AOAC OFFICIAL METHODS OF ANALYSIS (2012) hromatograph acids individually by using S mLaliquots of each sandard solution and $ mL aliquots of $-fold dilutions of each ‘todd acid solution. Combinations approximating compositions in ‘et prodts may also be prepared, (@ Column separation—{(Good separation and yields depend tno transfer of tet solution to column with amount of solvent _pcifed,) To dry residue of sodium salts add 2 mL 1% (v/v) butanol CHCl, solution and while stising with glass rod add H,S0, (I + !)dopwise uni all sodium salts are converted free acids (acid to Omgo red paper). Add I g anhydrous Na,SO, Acids elute in following order: valerie, butyric, propionic, and ‘exc. Place 50 mL. graduate under column as receiver, Decant sipemate onto coluran, pouring itslowly down sidef tube without -dsubing fevel surface of column. Apply pressute until solvent B ixces surface of gel. Wash beaker with I mL solvent, pour onto coun, and with stirring rod transfer residue in beaker to column, [feply pressure until solvent just disappears into Na,SO, layer. Wash beaker with another 1 mL solvent, transfer to column, wash inside of tbe with 1 ml. solvent, and apply pressure until solvent ut dsappears into Na,SO, layer. Fill ube with solvent and apply fressre. Each time front (lower edge) of a bund reaches point 25 mm above narrowest portion of constriction of tube, record solume collected and change receiver. For each acid, total cunalative volume is threshold volume used for identifying bands in succeeding runs, After propionic acid (third band) has been hed, il tube with 10% (¥/¥) butanol in CHC anduse this solvent lodte CH,COOH. (Propionic scid is eluted after ea $0 mL. 1% butanol in CHCl, has passed through column, Observe volume [scully used. In succeeding runs, change to 10% (viv) butanol in FC, a that volume whether or not propionic acid is presen.) ‘Transfer eluates to separate 125 mL. Evlenmeyers rinsing each taduate with three 5 mL portions H,0. Add 1 drop cresol red ictor solution and titrate with 001M alkali, As end point proaches, stopper Mask and shake vigorously to completely tact acids from solvent phase. Correct titration of each eluted ‘aol for blank as follows: Collect 28 mt. butanol-CHCL, mixture om column before any acids are transfered, add 15 mL boiled and Fed water, and titrate as sbove with 0.01M alkali Itbands are not clearly differentiated or recoveries are <90%, fect the silicic acid, (Additional standardization with respect to ehold volume may be desirable for ideification in some tances, ©) L Ientiication and Determination ‘Add | drop IM NaOH to neutralized distillate A obtained in ROVE (see 35.1.27) and evaporate to smal volume. Transfer to fi mL beaker, evaporate to dryness on steam bath, and proceed ‘asl in BOQ. itty acids by comparing their threshold volumes with those for jpoximstely same amounts and ratios of known acids found in dization operation, Threshold volume of given amount ofeach Ft wid is characteristic and reproducible under silat conditions lover ifeondtions change, such as use of diferentbatch of silicic Sor dfferent ameunt of samme batch, or diferent amount of H,0, cbold volume for each acid must be redetermined. (I present, Fisi AND OTHER MARINE PRODUCTS Chapter 3, p15 img Aci/100 g test portion mL 0.01M NaOH (corcected for blank) semolarty x F where F includes equivalent weights of scids, corrected for isk, ining i and blender jar wit methanol and adding rnsings to [fas Heat in water bath to 60°C and let stand 1S min at this ‘empaature. Cool to 25°C, dilate to volume with methanol, and fier tough fled pepe. Alcohol fate may be stored in refSgertor © several weeks (Light powdery precipitate separating on storage may beigne) Dit Sm filtrate to 100 mL. with H,O (disregard turbidity). Pipet Slaiquotnt 16150 mm glas-topperedtestube snd add I drop F beraaehyde (Cle) and 0.2 mi20% (wiv) NaOH. (pH aRerading Fh should beea 12 4-12.5) Shake vigorously ca25 times. Letstand ‘minand add S mL benzene-r-butanol mixture, Shake vigorously ca E tStimes nde stand Sin o separate emulsion forms, centifige Taser uper layer with fine-tp tube equipped with robber bu 0 {ously prepared CAS column, avoiding transfer of any aqueous ‘ase Reet aqueous solution with Sm benaane-butagol mintre ‘sb, shaking, leting stand $ mi, and transferring uppe layer 0 fblum, Rinse lip and sides of column with fine steam ofalobol from nak ote, syringing out CAS. Wash column with 3 iL. alcohol, -yige out, wash with two 3 mL portions H,0, and syringe out read solvents and washes, Ee hstine fiom CAS into 25 ml. glass-stopered Eslenmeyer Ey seahng down sides of tube with 2.0 ml. 0.20:4001M H,S0, (volume od concentration of acid ae critical followed by 3miL 0, _Sytiu oat afer dipping ceases. Coo eat in ice bath, weighting Mask with Lead ring or clamp fo prevent tipping, and let stand $-10 min. Add 0.5 mL cooled ‘jazoium reagent and lt stand 5 min in ice bath. Add 0.50 ml. ‘oulng bles (volume i eiical; Ostwald pipet is convenient) Eth continuous shaking or swirling to avoid localized alkalinity gflater addition of coupling buffer, $-6). Let stand 5 min in ice "fu, Sota solution with ca 0.25 g powdered Na,8,0;-108,0 {ded in one portion. Shake solution immediately and FISH AND OTHER MARINE PRODUCTS Chapter 35, 17 continuously ca 30s to ensure rapid and complete saturation (final pH ca 8.6) Let stand 15 min in ice bath, Pipet in 5.0 mL methyl isobutyl Ketone and shake vigorously 25 times. Immediately transfer both layers o 16 x150 mm test ube (Go not rinse) and let stand 10 min t room temperature to separate and to warm up. Transfer upper layer with fine-tip dropper to second, 16 «150 mm glast-stoppered test tube containing 5.0 ml. barbital buffer. Avoid transfering aqueous and solid phases if present (uansfer need not be quantitative). Shake vigorously ca 25 times (pH of barbital buffer after washing, ca 8,3-8.4),..et stand 10 min to separate, Transfer upper layer with finestip dropper to 1 em cell and determine 4 at 475 nm against methyl isobutyl ketone. Repeat determination on ests yielding 4 values >25 yg standard by diluting {Im methanol filtrate to 100 mL with H,O, Altematively, aquecus dilution may be eluted 1 +4 (or more) with H,0. Conduct standaid and blank through deterination as follows: Pipet S mi. 5 g/mL histamine standard solution into 16 «150 mm, alassstoppered test tube and pipet 5 mL $% (v/v) methanol into similar tube for blank. Proceed as in C, Determination, beginning, paragraph 2, line 2, add 1 drop benzaldehyde... Subtract blank 4 from 4 of standard (4) and of test solution (4) and calculate histamine in test aliquot: Ads Histamine, mg= 22 Reference: JAOAC 4, $92(1957) CAS-51-45-6 (histamine) Revised: March 2002 // (#) AOAC Official Method 977.13 Histamine in Seafood Fluorometric Method First Action 1977 Final Action 1987 fossai Codex-Adopted-AOAC Method” Caution: See Appendix B, Laboratory Safety for “Safe Handling of Allalies"—sodium hydroxide; “Safe Handling of Acids"-~phosphorc and hydrochlri acids; and “Safe Handling of Special Chemical Hazards"—methanol, Dispose of wast solvents in an approprite manner compatible with aplicable environmental rules and regulations ‘See Tables 977.13A and B for the results of the interlaboratory study supporting acceptance of the method, and Table 977.13C for recovery data A. Principle Product is extracted with 75% (v/v) methenol, Extract s passed ‘through ion exchange column, o-Phthaldialdehyde solution is added to eluate to form fluorescent histamine derivatives. Floorescent intensity of derivativesis measured using fluorometer and histamine ied using external standards ‘@2VZAOAC INTERNATIONALFist AND OTHER MARINE PRODUCTS CChaptor 35, p. 18 [AOAC OFFICIAL METHODS OF ANALYSIS 20 Table 977.438, collaborative study results of the original method) Interlaboratory study results for determination of histamine in tuna by fluorometric method (based on : Histamine.” Avg. histamine RSDq “Test sample “mg/i00g found, mgii00g RSD. % $e 36" Heat ‘Acceplatle skipjack packed in water 1 14 068 469 1009736 — ‘Skpjack with 25 mg histamine addea/100 test sample 28 258 0950 37 138354 sd Decomposed albacore packed in wator 30 16 205i 65 3473, 1100 Decompesed yellowin packed in water 2 198 oes 471720 87s ‘ecompased skipack packed inci 120 1286 6432 8.1 8.807 Decomposed yellowin packod in oi 200 198.8 4001 24 10.6555 ‘eproninate compositon 2B. Apparatus i Rinse al plastic and glass containers wth HCI (I + 3) and H,0) before use, (2) Chromatographic tube.—200 7 id mm polypropylene tube fited with small plastic stopeocks and ca 45 em Teflon tubing. Control flow rate st>3 mLminby adjusting height of column relative to mbing outlet. Alternatively, use 2-way valve in place of tubing. (©) Photofluorometer—With medium pressure Hg lamp with excitation at 350 nm and measuring emission at 444 nm, (©) Repipets 1 and 5 mL. ©. Reagents (a) Ton-exchange resin —Bio-Rad AG 1-X8, 50-100 mesh (Bio-Rad Laboratories, 1000 Alfred Nobel Dr, Hercules, CA94S47, USA; wwrwbiorad.com) or Dowex 1-X8, 50-100 mesh. Convert to OI form by adding ca 1S mL. 2M NaOH/g resin to beaker. Switl mixture and let stand <30min, Decant liquid and repeat with additional base. Thoroughly wash resin with H,O, surry ito fluted paper and wash again with 1,0, Prepare resin Iresh weekly and store under H,O. Place glass wool plug in base of tube, B(a), and slurry in enough resin to form 8 cm bed. Maintain H,0 level above {op of resin bed at all times. Do not regenerate resin in packed column; rather, use batch regeneration in beaker when nevessary ‘Wash column with ea 10 mi. H,0 before applying each extract. (b) Phosphoric acid—To prepare 1.19M as phosphoric acid, ile 73.33 mL 85% (15M H,PO,) 0 | L. For other concentrations of HyPO,, the volume required for | L 1.19M HPO, = 17 495/(density H,PO, x % H,PO,). Standardize 5.00 mL. by titration with 1.00M NaOH to phenolphthalein end point and wu concentration if necessary 7 (©) o-Phihaldialdehyde (OPT) solution —0.1% (wi). Diss 100 mg OPT in 100 mL distilled in-glass methanol, Store in amb bottle in refrigerator. Prepare fresh weekly. (a) Histamine standard solutions.—Store in reftigess (1) Stock solution—1 mg/m as free base. Accurately weigh a 169.1 mg histamine 2HCI (98%) into 100 mL volumetric Mas, ae lssolve and dilute to volume with 0.1M FICI Prepare fresh wos (2) Intermediate solution —10 jg/L. Piet I mi stock solution ins 100 mL volumetri flask, and dilute to volume with 0.1 M HCl. sue fiesh weekly. (3) Working solutions. —0., 1.0, and 15 ug/5 mL. Ppt 1,2, and 3 mt intermediate solution ino seperate 100 mL volume. ‘ass and dilute each to volume with 0. MHCI, Prepare es diy (6) Methanol.—75% (viv). Place 75 mL. MeOH (distil i lass) into 100 mL volumetric flask or stoppered graduated cylin, Dilute to volume with H,O. Swirl flask while adding HO. D. Preparation of Standard Curve a Pipet duplicate $ mi aliquots of each working standard solution it separae'50 mL glass or polypropylene Evlenmeyers, Pipe in 10 mi (0.1M HCI to each flask and mix. Pipet in 3 mL 1M NaOH and mix ‘Within 5 min, pipet in 1 mL OPT solution and mix immediately Ae, ‘exactly 4 mia, pipet in 3 ml. 1.19M TPO, anid mix immediatly. important to mix thoroughly after each addition anda lest once dig OPT reaction, (Ram 6-10 OPT reactions simultaneously by aig reagents to Enlenmeéyers in set order) Prepare blank by substtuig 5 mL 0.1M HCI for histamine solution, Within 1.5 h, reexd ‘uorescence intensity (D of working standard solutions with H,0 Table 977.138._Interlaboratory study results for determination of histamine in earned tuna and frozen mahimahi by fluorometric smothod (generated for modified method using 75%: (vy) methanol)" Testsample — Meangig —_S, ugl9 Sq. HO'9 RS0,,% SDR, % ie we HorRat 1 93896 0884 oats 901 92 Bais 2562 os 2 130 ano smo 13:7 saaz 4708 4708 12 3 5627 4.205 1295 haa 23.03 3374 3.826 188 4 7834 4.084 41008 13.84 ‘304 3.08 3.035 449 5 20.28 1340 2077 562 10.24 3.192 5016 401 6 58.02 ain 5466 364 942 sont 15:05 1.09 z __ 1584 657514050 415 887i 3.340 119 * Gonadanoaseceved ton Te abso © Rezbetn ‘SRT AONC INTERNATIONAL.OAC OFFICIAL METHODS OF ANALYSIS (2012) Table 977.13C. Recovery of histamine added to canned tuna (generated for modified method using 75% methanol Background, Found, Recovered, Recovery, Las? win yoga eae a 10.00 69.00 59.00 117.65 66.00 580011167 8 aes 630 53.45 106.58 6220 ©5235 104.39 ce 965 6700 87.35 114.36 406275 125.12 ° 895 55.00 46.05 - 91.62 se10 49.15 98.01 & 950 S840 4890. grt 55.10 4560” 9099 F 965 5810 48.45 96.61 5180 42:15 84.05 6 1185 61404985 99.40 S780 48259222 x 920 5410 44.90 80.53 54104490 80.53, 4 930 5560 46.30 82.32 5610 4880 © 93.2 « 1025 530042755 24 5300 4275 88.24 N 1010 5430 46.20 ea.14 5430 4420 88.14 ° 1000 5900 © 4900 97.71 5900 49.00 a771 P 1070 © 53304260 e958, 5390 4260 98.80 Po Seat F acta 015 pghitaminny Dat trom Laboratres and M wore excluded because resus forthe beelgrund or the some ware avons reference cel, using excitation wavelength of 380 nm and emission ‘wavelength of 444 nm, Plot J (corrected for blank) against ug isrine' ml aliquot, Determination Extract prepared 10 g test portion with 75% (w/) methanol asin 987.07C (see 35.131), paragraph 1, Pass 4-5 mL HO theough column, B(a) and discard eluate, Pipet 1 mL extract onto coluinn and add 4-5 mL H,0. Immediately initiate column flow into 50 mak, Yolumetric flask containing 5.00 ml. 1.00M HCl. When liquid level isca2mm above resin, add ca $ ml. H,O and let elute, Follow with H,Q in larger portions until ca 35 mL bas eluted. Sp column flv, lute to votome with H,O, stopper, and mix. Refrigerate eluate Pipet mL eluate into 50 mL Eslenmeyes, and pipet in 10 mL0.1M HCL Proceed asin D, beginning “Pipet in 3 mL IM NaOH...” If test sample contains >15 mg histamine/100 g product, pipet 1 mi test solution-OPT mixture into 10 mL beaker containing actly 2 mL blank-OPT mixture, and rx thoroughly. Read fuorescence of new solution. Dilute and mix aliquots with bank-OPT mixture as needed to obtain measurable reading. This Approximation indicates proper dilution of eluate requted prior to second OPT reaction needed for reliable quantitation of test soliton. Alternatively, use sensitivity range control of fkiorometer (if instrument has one) to estimate dilution, Use these ‘pproximations to prepere appropriate dilution of aliquot of eluate Fis AND OTHER MARINE PRODUCTS ‘Chapter 35,p. 19 with 0.1M HC, and proceed asin D, beginning “Pipetin 3 mL IM NaOH...” F. Caleulations Pot of F (measured by meter deletion or recorder response and corrected for blank) against ig histamine!S mi. test solution should be straightin passing trough origin withslope=m=(,/I 5) +h #28 J3. img Histamine/100 g fis LOyCeYCUI\ A) tig Histamine fish = 10 x (mg histamine/100 fish) where J, lly ane /= fluorescence fom tet solution, 1.5, 1.0, and 0.5 ug histamine standards, respectively; and F» dilution ctor (mL eluate +mL0.1MHCH/mLeluate. P= 1 for undiluted eluate calibration plot is not linear, use standard curve directly for quantitation. Each subdivision on abscissa should be <0.1 12 histamine/S mL tes solution. Read all values from curve to nearest, 0.05 ig histamine/S mL. test solution 1g Histamine/100 g test portion = (LOEW) tig Histamine’ test portion = 10 « (ng histamine/100 g test postion) where = ug bistamine/S mL. test solution as determined from, standard curve, Reference: JAQAC 60, 1125, 1131(1977) CAS-S1-45-6 (histamine) Revised: June 2003, September 2012 * Codex Stn 36-1981, Rew 11995, Codex Standard for Quick Frozen Finfish, Uneviseerted and Evisersted. Codex Stan 70-1981, Rew 1-1998) Codex Standard for Canned Tuna and Bonita. Codex Sian 94-1981, Rev. 1-1995, Codex Standard for Canned Sardines and Seidine-Type Products. Codex Stan 119-1981, Rev. 1-1995, Codex Standard for Canned Finish. Codex Stan 168-1989, Rev, 1-995, Codex ‘Standard for Quick Frozen Blocks of Fish Fill, Minced Fish Flesh, and ‘Mintures of Filles and Minced Fish Flesh Codex Sian 166-198, Rev 1-1995, Codex Standaed for Quick Frozen Fish ticks (Fish Fingers), Fish onions, and Fish FilltsDreaded orn Baer Codex Sta 190-1995, ole Genta Stee Quek roan Fak Ele 77 38.1.33 AOAC Official Method 948.17 Indole in Crabmeat, Oysters, and Shrimp Colorimetric Method Firet Action 1948 Final Action 1974 A. Apparatus and Reagents (a) Color reagent-—Dissolve 04 gp-dimethylaminabenzaldehyde in 5 mL CH,COOH and mix with 92 mL HyPO, and 3 ml. HCI. As purity of p-dimethylaminobencaldchyde affects intensity of reagent blank, party yellow commercial reagent as follows: Dissolve 100 g in 600 mL HCl (1 + 6). Add 300 ml H,O and precipitate aldehyde by slowly adding 10% (w/v) NaOH solution with vigorous stiring. As soon as precipitate aldehyde appears white, stop addition of NaOH solution, filter, and discard precipitate. Continue neutralization until practically all aldehyde is ‘SZ AOAC INTERNATIONAL