492.18 AOAC Official Method 991.31 Atiatoxins in Com, Raw Peanuts, and Peanut Butter Immunoatfinity Column (Afiatest) Method First Action 1991 Final Action 1904 AOAC-UPAC Method (Applicable t determination of aflatoxins By, By, G}, and Gp at 210 ng total aflatoxins/g in corn, raw peanuts, and peanut batter.) See Teble 991.314 for results of the interlaboratory study sup- porting acceptance af the method. A. Pilnciple ‘Test portion is extracted with CH;OH-H,O (7 +3). Extract is = ‘ered, diluted with water, and applied to aifinity column containing monoclonal antibody specifie for aflatoxins B,, B,, G,, and G,. ‘Afiatoxins are isolated, purified, und concentrated on column and removed from antibodies with CH,OH. Total aflatoxins are ‘quantitated by fluorescence measurement after reaction with bro- mine solution (SFB method). Individoal aflatoxins are quantitated by LC with fluorescence detection and postcolumn iodine derivatization (PCD method). B. Performance Standards tor immunoaffinity Column ‘The 1 mL. syringe-barrel column contains immobilized anti body to aflatoxins By, Bs, G,. and G, and is filled with preserved butfer solution. Column can be stored 1 year at room temperature without loss of column performance. Recoveries for a standard aflatoxin solution of 15 ml CH,OH-H,O (1 + 3) that contains 25ngB,, Sng B,, 15 ng G,, and 5 ag Gy should be at least 90, 80, 90, and 60%, respectively. C. Apparatus (a) Blender —High speed. With $00 mL. blender jar and cover. (b) Filter paper-—24 cm, prefolded (Whatman 2V performed satisfactorily). (©) Glass microfiber filter paper. —11 cm (Whatman 934AH per- formed satisfactorily). (d) Affinity cotumn.—See B. Aflatest P column (Vicam, 29 Mystic Ave, Somerville, MA 02145, USA) performed satisfactorily Fable 991.31A interlaboratory etudy results for atiatoxins corn, raw peanuts, and peanut butter : (©) Syringe —20 mt, Luer tp for sample reser, (f) Hand pump.—20 mL syringe barre and piunger wit lene stopper aitached to outlet. (h) Fluorometer—360 nm excitation filter and 450amenis filter (Sequoia Tumer Model 450 [Mountain View, CA. USA] performed satisfactorily). (@) Awomatie dispenser. —Amber 2 o2 bottle with 0 ml. matic dispense: (Tri-Continent Scientific, Inc. (Grass Val, 95945, USA] performed satisfactorily). (i) Volumetric flask —2 mL. (6) LC pumnp.—Suitable for flow rate at 10.005 mLmin (Q) Injection system —Syringe-loading injecion valve ‘50 UL loop or equivalent. Teflon tubing, and heating bath or postcolumn reactor (Ci Nos. F1A920 and F1A910(Rainin) performed satisfastori (0) Fluorescence detector—360 nm excitation fier >420 nm cutoff emission filter (Kratos Mode! 950 (R NJ 07446, USA] performed satisfactorily) D. Reagents (a) Solvents —Distilled-in-glass methanol, LC grade met acetoniuile, and water. (b) Extraction solvent —CH,OH-H,0 (7 +3), (©) Bromine developer solution —0.002% bromine in Prepare fresh daily by mixing 10 mL. 0.01% solution (Vi Agaeg, RSD, Food ng/g Se Ee Sr com 10 4.44 zo 3.07 30 5.43 16.57 6.55 Raw peanuts 10 3.17 20 2.68 12.79 3.22 30 3.93, Poanut butter 10 1.54 qwA75 177 20 4.38 so 3.65 LG postcoiumn derivatization Corn 10 3.66 20 0.78 30 1.79 7-31 2.86 Raw peanuts 10 4.06 20 oes 5.20 2.58 30 9.64 Peanut butter 10 1.45 17.22 254 20 a.74 30 5.07“Title 991.318 Preparation of working standard solutions ‘Working standard solution contains, 0. @ 0.750 0.188 0375 0.188 4 ©0500 0.125 0250 (0.125 20 0.250 0.063 0.125 0.063 F ‘zest concentrated developer) with 40 mi. H,O. Keep solution in (@) Fluorometer calibration standards —Use 0.05M HSO, a8 ‘Vank Use 34 mg quinine sulfate dihydrate (Ultrapure J.T. Baker, ‘ceqivalent) mL 0.05M@ H,SO, a8 20 ng aflatoxin/g standard. © (@) LC mobile phase —H,0-CH,CN-CH,OH (3+1+ 1), teased Fp Postcolunn reagent-—Dissolve 100 mg iodine in 2 mi © (HOH Add200mL 1,0, str 'h,and filter through 0.45 um filter. Eee cil. © © Maoxin standard solutions for LC—(D) Mixed flataxins © sek sendard solution —Prepare as in 9T1-22B-E (see 492.03) to © contain 500 ng B,, 125 ng B,, 250 ng Gy, and 125 ng G/mL ben- © ame CH,CN (98 + 2). (2) Working standard solutions —Transfer © extiqutity indicated, Table 991.31B, of stock solution J into series die 2m volumetric flasks. Evaporate solutions just to dryness ‘wit stream of nitrogen at room temperature. To each flask, add In CH,OH. mix, dilute to2mL with wate, and mix. Prepare daily. © E Preparation and Extraction of Samples See 971.16 (see 49.2.01) for sampling procedures. See 972-26 F (ee 49.2.14) for com, See 968:22C (see 49:2.08) for peanuts and pent bute. | Weigh 25 g test portion into blender jar. Add S g NaCl and 125 | eLexration solvent. Blend 2 min at high speed. Filter through © peilded paper. Pipet 15 mL. filtrate into 125 mL glass-stopper Eienmeyer flask. Add 30 mL HO, stopper, and mix. Filter diluted | mac hrough glass microfiber paper <30 min before affinity col- © tan chromatography. Fltrate should be clear. If not, refiltr. Pro- © ced immediately with column chromatography. ~.Afnty Column Chromatography ‘Secure syringe reservoir to aring stand. Remove top cap from col- ‘zm Cutofftip and use cap as connector between column and reser- ‘ei Pipe 1S mi. second filtrate (equivalent o Ig test portion) into ‘reservoir. Connect reservoir to air-filled hand pump. Remove end cap fom column, Push extract through column at flow rate of ca 2ézops(6 mL/min}, Disconnect hand pump from reservoir and fll © amp withair. Reconnect hand pump to reservoir and pass 2-3 ml. © artocugh column. Disconnect hand pump and add 10 ml HO to resrvois. Fillhand pump with air and reconnect. Push water through ‘alum at flow rate of 6 mLimin, Repeat with another 10 mL H,0. ‘Discard water washings. Disconnect hand pump and fill withair. Re- } comect and pass 2-3 mL air through column. Disconnect hand © pump and add 1.0 mL LC grade CH,OH to reservoir. Collect eluate inqropricte container. Fill hand pump with air. Reconnect and 8 CHLOH through column, Pass additional 2-3 mL air through ima, emma F> (a) For quantitation by SFB, collect CH,OH eluate in fowometer cuet. Immediately proceed with Fluorometric determi- wan. (Note: Conduct affinity column chromatographic determi nation with duplicate aliquots of second filtrate and determine fluorometer reading of both.) (b) For quantitation by PCD, collect CH,OH eluate in2 mL volu- metric flask. Dilute to volume with LC grade water, mix, and pro- ceed with LC quantitation. G. Solution Fluorometry (a) Calibration of luorometer.—Let fluorometer warm up 20 min. Insert blank, D(d), and use zero knob to adjust instrament to ead 0. Insert 20 ng aflatoxin/g standard, D(d), and use span knob to adjust instrument to read 20. Use proper gain setting to ensure that blank gives O reading and standard gives 20 reading. Calibrate fluorometer just before reading affinity column eluate to avoid pos- sible instrument drift. (b) Quantitation —Adid 1.0 mL diluted developer to eluate, F( inclean testtube, C(g). Vortex-mix Ss. Ifbubbles adhere to wall of test tube, tap lightly to dislodge. Insert tube in fluorometer and wait 60s. Record reading, which is equivalent ong total aflatoxins test portion H. LC Determination with Fluorescence Detection and Postcolumn Derivatization Connect LC column outlet to one armof stainless steel, low dead volume, using short piece of 0.01 in. (0.25 mm) id tubing. Connect outlet of second LC pump, which delivers the postcolumn reagents, to second arm of T. Connect one end of 610cm x0.5 mm idoil of Teflon tubing to third armof T and con- tect other end to detector. Using oven or constant temperature bath, maintain reaction coil temperature at 70°C. Set following flow rates: (1) mobile phase (column), 10 mL/min; (2) postcolumn reagent, 0.3 mL/min; and (3) total rote through reaction coil, 1.3 mL/min. Reaction coil volume is ca 1.2.cm? so that, with total Nlow rate of 1.3 mL/min, postoolumn reaction time is ca 55 s. Let entire system run 10-20 min to stabilize. I integrator is used, adjust sensitivity controls of fluorescence detector or integrator to give reasonable re- sponse (signal:noise = 5:1) for 0.125 ng aflatoxin G,/50 pL. If strip chart recorder is used, adjust fluorescence detector control to give 30-40% scale deflection with 0.125 ng aflatoxin G./50 pL. Inject 50 pL working standard mixture into injector, using 20-30 pL excess to ensure that 50 ul. loop is completely filled. Aflatoxinsclute inorder G;,G,,B>,B, withretention imesca6, 8,9, and 11 min, respectively, and should be baselin-resolved. Ifneces- sary, adjust retention times by changing CH,OH concentration of roobile solvent. Inject 50 ul. working standard solutions 1,2, and 3, ‘DigX2). daily and prepare standard curves for aflatoxins. Inject 50 pL test solution from F(b), into injector. Identify cach aflatoxin peak in chromatogram from analysis of tes solution by comparing retention times with those of comesponding reference standards, Determine quantity of each aflatoxin in eluate injected from corresponding standard curves. Calculate concentration of each aflatoxin in test portion, using following formulas: W=25 gx(15 mL/125 mL) x(15 mL/45 mL.) = 1g Aflatoxin, ng/g = A x (TY/I,) x(1/W) = A x40 ‘where W = weight of commodity represented by eluate, A=ng afla- toxin in eluate injected, T, = final eluate volume (2000 pL), and1, = cluate injected (50 pL), Add concentrations of the 4 aflatoxins to obsain total aflatoxin concentration. [Note: Soak all laboratory glassware in 10% solution of house- hold bleach, which generally contains 5.25% NaOCl, before reus- ing or discarding. See 990.32J (see 49.2.16) for further details on decontamination. ] Reference: JAQAC 74, 81(1991).