosphotungstic acid solution (40 mL is usually enough),
make to 300 g with H,O, shake vigorously ca 1 min, and filter
through 24 cm rapid folded paper.
E. Distillation and Computation of Volatile Acid Number
Pipet 150 mL prepared solution into distillation flask of apparatus
and make acid to Congo red paper with H,SO, (1 + 1). Steam distil as
in C. Collect 200 mL distillate, titrate with O.OlM NaOH to
phenolphfhalein end point, and designate as A. Collect second
200 mL portion distillate, titrate with 0.005M BatOH), solution to
phenolphthalein end point, and designate as B. To calculate volatile
acid number, multiply titration obtained on distillate A, corrected for
blank, by 4.
F. Determination of Formic Acid
(Caution: See Appendix B, safety notes on mercury salts.)
0 2000 AOAC INTERNATIONiii
FISH AND OTHER MARINE PRODUCTS
Chapter 35, p. 14
Add 2 drops saturated Ba(OH), solution to distillate B, E, and
evaporate to dryness on steam bath. Add ca 5 mL H,O to residue and
1 mL more of 1N HCl than necessary to liberate volatile acids. Filter
through small paper into 125 mL Erlenmeyer with standard taper
joint, and wash paper with HZ0 in such manner that total filtrate
equals 30-40 mL. Add 10 mL NaCH,CO&NaC1 solution and
10 mL 5% HgCl, solution. Connect flask with standard taper aircon-
denser and place on steam bath 2.5 h
With suction through glass siphon attached to funnel by rubber
stopper, transfer precipitate of Hg,Cl, to previously weighed
microfunnel, A(e), provided with glass fiber filter ca 2 mm thick.
Rinse flask with H,O followed by alcohol. Dry 30 min at 100°C.
cool, and weigh. Weigh funnel with another funnel, prepared with
glass fiber filter and treated similarly to one containing precipitate,
as counterpoise.
Weight Hg,Cl, (mg) x 0.0975 = mg formic acid in distillate. To
calculate total formic acid originally present in aliquot of test sample
in distillation flask before distillation, divide mg formic acid found
by 0.24 (fraction formic acid distilled in second 200 mL distillate)
and multiply by 4 to obtain formic acid in 100 g test sample being an-
alyzed. [Note: Factor 4 applies only to products prepared as in D. If
other test sample weights and aliquots are used as for eggs, 938.07A
(see 34.1.19), appropriate factor must be used.]
References: JAOAC 21,684, 688(1938); 25, 176(1942);
28,644( 1945); 33,848( 1950).
Biochemical Z. 51,253(1913).
CAS-64-18-6 (formic acid)
Revised: March 1996
35.1.28
AOAC Official Method 945.52
Fatty Acids (Volatile)
in Seafood
Chromatographic Separation
of C2 to C4 Saturated Fatty Acids
First Action 1945
Final Action 1965
A. Preparation of Partition Column
To 5 g silicic acid in glazed porcelain evaporating dish add 1 mL
Alphamine red R indicator solution and just enough 1M NH,OH to
give alkaline color of the indicator (1 drop is usually enough). Add
maximum amount of H,O that the silicic acid will hold without be-
coming sticky or agglomerating in the butanol-CHCl, solution.
(This amount must be determined for each batch of siltcic acid and
usually varies from 50 to 75% of weight of silicic acid.) Mix thor-
oughly with pestle until homogeneous. Add ca 25 rnL 1% (v/v)
butanol in CHCl,, and mix to form slurry that pours readily. Pour this
slurry into chromatographic tube containing small cotton plug in
neck of constricted end. To avoid air pockets, tilt tube slightly while
pouring. If air bubbles form while pouring, eliminate by stirring sus-
pension in tube with long glass rod.
Clamp tube vertically. In top insert l-hole rubber stopper fitted
with glass tube bent to 90” angle and held in place by Bunsen clamp
against pressure to be exerted. Connect bent glass tube to pressure
source, 938.09A(c) (see 35.1.27). Adjust pressure to 5-10 psi
(34.5-68.9 kPa) so that excess solvent is forced through column
dropwise.
0 2000 AOAC INTERNATIONAL
AOAC OFFICIAL METHODSOF ANALYSIS(2000)
During removal of excess solvent, gel packs down. As column
packs down, particles of gel adhere to wall of tube, but eventually gel
leaves wall of tube relatively clean. Tlus is point of optimum density
of column, and column is ready for use. Apply pressure until solvent
reaches surface of column. If solvent passes below surface, causing
drying or “cracking” of column, or if air pockets are present, extrude
packing from tube, reslurry with solvent, and repack column.
B. Test of Silicic Acid for Suitability and Standardization
of Column
(a) Preparation of standard acid solutions.-Pipet 1 mL of
each of following acids into separate 1 L volumetric flasks: Acetic,
propionic, butyric, and valeric. Dilute to volume with H,O, and
mix. Plpet 10 mL of each solution into separate 125 mL
Erlenmeyers and titrate with O.OlM NaOH, using cresol red indlca-
tor, to pink persisting ca 45 s.
mL O.OlM NaOH x normality x F
= mg acid/ml standard acid solution
where F= 6.01 for CH,COOH; 7.41, propionic; 8.81, butyric; and
10.2, valeric acid.
(b) Preparation of standard acid standards.-Pipet 50 ti of
each standard acid solution into same 250 mL volumetric flask and
dilute to volume with H,O. Concentration of each acid in standard
acid mixture = l/5 concentration in standard acid solution.
(c) Preparation of known samples.-Pipet 5 and 25 mL
aliquots of standard acid mixture into separate 50 mL beakers.
Just neutralize with O.lM NaOH, using phenolphthalein, and add
10 drops excess. Evaporate to dryness on steam bath. If desired,
chromatograph acids individually by using 5 mL aliquots of each
standard solution and 5 mL aliquots of 5-fold dilutions of each
standard acid solution. Combmations approximating compositions in
samples may also be prepared.
(d) Column separation-(Good separation and yields depend
upon transfer of test sample to column with amount of solvent speci-
fied.) To dry residue of sodium salts add 2 mL 1% (v/v) butanol in
CHCI, solution and while stirring with glass rod add H,SO, (1 + 1)
dropwise until all sodium salts are converted to free acids (acid to
Congo red paper). Add I g anhydrous Na2S04.
Acids elute in following order: valeric, butyric, propionic, and
acetic. Place 50 mL graduate under column as receiver. Decant
supemate onto column, pouring it slowly down side of tube without
disturbing level surface of column. Apply pressure until solvent
reaches surface of gel. Wash beaker with 1 mL solvent, pour onto
column, and with stirring rod transfer residue in beaker to column.
Apply pressure until solvent just disappears into Na$O, layer.
Wash beaker with another 1 mL solvent, transfer to column, wash in-
side of tube with 1 mL solvent, and apply pressure until solvent just
disappears into Na,SO, layer. F111tube with solvent and apply pres-
sure. Each time front (lower edge) of a band reaches point 2-5 mm
above narrowest portion of constriction of tube, record volume col-
lected and change receiver. For each acid, total cumulative volume is
threshold volume used for identifying bands in succeeding runs. Af-
ter propionic acid (third band) has been eluted, fill tube with 10%
(v/v) butanol in CHCl, and use this solvent to elute CH,COOH.
(Propionic acid is eluted after ca 50 mL 1% (v.v) butanol in CHCl,
has passed through column. Observe volume actually used. In suc-
ceeding runs, change to 10% (v/v) butanol in CHCl, at that volume
whether or not propionic acid is present.)
 AOAC OFFICIAL METHODS OF ANALYSIS (2002) FISH AND OTHER MARINE PRODUCTS
Chapter 35, p. 15

Transfer eluates to separate 125 mL Erlenmeyers, rinsing each 35.1.30

graduate with three 5 mL portions H,O. Add 1 drop cresol red indi- AOAC Official Method 954.04*
cator solution and titrate WI th O.OlM alkali As end point ap- Histamine in Seafood
proaches, stopper flask and shake vigorously to completely extract Biological Method
acids from solvent phase. Correct titration of each eluted band for First Action 1954
blank as follows: Collect 25 mL butanoXHC1, mixture from col- Final Action
umn before any acids are transferred, add 15 mL boiled and cooled Surplus 2000
H,O, and titrate as above with O.OlM alkali. A. Apparatus
If bands are not clearly differentiated or recoveries are <90%, re-
(a) Kymogruph.-With horizontal muscle lever arm having fric-
ject the silicic acid. (Additional standardization with respect to
tion or gravity writing point.
threshold volume may be desirable for identification m some m-
(b) Muscle b&.-At least 50 mL capacity, m 37°C constant
stances, C.)
temperature bath. See Figure 954.04. May be conveniently filled and
C. Identification and Determination emptied through 3-way stopcock; 1 tube connected to reservoir of
Add 1 drop 1M NaOH to neutralized distillate A obtained in Ringer-Locke solution through bulb or coil immersed in bath; other
938.093 (see 35.1.27) and evaporate to small volume. Transfer to tube connected to vacuum through suction flask as waste receiver.
50 mL beaker, evaporate to dryness on steam bath, and proceed ex- Bubble air, filtered through cotton, slowly and continuously around
intestine from fine capillary tube.
actly as in B(d).
Identify acids by comparing then threshold volumes with those for B. Reagents
approximately same amounts and ratios of known acids found in stan- (a) Sodium chloride stock solution.-1 80 g/L.
dardization operation. Threshold volume of given amount of each (b) Potassium chloride stock solurion.42 g/500 mL.
fatty acid is characteristic and reproducible under similar conditions. (c) Sodium bicarbonate stock solurion.-15 g/500 mL.
However, if condltlons change, such as use of different batch of silicic (d) Arropine sulfate stock solution.-1 .O g/500 mL.
acid or different amount of same batch, or cbfferent amount of H,O, (e) Calcium chloride stock solution.-24 g anhydrous
threshold volume for each acrid must be redetermined. (If present, salt/500 mL.
isobutyric acid is measured as is-butyric acid.) For further identifica- (f) Ringer-Locke solution.-NaCl, 0.9% (w/v); KCl, 0.042%
tion as characteristic salts, see JAOAC 28, 644( 1945). Acids may (w/v); CaCl,, 0.024% (w/v); NaHCO,, 0.015% (w/v); glucose, 0.1%
also be identified by paper chromatography, 965.24B (see 32.3.06). (w/v); and atropine sulfate, 0.001% (w/v). Add H,O to volume of ca

-
1800 mL and then 10 mL (e) while swirling. Add 2 g anhydrous glu-
mg Acid/100 g test sample = cose before use. Dilute to volume with H,O. Keep solution contain-
mL O.OlM NaOH (corrected for blank) ing glucose m refrigerator when not in use, discarding when it
xnormality x F becomes moldy.
(g) Histamine diphosphare standard solutions.“.1 mg hista-
mine diphosphate/mL boiled H,O. If stored in refrigerator when not
where F includes equivalent weights of acids, corrected for distilla-
in use, it will keep 23 months. Prepare diluted standards of
tion recoveries, and dilutions. F = 421 for CH,COOH; 366,
0.01 mglmL and 0.005 mglmL as required. If bath ~50 mL is used,
propionic; and 383, butync acid.
prepare dilutions of(g) with (f) to avoid dilution of bath when stan-
[Norer Factors given apply only to products prepared as in 938.09D
dards are added.
(see 35.1.27). If other sample weights and aliquots are used as for eggs,
938.07A (see 34.1.19), appropriate factors must be used.]

References: JAOAC 28,644(1945); 33,848(1950); 48,628(1965). To KYMOGRAPH

- AIR
CAS-64-19-7 (acetic acid)
CAS-107-92-6 (butyric acid)
CAS-79-09-4 (propionic acid) TO R-L n
CAS-109-52-4 (valenc acid) S,OiUTloN w

35.1.29
AOAC Official Method 973.26
Fatty Acids (Volatile) in Seafood
INTESTINE
Gas Chromatographic Method
First Action 1973
Final Action 1973

Proceed as in 971.11 (see 34 1.20), except substitute 938.09D (scse

35.1.27) for 971.11D (see 34.1.20). To calculate volatile acid num- otz3

ber, multiply mL O.OlN NaOH required to neutralize 200 mL distil- - CM l-i

late, corrected for blank, by 4.

Reference: JAOAC 56,271(1973). Figure 954.04-Muscle bath.

0 2002 AOAC INTERNATIONAL

FISH AND OTHER MARINE PRODUCTS
Chapter 35, p. 16
(h) Guinea pig inrest&.-Use gumea prgs weighing 300-400 g.
Starve pig 24 h, kill by blow on head, and remove intestine, sevenng
at point proximal to ileocecal junction, retaining ca 12 cm for first se-
ries of assays. Place remamder of intestine on cotton m Petri dish,
just covering wrth solution (f). Prop lid to admit air and store in re-
fngerator at ca 4-7°C (40-45”F). (Below 4°C intestine loses its ac-
tivity.) Use additional portions of intestine as required as long as
matenal shows enough response to histamine stimulus (usually
8 days). These additional portions of mtestme are not as sensitive as
ileum but give uniform response with barely noticeable pendulum
movements after storage.
C. Preparation of Test Sample
See 937.07 (see 35.1 .Ol). Freeze ground test sample for storage, rf
desired.
D. Assay
Weigh 10 g prepared test portion into small mortar. Add enough
H,O to make smooth paste while grinding with pestle. Transfer paste
with little additional H,O to 100 mL Kohhausch flask and add 1 mL
HCl (1 + 1). Add H,O to total volume of ca70 mL, mix well, and heat
flask ca 20 mm in boiling H,O bath. Remove from bath, cool, dilute
to volume with H,O, mix, and filter on Whatman No. 12 folded pa-
per, or equivalent. (Extract filters slowly but only ca 5 mL need be
collected for analysis.) Filtrate may be stored in refrigerator 10 days
without diminished acttvtty. Neutralize 1 mL filtrate with 2 mL 1%
(w/v) NaHCO, solution. Dilute to 10 mL with Ringer-Locke solu-
tion (without glucose).
Attach intestine to muscle lever and let stand at least 0.5 h in
50 mL Ringer-Locke solutron, (f), in constant temperature bath at
37°C. With fresh ileum, contractions and relaxations may be
nonrhythmic for ca 2-3 h with extreme and not always uniform re-
sponses to histamine stimuli. Determinations may be performed
during thts period, but it 1s necessary to add small and increasing
amounts of diluted standards to stabilize intestine response, and to
check responses several times until readings are reproducible.
Add known amount of dilute standard solution, (g), to bath, record
response, and remove writing lever from contact with drum. Drain
mner bath, add fresh 37°C solution, (f), to wash chamber and intes-
tine, remove, and refill with fresh solution. Let intestine rest 3 mm.
Estimate amount and dilution of neutralized fish extract that will
give approximately equal response and add to bath. Repeat record-
ing of response, washmg muscle chamber, and resting 3 min During
thrs interval, measure step heights with mm scale and calculate
amount standard necessary to match assay step height. Alternately
add extract and standard as above until exact match is obtained.
References: JAOAC 37,568(1954); 39,91,609(1956).
CAS-51-45-6 (histamme)
Revised: March 2002
35.1.31
AOAC Official Method 957.07+
Histamine in Seafood
Chemical Method
First Action 1957
Final Action 1968
Surplus 2000
(Use H,O redtstdled from glass for preparation of reagents and for
determmations. Do not clean glassware with soap; use fresh chromic
0 2002 AOAC INTERNATIONAL
AOAC OFFICIALMETHODSOF ANALYSIS(2002)
acid cleaning solution, rinsing well with tap H,O, then 3 times with
distilled H,O, and 3 times with redistilled H,O. Alcohol may be used
to soak or rinse glassware.)
A. Reagents
(a) Benzene-n-butanol mixture.-3 + 2 (v/v).
(b) Conon acid succinate.-Dissolve 5 g anhydrous NaCH,COO,
fused Just before use, and 40 g succmrc anhydride m 300 mL
CH@OH in 500 mL Erlemneyer. Immerse 10 g absorbentcotton, cut
mto strips, m solution; attach drying tube containmg drying agent, and
heat 48 h at 100°C. (Flask may be immersed to neck m active steam
bath) Filter; wash well with H20, HCl (1 + 9), H,O, and finally with al-
cohol. Dry in vacuum oven at 100°C
(c) Diazonium reagent.-Dissolve 0.1 g p-mtroanilme,
recrystallized from hot H,O, and ddute to 100 mL with 0.1M HCl. Store
in refrigerator. Dissolve 4 g NaNOs in H,O and dilute to 100 mL. Store
in refrigerator. Just before use, place 10 mL p-nitroamline solution in
ice bath 25 mm, add 1 mL NaNO, solution, mix, and let stand m bath
5 min before use.
(d) Coupling bu$er -Dissolve 7.15 g sodium metaborate (NaBO,)
and 5.7 g Na,CO, in H,O, and dilute to 100 mL. Store in polyethylene
bottle.
(e) Barbital bu@.-Dissolve 10 g sodium barbital in 1 L H,O and
adjust to pH 7.7 with CHsCOOH (1 + 15) (ca 25-30 mL), using pH me-
ter. Store in refrigerator to prevent mold growth. Dissolve any precipi-
tate by warming before use. (50-250 mL bottle of the buffer may be
kept at room temperature and replenished from mam supply when mold
growth is apparent.)
(f) Histamine standard solutions.-Dry histamine.2HCl (USP Ref-
erence Standard or material checked against Standard as m C) 2 h over
H2S04. Dissolve 0.1656 g dried histamine~2HCl in H,O and dilute to
100 mL (1 mL = 1 mg histamine). Dilute 10 mL of this stock solution to
1OOmL withH,O (1 mL= 100 pghistamine). Dilute 5 mLoftbisdilute
standard solution and 5 mL methanol to 100 mL with H,O (1 mL = 5 pg
histamine). Store in cold. Prepare fresh standards weekly.
(g) I-Methyl-2-pentanone (methyl isobutyl ketone).-Commer-
cially punfied grade (Eastman Kodak Co. No. 416 has been found satrs-
factory). To recover used ketone, wash once with saturated NaHCO,
solution and 3 times with H20, distill, retaming fractron boding at
115-l 18’C, and check A at 475 nm.
(h) BenzaMehyde.-Cl-free.
(i) Dilute sulfuric ncid.-O.20 * 0.005M. accurately standardized.
8. Preparation of CAS Column
Prepare column by firmly placing small plug of cotton acid
succinate (CAS; ca 50 mg) in column prepared by cutting off or
blowing out bottom of 15 mL centrifuge tube. Wash plug with three
15 mL portions H,O and two 3 mL portions alcohol. Let solvents
drip through CAS, syringmg out column by blowing out last por-
tion of each solvent, using 10 mL syringe with needle inserted
through rubber stopper. CAS plugs may be reused for months by
washing shortly after use with H,O and alcohol as above, and pro-
tecting from dust with inverted beaker.
C. Detetmhation
Transfer 10 g prepared test sample, 954.04C (see 35.1.30), to
semimicro container of high-speed blender, add ca 50 mL methanol,
and blend ca 2 min. Transfer to 100 mL glass-stoppered volumetric
flask, rmsing lid and blender ear with methanol and adding rmsings to
flask. Heat in H,O bath to 60°C and let stand 15 min at this temperature.
Cool to 25°C. llute to volume with methanol, and filter through folded
AOAC OFFICIALMETHODSOF ANALYSIS(2002) FISHAND OTHER MARINE PRODUCTS
Chapter 35, p 17

paper. Alcohol filtrate may be stored m refrigerator several weeks.
(Light powdery precipitate separating on storage may be ignored.)
Dilute 5 mL filtrate to 100 mL with H,O (disregard turbidity). Plpet
5 mL aliquot into 16 x 150 mm glass-stopperedtest tube, and add 1 drop
benzaldehyde (Cl-free) and 0.2 mL 20% (w/v) NaOH. (pH after addmg
alkali should be ca 12.4-12.5.) Shake vigorously ca 25 times. Let stand
2 min and add 5 mL benzene-n-butanol mixture. Shake vigorously ca
25 times and let stand 5 min to separate.Ifemulsion forms, centrifuge
Transfer upper layer with fine-tip tube equipped with rubber bulb Ito
previously prepared CAS column, avoiding transfer of any aqueous
phase.Reextract aqueoussolution with 5 mL benzene-butanol rmxture
as before, shakmg, letting stand 5 mm, and transfemng upper layer Ito
column. Rinse hp and sides of column with fine stream of alcohol from
wash bottle, synnging out CAS. Wash column with 3 mL alcohol; sy-
ringe out; wash with two 3 mL portions H,O, and synnge out. Discard
solvents and washes.
Elute histamme from CAS mto 25 mL glass-stoppered Erlemneyer
by washing down sides of tube with 2.0 mL 0.20 + 0.005M H,SO,
(volume and concentration of acrd are critical) followed by 3 mL H,O.
Syringe out after dripping CCiSeS.
Cool eluate m ice bath, weighting flask with lead ring or clamp to
prevent tlppmg, and let stand 5-10 mm. Add 0.5 mL cooled
diazomum reagent and let stand 5 min m ice bath. Add 0.50 mL
coupling buffer (volume 1s critical; Ostwald pipet 1s convenient)
with continuous shaking or swlrlmg to avoid localized alkalinity
(pH after addition of coupling buffer, 5-6). Let stand 5 mm in ice
bath. Saturated solution with ca 0.25 g powdered Na2B40,.10H,0
added m one portion. Shake solution unmedlately and continu-
ously ca 30 s to ensure rapid and complete saturation (final
pH ca 8.6). Let stand 15 min m ice bath.
Pipet in 5.0 mL methyl isobutyl ketone and shake vigorously
25 times. Immediately transfer both layers to 16 x 150 mm test tube
(do not rinse) and let stand 10 min at room temperature to separate
and to warm up. Transfer upper layer with fine-tip dropper to second
16 x 150 mm glass-stoppered test tube containing 5.0 mL barbital
buffer. Avoid transferring aqueous and solid phases if present
(transfer need not be quantitative). Shake vigorously ca
25 times (pH of barbital buffer after washing, ca 8.3-8.4). Let
stand 10 mm to separate.
Transfer upper layer with fine-tip dropper to 1 cm cell and deter-
mine A at 475 nm against methyl isobutyl ketone. Repeat determma-
tion on tests yleldmg A values >25 pg standard by diluting 1 mL
methanol filtrate to 100 mL with H20. Altematlvely, aqueous dilu-
tlon may be diluted 1 + 4 (or more) with H,O
Conduct standard and blank through determmatioa as follows:
Pipet 5 mL 5 pg/mL histamine standard solution mto 16 x 150 mm
glass-stoppered test tube and pipet 5 mL 5% (v/v) methanol into slm-
ilar tube for blank. Proceed as in C, Determination, beginning para-
graph 2, line 2, “. add 1 drop benzaldehyde .”
Subtract blank A from A of standard (A) and of sample (A) and cal-
culate histamme in sample ahquot:
Hlstamme, mg = e
AA’
Reference: JAOAC 40,892( 1957).
CAS-51-45-6 (histamine)
Revised: March 2002
35.1.32
AOAC Official Method 977.13
Histamine in Seafood
Fluorometric Method
First Action 1977
Final Action 1987
Caution. See Appendix B, Laboratory Safety for “Safe Handling of
Alkahes”-sodium hydroxide; “Safe Handhng of
Acids”-phosphoric and hydrochloric acids; and “Safe
Handling of Special Chemical Hazards”-methanol. DIS-
pose of waste solvents in an appropriate manner compati-
ble with applicable environmental rules and regulahons.
See Tables 977.13A and B for the results of the interlaboratory
study supporting the acceptance of the method, and Table 977.13C
for recovery data.
A. Principle
Sample 1s extracted with 75% (v/v) methanol. Extract is passed
through ion exchange column. o-Phthaldialdehyde solution is
added to eluate to form fluorescent histamine derivatives. Fluores-
cent intensity of derivatives is measured using fluorometer and his-
tamine 1s quantified using external standards.
6. Apparatus
Rinse all plastic and glass containers with HCl (1 + 3) and H,O be-
fore use.
(a) Chromarographic rube.-200 x 7 id mm polypropylene hibe
fitted with small plastic stopcocks and ca 45 cm Teflon tubing. Con-

Table 977.13A interlaboratory study results for determination of histamine in tuna by fluorometric method (based on collaborative
study results of the original method)
Histamineb, Avg. hlstamme
Test samplea mg/lOO g found, mg/lOO g Sr RSD,, % sR RSDR, 5%
_--.
Acceptable skipjack packed tn water 1 14 0.643 46 9 1.009 73.6
Skipjack with 25 mg histamine added/100 test sample 26 25.8 0.950 37 I ,383 5.4
Decomposed albacore packed in water 30 31 6 2.053 65 3.473 11.0
Decomposed yellowfin packed in water 20 19.8 0 941 4.7 1.729 a7
Decomposed skipjack packed iln 011 120 1256 6 432 51 a.807 7.0
Decomposed yellowfin packed In oil 200 198.8 4 801 2.4 IO 6555 54
a Blrnd duplicates
* Approximate composltlon

0 2002 AOAC INTERNATIONAL

FISH AND OTHER MARINE PRODUCTS AOAC OFFICIAL METHODS OF ANALYSIS (2000)
Chapter 35, p 18

Table 977.136 Interlaboratory study results for determination of histamine in canned tuna and frozen mahimahi by fluorometric
method (generated for modified method using 75% (v/v) methanol)a

Test sample ~~-

.____ Mean,a&t
____. Sr. wkl SR, Pglg RSD,, % RSDR, % P RC
1 9 896 0.884 0.915 9.01 9.32 2.475 2 562
2 13.0 1.710 1710 13.17 13.17 4788 4.788
3 5627 1 205 1 295 2142 23 03 3 374 3626
4 7.831 1084 1084 13.84 13.84 3035 3035
5 2028 1 140 2077 5.62 1024 3.192 5816
6 58.02 2.111 5466 3.64 9.42 5911 15.305
7 - - ~~~~ 158.4
.__~ -~~~ -~~6 575
---~~~~~~~~ 14050 4.15 8 87 18410 39340
a Based on results recerved From 16 laboratories
’ r=28xs,
c R=2axsR

trol flow rate at >3 mUmin by adjusting height of column relative to
tubing outlet. Alternatively, use 2-way valve in place of tubmg.
(b) Photofluorometer-With medium pressure Hg lamp with
excitation at 350 nm and measuring emission at 444 nm.
(c) Repipers.-1 and 5 mL.
C. Reagents
(a) Ion-exchange resin -Blo-Rad AG l-X8, 50-100 mesh
(Blo-Rad Laboratories, 1414 Harbour, South Richmond, CA 94804,
USA) or Dowex l-X8,50-100 mesh. Convert to -OH form by add-
ing ca 15 mL 2M NaOWg resin to beaker. Swirl rmxture and let
stand <30min. Decant liquid and repeat with additional base. Thor-
oughly washresm with H20, slurry into fluted paper and wash again
with H,O. Prepare resin fresh weekly and store under H,O. Place
glass wool plug in base of tube, B(a), and slurry m enough resin to
form 8 cm bed. Maintam Hz0 level above top of resin bed at all
times. Do not regenerate resin in packed column; rather, use batch
regeneration in beaker when necessary.Wash column with ca 10 mL
H,O before applying each extract.
(b) Phosphoric acid.-3.57N. Dilute 121.8 mL 85% H,PO, to
1 L. For other concentration H,PO,, volume required for 1 L 1.19M
acid = 17493/(density H3P0, x percent H,PO,). Standardize
5.00 mL by titration with l.OOM NaOH to phenolphthalein end
pomt, and adjust concentration if necessary.
(c) o-Phthaldiuldehyde (OPT) so/ufion.--O. 1% (w/v). Dissolve
100 mg OPT m 100 mL distilled-m-glass methanol. Store in amber
bottle in refrigerator. Prepare fresh weekly.
(d) Histamine standard solutions.-Store m refrigerator.
(I) Stock solution -1 mg/mL as free base Accurately weigh ca
169 1 mg histamme 2HC1(98%) mto 100 mL volumemc flask, and dls-
solve and &lute to volume with O.lM HCl. Preparefresh weekly (2) In-
fernwdiare solution -10 pg/mL Pipet I mL stock solution into 100
mL volumetric flask, and dilute to volume with O.lM HCl. Prepare
fresh weekly (3) Working solutions.-0 5, 1.O,and 1 5 pg/5 mL. Pipet
1, 2, and 3 mL intermediate solution into separate100 mL volumetric
flasks, and ddute each to volume with 0. 1M HCl. Prepare fresh daly.
(e) Mefhanol-75% (v/v). Place 75 mL MeOH (distilled m
glass) into 100 mL volumetric flask or stoppered graduated cylinder.
Dilute to volume with H20. Swirl flask while adding H,O
D. Preparation of Standard Curve
Pipet duplicate 5 mL aliquots of each workmg standardsolution into
separate50 mL glass or polypropylene Erlenmeyers. PIpet in 10 mL
O.lM HCl to each flask and mix. Pipet m 3 mL IM NaOH and mix.
Within 5 min, pipet in 1 mL OPT solution and mix unmedlately. After
exactly 4 min, plpet in 3 mL 3.57N H,PO,, and mix immechately. It IS
important to mix thoroughly after each addition and at least once during
OPT reaction. (Run 6- 10 OPT reactions simultaneously by adding re-
agentsto Erlenmeyers in set order.) Prepareblank by substltutmg 5 mL
O.lM HCl for histamine solution. Wahm 1.5 h, record fluorescencem-
tens&y (I) of working standardsolutions with H,O in referencecell, us-
ing excitation wavelength of 350 nm and emission wavelength of
44.4~1. Plot [(corrected for blank) against pg hlstamineR mL ahquot.
E. Determination
Extract prepared sample with 75% (v/v) methanol as m 957.07C
(see 35.1.3 l), paragraph 1. Pass 4-5 mL H,O through column, B(a),
and discard eluate. Pipet 1 mL extract onto column and add 4-5 mL
H,O. Immediatety initiate column flow into 50 mL volumetric flask
containing 5.00 mL 1 .OOMHCl. When liquid level is ca 2 mm above
resin, add ca 5 mL H,O and let elute. Follow with H,O m larger por-
tions until ca 35 mL has eluted. Stop column flow, dilute to volume
with H20, stopper, and mix. Refngerate eluate.
Plpet 5 mL eluate into 50 mL Erlenmeyer, and piper m 10 ti 0.1 M
HCl. Proceed as m D, beginning “Pipet in 3 mL 1M NaOH .“.
If test sample contains >15 mg histamme/lOO g fish, pipet 1 mL
test solution-OPT mixture into 10 mL beaker containing exactly
2 mL blank-OPT mixture, and mix thoroughly. Read fluorescence
of new solution. Dilute and mix ahquots with blank-OPT mixture as
needed to obtain measurable reading. This approximation mdlcates
proper dilution of eluate required prior to second OPT reactlon
needed for reliable quantltation of test sample Alternatively, use
sensitivity range control of fluorometer (if instrument has one) to es-
timate dilution. Use these approximations to prepare appropriate dl-
lution of aliquot of eluate with 0 1M HCI, and proceed as m D,
beginning “Plpet m 3 mL 1M NaOH .‘I.
F. Calculations
Plot of I (measuredby meter deflection or recorder responseand cor-
rected for blank) against pg tustamine/5 mL test solution should be
strrughtline passingthrough origin with slope = m = [(I,ll.5) + Ib+ 21,.]/3.
mg Hlstamme/lOO g fish = (lO)(F)(Ilm)(l,)
pg Histamine/g fish = 10 x (mg hlstammellO0 g fish)
where I,, I,, I,, and I, = fluorescence from test solution, 1.5, 1.0, and
0.5 pg histamine standards, respectively; and F = dilution factor =
(mLeluate + mLO.lM HCl)/mLeluate. F= 1 forundilutedeluate.

0 2000 AOAC INTERNATIONAL

AOAC OFFICIALMETHODSOF ANALYSIS(2000) FISH AND OTHER MARINE PRODUCTS
Chapter35, p. 19

Table 977.13C Recovery of histamine added to canned tuna 35.1.33

(generated for modified method using 75% AOAC Official Method 948.17
methanol)a
- lndole in Crabmeat,
Background, Found, Recovered, Recovery, Oysters, and Shrimp
Labb Id9 Id9 P&l % Calorimetric Method
-
A 10.00 69.00 59.00 117.65 First Action 1948
Final Action 1974
66.00 58.00 111.67
B 9.85 63.30 53.45 106.58
A. Apparatus and Reagents
62.20 52.35 104.39
(a) Color reagent.-Dissolve 0.4 g p-dimethylaminobenzaldehyde
C 9.65 67.00 57.35 114.36
in 5 mL CH,COOH and mix with 92 mL H,PO, and 3 mL HCl. As pu-
72.40 62.75 125.12 rity of p-dimethylaminobenzaldehyde affects intensity of reagent
D 8.95 55.00 48.05 91.82 blank, purify yellow commercial reagent as follows:
58.10 49.15 98.01 Dissolve 100 g in 600 mL HCl (1 + 6). Add 300 mL H,O and pre-
E 9.50 58.40 48.90 97.51 cipitate aldehyde by slowly adding 10% (w/v) NaOH solution with
vigorous stirring. As soon as precipitate aldehyde appears white,
55.10 45.60 90.93
stop addition of NaOH solution, filter, and discard precipitate. Con-
F 9.65 58.10 48.45 96.61 tinue neutralization until practically all aldehyde is precipitated, but
51.80 42.15 84.05 do not carry to completion because last 4-5 g may be colored. Filter,
G 11.55 61.40 49.85 99.40 and wash precipitate with H,O until washings are no longer acid.
57.80 46.25 92.22 Dry aldehyde, which should be practically white, in desiccator.
H 44.90 89.53
(b) Acetic acid, purified.-If this reagent turns pink with color
9.20 54.10
reagent, purify as follows: Add, in order specified, to standard taper
54.10 44.90 89.53
1 L round-bottom flask: 500 mL CH,COOH, 25 g KMnO,, and
J 9.30 55.60 46.30 92.32 20 mL H2S04. Distil in all-glass still not >400 mL.
56.10 46.80 93.32 (c) Dilute hydrochloric acid.-Dilute 5 mL HCl to 100 mL with
K 10.25 53.00 42.75 85.24 H,O.
53.00 42.75 85.24 (d) Indole standard solution.-Accurately weigh 20 mg indole
into 200 mL volumetric flask and dilute to volume with alcohol.
N 10.10 54.30 44.20 88.14
Keep refrigerated and discard after 2 weeks.
54.30 44.20 88.14
(e) Distillation apparatus.-Use separate steam generator for
0 10.00 59.00 49.00 97.71 each unit. Steam generator may be made from 1 L Erlenmeyer and
59.00 49.00 97.71 connected to all-glass steam distillation apparatus with minimum
P 10.70 53.30 42.60 84.95 use of rubber tubing. Distillation flask (capacity 2500 mL) is con-
53.30 42.60 98.89 nected to straight bore condenser through spray trap; 500 mL
96.41 _
Erlenmeyer is effective receiver. Foil-covered rubber stoppers may
Avg rec., 8
z Added 50.15 pg histamine/g.
be used in absence of all-glass apparatus. (Unprotected natural or
Data from Laboratories I, L, and M were excluded because results for the synthetic rubber connections and stoppers cause variable distillation
background or the spike were outliers blanks.)
Ensure absence of Cl in the H,O which may partly or entirely in-
hibit development of indole color.

If calibration plot is not linear, use standard curve directly for
quantitation. Each subdivision on abscissa should be SO.1 pg hista-
mine/5 mLtest solution. Read all values from curve to nearest
0.05 pg histamine/5 mL test solution.
mg Histamine/100 g fish = (IO)(F)(W)
pg Histamine/g fish = 10 x (mg histamine1100 g fish)
6. Preparation of Test Samp/e
Crabmeat, oysters, and shrimp.-For oyster meats, weigh
50 g; for drained crabmeat or peeled raw or cooked shrimp,
weigh 25 or 50 g (depending upon amount of indole expected).
Transfer weighed portion to high-speed blender, add 80 mLH,O
(if oysters or crabmeat) or 80 mL alcohol (if shrimp), and mix
several min until homogeneous. Quantitatively transfer mixture
to distillation flask, and rinse mixing chamber with minimum
amount of same solvent used for preparing mixture.

C. Determination
where W= pg histamine/5 mL test solution as determined from stan- Connect flask for steam distillation and gently apply steam until
dard curve. distillation is well started, using care not to pass in steam so vigor-
ously as to cause excessive foaming. Apply enough heat to distilla-
Reference: JAOAC60,1125, 1131(1977). tion flask to maintain volume of 80-90 mL. Collect 350 mL distillate
CAS-51-45-6 (histamine) in ca 45 min. (If alcohol was used in preparation of test sample, col-
lect 450 mL.) Wash condenser with small amount of alcohol and
Revised: March 1999 drain into receiving flask containing distillate.
FISH AND OTHERMARINEPRODUCTS AOAC OFFICIALMETHODSO F ANALYSIS(2000)
Chapter 35, p. 20

Transfer distillate to 500 mL separatorand add 5 mL dilute HCl and
5 mL saturatedNa$O, solution. Extract successively with 25,20, and
15 mL portions CHCl,, shaking vigorously 11 min each time. Com-
bine the 25 and 20 mL extracts in 500 mL separator and wash with
400 mL H20, 5 mL saturatedNa,SO, solution, and 5 mL dilute HCl.
Save wash H,O. Filter combined extracts through cotton plug into dry
125 mL separator.Wash 15 mL portion, using same wash H,O, and
combine with other portions in same 125 mL separator.
Add IO mL. color reagent to combined extracts, shake vigorously
exactly 2 min, and let acid layer separate as completely as possible.
Transfer 9.0 mL acid layer to 50 mL volumetric flask, dilute to vol-
ume with CH,COOH, mix well, transfer solution to suitable pho-
tometer cell, and determine A at 560 nm. Color solution may be
diluted with CH,COOH containing 9.0 mL color reagent/50 mLtest
solution, provided blanks are determined at same dilutions.
Prepare standard curve as above by steam distilling series of
freshly prepared dilutions of standard indole solution. Determine
distillation blank similarly, omitting addition of indole.
Reference: JAOAC 31,96, 507(1948).
CAS- 120-72-9 (indole)
35.1.33A
AOAC Official Method 996.07
Putrescine in Canned Tuna
and Cadaverine in Canned Tuna and Mahimahi
Gas Chromatographic Method
First Action 1996
(Applicable to determination of 20.3 g putrescine/g in canned tuna,
and 20.5 pg cadavenneig in canned tuna and mhmahi.)
See Tables 9%.07A and B for the results of the interlaboratory stud-
ies supporting the acceptance of this method. See Tables 996.07C
and D for recovery data.
Caution: See Appendix B, safety notes. Pentafluoropropionic an-
hydride causes severe bums. Avoid contact with skin
and eyes. Wear gloves and use effective fume removal
device.
A. Principle
Putrescine and cadaverine are extracted from test portion with
7 5 % methanol and then converted to fluorinated derivatives. Reac-
tion mixture is purified by passing through a solid-phase extraction
(SPE) column and derivatives are quantitated by electron capture
gas chromatography (GC) after separation on OV-225 column.
B. Apparatus
(a) Gas chromatograph.-With 63Ni pulsed electron capture de-
tector. Operating conditions. injection port, 210°C; detector,
320°C; GC column, 170-180°C; N flow, 25 mL/min; electrometer
range, lo-” (full scale). Detector makeup purge gas as needed (e.g.,
40 mL/min) .
(b) GCcobmn.-1800 x 2 mm id, glass column, packed with 3 %
OV-225 (50% cyanopropylphenyl 5 0 % methyl) on 100-120 mesh
Gas-Chrom Q, or equivalent capillary column. Condition 2 h with
gas flow at room temperature, increase temperature gradually (ca
6”CYmin) to 24O”C, and hold 16 h. Retention times of
pentafluoropropionic derivatives of putrescine, cadaverine, and
hexanediamine should be 7,9, and 12 min, respectively.
(c) SPE tubes.-3 mL, SupelClean LC-Alumina-N SPE tubes
(available from Supelco, Inc., Supelco Park, Bellefonte, PA
16823-0048, USA, or equivalent). Check activity of each lot of SPE
tubes as follows: Pass 2 mL petroleum ether-toluene solvent, C(k),
through SPE tube and then add to tube 50 pL Sudan I dye solution,
C(l). Add a few drops of petroleum ether-toluene solvent, C(k), to re-
move dye from top frit. Pass3 mL solvent through column. If band of
dye stays in the top 4 mm of SPE tube, the batch IS satisfactory for pu-
rification of cahbration standard and analyte derivatives.
(d) Analytical balance.
(e) Rotary evaporator.-Maintaining 50 + 5°C.
(f) Water bath.-Mamtaining 50 5 5°C and 60 + 5°C.
(g) Glassware.-Volumetric flasks, 100 mL (glass-stoppered)
and 1 L; round or flat bottom 24140flasks, 100 mL; graduated cylin-
ders, 100 and 500 mL (glass-stoppered).
(h) Blender.-For test sample preparation.
(i) Pipets.-Accurately delivering 1,2, 3, 5, 10, and 25 mL.
u) Micropipets.-Accurately delivering 50, 150, and 500 pL.
(k) Foldedfilter paper.
(I) Glass syringe.-1.0 mL, calibrated in 0.01 mL.
C. Reagents
(a) Pentafluoropropionic (PFP) anhydride.-Store refrigerated
and protected from moisture (available from Pierce Chemical Co.,
PO Box 117, Rockford, IL 61105-0117, USA).
(b) Ethyl acetate.--Distilled in glass. Suitable for GC.
(c) Toluene.-Distilled in glass. Suitable for GC.
(d) Hexane.-Suitable for GC.

Table 996.07A Interlaboratory study results for determination of putrescine (@g) in canned tuna by gas chromatographya

Test sample Mean, wfg Sr, P&J -3% l4Jg RSDr, % RSDR, % rb RC
1 0.323 0.053 0.064 16.49 19.75 0.148 0.179
2 0.184 0.028 0.080 15.57 43.6 0.078 0.224
3 0.524 0.089 0.141 17.09 26.86 0.249 0.395
4 0.778 0.058 0.072 7.51 9.23 0.162 0.202
5 2.606 0.196 0.209 7.51 8.03 0.549 0.585
6 4.545 0.234 0.399 5.17 8.77 0.655 1.117
z Based on results received from 14 laboratories.
r = 2 8 x s,.
c R=28xs,

0 2000 AOAC INTERNATIONAL

AOAC OFFICIALMETHODSOF AN,&LYSIS (2000) FISH AND OTHER MARINE PRODUCTS
Chapter 35, p. 21

Table 996.076 Interlaboratory study results for determlnatlon of cadaverine (&g) In canned tuna and mahlmahl by gas
chromatographya
Test sample Mean, ps/s srt PdLl SRSPd9 RSD,, % RSDR, % rb RC
Canned tuna 0.564 0.056 0.104 9.91 18.40 0.157 0.291
Canned tuna 1.064 0.082 0.104 7.71 9.80 0.230 0.291
Canned tuna 1.118 0.099 0.117 8.88 10.49 0.277 0.328
Canned tuna 2.573 0.076 0.111 2.98 4.55 0.213 0.311
Canned tuna 9.120 0.515 0.648 5.65 7.1 1.442 1.814
Canned tuna-spiked 5.171 0.779 0.914 15.06 17.68 2.181 2.559
Mahimahi 8.390 0.416 1.239 4.45 14.77 1.170 3.469
1 Based on results received from 14 laboratories
r = 2.8 x s,

(e) Hydrochloric acid.-1M (83.3 mL HCl diluted to 1 L with
water); O.lM (dilute 100 mL 1N HCl to 1 L with water).
(f) Ethyl acetate-toluene solvent.-30% Ethyl acetate in toluene.
Transfer 150 mL ethyl acetateto stopperedgraduatedcylinder, add
350 mL toluene, and mix.
(g) Methanol.-75% (v/v). Place 750 mL MeOH (distilled in
glass) into 1 L volumetric flask. Dilute to volume with H,O while
swirling.
(h) Hexanediamine internal standard solutions.-(l) Hexane-
diamine standard stock solution.-Dissolve 163.0mg hexanediamine
dihydrochloridein O.lM HCl in 100mL volumetric flask and dilute to
volume with O.lM HCI. (2) 20 pg/mL--Pipet 2 mL hexanediamine
standardstocksolution,(I), into 100mL volumetric flask and dilute to
volume with O.lM HCl. (3) Hexanediamine internal standard work-
ing solution.-5 pg/mL. Pipet 25 mL 20 pg/mL hexanediaminestan-
dard solution into 100 mL volumetric flask and dilute to volume with
O.lM HCl.
(i) Putrescine-cadaverine standard stock solution.-Dissolve
91.4 mg putrescine dihydrochloride and 171.3 mg cadaverine

Table 996.07C Recovery of putrescine added to canned Table 996.07D Recovery of cadaverlne added to canned
tunaa tuna’
-
Background, Found, Recovered, Recovery, Background, Found, Recovered, Recovery,
Laboratory K34 P&J Pg/g % Laboratory Ids Ids ld!J %
A 0.35 4.60 4.25 86.38 A 0.53 5.10 4.57 91.95
4.80 4.45 90.45 5.40 4.87 97.99
B 0.31 5.35 5.04 102.44 B 0.54 5.33 4.79 96.38
4.82 4.51 91.67 5.48 4.94 99.40
C 0.35 4.78 4.43 90.04 C 0.46 5.35 4.89 98.39
4.65 4.30 87.40 5.33 4.87 97.99
D 0.32 4.88 4.56 92.68 D 0.57 5.40 4.83 97.18
4.83 4.51 91.67 5.30 4.73 95.17
E 0.33 4.83 4.50 91.46 E 0.50 5.33 4.83 97.18
4.51 4.18 84.96 4.58 4.08 82.09
F 0.42 4.00 3.58 72.76 F 0.58 5.38 4.80 96.58
4.15 3.73 75.81 5.32 4.74 95.37
G 0.24 4.66 4.42 89.84 G 0.59 5.43 4.84 97.38
4.74 4.50 91.46 5.50 4.91 98.79
H 0.31 4.71 4.40 89.43 H 0.72 5.38 4.66 93.76
4.59 4.28 86.99 5.66 4.94 99.40
I 0.26 4.57 4.31 87.80 I 0.60 5.52 4.92 98.99
3.74 3.48 70.73 4.41 3.81 76.66
K 0.40 4.61 4.21 85.57 J 0.71 6.13 5.42 109.05
4.42 4.02 81.71 6.27 5.56 111.87
Avg. rec., % 87.05 Avg. rec., % 96.58
Added 4 92 pg putrescine/g tuna a Added 4.97 pg cadaverine/g tuna.

0 2000 AOAC INTERNATIONAL

FISHAND OTHER MARINEPRODUCTS
Chapter 35, p. 22
Table 996.07E Preparation of putrescinecadaverlne calibration standard solutions
Putrescine-cadaverine Putrescine-cadaverine standard
calibrating standard solution working solution Volumea, mL
I B 0.5
II B 1.0
Ill B 5.0
IV A 1.0
il Added to 1 mL hexanediamine standard working solution.
dihydrochloride in 0. 1M HCI in 100 mL volumetric flask and dilute
to volume with O.lM HCI.
(j) Putrescine-cadaverine standard working solu-
tions.-(l) Solution A.-l0 pg cadaverine/mL and 5 pg
putrescine/mL as free base. Pipet 1 mL putrescine-cadaverine stan-
dard stock solution, (i), into 100 mL volumetric flask and dilute to
volume with 0. 1M HCl. (2) Solution B.- 1 pg cadaverine/mL and
0.5 kg putrescine/mL as free base. Pipet 10 mL solution A into
100 mL volumetric flask and dilute to volume with O.lM HCI.
(k) Petroleum ether-toluene solvent.4 + 1 (v/v). For checking
activity of SPE tubes.
(I) Sudan I dye solution.-For checking activity of SPE tubes.
Dissolve 0.01 g Sudan I dye (97%) in 8 mL petroleum ether-toluene
solvent, (k).
D. Preparation of Putrescine-Cadaverine Calibration Standard
Solutions
Prepare putrescine-cadaverine calibration standard solutions
I-IV (see Table 996.073) as follows: PIpet 1 mL aliquots of
hexanediamine internal standard working solution, C(h)(3), and in-
dicated volumes of putrescine-cadaverine standard working solu-
tions (see Table 996.07E) into separate 100 mL round- or
flat-bottom 24140 flasks. Add ca 0.5 mL 1M HCl into each flask and
evaporate to dryness on rotary evaporator at ca 50-60°C. (Note: Pro-
vide adequate chilling and vacuum to obtain evaporation.) Rinse
each flask with l-2 mL H,O and evaporate to dryness again to re-
move last trace of HCl.
To dried residue, add 1 mL ethyl acetate, C(b), and 300 pL PFP
anhydride, C(a), using glass syringe. Close flask with stopper, mix,
and heat 30 min at 50°C in water bath. (Note: Because of pressure in-
crease as PFP reactlon is heated, stoppers should be secured with re-
straming clip.)
Swirl solution at least once during reaction. After reaction, the re-
sulting reaction mixture should remain clear. Within 2 h after re-
moval from water bath, proceed to next step to purify reaction
mixture using SPE tube.
Add 2 mL toluene, C(c), to putrescine-cadaverine calibration
standard-PFP reaction mixture. Add 2 mL hexane, C(d), to each
SPE tube and let flow through by gravity. Discard hexane. Add
150 pL putrescine-cadaverine calibration standard-PFP-toluene re-
action mixture to top of SPE tube. Start collecting effluent when
mixture is added. Add 3 or 4 drops 30% ethyl acetate-toluene sol-
vent, C(Q, to tube. After mixture passesinto frit, add 2 mL 30% ethyl
acetate-toluene solvent. Add additional 6 mL 30% ethyl ace-
tate-toluene solvent (total of 8 mL) and collect entire effluent. Efflu-
ent is stable at least 1 week stored in refrigerator in the dark.
0 2000 AOAC INTERNATIONAL
AOAC OFFICIALMETHODSOF ANALYSIS(2000)
Equivalent cadaverine level in Equivalent putrescine level in
test sample, &g test sample, &g
0.5 0.25
1 0.5
5 2.5
10 5
E. Isolation of Analyte
Extract product with 75% methanol as follows: Transfer 10.00 g
product to blender bowl and add ca 60 mL 75% methanol, C(g).
Blend ca 2 min at high speed. Transfer to 100 mL glass-stoppered
volumetric flask, rinsing lid and blender jar with 75% methanol and
adding rinsings to flask. Place flask in 60°C water bath and maintain
15 min. Cool to room temperature and dilute to volume with 75%
methanol. Mix by inverting and filter through folded filter paper.
Methanol extracts are stable at least 4 months in refrigerator.
Pipet 10 mL methanol extract into 100 mL round- or flat-bottom
flask, add 1 mL hexanediamine internal standard working solution,
C(h)(3), and ca 0.5 mL 1M HCl. Evaporate to dryness on rotary
evaporator at ca 5%60°C. (Note: Extracts must be evaporated to
dryness. Provide adequate chilling and vacuum to obtain evapora-
tion.) After all solvent is evaporated, add 2-3 mL H,O, swirl, and
evaporate again. (Note: This will eliminate the last trace of HCl and
ensure that extract is dry.) Dried residues of methanol extracts (be-
fore reaction with PFP anhydride) are stable at least 3 days.
To dried residue, add 1 mL ethyl acetate and 300 pL PFP anhy-
dride using glass syringe. Close flask with stopper, mix, and heat
30 min at 50°C in water bath. (Note: Because of pressure increase as
PFP reaction is heated, stoppers should be secured with restraining
clip.)
Swirl solution at least once during reaction. After reaction, the re-
sulting analyte reaction mixture will turn yellow with most fishery
products. (Note: Clear reaction rmxture Indicates presence of H,O in
residue of methanol extract. In such case, repeat evaporation step
and then proceed with analysis.) Within 2 h after removal from water
bath, proceed to next step to purify reaction mixture using SPE tube.
Proceed as in D, beginning “Add 2 mL toluene.. .,” using
analyte-PFP reaction mixture instead of putrescme-cadaverine cali-
bration standard-PFP reaction mixture.
F. GC Determination
Adjust GC system to give full scale recorder response for mjec-
tion of 1 pL derivatized putrescine-cadaverine calibration standard
solution IV from D. For products containing lower levels of analytes
(e.g., O-5 pg cadaverinelg), set derivatized putrescine-cadaverine
calibration standard solution III (5 yg/g) full scale.
Inject l-2 pL effluent from E onto GC system. (Note: Full cah-
bration of GC system with duplicate injections of each of
putrescine-cadaverine calibration standard solutions is required
only at the beginning of analysis unless instrument is shut down be-
tween analyses. When analyzing test portions on succeeding days,
rerun derivatized putrescine-cadaverine calibration standard solu-
tions II and III through GC system to ensure that calibration of m-
strument is stable.)
AOAC OFFICIAL METHODS OF ANALYSIS (2000) FISH AND OTHER MARINE PRODUCTS
Chapter 35, p. 23

G. Calculations 35.1.34
Perform following calculations: AOAC Official Method 982.20
(I) Calculate response ratio of cadaverine (RS,) in each lndole in Shrimp
putrescine-cadaverine calibration standard solution as follows: Gas Chromatographic Method
First Action 1982
RS=ps,
c PS,
Final Action 1996

where PS, = peak height of PFP cadaverine in putrescine-cadaverine
calibration standard solution and PS, = peak height of PFP
hexanediamine in putrescine-cadaverinecalibration standardsolution.
Plot RS, vs pg cadaverine derivatized (W,).
(2) Calculate response ratio of putrescine (RS,) in each
putrescine-cadaverine calibration standard solution as above, using
peak height of PFP-putrescine (PS,).
Plot RS, vs pg putrescine derivatized (W,,).
(3) Calculate response ratio of cadaverine in test portion (RT,) as
follows:
RT =pT,
’ PT,
where PT, = peak height of PFP-cadaverine in test and PT,, = peak
height of PFP-hexanediamine in test.
(4) Calculate response ratio of putrescine in test (PT,) as above,
using peak height of putrescme in test (PT,,).
(5) Determine WC and Wp for tests from calibration graph.
(6) Calculate amount of cadaverine in test portion as follows:
pg Cadaverinelg = wc y ‘1’
L volumetric flask and dilute to volume with ethyl acetate.
where F = attenuation (or dilution) factor, if required, and W, :=
weight of test portion, g.
(7) Calculate amount of putrescine in test portion as above, using
Wp value. Report all results to 0.1 pg/g.
If values obtamed for putrescine and cadaverine in test portion are
higher than those obtained for the most concentrated
putrescine-cadaverme calibration standard solution, quantitate
putrescine andcadaverine levels in test portion by analyzing smaller
aliquot of 75% methanol extract (e.g., 1 mL), and multiply by appro-
priate factor to calculate values in pg/g. Smaller volume of test solu-
tion may be inJected or instrument attenuation may be adjusted to
estimate putrescme and cadaverine content.
Reference: J. AOAC Inr. 80,591(1997).
Revised: March 1999
A. Reagents
(a) Purijied water.-Distilled HZ0 treated by Mini-Q water puri-
fication system (Millipore Corp., Bedford, MA 01730, USA), or
equivalent.
(b) Solvents-Ether, anhydrous (containing 0.05% (v/v) alco-
hol), ethyl acetate, and hexane, distilled from glass.
(c) Carbonate buffer.-pH ca 9.6; 0.2M each Na,CO, and
NaHC4. Dissolve 21.2 g Na,CO, and 16.8 g NaHCO, in H,O and di-
lute to 1 L.
(d) Silica gel.--Dry IO-230 mesh silica gel 60 (EM Science, No.
7734) 2 h at 125°C in open vessel in layers 2 cm deep. Add 3.0 g H,O
per 25.0 g dried silica gel and place on wrist-action shaker 4 h. Store in
air-tight container.
(e) Indole standard solutions.-Stock solution L-l.00 mg/mL.
Dissolve lOO.Omg indole in alcohol in 100 mL volumetric flask and di-
lute to volume with alcohol. lndole standard solution IL-20 ~g/mL.
Pipet 2.0 mL stock solution into 100 mL volumetric flask and dilute to
volume with ethyl acetate. lndole standard solution III.-5 pg/mL.
Pipet 25 mL standard solution II into 100 mL volumetric flask and di-
lute to volume with ethyl acetate. Zndole standard solution
IV.-2 pg/mL. Pipet 10 mL standard solution II mto 100 mL volumet-
ric flask and dilute to volume with ethyl acetate.Zndole standard solu-
tion V.-l pg/mL. Pipet 5 mL standard solution II into 100 mL
(f) 2-Methylindole solution.--Stock solution I.-l mg/mL. Dis-
solve 100 mg 2-methylindole in alcohol in 100 mL volumetric flask and
dilute to volume with alcohol. Dilute stock solution IL-50 @IILL.
Pipet 5 mL stock solution into 100 mL volumetic flask and dilute to
volume with alcohol. Working standard solution Ill.-10 pg/mL. Pipet
20 n&dilute stock solution II into 100 mL volumetric flask and dilute to
volume with alcohol.
B. Apparatus
(a) Gas chromatograph.-With N-specific detector, such as
Perkin-Elmer Model Sigma 2 (Perkin-Elmer Corp., Norwalk, CT
06856, USA), or equivalent, and 6 ft (1.8 m) x 2 mm id glass col-
umn packed with 10% neopentylglycol adipate (NPGA) or
Superpak 20M [Analabs Inc., see (b) below], or equivalent. Adjust
bead current, air flow, and H, flow to obtain 150% full scale deflec-
tion for 5 pg indole (3 FL calibration solution 4, Table 982.20). Ad-

Table 982.20 Calibration solutions for GC determination of indole in shrimp

Calibration solution Standard solution Concentration, &mL mL w 2-Mel, apL Ethanol, mL Total, mL _
1 V 1 1 1 1 1 3
2 V 1 2 2 1 0 3
3 IV 2 2 4 1 0 3
4 III 5 1 5 1 1 3
5 III 5 2 10 1 0 3

2-Methylindole working standard solution, 10 &mL

0 2000 AOAC INTERNATION~

FISH AND OTHER MARINE PRODUCTS
Chapter 35, p. 24
just chromatographic conditions so that retention times for indole
and 2-methylindole are ca 6.8 and 9.4 min, respectively. Baseline
resolution should be obtained for tri-n-butyl phosphate (ca 5 min),
mdole, skatole (ca 8 min), and 2-methylindole.
(b) CC coZnrnns.-(I) or (2) below:
(I) NPGA-Dissolve 1.2 g NPGA (HI-EFF-3a,
Alltech-Applied Science Laboratories, Inc.) in 100 mL CHCl, in
400 mL beaker. Add 10.8 g 80-100 mesh acid-washed
Chromosorb W (Manville Filtration and Minerals) with stirring.
Evaporate solvent on steam bath with stirring. Transfer powder to
rotary evaporator and remove last of solvent at 40-50°C with vac-
uum. Fill clean, dry glass column, (a), with 5% (w/v) solution of
dimethyldichlorosilane in toluene and let stand ca 5 min. (Cau-
rion: Dichlorodimethylsilane is toxic. Avoid contact with skin or
eyes. Use effective fume removal device.) Rinse column with ca
50 mL methanol and dry under N,. Pack column with coated sup-
port and purge 22 h withN, at room temperature. Condition 24 hat
220°C with 10 mL N,/min.
Representative conditions are: temperatures-column 195”C,
injector220”C, detector250”C; carriergasflowca30mLNZ/min.
(2) Super Pak 20M.-Fill clean, dry glass column with 5%
(w/v) solution of dimethyldichlorosilane in toluene and let stand
ca 5 mm. Rmse with ca 50 mL mixture and dry under N,. Pack col-
umn with SuperPak 20M and purge 23 h with He at room tempera-
ture. Condition column 24 h at 220°C with He flow of 20 ml/min.
Typical GC operating conditions are: temperatures-column
16O”C, injector 22O”C, detector 250°C; carrier gas 30 mL He/min.
(c) Whatman 1 PS phase separating filter paper.
C. Calibration
Prepare calibration solutions containing volumes as indicated in
Table 982.20. Pipet indicated volumes into separate vials with Tef-
lon-lined screw caps and mix. Store in refrigerator.
Make duplicate injections (ca 3 pL) of each solution. Measure
peak heights and calculate peak height ratio, R = height indole
peak/height internal standard peak. Prepare standard curve by plot-
ting R against pg indole in standard solution.
D. Determination
Weigh 25.0 g well-mixed test sample into blender bowl, add
100 mL carbonate buffer, and blend 2 min at high speed. Quantita-
tively transfer slurry to 500 mL separator. Rinse blender with 25 mL
H,O from squeeze bottle and add rinse to separator. Add 200.0 mL
ethyl acetate to separator and shake vigorously 2 min.
Let layers separate and drain lower layer into beaker. If ethyl ace-
tate (upper layer) is <150 mL, centrifuge lower layer, separate, and
combme resulting organic layers. Pass organic layer through
Whatman phase-separating paper. Measure exactly 150 mL filtrate
into glass-stoppered flask and add 1 .OOmL 2-methylindole working
solution and 10 g anhydrous Na2S04. Shake 1 min.
Decant extract and concentrate to ca 5-10 mL, using rotary evapo-
rator and ca 35-4O”C H,O bath. Do not evaporate to dryness. (Alter-
natively, concentrate under stream of N2 in 50°C H,O bath.)
Transfer extract to vial and concentrate to ca l-l.5 mL under N,.
Add 2 mL hexane and mix. Add 1.5 g anhydrous Na,SO, and shake
vigorously 2 min.
Prepare cleanup column by firmly placing plug of glass wool in
bottom of 10 cm x 15 mm or 10.5 mm id glass chromatographic tube.
Add 6 g sihca gel and tap firmly. Place ca 0.5 cm anhydrous Na2S04
on top of silica gel bed. Pipet ca 2 mL hexane on top of column. Im-
mediately after hexane is absorbed, transfer concentrated extract
0 2000 AOAC INTERNATIONAL
AOAC OFFICIALMETHODSOFANALYSIS(2000)
(containing added hexane) to column with minimum disruption of
bed. When liquid level reaches top of Na,SO,, add 10 mL hexane.
Similarly, add 10 mL ether-hexane (15 + 85) and 40 mL
ether-hexane (15 + 85). Collect all column effluent. Concentrate to
ca 1.5 mL at 50°C under N,. Do not evaporate to dryness.
Make duplicate inJections, measure peak heights, and calculate R
values. Determine I (pg indole) test samples from calibration
plot. Calculate amount of indole in each test sample:
I xl00
pg Indole/lOO g test sample =
g test sample x (150 / 200)
Calculate average amount of indole from duplicate injections.
References: JAOAC57, 813(1974); 65, 842(1982).
CAS-120-72-9 (indole)
Revised: March 1997
351.35
AOAC Official Method 981.07
lndole in Shrimp
Liquid Chromatographic Fluorometric Method
First Action 1981
Final Action 1982
Caution: See Appendix B, safety notes on blenders and methanol.
A. Principle
Test sample is blended with methanol and 2-methylindole internal
standard. Filtered extract is analyzed by LC on ODS reverse phase col-
umn using methanol-H,0 (60 + 40) and spectrofluorometric detection.
6. Reagents
(a) Purified water.-Distilled HZ0 treated by Milli-Q purification
system (Millipore Corp.), or equivalent. Use throughout.
(b) Methanol.-Distilled from glass, or equivalent.
(c) lndole standard solutions.-Solution A.-l.20 mg/mL. Dis-
solve 120 mg indole in methanol in 100 mL volumetric flask and dilute
to volume with methanol. Solution B.-l2 &nL. Pipet 1.0 mL Solu-
tion A into 100 mL volumetric flask and dilute to volume with metha-
nol. Solution C.-l.2 pg/mL. Pipet 5.0 mL Solution B into 50 mL
volumetric flask and dilute to volume with methanol.
(d) 2-Methylindole internal standard solution.-SoZu-
tionA.-1.25 mg/mL. Dissolve 125 mg 2-methylindole in methanol in
100 mL volumetric flask and dilute to volume with methanol. Solu-
tion K-62.5 pg/mL. Pipet 5.0 mL Solution A into 100 mL volumetric
flask and dilute to volume with methanol. Solution C.-6.25 @mL.
Pipet 10.0 mL Solution B into 100 mL volumetric flask and dilute to
volume with methanol.
(e) Mobile phase.-Dilute 600 mL methanol to 1.O L with H,O.
C. Apparatus
(a) Liquid chromatograpk-Model 203 spectrofluorometer
equipped with flow cell (Perkin-Elmer Corp.), or equivalent; excitation
wavelength 280 nm and emission wavelength 330 nm. Model 6000 sol-
vent delivery system (Waters Associates, Inc.), or equivalent Valco
7000 psi injection system (Valco Instruments Co., Inc., PO Box 55603,
Houston, TX 77255, USA), or equivalent
(b) LC columns.-Analytical column-30 cm x 4 mm id, packed
with 10 pm ODS, reverse phase, or equivalent. Use column and chro-
AOAC OFFICIAL METHODS OF ANIALYSIS(2000)
Table 981.07 Calibration solutlonsator LC tluorometric determination of indole in shrimp
lndole standard solution C (1.2 vg/mL)
Calibration solution mL IQ
1 0.5 0.6
2 1.0 1.2
3 2.0 2.4
4 3.0 3.6
5 4.0 4.6
Final volume 100 mL in methanol
matographic conditions that will produce resolution between peaks 1.2.
Guard column.-7 cm x 4 mm id, packed with 30 pm Co:Pell ODS
(Whatman, Inc.), or equivalent. Use replaceable 2 pm frit on outlet. .At
mobile phase flow rate of 1.O mIJmin, representative retention times
are indole 8.6 min, 2-methylindole 12.1 min. Flow rate may be varied
from 0.7 to 1.3 mUmin to obtain best separation.
D. Calibration
Prepare calibration solutions indicated in Table 981.07. Pipet
indicated volumes into separate 100 mL volumetric flasks and
dilute to volume with methanol. Store in refrigerator.
Adjust instrument to give 150% full scale recorder deflection for
720 pg indole (20 PL of calibration solution no. 4). Inject 20 pL
aliquots of indole calibration solutions, measure peak heights, and
calculate peak ratio R = height indole peak/height internal standard
peak. Plot R against c(g indole (W) in calibration solution.
E. Determination
Weigh 10.0 g test portion into blender and add 50 mL methanol.
Pipet 1.00 mL internal standardsolution C (6.25 pg) into blender by let-
ting pipet drain onto side wall above liquid level. Rinse blender wall
with ca 5 mL methanol. Blend slowly 1 min to ensure even distribution
of internal standard. Avoid splattering onto blender walls. Place Al
foil-lined cap on blender and operate at high speed3 min. Rinse walls of
blender bowl with ca 25 mL methanol and blend combined liquids (ca
80 mL) 1 mm at high speed.Filter extract and inject ca20-30 pL filtrate
into liquid chromatograph. Measure peak heights, and calculate R a
above. Determine W (pg indole) from calibration curve.
1.18Indole/lOO g test portion = 1OW
Reference: JAOAC 64,592(1981).
CAS-120-72-9 (indole)
Revised: March 1996
35.1.36
AOAC Official Method 986.12
Ethanol in Canned Salmon
Headspace Gas Chromatographic Method
First Action 1986
Final Action 1996
A. Principle
Liquid from canned salmon is separated into oil and aqueous
phases. Aqueous phase is analyzed for ethyl alcohol by headspace
FISHAND OTHER MARINE PRODUCTS
Chapter 35, p. 25
2-Methylindole internal standard solution C
H20 mL (6.25 &mL), mL
10 1.0
10 1.0
10 1.0
10 1.0
10 1.0
gas chromatography with flame ionization detection. Ratios of peak
areas of ethyl alcohol to internal standard in test sample and standard
are compared.
B. Apparatus
(a) Gas chromarograph.-Model 5880A, equipped with
flame ionization detector (Hewlett Packard Co., Avondale Div.),
or equivalent. (Equivalent system must include electronic data
system or integrator capable of measuring peak areas for off-scale
peaks.) Representative operating conditions: temperatures-in-
jector 2 O O ”C, detector 25O”C, column 150°C; gas flows-N, car-
rier gas, 50 mL/min, H, 45 mUmin, air 500 mL/min. Suggested
sensitivity: Choose attenuation so that injection of 5 mL
headspace from ca 11 ppm headspace standard (contains ca
11 ppm ethyl alcohol and 4.2 ppm tert-butanol; see preparation of
headspace standards) gives terr-butanol peak 250% ~100% FSD.
Ethyl alcohol peak will then be 225% FSD.
(b) Gas chromatographic column.-6 ft x4 mm id glass, packed
with 80-100 mesh Super Q (Alltech Associates, Inc., 2051
Waukegan Rd, Deerfield, IL 60015, USA). With column in place
and connected to detector, purge with N, carrier gas at 50 mUmin at
33°C (ambient temperature) for 30 min. Increase temperature at
3/min to 225°C and hold for 2 h. Decrease to operating temperature,
let column stabilize, adjust carrier gas flow to 50 mUmin, and let
column further stabilize overnight.
System &e&.-For operating conditions given in (a), retention
times are ca 3.5-3.8 min for ethyl alcohol; and ca 10.4-l 1.5 min for
tert-butanol. However, adjust column temperature for adequate res-
olution between ethyl alcohol and tert-butanol peaks, and any signif-
icant product peaks. For some products, small peak may occur at
retention time ca 8.4-8.8 min; this should not significantly overlap
tert-butanol peak.
(c) Syringes.-Gas-tight, Hamilton No. 1005-LTN (5.0 mL ca-
pacity) or No. lOlO-LTN (10.0 mL capacity).
(d) Headspace vials.-Glass, Kimble Glass Cat. No. 60910-L, 23
x 85 mm, 6 dram (ca 22 mL) capacity (Ace Glass, Inc.) fitted with
perforated screw cap (Cat. No. 95053) with Teflon-faced liner (Cat.
No. 9522) (both screw cap and liner from Alltech Associates, Inc.)
(e) Continuously adjustable digital microliter pipet.-Pipetman
Model P-200, 20-200 PL range (Cat. No. P-200, Gilson Medical
Electronics, 3000 Beltline Hwy, PO Box 27, Middleton, WI 53562,
USA), equipped with disposable microliter pipet tips (Rainin Cat.
No. RC-20; both from Rainin Instrument Co., Inc., Mack Rd,
Wobum, MA 01801, USA), or equivalent.
-
0 2000 AOAC INTERNATIONAL
FISH AND OTHER MARINE PRODUCTS
Chapter 35, p, 26
C. Reagents
(a) Sodium chloride crystal.-Baker Analyzed Reagent (J.T.
Baker, Inc., No. 3624).
(b) tert-Bufanol internal standard solutions.-(l) Stock solu-
tion.-Approximately 6000 ppm. Into tared 250 mL volumetric flask,
pipet 2.0 I-I-ILliquid tert-butanol (99.5%, Aldrich Chemical CO., Inc.).
Reweigh stoppered flask and its contents to determine weight of
tert-butanol (ca 1 SO g). Dilute to volume with H,O. (2) Working solu-
tion.-Approximately 108 ppm. Pipet 9.0 mL stock solution into
500 mL volumetric flask and dilute to volume with H,O.
(c) Ethanol standard solutions.-(l) Stock solution.-Approxi-
mately 15,600 ppm. Into tared 100 mL to volumetric flask, pipet
2.0 mL absolute alcohol (USP) (Aaper Alcohol and Chemical Co.,
Inc., P.O. Box 339, Shelbyville, KY 30065; +I-800-456-1017).
Reweigh stoppered flask and Its contents to determine weight of
ethyl alcohol (ca 1.56 g). Dilute to volume with H,O. (2) Zntermedi-
ale solution.-Approximately 1090 ppm. Pipet 7.0 mL stock solu-
tion into 100 mL volumetric flask and dilute to volume with H,O.
(3) Working solutions.-Solutions A, B, C, and D, ca 22,44,76, and
109 ppm, respectively. Pipet 2.0,4.0,7.0, and 10.0 mL intermediate
solution into separate 100 mL volumetric flasks and dilute each to
volume with H,O. Solution E, ca 11 ppm. Pipet 10.0 mL solution D
(ca 109 ppm) into 100 mLvolumetric flask and dilute to volume with
H20.
Prepare fresh ethyl alcohol standard solutions weekly.
0. Check for Possible Contaminafion
(a) Air.-Using gas-tight syringe, inject 5 mL air (in location
where aliquots will be withdrawn from headspace standards and test
samples) into GC apparatus to ensure that syringe is not contami-
nated and that air does not contain compounds that significantly in-
terfere with ethyl alcohol and tert-butanol peaks in analysis.
(b) Blank.-Pipet 5.0 mL HZ0 into glass headspacevial. Add 3.0 g
NaCl and seal. Hold vial in vertical position so as not to wet Tef-
lon-faced liner while swirling. Swirl contents vigorously 2 min, and
then let stand 5 min. Withdraw 5 mL aliquot from headspace into
gas-tight syringe by withdrawing plunger in single, slow, continuous
action; then inject it into GC apparatus to determine any significant in-
terference with ethyl alcohol and terf-butanol peaks in analysis. This
should be done in same location where air is analyzed for contamina-
tion, (a).
When air contains low levels of interfering compounds, analysis
of blank may reveal that levels are too low to cause significant inter-
ference with headspace analysis. However, analysis of blank may
indicate that headspace must be withdrawn from headspace vials
where atr is free from interfering compounds. If air in room is con-
taminated with ethyl alcohol, headspace standards and test samples
can be prepared m that room, but headspace must be withdrawn in
area free of ethyl alcohol contamination. If it is then brought back
into contaminated room and immediately injected into GC appara-
tus, analysis will not be contaminated with ethyl alcohol.
E. Preparation of Headspace Standards
Pipet 5.0 mL ethyl alcohol working standard solution into glass
headspace vial. Then add 200 pL terr-butanol working internal stan-
dard solution (ca 108 ppm), using adjustable microliter plpet
(Model P-200) to give tert-butanol concentration of ca 4.2 ppm.
Gently mix 3-4 s, add 3.0 g NaCl, and seal vial. Holding vial in verti-
cal positlon so as not to wet Teflon-faced liner while swirling, swirl
contents vigorously 2 min, and then let stand >5 min. Inject ca 5 mL
aliquot of headspace mto GC apparatus (see below),
0 2000 AOAC INTERNATIONAL
AOAC OFFICIAL METHODS OF ANALYSIS (2000)
F. Preparation of Calibration Curve
Prepare headspace standards as previously described using ethyl
alcohol working standard solutions A, B, C, D, and E (ca
1 l-109 ppm). Analyze each headspace standard as follows: With-
draw 5 mL aliquot of headspace from vial into gas-tight syringe by
withdrawing plunger in single, slow, continuous action; then inject it
into GC apparatus. If for any reason a second injection is required,
prepare new headspace standard. Between injections, pump syringe
210-15 times to eliminate gases and HZ0 vapor from previous injec-
tion to avoid contammation.
Use peak areas only for quantitation. To determine peak area, use
tangent skimmmg for ethyl alcohol peak, on tail of air peak. For each
headspace standard, calculate peak area ratio, R = area ethyl alcohol
peak/area tert-butanol peak. Prepare calibration curve as follows:
For each headspace standard, plot R against concentration of ethyl
alcohol working standard solution used to prepare that standard.
Draw best curve that fits points on graph, or use automated curve fit-
ting or multi-level calibration if instrument is so equipped.
G. Canned Salmon Aqueous Phase
Open can and drain liquid into 250 mL beaker while pressing lid
against contents. Retain salmon for sensory analysis if appropriate.
Transfer liquid to 250 mL separator and let oil and aqueous phases
separate. Drain aqueous phase into glass-stoppered cylinder and
store until analysis.
Ii. Preparation of Headspace Test Solutions
Use same procedure given for preparation of headspace stan-
dards, except transfer 5.0 mL test sample solution into headspace
vial with accurate 5 mL Mohr-style pipet. For test sample solution,
use either undiluted canned salmon aqueous phase, or, when neces-
sary for GC analysis (see below), canned salmon aqueous phase ac-
curately diluted with H,O.
1. Analysis of Headspace Test Solutions
Use same procedure given for analysis of headspace standards
(see preparation of calibration curve) and inject 5 mL aliquot of
headspace into GC apparatus. Analysis time for test solutions is ca
38 min because of late-eluting peak at ca 35 min.
Calculate peak area ratio, R, and determine ethyl alcohol concen-
tration in test sample solution from calibration curve.
If ethyl alcohol concentration is higher than that of most concen-
trated standard, accurately dilute original canned salmon aqueous
phase with H,O to give concentration within calibration limits, and
reanalyze diluted test solution. Multiply by dilution factor to obtain
concentration for original undiluted test solution.
Reference: JAOAC 69,524(1986).
Revised: March 1997
35.1.37
AOAC Official Method 959.08
Paralytic Shellfish Poison
Blological Method
First Action 1959
Final Action
See 959.08 (49.10.01).
Revised: June 2000
AOAC OFFICIAL METHODS OF ANALYSIS (2000)
351.38
AOAC Official Method 985.12
Parasites in Fish Muscle
Candling Procedure
First Action 1985
Codex-Adopted-AOAC Methos’
A. Apparatus
(a) Candling table.--I2lgid framework to hold light source below
rigid working surface of whte, translucent acrylic plastic or other smt-
able matenal with 45-60% translucency.Length and width of workmg
surface should be large enough to examme entire test fillet, e.g.,
30 x 60 cm sheet, 5-6 mm thick.
(b) Lqht source.-“Cool white” with color temperatureof 4200°K.
At least two 20 watt fluorescent tubes are recommended. Tubes and
their electrical connections should be constructedto prevent overheat-
ing of light source.
Average light intensity above working surface should be
1500-1800 lux as measured 380cm above center of acrylic sheet.
Dlstnbution of illumination should be in ratio of 3: 1:O.1, i.e., bright-
ness directly above hght source should be 3 times greater than that of
outer field and bnghtness of outer hrmt of visual field should be not
more than 0.1 that of inner field.
Overhead illummatlon (indirect light) in vicmity of candling table
should be .>500 lux.
B. Procedure
Place skinned test fish fillets in single layer on hghted working
surface and examine visually for parasites. Normally, test fillets are
not cut into pieces before examination, but if presence of parasites is
suspected, thick test fillets may be cut for further examination.
Reference: JAOAC 68,549(1985).
Revised: March 1997
* Adopted as a CodexDefining Method (Type I) for visual examinationof
parasites in quack frozen fish sticks (fish fingers) and fish
portions-breaded or m batter.
35.1.39
AOAC Official Method 962.15
Identification of Fish Species
Starch Gel-Zone Electrophoresis Method
First Action 1962
Final Action 1965
Caution: Cover for electrophoresischamber should have switch to
disconnect current to electrodeswhen chamber 1sopen.
Shield contacts and electrodes against body contact.
A. Apparatus
[A m. (6.4 mm) Lucite or Plexiglas is suitable plastic for trays,
cabinet, and containers; Figure 962.15.1
(a) Trays.-Plastic; 20 x250 mm, 6 mm deep, to hold starch gel.
(b) Cabinet.-Plastic; 3 x 8 x 14 in. (7 5 x 20 x 36 cm) with flat
cover. Make slots m rear for msertmg electrodes.
(c) Buffer and electrode conrainers.-Plastic; 2 x2 x7 m. (5 x 5
x 18 cm) with electrode compartment 2 x2 x lm. ( 5 x5 x2.5 cm) at
one end.
FISH AND OTHER MARINE PRODUCTS
Chapter 35, p 27
(d) Electrodes.-18 gage Ag wire. Make tight co11at one end
and insert in electrode compartment; place other end through glass
tube and attach to power supply.
(e) Filter paper strips.--Whatman No. 3; 6 x 19 mm for sam-
ples; 18 x 75 mm for connecting gel to buffer and buffer to elec-
trode compartment.
(f) Power supply.-To supply constant 15 mA at
190-210 volts, e.g., Heathklt or Constat.
B. Reagents
(a) Borate bu#er.-pH 8.65. Dissolve 18.5 g H,BO, and 4.8 g
NaOH in H,O and dilute to 1 L. Adjust with 10% (w/v) NaOH solu-
tion, if necessary. Dilute 1 + 9 for prepanng starch gel.
(b) Sodium chloride solution.-Prepare saturated solution and
dilute 1 + 2 for use m electrode compartment.
(c) Hydrolyzed potato starch.-Hydrolyze 300 g potato starch
1 h at 40°C in 600 mL acetone containing 6 mL HCl. Stop reaction
by addition of 150 mL 1M NaCH,C00,3H,O (136 g/L) and filter
with suction. Wash precipitate starch with 2-3 L H,O. Resuspend
starch in 1.5 L H,O and stir 1 h. Refilter and wash with 2 L HzO, and
dry overnight at 50°C. Grind to fine powder and test for correct gel
consistency by preparmg test lots containing 12,13, 14%. etc. starch
in pH 8.65 buffer, heat to just below bp, and pour into starch gel trays
to cool and harden. Prepare night before use and store m refrigerator.
Using known protein extract, determine starch concentration giving
best pattern separation m determmatlon. Use this concentration for
all determinations with d-usbatch of starch. (Prepared starch avail-
able from Fisher Scientific Co., S-676.)
(d) 5-5-I solvent.-Mix 5 parts H20, 5 parts methanol, and 1 part
CH,COOH.
(e) Amido black IOB.-1 .O% Dissolve 2 g dye in 200 mL 5-5-l sol-
vent. (ICN Fbnnaceutds, Inc., Costa Mesa, CA 92626, USA; num-
ber 100563).
C. Determination
Weigh 20-30 g minced test portion into blender cup. Add
40-60 mL H,O and blend 2 min at high speed. Pour mixture into
50mLcentnfuge tubes and centrifuge at 18OOrpmuntil separation is
complete (10-15 min). Filter supernate through folded Whatman
No. 1 paper or on Btichner with suction to obtain protein extract.
Dip small filter paper strips into protein extract and insert into
starch strips by cutting starch crosswise at midpoint of length of
strip. Place loaded trays m cabmet (Figure 962.15). Place one end of
Figure 962.15-Electrophoresis cabinet (scale: 1 in. =
10 in.).
6 26%ii6%ii%4TERNATIONAL
FISH AND OTHER MARINEPRODUCTS
Chapter 35, p. 28
large paper stnp saturated with pH 8.65 buffer on each end of starch
tray and dip other end into tray of pH 8.65 buffer. Connect buffer
compartment to electrode compartment contaming diluted NaCl so-
lution with similar strip saturated with the NaCl solution. hnmerse
Ag electrodes in both electrode compartments. Pass constant 15 mA
current (190-2 10 volts) through system 5 h.
Remove trays andimmerse starch strips 5 min m dye solution. Re-
turn excess dye to stock solution (may be used 6-9 times). Wash
dyed strips with several portrons 5-5-l solvent to remove dye from
unstained portrons of starch, leaving protein fracttons permanently
stained as blue bands.
Compare pattern with patterns of authentic materials and
standards run simultaneously.
References: JAOAC 45,275(1962); 48, 123(1965).
35.1.40
AOAC Official Method 967.14*
Identification of Fish Species
Actylamide Disc Electrophoresis Method
First Action 1967
Final Action 1974
Surplus 2000
A. Apparatus
(a) Disc eZecrruphoresis.-Assemble apparatus shown in Fig-
ure 967.14, consisting of regulated power source (60 mA, 500 volts,
DC); 2 plastic reservoirs, 5 m. (ca 13 cm) diameter; C or Pt elec-
trodes; tube grommets (standard electric grommets); glass tubes,
65 x 5 mm id. “Top view” represents base of upper reservoir show-
mg holes lined with rubber grommets. Gel tubes protrude from base
of upper reservoir and dip into lower reservoir buffer. (See Caurion
above 962.15A [see 35.1.391.
Polyacrylamide gel column is composed of 3 layers: upper,
large-pore (stacking) gel containing test sample ions m which elec-
trophoretic concentratron of ions is initiated; middle, large-pore gel
(spacer gel) in which electrophoretic concentration of test sample
ions IS completed; and lower, small-pore gel in which electropho-
retie separation occurs.
Thoroughly clean and rinse apparatus after each determmatron.
Wash test sample tubes and soak in chromic acid cleaning solution
followed by thorough rinsing wtth H,O. When apparatus is not in
use, remove buffer, B(b)(7), from upper and lower reservoirs and
store in separate containers in refrigerator. Upper buffer is usually
good for ca 6 determmattons. Discard buffer when pHdrops to <8.2.
(b) Photopolymerizing light.-15 watt fluorescent light,
15 tn. long.
B. Reagents
(a) Relatively stab/e.-Acrylamide monomer,
NJ’-methylene-bisacrylamide (Bts), 2-amino-2-hydroxy-
methyl-1,3-propanedtol (Tris), N, N,N’,N’-tetramethylethylene-
diamme (TEMED), ammonium persulfate, riboflavin, glycine
(NH,-free), amhne black.
(b) Relatively unstable (solutions).-Stable ca 6 months; refrigerate
solutions l-6. (I) 48 mL 1M HCI, 36.3 g Tns, 0.23 mL TEMED, Hz0
to 100 mL (pH 8.8-9.0). (2) 25.6 mL 1M H,PO,, 5.7 g Tris, 0.46 mL
TEMED, Hz0 to 100 mL (pH 6.6-6.9). (3) 30.0 g acrylamide, 0.8 g Bis,
H,O to 100 mL (4) 10.0 g acrylamide, 2.5 g Bis, Hz0 to 100 mL. (5)
4 0 mg riboflavin, H,O to 100 mL. (6) Cutulysr -0.14 g (NH&O,,
~__-
0 2002 AOAC INTERNATIONAL
AOAC OFFICIALMETHODSOF ANALYSIS(2002)
Hz0 to 100 mL (mrx fresh weekly). (7) Bujfer.-3.0 g Tns, 14.4 g
glycine, Hz0 to 1 L (pH 8.8-9.0). (8) Specimen stain.-1.0 g aniline
black, 7.5% (v/v) CHsCOOH to 200 mL. (9) Trucking dye.-O.O05%
(v/v) aqueous bromophenol blue.
(c) Working solurions.-Prepare fresh for each determination.
(1) Lower gel (separating gel) -MIX solutions (b)(l), (3), and
Hz0 (1 + 2 + l), pH 8.8-9.0. To form gel, mix with catalyst solu-
tion (6) (1 + 1). (2) Upper gel (stacking gel).-Mix soluttons
(b)(2), (4), and (5) (1 + 2 + 1). pH 6 6-6.8. To form gel, expose to
fluorescent light, e.g., 973.24B(b) (see 35 1 05).
C. Preparation of Column
(a) Lower and spacer gels.-Cap base of glass tube and set
tube in rack. Prepare separatmg gel with catalyst solution,
B(c)(l), in 20 mL syringe and add mixture to tube to ca 12 mm
from top. Tap tube occasionally during filling to avoid trapping
air bubbles under gel surface. Place few drops tracking dye in ca
20 mL Hz0 and with eye dropper cauttously layer 6 dye solutton
on top of separating gel without disturbing gel surface by touch-
ing dropper to tube edge (dye solution aids m dtfferentiatmg Hz0
and gel layers). Discard tubes in which drstmct, sharp boundary
line is not visible. Let gel polymerize 30 min. (Polymerization
time is based on reaction at 24°C Let refrigerated reagents stand
at room temperature ca 20 mm before usmg.) Gently shake off
Hz0 layer and add ca 0.1 mL stacking gel solutton, B(c)(2), to top
of column (functionally this 1s “spacer gel layer”). Cautiously
layer 3 mm Hz0 atop gel. Set photopolymerizing light 7-13 cm m
front of gel column rack to polymerize spacer gel in lo-15 mm.
Polymerization IS complete when spacer gel changes from light
green to translucent white. Shake off top Hz0 layer.
(b) Sample gel.-Prepare test sample extract as in first paragraph,
%2.1X (see 35.1.39), and mix extract, H,O, and solutions, B(b)(2),
(4), and@) (1 + 3 + 1 + 2 + 1) (test sample stacking rntxture). If inade-
quate (no bands or only very faint band appears in determinatron), pre-
pare new test sample stacking rmxture (2 + 2 + 1 + 2 + 1).
Add 0.1 mL test sample stacking mixture to tube prepared m (a)
and polymerize, using fluorescent light as in (a). Remove base
cap from tube by pressing at bottom and peeling off edge to
break vacuum. Avoid displacement of gel column from tube
wall. Do not touch gel or sharp glass edge of column.
Cut-Away Side View
Figure 967.14-Disc electrophoresis apparatus.
 AOAC OFFICIAL METHODS OF ANALYSIS (2002) FISH AND OTHER MARINE PRODUCTS
Chapter 35. p 29
-

D. Determination 35.1.41
Pour ca 500 mL buffer solution, B(b)(7) (containing enough track- AOAC Official Method 980.16
ing dye solution to produce definite blue tinge), into lower bath or Identification of Fish Species
enough to cover lower electrode 6 mm. Insert test sample end of col- Thin-Layer Polyacrylamide Gel
umn (top) into rubber holes in underside of upper bath until tops of Isoelectric Focusing Method
glass tubes are flush w~tb tops ofrubber holes. Plug unused holes with First Action 1980
Final Action
stoppers to avoid top buffer leakage. Place upper bath over lower bath.
Slowly pour ca 250 mL buffer solution (containing tracking dye) Caution: Polyacrylamide gels may contain small amounts
into middle of upper bath. Fill slowly to avoid cross contamination acrylamide monomer which is harmful if absorbed
of test samples. Take extra caution to liberate all air entrapped be- through skm. Wear disposable vinyl gloves when han-
tween sample gel and buffer to permit electric current to flow by dling gels.
gently introducing buffer soltmon into top of each test sample tube,
using synnge to drive out air. !<et polarity of electrodes so that test A. Apparatus
sample ions migrate to lower bath (positive electrode). Turn on cur- (a) Thin-layer isoelecrricfocusing-Multiphor for Electrofocusmg
rent and adjust power unit to give 5 mA/test sample tube (5 mA xno. (Amersham Pharmacia Biotech, Inc., 800 Centennial Ave.,
of test sample columns = total mA adjustment of power supply). plscataway, NJ 08854, USA, or equivalent isoelectric focusing appara-
Turn off current when “front” band IS within 5 mm of bottom of te,st tus with sirmlar sized cooling platform and electrode geometry.
sample column (ca 30-40 mm). (b) Power sup&.-Constant-power type capable of maintain-
Transfer gel column (“test sample end” or “top” first) to test sam- ing constant power of 21-30 watts. Constant-voltage type may be
ple tube, s full with specimen stam diluted with H,O (1 + l), tmme- used provided voltage is manually increased at intervals to maintain
constant power level.
diately after electrophoresis to stam and fix. Protein fractions m
(c) Constant temperature circulator.-Capable of cuculatmg
gel diffuse if not properly fixed or stamed. Remove gel column H,O or antifreeze solution through cooling platform at 0-10°C.
from tube under Hz0 in pan as follows Fill 10 or 20 mL syringe (d) Trays.-Plastic or metal covered. Minimum size
with cold H,O and insert gel-removing needle at test sample end 125 x260 x20 mm, to hold fixing, staining, destammg, and preserv-
of column between glass and gel so that needle tip reaches sepa- ing solutions.
rating gel Keeping needle flat against glass surface to avoid (e) Glussplntes-125 x260 mm, to support gels durmg drying.
(f) LKB Ampholine PAGplates.-pH 3.5-9.5 (Amersham
scratching gel, rotate needle completely around circumference of
Pharmacia Biotech, Inc.).
gel. Remove needle and insert it from other end to depth of ca 1 cm,
B. Reagents
a holding it against inner surface of glass column and at same time
forcing stream of H,O through needle. If these steps are carefully (a) Fixing so&ion.-Mix 150 mL methanol and 350 mL H,O (or
500 mL H,O. Add 17.25 g sulfosabcylic acid and 57.50 g
performed, entire intact gel will come free and can be slipped out of trichloroacetic acid. Discard after one use.
tube. If necessary, little air pressure from rubber bulb will assist in (b) Destaining solurion.-Mix 500 mL alcohol and 160 mL
getting column. CHsCOOH. Dilute to 2 L with H,O.
While gel is being stained (above), prepare 75 x 7 mm id glass (c) Staining sol&on.-Dissolve 0.230 g Coomassie Brilliant
destain tube as follows: Place fire pohshed end of tube in large diam- Blue R-250 (Bto-Rad Laboratones, 2000 Alfred Nobel Dr. Hercu-
eter base cap. Add ca 1 cm separating gel with catalyst to bottom of les, CA 94547, USA) in200 mL reagent (b) Heat to 50-60°C m cov-
ered container in H,O bath before use. Discard after one use.
column and let polymerize. When staining is complete (ca 15 min),
(d) Preserving solution.-Add 50 mL glycerol to 500 mL re-
decant stain by placing lip of test tube against inside lip of beaker to agent (b).
retain gel m tube. Rinse twice with 7 5% (v/v) CH,COOH (v/v) to (e) Anode solurion.-1M HsPO,.
remove excess stam. Transfer ,gel bottom “front” first into destain (f) Cathode solution.-1M NaOH.
tube in which bottom plug of gel has been cast and cap removed. (g) lnsulnring fluid.-Light paraffin oil (Saybolt viscosity
Place destaining tubes in rubber holes m underside of upper bath 1251135). (Kerosene also works well.)
Plug any vacant holes. Pour ca 500 mL 7 5% (v/v) CH,COOH in lower C. Preparation of Test Sample
bath and ca 250 mL mto upper bath. As prevtously, use syrmge con- Extract sarcoplasmic proteins test sample as in 962.1X (see
taining 7.5% (v/v) CHsCOOH to ensure no air bubbles exist between 35.1.39), first paragraph, or as in 970.32C (see 35.1 42). Centnfuge
gel and CHsCOOH bath. Turn on current, adjusting to 5 mA/column all extracts, 1500 xg for 15 min, at 4°C or filter through Whatman
(5 x No. of columns = total mA). Gels will be destained in ca No. 1 paper in refrigerator.
45-60 min When gel part contaming no protein is clear of stain, or
has pale blue color, remove specimens, and place in small test tube D. Determination
containing 7.5% (v/v) CHsCOGH for viewmg and storage. Visuall,y Step I .-Place template supplied with Ampholme PAGpIates on
compare finished tubes with patterns prepared from authentic test cooling platform over thm layer of msulatmg fuild, B(g). Avoid air
samples and standards run simultaneously. bubbles.
Step 2.--Gpen PAGplate package by cutting around edge. Do not
Reference: JAOAC SO, 282( 1967). cut or crush gel. PAGplate may be cut in X or A, tf less than whole gel
is required, and remamder may be resealed in package for later use.
Revised: March 2002 Leave clear plastic film covering PAGplate.

- _~-.-
0 2002 AOAC INTERNATIONAL
FISH AND OTHER MARINE PRODUCTS
Chapter 35, p 30
Srep 3.--Lift PAGplate by ends of protruding plastic support.
Place PAGplate on cooling platform, over template, using additional
oil (kerosene), B(g). Keep gel surface clean. Remove clear plastic
film covering gel surface
Step I.-Place electrode strip (supplied with PAGplates) on clean
glass plate. Saturated stnp with 1M H,PO,, B(e). Strip should show
wet surface. Place wet stnp on PAGplate surface at anode positIon
marked on template. Cut protruclmg ends of stnp with clean sharp
scissors.
Step 5.-Saturate another electrode strip as in step4, but with 1M
NaOH, B(f). Place wet strip on PAG plate surface at cathode posi-
tion marked on template. Cut protrudmg ends of stnp with clean
sharp scissors.
Step 6.-Pick up test sample extract apphcatlon piece with clean
forceps. Apply 20 pL test sample extract to piece, using mlcropipet or
by dlppmg application piece into extract. Place piece on PAGplate
surface within sample application area marked on template at cathode.
Sixteen test sample extracts authentic species standards,and pl cah-
bration standards may be applied if long side (10 mm) of pjece is
placed parallel to cathode strip. Twenty-four test sample extracts, au-
thentIc speciesstandards,and pI cahbration standards may be apphed
if short side (5 mm) of piece IS parallel to cathode strip Leave mim-
mum of 5 mm between adjacent test sample extracts.
Step 7 -Place electrode lid (for electrofocusing across width of
gel) on gel with Pt wires centered on electrode strips. Connect leads
to electrofocusmg apparatus terminals. Observe proper polarity.
Srep 8.-Place safety cover m place and connect leads to power
supply. Observe proper polarity.
Step 9.-Set power supply con&tions.-(l) For constant-power
power supply.-Power, 30 watts; maximum voltage, 1.5 kV; maxi-
mum current, 50 mA; for 1.5 h. Or for overt& focusing: power, 1 watt;
maxmm voltage, 1 S kV; maximum current, 50 mA; for 16 h.
Reduce power level to 10-15 watts for 2 hif cooling platform tem-
perature is lO-25”C, or If less than whole PAGplate is used. When
using other power supply conditions, use maximum voltage, cur-
rent, and time to ensure all proteins have reached equilibrium.
(2) Consranf-volfuge power supply.-Set to constant-voltage
mode, initial setting 300 V, and increase voltage by 100 V at 30 min
intervals. Experimentally determme maximum voltage and expen-
ment time for each set of conditions.
Usmg either constant-power or constant-voltage supplies, mim-
mum expenment time 1stime required for all proteins to reach their
isoelectric points. Apply ca 10 pL protein calibration standards
(brand p1 calibration kit [pH 3-101 [Amersham Pharmacla].
Step IO.--Switch off power supply after 30 mm; remove safety
cover and electrode lid. Remove sample application pieces. Replace
safety cover and lid and continue separation.
Step 11 .-Switch off power supply at end of separation and re-
move safety cover and electrode lid. Remove electrode stnps from
gel surface gently with forceps. If stnp sticks to gel, cut through gel
(not plastic support) at inner edges of electrode strips. Discard strips.
Step 12.--Place PAGplate immediately m fixing solution, B(a),
30 min.
Step 13 -Rinse PAGplate m destaining solution, B(b), 5 min.
Step 14.-Stain PAGplate 10 mm m hot (60°C) staining solu-
tion, B(c).
Step I5.-Destain PAGplate with several changes of reagent (b).
Gel background should be clear after destaining overnight. Gently
remove any precipitate dye from PAGplate surface with cotton wool
soaked m reagent (b).
~. .-
0 2000 AOAC INTERNATIONAL
AOAC OFFICIALMETHODSOF ANALYSIS(2000)
Step 16.-Compare protein patterns on destained PAGplates with
patterns of authentic species. Patterns may also be compared after
step 19.
Step I7.-Place destained PAGplate m preserving solution, B(d),
60 min.
Step 18.-Dry PAGplate on glass plate at room temperature over-
night. Protect gel surface from dust, etc.
Step 19.-Roll sheet of plastic (supplied with PAGplates) onto
dried PAGplate, avoiding au bubbles. Store dried PAGplate in dark.
Dried PAGplate may be stapled into notebook.
Reference: JAOAC 63,69( 1980); corr. 684( 1980).
35.1.42
AOAC Official Method 970.32
Identification of Fish Species
Cellulose Acetate Strip Method
First Action 1970
Final Action 1971
A. Principle
Fish protein is applied to cellulose acetate supporting medium and
travels when fixed voltage is applied for definite length of time. Fin-
ished strips are stained, washed, and dried to fix patterns.
6. Apparatus and Reagents
(a) Electrophoresis cabmer.--Divided into 2 compartments with
adjustable constant voltage power supply with 300 V minimum out-
put voltage; with voltammeter which permits reading and adjusting
voltage to produce desired current (see Caution, 962.15 [see
35.1.391).
(b) Gelmun applicator.
(c) Cellulose acetate sfrips.dx x 1 in.
(d) Capillary tubes.-1.6-l .8 x 100 mm.
(e) Filterpaper.-Whatman No. 1 sheetscut to convenient size.
(f) Verona1 b@er.-pH 8.6, lomc strength 0 04; sodium
dlethylbarbiturate 8.64 g, diethylbarblturic acid 1.2 g, and H,O
to 1 L.
(g) Stain.-Dissolve 200 mg Ponceau S stam rn 100 mL 5%
(w/v) tnchloroacetic acid.
(b) Wash solution.-5% (v/v) CH,COOH.
C. Preparation of Samples
(a) Fresh.-Grind ca 3 g fish flesh in mortar with 3 mL H,O and
squeeze through several tlucknesses of cheesecloth. If centrifuge is
available, place pieces of fish m centrifuge tubes and express fluid
by centrifuging; no added H,O is required.
(b) Frozen.-Thaw test sample and use drip that forms undiluted.
(c) Freeze-dried.-Reconstitute test samples with H,O and treat
as in (a).
(d) Breaded raw sticks and portions--Remove breading by
soaking for few s m H,O and scraping off breading with spatula.
Treat in same manner as in (a) or (b).
(e) Precooked sticks and porrions.-Trim breading and all sur-
face meat until internal center section remains. Treat as in (a).
D. Determination
Soak cellulose polyacetate strips 30 min in buffer (soaking is re-
quired to bring strips back to origmal gel structure). Use new buffer
AOAC OFFICIAL METHODS OF AN,I\LYSIS (2000)
supply each time. Since 6 test strips can be run simultaneously, each
with different test sample, identify each strip with pencil notation
before soaking.
Add chilled (1°C; 34°F) buffer to each chamber of cabinet and
level to point slightly below compartment dividers. Remove 1 strip
from buffer and gently blot between sheets of filter paper to remove
excess buffer.
Take up tissue fluid into capillary tube and transfer to applicator.
Draw capillary tube along applicator to within 6 mm of both ends.
Then press applicator firmly against strip about 5 cm from one end.
Place strip containing test portion across cabinet dividers so that test
portion is on cathode side and both ends are immersed in buffer in
the 2 outer chambers. Secure acetate strips at each end with magnets
or glass wedges to prevent slippage. Keep taut. Repeat test portion
application with each strip, working quickly to prevent strips from
drying out.
Put cabinet cover in place, connect electrodes, and set power sup-
ply to give constant voltage. Adjust voltage (between 200 and
300 V) to produce initial current of 1.5 mA/strip. Maintain this volt-
age 30 min. Shut off power supply, remove strips from cabinet, and
place in Ponceau S stain 5 min. Immerse strips in series of 3 rinsing
solutions of 5% (v/v) CH,COOH to remove excess dye. Rinse strips
until only protein bands are left stained and remainder of strip is free
from dye. Finally, blot strips and dry between several sheets of filter
paper. Compare protein patterns of test portions with those of au-
thentic flesh.
References: JAOAC 52,703(1969); 53,7(1970).
35.1.43
AOAC Official Method 981.08
Generic Identification
of Cooked and Frozen Crabmeat
Thin-Layer Polyacrylamide Gel Isoelectric Focusing
First Action 1981
Final Action 1998
Caution: Inhalation, ingestion, or absorption of acrylamide may
cause nervous system disorders. Wear hsposable
gloves and mask when preparing and handling gels.
A. Principle
Urea-extracted proteins are exposed to pH gradlent created by
isoelectric focusing on thin layer of polyacrylamide gel. Pattern of
test sample is compared with those of known genera.
8. Apparatus
(a) Thin-layer isoelectric focusing equipment (TLZEF).-See
method 980.16 (see 351.41).
(b) Constant temperature circulator.-Any that can maintain
o-10°C.
(c) PowersuppZy.-Constant power-type capable of maintaining
constant power of 1 watt up to minimum of 500 V.
C. Reagents
(a) Anolyte solution.-O. 1% I(V/V)pH 7-9 ampholyte. Store in re-
frigerator.
(b) Catholyte solution.- 1.0% (v/v) pH 9-l 1 ampholyte. Store
in refrigerator.
(c) AmphoZyte solution.-Mix 2 parts ampholyte pH 7-9 (dry
content 40%) and 1 part ampholyte pH 3.5-10 (dry content 40%).
FISH AND OTHER MARINE PRODUCTS
Chapter 35, p. 31
(Amersham Pharmacia Biotech, Inc., 800 Centennial Ave,
Piscataway, NJ 08854, USA). Store in refrigerator.
(d) Urea.-1OM ultra pure (Schwa&Mann, Orangeburg, NY
10962, USA); prepare fresh daily.
(e) Ammoniumpersulfatesolution.-10% (w/v); prepare fresh
weekly.
(f) Polyacrylamide mixture.-Dissolve 20 g acrylamide and
0.8 g N,N-methylene-bis-acrylamide (Bis) in H,O and dilute to
100 mL. Store in refrigerator.
(g) Stain L-0.1% (w/v) anhydrous CuSO, and 0.5% (w/v)
Coomassie Brilliant Blue R-250 in CH,COOH-ethyl alco-
hol-H,O (10 + 30 + 60).
(h) Stain II.-0.1 % (w/v) Coomassie Brilliant Blue R-250 in
CH,COOH-ethyl alcohol-H,0 (10 + 25 + 65).
(i) Destain.-CH&OOH-ethyl alcohol-Hz0 (10 + 10 + 80).
D. Preparation of Test Sample
Blend g thawed crabmeat equal to mL 10M urea 2 min using me-
chanical blender, or until well mascerated using hand tissue grinder.
Blender method yields darker bands after staining. Centrifuge at
3000-13,000 xg, draw off supemate, and refrigerate for same day use.
E. Preparation of Gel
Prepare gel mold according to specifications of ampholyte manu-
facturer, using 1 mm spacer bar. Prepare gel fresh daily by addmg
following reagents sequentially to flask for 250 x 110 x 1 mm gel:
16.4 mL 10M ultra pure urea, 6.0 mL 50% (v/v) glycerol, 10.0 mL
polyacrylamide mixture, and 2.4 mL ampholyte solution. Degas un-
der vacuum 3 min. Add 100 pL 10% (w/v) ammonium persulfate
and degas additional 1 min. Quickly transfer gel to mold with Pas-
teur pipet. When gel has polymerized (ca 30 min), refrigerate mold
15 min, and carefully remove template and spacer, leaving gel ad-
hered to glass plate. Place plate on cooling platform over thin film of
light paraffin oil.
F. Determination
Thoroughly wet electrode strip (supplied by TLIEF manufac-
turer) with anolyte solution and align on gel surface with anode.
Wet second strip with catholyte solution and align on gel with
cathode. Place these wicks on edges of gel, ca 90 mm (center to
center) apart and aligned such that Pt electrodes embedded in slab
cover plate rest on wicks and provide electric contact. Place
5 x 10 mm wicks of Whatman 3 MM paper close to, but not in con-
tact with, anode wick. Pipet 20 pL crabmeat test extract onto each
test sample wick. Two wicks of 20 pL test extract each can be laid
on top of each other to obtain darker protein pattern after staining.
Cool platform to 0-10°C and connect focusing equipment to
power supply. Observe proper polarity. Apply 1 watt constant
power up to maximum of 500 V. Continue focusing at 500 V con-
stant voltage ca 20 h.
Switch off power and remove gel from coolmg slab. Clean par-
affin oil from plate and put elastic bands around glass plate and
electrode wicks. Stain protein at room temperature as follows:
stain I, 4 h; stain II, 4 h; destain, 1 h. Identify unknown test ex-
tracts immediately after destaming by comparing patterns with
known test extracts.
Reference: JAOAC 64,670(1981).
Revised: March 1997
0 2000 AOAC INTERNATIONAL
FISH AND OTHER MARINE PRODUCTS AOAC OFFICIAL METHODS OF ANALYSIS (2000)
Chapter 35, p. 32

35.1.44
AOAC Official Method 979.15
Identification of Canned Pacific Salmon
Microscopic Method
First Action 1979
Final Action 1990
A. Apparatus and Reagent
(a) Microscopes.-(I) W ide-field; and (2) compound, with vari-
able iris diaphragm.
(b) Glycerol jelZy.-To 60 mL H,O add 5 g gelatin and heat gently
until completely dissolved. Add 40 mL glycerol and 0.5 g phenol, and
mix. Cool and store in closed container at room temperature.
8. Method
Empty entire contents of can into pan. Remove as much skin as
possible from meat with aid of spatula and place skin in Petri dish.
Keep skin surface moist with Hz0 at all times; if skin is allowed to
dry, scales will curl, become brittle, and be unsuitable for mounting.
Examine skin under wide-field microscope, using ca loxmagnifica-
tion. Scales generally will be inside folds of skin (scale pockets) with
portion of one end of scale protruding from pocket. Scan entire sur-
face of all skin, and select largest and most nearly intact scales avail-
able. This is very important; rf size difference is seen on same piece
of skin, larger scales will have the most intact clear areas, and also in-
ternal scale pattern will be more complete.
Gently insert thin-tipped spatula well underneath whole scale be-
tween bottom side of exposed portion of scale and skin. Completely
fold back this entire portion to make whole scale visible. Again,
gently insert spatula between scale and skin and remove scale with
back-and-forth motion. While keeping scale moist with H,O, rub
very gently with small tool, such as artist’s brush, to remove any ad-
hering skin or foreign material. Place small amount glycerol jelly on
microscope slide and melt by placing slide on heat source. Pick up
scale with spatula and remove any excess H,O by touching gently to
towel. Place scale in jelly and cover with cover glass. If scale curls
durmg mounting, invert, uncurl, and cover quickly. Examine under
compound microscope at 25-505 using transmitted light. Adjust
light intensity for optimum contrast of scale patterns.
Examine whole scales with intact clear areas, avoiding undevel-
oped, distorted, or damaged scales. Intact clear area must be present
for accurate identification (except pink salmon). Determine salmon
species by using key in Figure 979.15A and by comparing to scales
in Figures 979.15B-F with pertinent features noted. Examine suffi-
cient number of scales to ensure positive identification.
Precautions.-Use only scales that contain distinct wave
striations and reticulations for comparison with key and photo-
graphs.
Scale patterns of coho and chinook species are somewhat similar.
Circuli extending into clear area (coho), and wave striations in clear
area (chinook) can be mistaken for each other. Note differences in
Figure 979.15C andD. Chinook scales may have only few striations.
Follow key (Figure 979.15A) carefully.
Chum and sockeye scales are both reticulated, but sizes and
shapes of this feature differ. Under low (30x) power, chum
reticulations appear globular, or like small round air bubbles; in
sockeye they are netlike, 4-6 sided, and larger.
W h e n pink salmon scales occur with reticulations and intact clear
area, they may be differentiated from sockeye by their length. Vertl-
cal axis of pink is 3.5 mm; of sockeye, >3.5.

3.5 m m or less 3.5 m m or greater

(vertical axis) (vertical axia)

Oncorhynchus

I
L
gorbusche
/Pink)
JI I
Retlcuiations

I
1. Circuli pattern new focus PreLnt Not p:esent
does not change sbruptlv
but is nearly uniform.
wave S~ristions Wave Siriatio”*
2. Rsticulations and clear
areas may or may not be I L I -I-
Present Not present Present Not present
7 1 I I I

1. Circuli pmtan “ear focus 1. Circuli pattern near focus 1. Circuli patter” new focus 1. Circuli patter” near focus
4. Size usually 2-3 mm does not change abruptly chanaa abrwtlv. (Cimuli mav or mav not chanps changes abruptly. (Circuli
(vertical axis). but is nearly uniform. cio0& apecsb amund abr&alv. Itnot. it is n&rly clcnelv spaced around
focus. then abruptlv become uniform. focus, then abruptly become
more widely spaced.) more wldelv spaced.)

2. Reticulations usually 2. Reticulations netlike, 2. Generally 3-12 complete 2. Generally 8-25 complete
globular shaped. having 4-6 sides. and broken circull below and broken eirculi below
focus. focus.

3. Circuli do not extend into 3. Circuli below focus 3. Circuli below focus
clear area. usually etiend to or slightly usually e-end extensively
into clear are*. into clear area.

3. Sne usually 5-5 mm 4. size uruslly 4-5 mm 4. Size usually 6-B m m 4. Sirs uluslly 4-5 mm
(verllcal axis). (vertical axis). (vertical axis). (vertical axis).

Figure 979.15A-Key to identification of canned salmon species by scale characteristics.

0 2000 AOAC INTERNATIONAL

AOAC OFFICIAL METHODS OF ANALYSIS (2000) FISH AND OTHER MARINE PRODUCTS
Chapter 35, p 33

Figure 979.156-Sockeye (red) salmon scale Figure 979.15~Coho (silver) salmon scale
(Oncorhynchus nerka), 10x. A, Reticulations: net-like di- (Oncorhynchos kisutch), 10x. A, Circuli (concentric
visions at junction of circuli and clear area; B, clear rings).
area.

Figure 979.1X-Chinook (King) salmon scale

(Oncorhynchus tshawytscha), 10x. A, Focus (the area Figure 979.15E-Chum salmon scale (Oncorhynchus
within first circulus); B, wave striations (irregularly keia), 10x. A, Wave striations (irregularly shaped nearly
shaped nearly horizontal wavy lines in clear area). horizontal wavy lines in clear area).

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0 2000 AOAC INTERNATIONAL
FISHANDOTHERMARINEPRODUCTS AOAC OFFICIALMETHODS OF ANALYSIS (2002)
Chapter 35, p 34

(2) Check repeatability relative standard deviation of instruments

tion using the following (1): (a) Run replicate analysis of one test
sample within the 8-80 mg NH,/100 g range. Maximum allowable
RSD, is 14%. (b) For further confirmation of instrumentation RSD,,
run different test samples in duplicate at various ammonia concep
trations within the levels 8-80 mg/lOO g andcalculate S, as follows:

RSD, = X($/m)

where S, = instrumentation standard deviation; 2 = from duplicate analy-

Figure 979.15F-Pink salmon scale (Oncorhynchus
gorbuscha), 10x.
Relative size of scales (as length of vertical axis) is important in
ldentlficatlon of all 5 species: chinook andchum have largest scales (7
and 6 mm, respectively), coho and sockeye scales are 4-5 mm, and
pmk salmon scales (easily recogmzed by their small size) are 3.5 m m
Disregard scales whch are damaged, have hard rib-hke structure,
or are distorted (all clrcuh surrounding focus are completely mlssmg
with this void extending about half way to scale edge).
References:JAOAC 52,696(1969); S&648(1972),
62,722(1979).
35.1 A5
AOAC Official Method 999.01
Volatile Bases in Fish
Ammonia Ion Selective Electrode Method
First Action 1999
[Applicable to the determination 8-80 mg ofvolatile bases/100 g m
seafood reported as NH,.]
See Table 999.01 for the results of the interlaboratory study sup-
porting acceptance of the method.
A. Principle
Ammonia and volatde amines (primarily trimethylamme) are hber-
ated from homogemzed fish tissues by addition of an alkalme ion
strength ad@mg (ISA) solution. The volatilized bases permeate a hy-
drophobic membrane until an equilibrium is reached with an ammo-
mum ion selective electrode (SIE). The quantity of volatile bases IS
dlsplayed in terms of the precahbrated ammoma response whch 1scon-
verted to mg (relative) ammonia/100 g based upon dilution factors.
B. Apparatus
(a) pH merer.-Orion Model 290A portable pH/ISE meter with
British naval connector, or equivalent.
(b) Ammonza electrode.--Orion Model 95-12, or equivalent am-
moma gas sensmg electrode with ammonia sensor membranes.
(c) Apparatus valldutron -Procedure to validate meter and am-
monia electrode operation: (I) Verify standard calibration checks
per manufacturer’s instructions Check troubleshooting section of
meter and/or electrode manual when values are out of range.
ses; K = number of sets of duplicates (materials); D = difference between
duplicate determinations; and m is the mean of all 2 k values. The maxk
mum allowable RSD, is 10% based on a l-sided 95% upper confidence
interval. This SD, is more reflective of the true variation. (3) Check ac-
curacy by spllang various test samples at the 10 mg NH,/100 g
(100 ppm) level. The acceptable recovery range is 85-l 10%.
C. Reagents
(a) Ammonia standard solutions --(I) IO00 ppm stock solu-
tlon.-Weigh 0.315 g NH&I into 100 mL volumetric flask and di-
lute to volume with distilled water. Alternatively, pipet 58.8 mL
O.lM N H @ (1700 ppm NH,), (c), into 100 mL volumetric flask.
Dilute to volume with distilled water Solution can be stored for
1 week refrigerated. Keep stock standard solutions refrigerated
when not m use (2) Calibration solutlons.+a) 50 ppm solu-
tzon -Pipet 5 mL 1000 ppm NH, solution into 100 mL volumetric
flask and dilute to volume with distilled water. (b) 20 ppm solu-
tron --Pipet 2 mL 1000 ppm NH, solution into 100 mL volumetric
flask and dilute to volume with distilled water. (c) 5 ppm solu-
tion -Pipet 0.5 mL 1000 ppm NH, solution into 100 mL volumetric
flask and dilute to volume with distilled water.
(b) lomc strength adjuster (ISA).-5M NaOH, 0.05M dlsodium
EDTA, and 10% (v/v) methanol in distilled water. To prepare
200 mL ISA solution, weigh 3.72 g disodium EDTA into 200 mL
volumetric flask and add 20 mL methanol. Weigh 40 g NaOH m
500 mL beaker and dissolve in 100 mL distilled water. Quantita-
tlvely transfer to 200 mL volumetric flask containing EDTA and
methanol solution and dilute to volume with distilled water.
(c) Electrode filling solution.-4 1M NH&I. Dissolve 5 349 g
NH&I m water and dilute to 1 L with distilled water.
D. Preparation of Ammonia Electrode
Prepare electrode according to the manual. Place membrane over
outer tip of electrode casing similar to placing Parafilm over test tube
opening. Avoid touchmg the membrane that will come in contact
with the mner reference solution and the external test and standard
solutions. Tweezers may help Screw on external protector tip that
holds membrane m place. Usmg Pasteur pipet, add NH, electrode
filling solution, C(c), to inner electrode. Insert reference electrode
mto casing with membrane on the tip and screw electrode into casing.
Some filling solution will overflow but this ensures enough solution
has been added. Allow membrane to equilibrate overnight in 100 ppm
NH, solution prepared by diluting 1OmL reagent, C(a)(/), to 100 mL
with distilled H,O in volumetric flask.
E. Calibration of ISE Meter
Cahbrate ISE meter according to manufacturer’s mstruction us-
mg separate 100 mL ahquots of the 5 and 50 ppm calibration solu-

0 2002 AOAC INTERNATIONAL

AOAC OFFICIAL METHODS OF ANP~LYSIS (2002) FISH AND OTHER MARINE PRODUCTS
Chapter 35, p. 35

Table 999.01 Results of interlaboratory study for volatile bases

No. Mean NHs Mean % Horw~tz

Sample type No. labs outliers mgll00 g recovery Sr SR RSD, RSDn ---- P .~ R* ratioC
Codd 9 0 15.4 1.28 2.20 a.3 14 3.58 6.15 1.9
Monkfishd 6 3 11.7 88.6 1 32 1.32 11 11 3 70 3.70 14
Soled a 1 8.43 0.87 0.87 10 IO 2.44 2.44 1.2
Halrbutd 9 0 28.0 3.56 3 56 13 13 9.97 9.97 1.9
Squidd 9 0 25.6 107 2.00 2.25 7.8 8.8 5.59 6.29 1.3
Codd a 1 62.3 128 2.58 6.02 4.2 9.7 7 24 16.9 16
Squidd 9 0 13.1 1.32 1.59 10 12 3.71 4.46 16
Monkfishd 9 0 20.5 1 41 2.14 6.9 10 3.96 5.99 1.4
Mackerel’ 9 0 17.3 0.65’ 1.94 3.8 11 1.82 5 43 1.5
Dog/squide 9 0 23.3 3.98’ 4.97 17 21 11 1 13.9 3.0
Monkfish 9 0 37.4 4.21 11 1.7
Squid 9 0 82.4 15.0 18 3.1
Dogfish-- 9 0
-__ 48.1 6.33- __~ 13 21
a 20xs,

b 28~s~

How~iz ratros between 0 5-2.0 lndlcate acceptable reproducibility precision

d Blrnd dupltcates
’ Youden matched parrs
’-.- s L--.---
as calculated for Youden matched pairs
Percent recovery (10 mg added NH3/1 00 gfi.sJ tissue)
- __-
Mean %
ID--. .~ 1 2 3 4 5 6 7 a 9 !e~-.- se RSDn, %
Cod 118 104 94.1 94.0 156 178 136 Not done 140 128 30.4 23.8
Monkfish 92.0 64.0 92.0 97.0 78.0 104 91.0 Not done 71.0 88.6 10.6 12.0
%JUid 110 88.0 __ 106 96.0 118
-__- 128 96.0 Not done 110 - 107 13.0 12.1

tions, C(a)(Z)(b), in 250 mL beakers followed by 100 mL of a pipet 1 mL of 1000 ppm NH, standard solution, C(a)(I), to representa
20 ppm control solution. Recalibrate the instrument if readmg is out- 20 mg/lOOg spike. Blend for 2 min at high speedand continue as above.
side 18-22 ppm.
H. Calculations
F. Preparation of Test Material
mg relative NH,/100 g =
Fillet whole fish without skin. Grmd or blend a representative
(lOO/Wt)(ppm)(l mg/lOOO ppm)(lOO) =
number of thawed fillets m a food processor or grinder until thor-
2 ppm if Wt = 5.0 g cornminuted fish tissue
oughly minced (paste-like). If fish are frozen, run cold water over fil-
lets placed m a plastic tray until no longer rigid, then grind. Weigh
5 g test portion for analysis or store refrigerated (3-5°C) in a sealable where Wt = weight of test portion m g (5.0); ppm = direct ISE meter
plastic bag for testing later that day. Freeze test portion m bag at reading; 1000 = conversion of ppm to mg; 100 = conversion to 100 g
-15°C if testing cannot be accomplished on same day. test portion.

G. Determination Recovery, % =
Weigh 5.0 f 0.1 g comminuted fish in 250 mL blender. Record (mg NH, / 100 g spike)-(mg NH, / 100 g background) xloo
weight to 0.1 g. Add 95 mL distilled water. Blend at high speed foi S
2 mm. Transfer test mixture into 250 mL beaker and cover beaker
with aluminum foil or Parafilm until measurement. Prepare 4-5 tests where S = spike equivalent in mg NH,/1 00 g; S = lOO/Wt when using
at a time for analysis. Run a 20 ppm control after every fifth test. If read- 1 mL 1000 ppm spiking standard.
mg of control is between 18-22 ppm, more tests can be analyzed with- The term “relative” NH, indicates that the value reflects ammonia
out recalibration. If reading is outside these values, recalibrate witb plus any TMA that permeates the membrane and is sensed by the
freshly prepared 5 and 50 ppm standards and verify with fresh 20 ppm electrode. Therefore, the electrode acts as a total volatile base probe
control. Add 2.0 mL ISA one test portion at a time before placing elec- measuring ammonia and trimethylamine.
trode in test solution. Do not add ISA to all blended suspensions prior to
References: (1) .I AOACZnt. 81, lOlI(1998).
batch analysts; otherwise, NH, will be given off prematurely.
For routine percent recovery determmation, weigh 5.0 + 0.1 g J. AOAC Int. 83, 933(2000).
comminuted fish m 250 mL blender jar with 94 mL detomzed water, then Revised: March 2002

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0 2002 AOAC INTERNATIONAL