496.04 AOAC Official Method 2000.03 ‘Ochratoxin Ain Barley Immuneatfinity by Column HPLC First Action 2000 (Applicable to the determination of osbatoxin A in barley at D1 apis) ‘See Table 2000034 for results ofthe inteslaboratory study supe porting the acceptance of the method. A. Principle ‘Test potion is extrncted by blending with acetonitrle-water. The ‘extra is cleaned up by passing through au immunoaffinity column (Ockratoxin A (OTA) is eluted with methanol, further parifed and identified by LC, and quantified by fluorescence. 8. Apparatus (2) Silomzed glaze vials Needed to ensue stability of OTA in aqueous solvents, Prepare vials by filling them with silanizing re ‘agent and leaving this seagent in the vial for 1 min, Rinse vil with, solvent of low polarity (eg. ‘olvene) and then with methanol. Finally, wash vials twice with distilled wate, and dry before use (b) Analytical balance —Accurate to 10 mg (@) Blender —1 1 jar and cover, explosion-proof, (@) Displacement pipers —$ mL, | ral, 200 ZL with appropsiate pipet tips (©) Vacuum manifold—To ascommodate immancsfEnity col- (8 Reservoirs and attachments —To fit toi nanoaffinity cole (8) Vacuum punp —Producing a vacuum of 1.0 mPa and pumpe ing 18 Linin. (ib) Filter papers. Whats No.4, or equivalent. (@ Disposable syringe filters —0.2 Cm pore size, 25 mm dian ter polysalfone membrane, @ LC apparatuz—(1) Pump —Mobile phase pusup (isocratic) ppomping 1 mL/min pulsefree. (2) Injetion system —Valve injec tion system with 100 TL injection loop. (3) Separation col tunins C18 seversedaphase, $m ODS (equivalent to ODS | or 2) with 11% carbon loading, filly end-capped (pore size 10 am), with, ‘comsesponding reversed-phare guard ealuann. (4) Detector —Fluo= rescence detector wit flow cel with emission wavelength 460 am, ‘excitation wavelength 333 nm, and data collestion system. (3) Col unr oven—Controlling column at 45.00.50. Maintain at a constant tempenntire,slthonightne specified termpersture is ast eit cal (k) UF spectrophoromerer—For checking concentration of standard. Calibrarion—Determine absorbance (4) of the 3 solutions of K,Cn0> in H:S0,, C(s). C(O), and C(u), at maximum absorption near 350: against 0.009M H,SO, (ea I mil H,S0, diluted to 21) as solvent blank. Caleulate molar absorptivity (2 at each concentra- sion ae follons oy 471000 c where 4 = absorbance at 350 nm, C= mM concentration of sole Ifthe 3 values vary by more than guaranteed accuracy of 4 scale, check either tesnique or instrument. Average the 3 Cvalues to ob- tain Q, Determine correction factor (CF) for particular instrument sind celle by abtiting in equations where 3160 ~ value for Sof K,Ci0> solutions, 5, ~ average ofthe 3 Dales calculated above. IECE is 198 of ™1.08, ches either tecanigque or instrument to determine and eliminate cause. (Use same set of cells in calibration sad determination of pity.) (D Inmanoafinty columns—Spesifie for OTA cleanup. The imsnmnostfnity column shotlé contain sutibadies raised agsinst ty of 00 ng OTA. sd recover “BS% OTA witen applied as a standard selution ia CH,OH-PBS (piuosphate buffered saline solution, Clam]: 1+ 10. iv) containing Sng toxin, (Colnmns fiom View LP [313 Pleasant St, Watertown, MA 02472, USAJ. and Rhone Diagnostics [West of Sectland Science Pass, Unit 3.06 Kelvin Canapnis, Glasgow G20 SP, UK] have met these eriteri.) ©. Reagents (@) Ocinatoxin 4 (OT4) standard solution A, 10 ygink —OTA standard in taluene-aeetic seid (09 + Ive). The abacnpivity (= 5440 mimo. (See970-44B [49.202] for sources ofmycotoxin stan- ddanks) OTA. The column show have maxis ea Table 2000.03. interlaboratory study results for ochratoxin A in barley o s.ngig _Nooflabs? __S, RSD,» RSDq.% __HORRAT Rec. %6 4ngi9 37 20) 0.16 4 043 2 os 9210 nc’ (af ot 14(1) 004 8 10 n 12 — cto) 19 150) 092 24 oa 38 os = nc) 30 140) oar 2 052 7 05. ~ nea) 45. 132) oa 4 067 18 ow — * Number eats retaned ater eimnating outers the suber of boraonies removed is india in parerheses © nc nawraty conta © Laporaones recanes #cupeate est samples abeted aD, an 7007 AOAC INTERNATIONALTable 2000038. LC calibrants Description caP t cal2 Cala cal cals Fiterod injection sovent, 4875 4750 4500 2780 2500) 20:ngiml OTA, 125 250 500 1250 200 Final concentration, agin OTA 05. 1 2 50 100 Determine concentration as follows: Record UV spectrum of coeliatoxin A solution aginst solvent used for solution in reference call, Determine concentration of ochratexin A solution by measire ing dat wavelength of maximom absoxption close to 333 am anduse ing following equation Te gOstnio a= AMID Om CF vere A~ absorbance, MIF is molecular weight of ochrtonin A (403), CF conection factor bined inB(k), = molar aboopii {9 (5440 in toluene-aoei ac, 99 v8) (b) Aeetntrle CHC) 908%, LC grade (©) Exracton solvent (vs) Mix 6 pats seston) with pars water (@) Glacial acetic aed (CHCOO8. (Methanol 99.9% LC grads (6) jection solve) Mx 30 parts methanol () with 70 rts wate and I pt eet acd) (@) Sodom chord (h) Disecu hydrogen orthophosphate. (D Potassium dikydrogen phosphate. (j) Potassium chloride (k) Sodium dvoid. (© Sadium iyrovie, 0.2 te: (im) Phosphate buffered saline (PBS)—Dissolve ‘aC (g), 1.16 gNa;HPO, th), 0.2 g KH;PO, (0), and 0.2 ¢ KCI (]) in 1 L water, Adjust pH to 74 with 02M NeOH (0. (u) Helaun ge. (6) Le mote phase (v4) —Mx 102 pata water with 96 parts CH,CN (ban 2 prt CH,COOH (2: iter toh 0.2 Sn filer ‘B(i) and degas (e.g., with He [n]). (0) Toluene (@) Toluene-glcial acetic acid mitre). —Mix9 ps tolae coe p) wih | pat CHC OOH (4) (© Silanizing reagent (vs)-—Saxioe silconizing Suid, $% (0+ s)sohtion euchan SursSl fom Pierce Chemieal Co, PO Box 117. Rockford TL 61105,USA, No 42800) Mix par Seri with 19 parts toluene (p). (8) Pocassnm dichromate, ca 0.25mM—Acewately weigh co 78 mg K,Cr0; (primary standard) and dissolve in 1.0 L 0.009M. HO, (ie tak $0 to 2 withwater:caleuste Ato 3 ip nile fgmes (MW K,C,0,~2942), (0) Potassium debra, ca.0125nM—Dilte nL. 0 25a 01:0; (to 50 nL. with 0.009180, in volumeti Mask. (Dissolve 8 g NaOH (kin IL wae (w) Poiassium dichromate, ca 0.0625mM— Dilute 25 mil 0.125mM C10; (t) to $0 mi. with 0.009M #80, in volumetne Ask D. Extraction ‘Weigh. to:nearest 01g. 0n25 gest potion of barley inta blender jar Bie). Add 100 mL extretion solvent C(e). Cover and seal blender: blend for 3 ani, Filter extract through filter paper B(h). E. Immuneaffinity Column Cleanup Pipet 4m. filtrate into 100 iL glass beaker (or similar) and dilute with 44 mL PBS C(m). Connect immunoaffinity columa B(D) to vac- ‘mm snanifold B(e), snd attach reservoir) t inannoadtnity cole, tamu. Add diluted extract to reservoir and pass through immunoaffinity column at $ mL/min flow rate. The imuamoeflinity coluna must act be allowed to nun dry. Wash, beaker and column with 10 mL water, remove from vacuum mnie fd, and place over silnized vil B(a. Elite OTA into silanized vial with four 1 ma portions methanal C(e). Evaporate eluate to dryness over steam bath, under N, ‘Redissolve in | mL injection solvent C(f), which has been filtered ‘Brough 0.2 Ga filter, Transfer to LC vial F. Lc Determination (@) Calibration graph—Prepace calibration graph at beginning of each day test portions ae analyzed. (b) Preparation of standards —Pipet 200 “E10 Tim OTA calitrant solution C(a)nto glass vial and ddureto I mm. with 800 “Z toluene-acetic acid C(q) to give 2 CpimL OTA solution. Pipet 1001.2 Gein OTA solution int silanized plas vial Bla)-Bvapo rate solvent tnder steam of N, Redissolve in 10 ail injetion sol ‘vent C(f) that has been filtered through 0.2 Gu filter. This gives 20ngiml. OTA solution, From this solution, prepare SLC calibransin separate Sm volue trie flasks aevording 0 Table 2000.03B, Dilute each calibrant (Cal) to volume (5 miL) with filtered injection solvent C(9, (€) Operating condtions.—When column BA) ad mobile pase specified C(o) were used, the following settings were appropriate: Flow rate, 1 mini: cokama oven temperate, 45 21-0: fluores cence detection, 460 am emission waveleagth, 333 um excitation wavelengths injection volume, 100 CL. (@) Evaluorion.—Determine, from calibration graph, masses in ‘ag of OTA in aliquot of test solution injected onto the LC solu, My CCaleulate mass fraction, Wors, of OTA in mek using the equae NALwhere M=niss of OTA intesteolition extract, ng: Vj Textmstion solvent, ml (100 mL); P= scetonitrle-water filtrate passed tHyough iammumoaffinity column, mL (4 mL): 7 = test solution (Lm); F=test solution injected, mL: Me=testportion extracted, & (@5 9): 1000= conversion factor for gg: 1000" = conversion fae tioa foe ag to, Reference: J. AOAC In. 88, 1377(2000) 7007 AOAC INTERNATIONAL