AOAC Official Method 956.03

50.1.04AOAC Official Method992.03Vitamin E Activity(All-rac-a-Tocopherol)in Milk-Based Infant FormulaLiquid Chromatographic MethodFirst Action1992Final Action1996Codex-Adopted–AOAC Method* (Applicable to determination of vitamin E activity in milk-based in-fant formula.)Results of the interlaboratory study supporting the acceptance of the method(milk-based liquid,ready-to-feed):Mean recovery=24.14IU vitamin E/L infant formulas r=2.04;s R=2.82;RSD r=8.46%;RSD R=11.69%A.PrincipleVitamin E activity in test portions of infant formula is determined by saponification of all-rac-α-tocopherol,partitioning with organic solvent,separation from product matrix,and quantification by liquid chromatography.B.Apparatus(a)Liquid chromatograph(LC).—Capable of pressures up to 3000psi with injector capable of100µL injections.Operating con-ditions:eluent flow rate2.0±0.2mL/min;temperature ambient.(b)Detector.—Capable of measuring absorbance at280nm,with sensitivity0.02AUFS.(c)Precolumn.—2mm id×2cm stainless steel,packed with 40µm pellicular reversed-phase C18(Alltech28551is suitable). (d)Column.—4.6mm id×25cm stainless steel,packed with 5µm silica(Hypersil Silica is suitable).(e)Shaking water bath.—Capable of maintaining70±2°C,with vari-able speed capable of60oscillations/min,ca11×14in.(28×36cm)test sample area(Precision Scientific Model25is suitable).(f)Glassware.—(1)125mL separatory funnels.(2)5mL volu-metric flasks.(3)100mL low-actinic volumetric flasks.C.Reagents(a)Mobile phase solution.—Hexane–isopropyl alcohol(99.92+0.08,v/v),HPLC grade solvents.Degas2–5min under vacuum.(b)Wash solution.—H2O–absolute ethanol(3+2,v/v).(c)Extraction solution.—Hexane–methylene chloride(3+1, v/v),HPLC grade solvents.(d)Saponification solution.—10.5M potassium hydroxide (KOH).Dissolve673g KOH in1L H2O.(e)Antioxidant solution.—1%pyrogallol.Dissolve5.0g pyrogallol(1,3,5-trihydroxybenzene,98%;Aldrich is suitable source)in500mL absolute ethanol.(f)Standard solutions.—(1)Stock standard solu-tion.—0.5mg/mL all-rac-α-tocopheryl acetate in hexane.Accu-rately weigh ca50mg all-rac-α-tocopheryl acetate(USP Reference Standard)into100mL low-actinic volumetric flask and dilute to volume with hexane(HPLC grade).Shake well to dissolve.Make fresh every3weeks.Store at–20°C in explosion-proof freezer when not in use.(2)Working standard solution.—10µg/mL all-rac-α-tocopheryl acetate.Pipet2mL stock standard solution,(1),into100mL low-actinic volumetric flask.Evaporate to dryness under nitrogen. Dissolve residue in antioxidant solution,C(e),and dilute to volume. Prepare fresh daily.(g)Suitability test solution.—Approximately15µg/mL all-rac-α-tocopherol(USP Reference Standard)and all-rac-α-tocopheryl acetate(USP Reference Standard)in hexane (HPLC grade).D.Extraction of Standard and Test PortionsPipet10.0mL working standard solution,C(f)(2),or test portion containing ca0.095IU vitamin E activity(10mL for ready-to-feed formulas)into150mL centrifuge tube.Bring test solution volume to 10mL with H2O,if necessary.To standard tubes,add10mL H2O, 20mL antioxidant solution,C(e),and5mL saponification solution, C(d).To sample tubes,add30mL antioxidant solution and5mL saponification solution.Cap tubes and swirl briefly to mix.Place tubes in70°C shaking H2O bath(ca60oscillations/min)for25min. Remove tubes and place in ice5min,or until contents cool to room temperature.Quantitatively transfer contents to separate125mL separatory funnels.Wash remaining test portion solution or standard from tube into funnel with30mL H2O.Pipet30.0mL extraction sol-vent,C(c),into funnel and shake ca2min.When layers separate, discard aqueous(lower)layer.Add30mL wash solution,C(b),to funnel and shake very gently30s,venting frequently.Let phases separate and discard aqueous layer.Repeat wash step3×.Pipet 20.0mL portion from funnel into50mL tube and evaporate to dryness under nitrogen.Transfer residues quantitatively to sepa-rate5mL volumetric flasks and dilute to volume with mobile phase solution,C(a).Inject100µL standard or sample into LC.E.System Suitability TestInject100µL suitability test solution,C(g),into LC.Typical peak retention times for tocopherol and tocopheryl acetate are6.0and 5.0min,respectively.Calculate resolution(R)factor between tocopherol and tocopheryl acetate as follows:R=22112()t tW W−+where t1and t2=retention time measured from injection time to elu-tion time of peak maximum of tocopherol and tocopheryl acetate,re-spectively,and W1and W2=width of peak measured by extrapolating relatively straight sides to baseline of alcohol and ace-tate,respectively.If R factor is>1.0,proceed with test portion analysis;if R factor is<1.0,decrease amount of isopropyl alcohol added per liter[mo-bile phase solution,C(a)]by ca0.01%.Inject working standard solution,C(f)(2),5×.Calculate reproducibility of replicate injec-tions in terms of standard deviations(per USP),which should be ≤2%.Typical relative standard deviation values for peak height are±1.5%.F.Liquid ChromatographyInject100µL standard or test solution into LC.G.CalculationsMeasure peak heights or peak areas of all-rac-α-tocopherol in test solution and standard chromatograms.Calculate IU per reconsti-tuted quart of vitamin E activity(A)as follows:A=H sam/H std×C std×0.001IU/µg×946.33mL/quart where H sam=peak height of test portion solution;H std=peak height of standard;C std=concentration of standard,µg/mL. Reference:J.AOAC Int.76,398(1993).CAS-59-02-9(α-tocopherol)CAS-7695-91-2(α-tocopheryl acetate)Revised:March1997*Adopted as a Codex Reference Method(Type II)for liquid chromatogra-phy of vitamin E(milk based infant formula)in special foods.。

AOAC Official Method 2007.01Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium SulfateGas Chromatography/Mass Spectrometry and Liquid Chromatography/Tandem Mass SpectrometryFirst Action 2007[Applicable for the following pesticides in grapes, lettuces, and oranges: atrazine, azoxystrobin, bifenthrin, carbaryl, chlorothalonil, chlorpyrifos, chlorpyrifos-methyl, l -cyhalothrin (incurred in lettuces), cyprodinil, o,p ¢-DDD, dichlorv os, endosulfan sulfate,ethion (incurred in oranges), imazalil, imidacloprid,kresoxim-methyl (incurred in grapes), linuron, methamidophos,methomyl, permethrins (incurred in lettuces) procymidone,pymetrozine, tebuconazole, thiabendazole (incurred in oranges),tolylfluanid (degraded in lettuces), and trifluralin. These were representative pesticide analytes chosen in representative matrixes,and the method is expected to be applicable to many other similar pesticides and matrixes. Limits of quantitation were demonstrated to be <10 ng/g.]See Tables 2007.01A–E for the results of the interlaboratory study supporting acceptance of the method.A. PrincipleThe QuEChERS (quick, easy, cheap, effective, rugged, and safe)method uses a single-step buffered acetonitrile (MeCN) extraction and salting out liquid–liquid partitioning from the water in the sample with MgSO 4. Dispersiv e-solid-phase extraction (dispersive-SPE) cleanup is done to remove organic acids, excess water, and other components with a combination of primary secondary amine (PSA) sorbent and MgSO 4; then the extracts are analyzed by mass spectrometry (MS) techniques after a chromatographic analytical separation. Figure 2007.01 outlines the protocol in a box format. In brief, a well-chopped food sample alongwith 1 mL of 1% acetic acid (HOAc) in MeCN and 0.5 g anhydrous MgSO 4/NaOAc (4/1, w/w) per g sample are added to a centrifuge tube or bottle, which is shaken and centrifuged. A portion of the MeCN extract (upper layer) is added to anhydrous MgSO 4/PSA sorbent (3/1, w/w; 200 mg per 1 mL extract), mixed, and centrifuged. This final extract is transferred to autosampler vials for analysis by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) to identify and determine a wide range of pesticide residues. To achieve <10 ng/g detection limits in modern GC/MS, large volume injection (LVI) of 8 m L is typically needed, or the final extract can be concentrated and solvent exchanged to toluene (4 g/mL), in which case 2 m L splitless injection is used.Both GC/MS and LC/MS/MS techniques are prone to matrix effects in pesticide residue analysis, albeit for different reasons [Erney, D.R., Gillespie, A.M., Gilvydis, D.M., & Poole, C.F. (1993)J. Chromatogr. 638, 57–63; Hajslova, J., & Zrostlikova, J. (2003) J.Chromatogr. A 1000, 181–197; Alder, L., Luderitz, S., Lindtner, K.,& Stan, H.J. (2004) J. Chromatogr. A 1058, 67–79]. To account for these effects, matrix-matched calibration was conducted (calibration standards in solvent solution may also be used if matrix effects are shown not to occur). Due to the situation that some laboratories had LVI capability and others did not, the necessary amounts of matrix blank(s) and final extract volume was different for some laboratories than others. Depending on the water content of the matrix, a 15 g sample typically yields 11–14 mL of initial MeCN extract after centrifugation. In dispersive-SPE, roughly half of the extract is lost to the powders, thus about 6–7 mL of final extract can be expected for a 15 g sample. Two options were provided in the protocol to account for the different situations among the laboratories.ã 2007 AOAC IN T ER N A T IONALTable 2007.01A. Interlaboratory study results for incurred pesticides (and chlorpyrifos-methyl)AnalyteMatrix Avg. concn s raRSD r b, %S R c, ng/g Rec., %RSD R d, %HorRat No. of labsOutlierlabs e Chlorpyrifos-methylGrapes 165 14 8.535 8321 1.00116-C, 4-C Lettuces 178 20 11 30 89170.811011-SGOranges174 25 14 36 87200.9812Kresoxim-methyl Grapes 9.2 1.921f3.2NA 35f1.0912Cyprodinil Grapes 112 NAgNA 18 NA 160.7313l -Cyhalothrin Lettuces 58 6.111 11 NA 200.80 911-C Permethrins Lettuces 112 9.88.741 NA 36f1.63 96-C, 1-C Imidacloprid Lettuces 12NA NA 1.6NA 140.4411Ethion Oranges 198 23 12 36 NA 180.891111-C Thiabendazole Oranges 53 3.87.27.6NA 140.5812ImazalilOranges13NANA4.7NA35f1.1587-SGa s r = Standard deviation for repeatability (within laboratory).b RSD r = Relative standard deviation for repeatability.c s R = Standard deviation for reproducibility (among laboratories).d RSD R = Relative standard deviation for reproducibility.e C = Cochran outlier; SG = single Grubbs outlier.f RSD r >15%; 120% < Rec. < 70%; RSD R >25%; HorRat >1.2; and fewer than 8 laboratories in an assessment.gNA = Not applicable.Analyte Avg. C, ng/g s r, ng/g RSD r, %s R, ng/g Rec., %RSD R, %HorRat No. of labs Outlier labs Atrazine9.30.6 6.9 2.093210.651345 3.27.1 5.790130.4913365 23 6.271 9119 1.0413Azoxystrobin9.40.6 6.6 2.094210.641392 8.79.411 92120.51128-SG182 17 9.226 91140.70128-SG Bifenthrin7.80.811 2.37830b0.89112-C, 10-C86 5.9 6.914 86170.73126-C923 71 7.7136 92150.9113Carbaryl12 1.211 2.8104 27b0.85125-SG50 6.413 11 100 220.87131003 70 7.0189 100 19 1.18125-C Chlorothalonil 6.30.914 2.163b33b0.97 810-C59 8.314 13 79230.9310140 19 13 38 7027b 1.27b10Chloropyrifos8.1 1.519b 3.08137b 1.121268 8.312 14 84200.8413396 25 6.450 79120.681211-SG Cyprodinil c123 13 10 26 101 210.9513240 20 8.363 9226b 1.32b13581 42 7.3110 9519 1.0913o,p¢-DDD8.9 1.416b 3.28936b 1.091242 3.17.37.084170.6512445 32 7.147 89100.58116-C Dichlorvos7.2 1.014 1.372180.53118-SG85 7.48.715 85180.77114-C294 25 8.562 9821 1.1012Endosulfan sulfate8.60.910 1.586170.52 7b10-C 115 14 12 21 77180.8111415 56 14 111 8327b 1.47b11Imazalil7.60.89.8 3.17641b 1.22b1150 2.5 4.915 67b30b 1.19108-C432 53 12 161 7837b 2.06b11Imidacloprid8.80.88.9 3.08834b 1.041345 3.57.78.999200.7813218 18 8.224 97110.56128-SG Linuron9.9 1.717b 2.99929b0.901199 7.47.415 99150.6712971 65 6.7191 9720 1.23b12Methamidophos10 2.929b 3.0101 30b0.95 95-SG80 8.010 14 80180.7712852 72 8.4119 85140.85118-SG Methomyl9.3 1.212 2.99332b0.981250 3.3 6.79.3100 190.7413204 10 4.926 102 130.6313Procymidone8.20.78.2 2.082240.74115-SG64 6.09.416 8524 1.0113428 16 3.870 86160.90129-C Pymetrozine 6.2 1.220b 1.662b27b0.771147 3.1 6.79.662b200.8111341 20 5.859 68b170.9211Tebuconazole9.2 1.112 1.292130.41123&4-DG63 5.58.78.884140.5813439 29 6.784 8819 1.0613Tolylfluanid7.9 1.012 3.17939b 1.191334 4.313 13 67b37b 1.41b13144 13 8.842 7229b 1.37b13Trifluralin7.80.78.5 1.878230.681210-C58 3.7 6.414 7725 1.0213379 19 5.148 76130.69106-C, 4-C, 11-SG aC = Cochran outlier; SG = single Grubbs outlier; DG = double Grubbs outliers.bRSD r >15%; 120% < Rec. < 70%; RSD R >25%; HorRat >1.2; or fewer than 8 laboratories in an assessment.cCyprodinil was incurred in the grapes and affected quantitation.ã 2007 AOAC IN T ER N A T IONALAnalyte Avg. C, ng/g s r, ng/g RSD r, %s R, ng/g Rec., %RSD R, %HorRat No. of labs Outlier labs Atrazine9.9 1.515 1.899180.561170 7.9111593210.8812930 50 5.4166 9318 1.11115-C Azoxystrobin10 0.8 7.6 1.8102 180.561247 2.4 5.2 6.593140.5512531 32 6.188106 170.9412Bifenthrin9.10.8 9.1 1.491160.481166 8.012 9.488140.5911217 27 123387150.7711Carbaryl9.4 1.112 2.094220.671292 6.1 6.7 9.092 9.80.43118-SG589 38 6.4127 9822 1.24b12Chlorothalonil 6.20.814 2.062b32b0.93 6b28 10 37b147048b 1.77b 7b684 134 20b205 68b30b 1.77b 6bChloropyrifos9.0 2.124b 2.39026b0.79 912-SG, 10&11-DG86 9.411208623 1.011111-SG179 18 103090170.821111-SG Cyprodinil9.7 1.010 1.497140.441111-SG44 2.7 6.1 8.989200.791111-SG848 61 7.2117 85140.84108-SGo,p¢-DDD8.90.6 7.0 1.989210.66 881 4.8 5.91281150.63 911-C214 19 8.72786130.6210Dichlorvos 5.2 1.020b 2.452b45b 1.29b1258 6.6111277200.8112838 50 6.0224 8427 1.63b11Endosulfan sulfate 5.6 3.359b 2.556b45b 1.28b 2b38 9.625b157539b 1.48b 7b769 330 43b312 7740b 2.44b 7bImazalil7.60.3 3.5 3.57639b 1.18 82-C72 3.7 5.22457b33b 1.39b11589 47 7.9229 59b39b 2.25b11Imidacloprid c22 1.3 6.2 1.7100 7.90.28118-SG84 6.2 7.4 8.197 9.60.4112515 21 4.253101 100.58115-C Linuron8.6 1.112 1.586170.531146 2.2 4.9 7.491160.63102-C234 14 5.82594100.5311Methamidophos8.80.8 8.5 1.388150.46 86-C66 4.5 6.91282180.7211538 37 6.86384120.67 95-C, 8-SG Methomyl9.70.8 8.6 1.096100.321099 8.0 8.1 6.499 6.50.29102-SG997 24 2.4168 100 17 1.0511Procymidone10 0.6 6.2 2.2101 220.68 82-C92 8.5 9.21592170.7311967 118 12129 97130.8311Pymetrozine 6.90.4 6.1 1.469b200.591033 1.6 4.7 4.667b140.51 911-C127 8.5 6.71763b130.6110Tebuconazole9.70.7 6.9 1.297130.40114-C89 6.8 7.71189120.5212948 42 4.4226 9524 1.48b114-C Tolylfluanid 3.7 1.130b 2.237b59b 1.59b 4b9.3 3.740b 4.1 9.3b44b 1.37b 83-SG, 8-SG142 22 158614b61b 2.84b 812-C, 3&8-DG Trifluralin10 1.413 1.7103 170.541142 4.511 9.084220.8311169 25 153084180.8411aC = Cochran outlier; SG = single Grubbs outlier; DG = double Grubbs outliers.bRSD r >15%; 120% < Rec. < 70%; RSD R >25%; HorRat >1.2; or fewer than 8 laboratories in an assessment.cImidacloprid was incurred in the lettuces unbeknownst to the SD.ã 2007 AOAC IN T ER N A T IONALAnalyte Avg. C, ng/g s r, ng/g RSD r, %s R, ng/g Rec., %RSD R, %HorRat No. of labs Outlier labs Atrazine 8.9 1.011 1.989210.65112-C 908.29.11290130.5712187 19 10 2793140.6912Azoxystrobin 8.4 1.316b 1.884210.651165 5.28.0 8.186120.5212853 35 4.18285 9.60.591111-C Bifenthrin 9.7 2.324b 2.397240.75109-SG45 2.5 5.6 6.891150.59102-C488 51 10 7698160.8712Carbaryl 8.40.67.3 2.184250.771066 5.07.51488210.8811172 8.8 5.13486200.9512Chlorothalonil 4.80.816b 2.748b56b 1.57b 3b7014 20b297042b 1.74b 6b330 137 42b131 66b40b 2.09b 7bChloropyrifos11 1.614 5.0111 45b 1.58b 92-C82 4.5 5.61282150.64109-C953 97 10 284 9530b 1.85b1211-C Cyprodinil 8.70.910 2.087230.721256 4.58.0 9.075160.6512199 12 6.23580180.8612o,p¢-DDD 9.10.67.2 1.891200.60 99-C74 5.1 6.9 9.899130.561010-C967 81 8.41919720 1.22b11Dichlorvos 9.30.88.1 1.093110.35 7b12-C43 2.2 5.2 8.085190.73 812-SG446 22 5.05489120.6810Endosulfan sulfate12 5.444b 5.4124b43b 1.40b 4b3-SG 8319 23b198323 1.0110240 35 15 618025 1.28b10Imazalil c22 1.77.7 6.29628b0.98 87-C58 4.37.41392220.91 9186 9.7 5.2418722 1.0610Imidacloprid10 1.110 2.8104 27b0.861193 6.57.01293130.5711989 64 6.5124 99130.7811Linuron 7.8 1.317b 2.77835b 1.041160 3.0 5.01386210.8611387 26 6.64279110.59 9111-DG Methamidophos 9.2 1.112 1.592160.49 89-C42 3.58.2 5.685130.52 84-C211 12 5.53185150.73 94&9-DG Methomyl 8.50.88.9 2.88533b0.99 97-C68 4.87.0 8.791130.5412492 19 3.96098120.6912Procymidone110.98.1 3.9108 36b 1.15 812-C43 3.58.0 5.886140.531010-C170 16 9.72585150.7111Pymetrozine 7.5 1.318b 2.17528b0.821077 5.97.71077140.5710789 38 4.8117 79150.89 912-C Tebuconazole 8.70.78.0 1.287140.421141 2.2 5.4 6.282150.5812177 14 7.92888160.7612Tolylfluanid 5.8 1.220b 1.458b240.69 911-SG 467.516b1461b31b 1.21b119-C356 54 15 134 7138b 2.02b12Trifluralin 8.60.4 4.5 2.48628b0.87 99-C 928.69.41192120.5412915 60 6.5194 9221 1.31b116-CaC = Cochran outlier; SG = single Grubbs outlier; DG = double Grubbs outliers.bRSD r >15%; 120% < Rec. < 70%; RSD R >25%; HorRat >1.2; or fewer than 8 laboratories in an assessment.cImazalil was incurred in the oranges unbeknownst to the SD.ã 2007 AOAC IN T ER N A T IONALIn Option A, if the laboratory had LVI capability, then 1 or 2 mL extracts were taken for dispersive-SPE (the volume depended on the analyst preference and the type of centrifuge and tubes available in the laboratory). The final extract volume was 0.5 mL if 1 mL was taken for dispersive-SPE, and 1 mL if 2 mL underwent the cleanup step. In either case, two 15 g blank samples were used for the matrix blank (0-standard) and 6 matrix-matched calibration standards (5, 10, 50, 100, 250, and 1000 ng/g equiv alent concentrations). For dispersive-SPE of the matrix blanks, either 7 separate tubes using the same 1–2 mL extract volumes as the test samples could have been used, or 1–2 dispersive-SPE tube(s) with 7-fold greater extract volume(s).In Option B, if LVI is not available for GC/MS, then »30 mL of matrix blank extract was needed after dispersive-SPE cleanup to prepare the matrix-matched calibration standards (or $60 mL initial extract). In this case, 6 matrix blanks of 15 g each were extracted along with the test samples to provide enough blank extract volume, which were combined, and seven 8 mL aliquots were distributed to 7 dispersive-SPE tubes containing 0.4 g PSA + 1.2 g anhydrous MgSO4.B. Apparatus and ConditionsNote: Tables 4 and 5 of the collaborative study [J. AOAC Int. 90, 485(2007)] list the analytical instrumentation and sources of sample preparation materials used by each laboratory in the study. Further information appears in the full report. Since the time of the collaborativ e study, at least 3 v endors, United Chemical Technologies (Bristol, PA, USA), Restek (Bellefonte, PA, USA) and Supelco (Bellefonte, PA, USA) hav e introduced commercial dispersive-SPE products for QuEChERS and other applications. See Table 4 [J. AOAC Int. 90, 485(2007)] for sources of analytical instruments.(a) Gas chromatograph/mass spectrometer.—An ion trap, quadrupole, time-of-flight (TOF), or other GC/MS instrument may be used with electron impact (EI) ionization, an autosampler (AS), and computerized instrument control/data collection. Either LVI of 8 m L for a 1 g/mL MeCN extract (e.g., 75°C ramped to 275°C at 200°C/min) or 2 m L splitless injection of 4 g/mL extracts in toluene a t250°C m a y b e u s e d.A3–5m,0.25m m i d, phenylmethyl-deactivated guard column must be used as a retention gap in either case. The analytical column is a 30 m, 0.25 mm id, 0.25 m m film thickness (5%phenyl)-methylpolysiloxane (low bleed) analytical column (DB-5ms or equiv alent). Set He head pressure on the column to be 10 psi or constant flow to be 1.0 mL/min with systems capable of electronic pressure/flow control. After an appropriate time for solv ent delay, use an appropriate ov en temperature program, for example, starting at 75°C for MeCN extracts or 100°C for toluene ramped to 150°C at 25°C/min, then to 280°C at 10°C/min, and hold for 10 min. All collaborators had much experience in pesticide residue analysis and were free to use their own analytical conditions provided that peak shapes were Gaussian, peak widths at half heights were <5 s, and signal-to-noise ratio (S/N) of the quantitative ion for the pesticides at 10 ng/g equivalent concentrations in the sample were >10. For qualitative purposes (which were not the focus of this study), at least 3 ions yielding relativ e abundances that reasonably match a contemporaneously analyzed reference standard are typically needed to make an analyte identification.(b) Liquid chromatograph/tandem mass spectrometer.— A triple quadrupole, ion trap, or other LC/MS/MS instrument may be used provided it is capable of electrospray ionization (ESI) in the positive mode with computerized instrument control/data collection and has an AS. An injection volume (5–100 m L) will be determined for each instrument to achieve S/N > 10 for the quantitation ion for a 10 ng/g equivalent sample concentration. As in GC/MS, the collaborators had much experience in the analysis of pesticides and were free to use their own conditions. Suggested LC conditions, howev er, include a 15 cm long, 3.0 mm id, 3 m m particle size C18 column, flow rate of 0.3 mL/min, and gradient elution with an initial condition of 25% MeOH in 5 mM formic acid solution taken linearly in 15 min to 90% MeOH in 5 mM formic acid solution and held for 15 min. A short C18 guard column must be used to protect the analytical column, and a bypass valve must be used before the MS instrument to av oid introduction of the early and late eluting nonanalyte components into the detector. The MS/MS conditions were optimized in each laboratory using direct infusion into the ESI source to prov ide highest S/N for the quantitation ion of each LC-type analyte from a single MS/MS transition. A second transition with reasonably matching relative abundance ratios vs a contemporaneously analyzed reference standard is typically needed for qualitative purposes.(c) Centrifuge(s).—Capable of holding the 50 mL centrifuge tubes or bottles used for extraction and 10–15 mL graduated centrifuge tubes or 2 mL mini-tubes used in dispersiv e-SPE. Determine the rpm settings that yield a given relative centrifugal force (RCF), and ensure that maximum ratings of the centrifuge, tube/bottles, and rotors for the instrument are not exceeded.(d) Balance(s).—Capable of accurately measuring weights from0.05 to 100 g within ±0.01 g.(e) Freezer.—Capable of continuous operation <–20°C.(f) Furnace/oven.—Capable of 500°C operation.ã 2007 AOAC IN T ER N A T IONALTable 2007.01E. Averaged interlaboratory study results for the fortified and incurred pesticides aMatrix Recovery, %RSD r, %RSD R, %HorRat No. of labs (n) Grapes86 ± 1110 ± 422 ± 80.90 ± 0.2912 ± 1 Lettuces87 ± 1210 ± 720 ± 90.83 ± 0.4510 ± 1 Oranges87 ± 1510 ± 620 ± 80.84 ± 0.3710 ± 2 Overall87 ± 1110 ± 621 ± 80.86 ± 0.3711 ± 2Incurred NA b12 ± 422 ± 80.92 ± 0.3011 ± 2aData from fewer than 7 laboratories in an assessment were excluded.bNA = Not applicable.(g ) Food chopper and/or bl ender.—Preferably an s-blade vertical cutter (e.g. Stephan, Robotcoupe) and and probe blender (e.g. Ultra-Turrax, Propsep).(h ) Sol vent evaporator (optional ).—For the ev aporation of MeCN extracts, if LVI is not used in GC/MS.C. Reagents[See Table 5 [J. AOAC Int. 90, 485(2007)] for sources of chemicals.](a ) Anhydrous magnesium sul fate (MgSO 4).—Powder form;purity >98%; heated in bulk to 500°C for >5 h to remove phthalates and residual water.(b ) Acetonitril e (MeCN).—Quality of sufficient purity that is free of interfering compounds.(c ) Acetic acid (HOAc).—Glacial; quality of sufficient purity that is free of interfering compounds.(d ) 1% HOAc in MeCN.—Prepared on a v/v basis (e.g., 10 mL glacial HOAc in a 1 L MeCN solution).(e ) Anhydrous sodium acetate (NaOAc).—Powder form (NaOAc·3H 2O may be substituted, but 0.17 g per g sample must be used rather than 0.1 g anhydrous NaOAc per g sample).(f ) Primary secondary amine (PSA) sorbent.—40 m m particle size (Varian Part No. 12213024 or equiv alent). (Note : Premade dispersive-SPE tubes are now available from at least 3 vendors.)(g ) C 18 sorbent (optional ).—40 m m particle size, if samples contain >1% fat.(h ) G r a p h i t i z e d c a r b o n b l a c k (G C B ) s o r b e n t (optional ).—120/400 mesh size, if no structurally planar pesticides are included among the analytes.(i ) Helium.—Purity that has been demonstrated to be free of interfering compounds in GC/MS.(j ) Toluene (optional ).—Quality of sufficient purity that is free of interfering compounds; only needed if LVI is not used in GC/MS.(k ) Methanol (MeOH).—Quality of sufficient purity that is free of interfering compounds in LC/MS/MS prepared in mobile phase solution.(l ) Water.—Quality of sufficient purity that is free of interfering compounds in LC/MS/MS.(m ) Formic acid.—Quality of sufficient purity that is free of interfering compounds in LC/MS/MS prepared in mobile phase solution.(n ) Pesticide standards .—High purity reference standards of the pesticide analytes, and quality control (QC) and internal standards (ISs) prepared at highly concentrated stock solutions (e.g.,2000 ng/m L) in MeCN with 0.1% HOAc. Stored in dark vials in the freezer. Check annually for stability.(o ) Standard sol utions.—Prepared in MeCN for all collaborators: IS solution = 40 ng/m L of both d 10-parathion and d 6-a -HCH in MeCN; triphenylphosphate (TPP) solution = 2 ng/m L TPP in 1% HOAc in MeCN solution; QC-spike solution = 40 ng/m L of the 27 pesticide analytes in 0.1% HOAc in MeCN; and individual test solutions = 10 ng/m L of each of the 30 compounds to be detected (except 40 ng/m L TPP) in 0.1% HOAc in MeCN solution.Collaborators prepared a test mix and calibration standard spike solutions from those provided as described in E .(p ) Bl ank sampl e .—Verified to be free of analytes abov e the detection limit.(q ) Other reagents.—Certain instruments may require nitrogen or other materials/devices for their operation.D. Materials(a ) Fl uorinated ethyl ene propyl ene (FEP) centrifugetubes.—50 mL; e.g., Nalgene Part No. 3114-0050 or equivalent for <16 g sample (or 250 mL FEP centrifuge bottles for 16–75 g sample size).(b ) Spatul a/spoon and funnel .—For transferring sample into centrifuge tubes.(c ) Solvent dispenser and 1–4 L solvent bottle.—For transferring 15 mL 1% HOAc in MeCN per 15 g sample in FEP centrifuge tubes or bottles.(d ) Centrifuge tubes (optional ).—10–15 mL graduated. For evaporation and/or dispersive-SPE.(e ) Mini-centrifuge tubes (optional).—2 mL. For dispersive-SPE (use tubes with o-ring-sealed caps to avoid leaks).(f ) Syringes/pipets.—Capable of accurate sample introduction of 2 or 8 m L volume into GC/MS and appropriate volumes of matrix spike, IS, and calibration standard solutions (12.5–300 m L).(g ) Repeating or vol umetric pipets.—Capable of accurately transferring 0.5–8 mL solvent.(h ) Containers .—Graduated cylinders, volumetric flasks, weigh boats, v ials, and/or other general containers in which to contain samples, extracts, solutions, standards, and reagents.E. Preparation of Reagent Materials and Comminuted Sample(1) Prepare the necessary number of sealable vials/cups containing 6.0 ± 0.3 g anhydrous MgSO 4 + 1.5 ± 0.1 g anhydrous NaOAc (or 2.5ã 2007 AOAC IN T ER N A TIONALFigure 2007.01. Outline of the QuEChERS protocol used in the collaborative study.± 0.2 g NaOAc·3H2O) per 15 g sample. Scoops of appropriate volume can be used to speed the process, but weighing should still be done to check consistency. The containers should be sealed during storage and can be refilled and re-used without cleaning in between usages.(2) Prepare the necessary number of appropriate centrifuge tubes (2 mL mini-centrifuge tubes or 10–15 mL centrifuge tubes) containing 0.05 ± 0.01 g PSA sorbent + 0.15 ± 0.03 g anhydrous MgSO4 per 1 mL extract taken for dispersive-SPE cleanup. (Note: At least United Chemical Technologies, Restek, and Supelco now provide dispersive-SPE products commercially to replace this step.) If LVI is not available for GC/MS, then evaporation of the extracts will be needed, and 8 mL extract will be transferred to 10–15 mL sealable centrifuge tubes containing 0.40 ± 0.08 g PSA sorbent + 1.20 ± 0.24 g anhydrous MgSO4. For matrixes that contain >1% fat, add an additional 0.05 ± 0.01 g C18 sorbent per mL extract to the container. If no planar pesticides are among the analytes (e.g., thiabendazole, terbufos, quintozene, and hexachlorobenzene), then 0.05 ± 0.01 g GCB sorbent per mL extract can also be added to the tube. (Note: Final extract volume may have to be reduced to 0.4 mL per 1 mL aliquot in dispersive-SPE if all 4 powders are used.) (3) Prepare 1% HOAc in MeCN in dispenser bottle by adding 10 mL HOAc to 990 mL v olume of MeCN or different desired amount in the same ratio.(4) Label all vials and tubes appropriately that will be used in the method.(5) Note: Step 5 was conducted by the Study Director (SD) when preparing the test samples. An appropriate chopper must be used to comminute large, representative sample portions. An uncommon or deuterated pesticide standard may be spiked into the sample during homogenization to determine the effectiv eness of the procedure. Blend the sample until it gives a consistent texture. Transfer .200 g to a sealable container for freezer storage after further homogenization with a probe blender. Blend this subsample with the mixer until it is homogeneous. The test portion (e.g., 15 g) is taken for extraction immediately, and the container is then sealed and stored in the freezer in case re-analysis is necessary. The advantages of this approach are that the 15 g portion is highly representative of the original sample, the sample is well-comminuted to improv e extraction by shaking, less time is spent on the ov erall homogenization process than trying to prov ide equiv alent homogenization of the large initial sample with the chopper, and a frozen subsample is available for re-analysis if needed.To provide the most homogeneous comminuted samples, frozen conditions, sufficient chopping time, and appropriate sample size to chopper volume ratio should be used. Use of frozen samples also minimizes degradative and volatilization losses of certain pesticides. In this case, cut the sample into 2–5 cm3 portions with a knife and store the sample in the freezer prior to processing. Cryogenic blending devices, liquid nitrogen, or dry ice may also be used (but make sure all dry ice has sublimed before weighing samples and ensure that water condensation is minimal, especially in a humid environment). (6) For laboratories with LVI in GC/MS, prepare a test mix of the pesticides in MeCN + 0.1% HOAc to determine the retention times (t R) and MS quantitation/diagnostic ions at the particular GC/MS conditions to be used in the analysis [see Table 2 of the collaborative study (J. AOAC Int. 90, 485(2007)].The preparation of the test mix and calibration spiking standards are described as follows:(1) Test mix in MeCN + 0.1% HOAc.—4 ng/m L in 10 mL of all 30 compounds to be analyzed. Add 1 mL each of QC-spike solution + IS solution + TPP test solution + 1% HOAc in MeCN and fill to 10 mL with MeCN. Calibration spike standards in MeCN for 27 pesticide analytes (make 10 mL each in volumetric flasks, then transfer to 15 mL dark glass vials and store in freezer).(2) Cal-standard-1000.—20 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 5 mL QC-spike solution + 1 mL IS solution + 1 mL 1% HOAc in MeCN and fill to the mark with MeCN.(3) Cal-standard-250.—5 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 1.25 mL QC-spike solution + 1 mL IS solution + 1 mL 1% HOAc in MeCN and fill to the mark with MeCN.(4) Cal-standard-100.—2 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 500 m L QC-spike solution + 1 mL IS solution + 1 mL 1% HOAc in MeCN and fill to the mark with MeCN.(5) Cal-standard-50.—1 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 250 m L QC-spike solution + 1 mL IS solution + 1 mL 1% HOAc in MeCN and fill to the mark with MeCN.(6) Cal-standard-10.—0.2 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 50 m L QC-spike solution + 1 mL IS solution + 1 mL 1% HOAc in MeCN and fill to the mark with MeCN.(7) Cal-standard-5.—0.1 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 25 m L QC-spike solution + 1 mL IS solution + 1 mL 1% HOAc in MeCN and fill to the mark with MeCN. For laboratories without LVI in GC/MS, the preparation of the test mix and the calibration spiking standards are described below: (1a) Test mix for GC in tol uene.—4 ng/m L in 10 mL of all 30 compounds to be analyzed. Add 1 mL QC-spike solution + 1 mL IS solution + 1 mL TPP test solution and fill to 10 mL with toluene. Calibration spike standards in MeCN for LC/MS/MS (in dark glass AS vials stored in freezer).(2a) Cal-standard-1000.—20 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 500 m L QC-spike solution + 100 m L IS solution + 100 m L 1% HOAc in MeCN + 320 m L MeCN.(3a) Cal-standard-250.—5 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 125 m L QC-spike solution + 100 m L IS solution + 100 m L 1% HOAc in MeCN + 695 m L MeCN.(4a) Cal-standard-100.—2 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 50 m L QC-spike solution + 100 m L IS solution + 100 m L 1% HOAc in MeCN + 770 m L MeCN.Dilute QC-spike solution.—4 ng/m L. Transfer 100 m L QC-spike solution to AS vial and add 900 m L MeCN.(5a) Cal-standard-50.—1 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 250 m L dilute QC-spike solution + 100 m L IS solution + 100 m L 1% HOAc + 570 m L MeCN.(6a) Cal-standard-10.—0.2 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 50 m L dilute QC-spike solution + 100 m L IS solution + 100 m L 1% HOAc and 770 m L MeCN.(7a) Cal-standard-5.—0.1 ng/m L of each pesticide + 4 ng/m L IS in MeCN + 0.1% HOAc. Add 25 m L dilute QC-spike solution + 100 m L IS solution + 100 m L 1% HOAc + 795 m L MeCN.Calibration spike standards in toluene.—Make 10 mL each in volumetric flasks, then transfer to 15 mL dark glass vials and store in freezer.(8a) Cal-standard-1000-tol.—20 ng/m L of each pesticide + 4 ng/m L IS in toluene. Add 5 mL QC-spike solution + 1 mL IS solution and fill to the mark with toluene.ã 2007 AOAC IN T ER N A T IONAL。

39.1.08AOAC Official Method 991.36 Fat (Crude) in Meat and Meat ProductsSolvent Extraction (Submersion) MethodFirst Action 1991Final Action 1996[Applicable to meat and meat food products that can be analyzed using 960.39 (see 39.1.05), 976.21 (see 39.1.06), and 985.15 (see39.1.07).]Results of the interlaboratory study supporting acceptance of the method:x—, 4.34% fat: s r= 0.106; s R = 0.112; RSD r= 2.44%; RSD R = 2.59%x—, 27.29% fat: s r= 0.534; s R = 0.637; RSD r= 1.95%; RSD R = 2.33%x—, 27.95% fat: s r= 0.648; s R = 0.739; RSD r= 2.32%; RSD R = 2.84%x—, 34.51% fat: s r= 0.764; s R = 0.799; RSD r= 2.21%; RSD R = 2.31%x—, 33.57% fat: s r= 0.340; s R = 0.516; RSD r= 1.01%; RSD R = 1.53%x—, 26.20% fat: s r= 0.406; s R = 0.631; RSD r= 1.55%; RSD R = 2.34%A. Apparatus装置(a) Extraction system.—Capable of simultaneous extraction of 6 test portions. Extraction unit for solvent addition to cups, 2-stage extraction process, and solvent recovery cycle. Service unit to supply hot oil through insulated tubing to extraction unit and to pump air for evaporation of last traces of solvent from cups (Soxtec System meets these specifications).(b) Thimbles and stand.—26 *60 mm, cellulose thimbles, and stand to hold 6 thimbles.(c) Extraction cups.—Al, 44 id, 60 mm height.(d) Glass beads.—3–4 mm diameter.(e) Mechanical convection oven. — Maintaining 125° ± 1°C.Items (a)–(c) are available as Soxtec sys tem from Perstorp Analytical/Tecator, Inc. (2875 C Towerview Rd, Herndon, V A 22071, USA).B. Reagents(a) Petroleum ether. — To meet specifications in 945.16A (see 27.4.04).(b) Sand.—<0.004 g extractables/5 g.(c) Cotton.—Defatted.C. DeterminationAccurately weigh ca 3 g test portion into thimble. Add sand to test portion and mix with glass rod. Place thimble in thimble stand and dry 1 h in 125°C oven. Remove from oven and let cool. Loosen test portion/sand mixture using glass rod. Wipe glass rod with small amount of cotton and place cotton in top of thimble. Transfer thimble to extraction unit. Accurately weigh extraction cup containing a few glass beads.Extract thimble with dried mixture with 40 mL petroleum ether in boiling position for 25 min and in rinsing position for 30 min. Adjust temperature of extraction unit to ensure condensation rate≥5 drops/s. At completion of extraction, close condenser valves and recover ether.Dry cup and contents 30 min in 125°C oven. Cool and weigh.D. CalculationsCalculate percent fat in test sample as follows:Fat content, % = (B -C) *100Awhere A = g test portion weight, B = g weight of extraction cup afterdrying, and C = g weight of extraction cup prior to extraction.Reference: J. AOAC Int. 75, 289(1992).39.1.07AOAC Official Method 985.15Fat (Crude) in Meat and Poultry 家禽Products Rapid Microwave-Solvent Extraction Method First Action 1985Final Action 1991A. Reagents and Apparatus(a) Automated solvent extractor.—Enclosed, self-contained, thermostatically controlled 恒温控制fat extraction and solvent recovery system with 0.5 mg fat sensitivity and 0–100% fat measurement range (CEM Corp., PO Box 200, Matthews, NC 28106, USA), or equivalent.(b) Methylene chloride.二氯甲烷—Reagent grade (Fisher Scientific Co., No. D-37) , or equivalent.(c) Glass fiber pads.玻璃纤维垫子—9.8 *10.2 cm rectangular 矩形and 11 cm round (CEM Corp.), or equivalent.(d) Microwave moisture analyzer.—0.2 mg H2O sensitivity, moisture/solids range of 0.1–99.9%,0.01% resolution分辨率. Includes automatic tare electronic balance, microwave drying system，and microprocessor digital computer control. Electronic balance pan is located inside drying chamber. (Balance sensitivity: 0.2 mg at 15 g capacity or 1.0 mg at 40 g capacity [CEM Corp., or equivalent].)B. DeterminationPrepare test samples as in 983.18 (see 39.1.01). Place 2 rectangular and one round glass fiber pad on balance pan inside microwave moisture analyzer, and tare. Remove rectangular pads and evenly spread ca 4 g well-mixed test portion onto rough side of one pad, cover with second pad, and place together with round pad on balance pan. Dry 3–5 min at 80–100% power, depending on product type. At end of drying cycle, remove from balance pan. Fold rectangular pads, with dried test portion, in half and place in automated solvent extractor chamber. Place round pad in recessed area at top of extractor chamber, close and latch lid. Start extraction cycle (test portion and rectangular pads are blended at this time with sufficient CH2Cl2to extract fat). After completion of extraction cycle, remove round pad with residue, and place on balance pan in microwave moisture analyzer. Redry pad and residue to constant weight (ca 30 s at 80–100% power) to re move residual solvent or moisture. Weight loss due to solvent extraction is converted to % fat by microprocessor and displayed on digital read out panel.Certain product classes require addition of adjustment factors to read out for accurate results, as follows: fresh meats, pre-blends, emulsions, cured/cooked meats, factor = 0.40; cooked sausages, factor = 0.80.Reference: JAOAC 68, 876(1985).39.1.06AOAC Official Method 976.21Fat (Crude) in Meat Rapid Specific Gravity MethodFirst Action 1976Final Action 1979A. Apparatus and Reagents(a) Foss-let fat analyzer.—Includes orbital shaker, specific gravity readout unit, solvent dispenser, reference standard oil (specific gravity at 23°C = 0.915; for periodic check of potentiometer calibration), stainless steel cup with cover and 8 mm bore brass hammer, pressure filtration device, and conversion chart (Foss Food Technology Corp.).(b) Drying agent.—Plaster of Paris (available locally through paint, hardware, or building supply dealers), 8 mesh Drierite, or an hydrous CaSO4.(c) Tetrachloroethylene.—Technical grade C2Cl4 (distributed locally through dry cleaning sup pliers or Fisher Scientific Co.,No. C-182).B. Deter mi na tionPrepare test sam ples as in 983.18 (see 39.1.01). Check calibration of Foss-let potentiometer daily by us ing C2Cl4 alone to set zero point.Use mixture of 22.5 g reference standard oil and 120 mL C2Cl4(specific gravity of mixture at 37° = 1.4763) to set 50% fat point at 850.0.Using either top-load or triple-beam balance with 0.1 g sensitivity, tare Foss-let cup after setting brass ham mer on itsspin dle. To an a lyze prod ucts con tain ing £60% fat, weigh 45.0 gtest sam ple into cup; for prod ucts con tain ing >60% fat, weigh22.5 g. Add ca 80 g Plas ter of Paris (or ca 60 g an hy drous CaSO4).Dis pense 120 mL C2Cl4 into cup. Press cover onto cup and in stall inor bital shaker. Set shaker timer for 2 min and turn unit on. Whileex trac tion pro ceeds, as sem ble pres sure fil tra tion de vice by plac inginto per fo rated base 7 cm fil ter pa per. To pro duce clear fil trate freeof mois ture drop lets (for very wet test sam ples), first place highre ten tion pa per, Whatman No. 50, or equiv a lent, and then phasesep a rat ing pa per, Whatman No. 1PS, or equiv a lent. Af ter 2 minex trac tion, re move cup from shaker, lift cover, and re move brassham mer from cup. Im merse cup in ice-water bath ca 0.4 min whilestir ring con tents with ther mom e ter to cool con tents from 47°–52°Cto ca 40°C. Wipe H2O from outer sur face of cup and pour con tentsinto as sem bled fil ter. Place pis ton at top of fil tra tion de vice andslowly press ex tract through mea sur ing sys tem. De press drain valvebut ton when ex tract ap pears in over flow tube and let cham ber drain;then re lease valve but ton. Re peat fill ing and drain ing 2 more timesun til 40–50 mL ex tract has flowed through, re tain ing fi nal 10 mLex tract in mea sur ing cham ber. Re move fil tra tion de vice, slideview ing lens into po si tion, ro tate con trol of read out po ten ti om e terclock wise un til hy drom e ter rises, and re cord read ing. Es tab lish thatex tract is at cham ber tem per a ture by re peat ing read ing 3–4 times.Av er age read ings and con vert into % fat by means of con ver sion chart. (Mul ti ply chart % fat by 2 if a 22.5 g por tion of high-fat product was used.)Ref er ences: JAOAC 58, 1182(1975); 60, 853(1977);68, 240(1985).。

45.2.01AOAC Official Method960.46Vitamin AssaysMicrobiological MethodsFirst Action1960Final Action(Throughout all stages,except where otherwise directed,protect so-lutions from undue exposure to light.)A.Stock Solutions for Basal Media(Store all solutions in dark at ca10°C.Store all solutions except those containing alcohol under toluene.Proportionate amounts may be prepared.)(a)Acid-hydrolyzed casein solution.—Mix400g vitamin-free ca-sein with2L constant-boiling HCl(ca5M)and either reflux8–12h or autoclave8–12h at121–123°C.Remove HCl from mixture by distil-lation under reduced pressure until thick paste remains.Redissolve paste in H2O,adjust solution to pH3.5±0.1with ca10%NaOH solu-tion,and dilute with H2O to4L.Add80g activated charcoal,stir1h and filter.Repeat treatment with activated charcoal.Filter solution if precipitate forms upon storage.(Some commercial sources of vita-min-free acid-hydrolyzed casein have been found satisfactory.) (b)Adenine–guanine–uracil solution.—Dissolve1.0g each of adenine sulfate,guanine⋅HCl,and uracil in50mL warm HCl(1+1), cool,and dilute with H2O to1L.(c)Asparagine solution.—Dissolve10g L-asparagine⋅H2O in H2O and dilute to1L.(d)Cystine solution.—Suspend2g L-cystine in ca750mL H2O, heat to70–80°C,and add HCl(1+1),dropwise,with stirring,until solid dissolves.Cool,and dilute with H2O to1L.(e)Cystine–tryptophan solution.—Suspend8g L-cystine and2g L-tryptophan(or4g D,L-tryptophan)in ca1.5L H2O,heat to 70–80°C,and add HCl(1+1),dropwise,with stirring,until solids dissolve.Cool,and dilute with H2O to2L.(f)Manganese sulfate solution.—Dissolve2g MnSO4⋅H2O in H2O and dilute to200mL.(g)Photolyzed peptone solution.—Dissolve100g peptone in 625mL H2O,add solution of50g NaOH in625mL H2O,and mix in vessel(such as crystallizing dish)of such size that depth of solution is1–2cm.Place100–500watt bulb,fitted with reflector,ca 30–50cm from solution,and expose solution,with occasional stir-ring,to light from bulb until riboflavin is destroyed(4–10h may be enough).Maintain solution at≤25°C during this treatment.Adjust solution to pH6.0–6.5with CH3COOH,add18g anhydrous NaCH3COO,stir until solid dissolves,dilute with H2O to2L,and fil-ter if solution is not clear.(h)Polysorbate80solution.—Dissolve25g polysorbate80 (polyoxyethylene sorbitan monooleate)in alcohol to make250mL.(i)Salt solution A.—Dissolve50g anhydrous KH2PO4and50g anhydrous K2HPO4in H2O,dilute to1L,and add10drops HCl. (j)Salt solution B.—Dissolve20g MgSO4⋅7H2O,1g NaCl,1g FeSO4⋅7H2O,and1g MnSO4⋅H2O in H2O,dilute to1L,and add 10drops HCl.(k)Tryptophan solution.—Suspend2.0g L-tryptophan(or4.0g D,L-tryptophan)in700–800mL H2O,heat to70–80°C,and add HCl(1+1),dropwise,with stirring,until solid dissolves.Cool,and dilute with H2O to1L.(l)Vitamin solution I.—Dissolve25mg riboflavin,25mg thia-mine⋅HCl,0.25mg biotin,and50mg niacin in0.02M CH3COOH to make1L.(m)Vitamin solution II.—Dissolve50mg p-aminobenzoic acid, 25mg calcium pantothenate,100mg pyridoxine⋅HCl,100mg pyridoxal⋅HCl,20mg pyridoxamine⋅2HCl,and5mg folic acid in 25%alcohol to make1L.(n)Vitamin solution III.—Dissolve10mg p-aminobenzoic acid, 40mg pyridoxine⋅HCl,4mg thiamine⋅HCl,8mg calcium pantothenate,8mg niacin,and0.2mg biotin in ca300mL H2O.Add 10mg riboflavin dissolved in ca200mL0.02M CH3COOH.Then add solution containing1.9g anhydrous CH3COONa and1.6mL CH3COOH in ca40mL H2O,and dilute with H2O to2L.(o)Vitamin solution IV.—Dissolve20mg riboflavin,10mg thia-mine⋅HCl,and0.04mg biotin in0.02M CH3COOH to make1L. (p)Vitamin solution V.—Dissolve10mg p-aminobenzoic acid, 20mg calcium pantothenate,and40mg pyridoxine⋅HCl in25%al-cohol to make1L.(q)Vitamin solution VI.—Dissolve10mg p-aminobenzoic acid, 50mg niacin,and40mg pyridoxine⋅HCl in25%alcohol to make 1L.(r)Xanthine solution.—Suspend1.0g xanthine in150–200mL H2O,heat to ca70°C,add30mL NH4OH(2+3),and stir until solid dissolves.Cool,and dilute with H2O to1L.(s)Yeast supplement solution.—Dissolve20g H2O-soluble yeast extract in100mL H2O,add solution of30g lead subacetate in 100mL H2O(solution is turbid),and mix.Filter,and adjust filtrate to pH10with NH4OH(1+2).Filter,and adjust filtrate to pH6.5with CH3COOH.Precipitate excess Pb with H2S,filter,and dilute filtrate with H2O to200mL.B.Culture and Suspension Media(a)Liquid culture medium.—Dissolve15g peptonized milk,5g H2O-soluble yeast extract,10g anhydrous glucose,and2g anhy-drous KH2PO4in ca600mL H2O.Add100mL filtered tomato juice, and adjust to pH6.5–6.8with NaOH solution.Add,with mixing, 10mL polysorbate80solution,A(h),and dilute with H2O to1L. Add10mL portions solution to test tubes,cover to prevent contami-nation,sterilize15min in autoclave at121–123°C,and cool tubes as rapidly as practicable to keep color formation at minimum.Store in dark at ca10°C.(Lactobacilli Broth AOAC,Difco Laboratories,has been found satisfactory.)(b)Agar culture medium.—To500mL liquid culture medium, (a),add5.0–7.5g agar,and heat with stirring on steam bath until agar dissolves.Add ca10mL portions hot solution to test tubes, cover to prevent contamination,sterilize15min in autoclave at 121–123°C,and cool tubes in upright position as rapidly as practica-ble to keep color formation at minimum.Store in dark at ca10°C. (Difco agar culture medium for AOAC microbiological assays [Lactobacilli Agar AOAC]has been found satisfactory.)(c)Suspension medium.—Dilute measured volume appropriate basal medium stock solution,Table960.46A,with equal volume H2O.Add10mL portions diluted medium to test tubes,cover to pre-vent contamination,sterilize15min in autoclave at121–123°C,and cool tubes as rapidly as practicable to keep color formation at mini-mum.Store in dark at ca10°C.C.Stock Cultures of Test OrganismsFor appropriate test organism,designated below,prepare stab cul-ture in≥1tubes of agar culture medium,B(b).Incubate6–24h at any selected temperature between30and40°C held constant to within ±0.5°C,and finally store in dark at ca10°C.Before using new cul-ture in assay,make several successive transfers of culture in 1–2week period.Prepare fresh stab culture≥1time weekly and do not use for pre-paring inoculum if>1week old.Activity of slow-growing culture may be increased by daily or twice-daily transfer of stab culture,and is considered satisfactory when definite turbidity in liquid inoculum can be observed2–4h af-ter inoculation.Slow-growing culture seldom gives suitable re-sponse curve and may cause erratic results.(a)Lactobacillus leichmannii.—ATCC No.7830.For use in as-say of cobalamin.(b)Streptococcus faecalis(faecium).—ATCC No.8043.For use in assay of folic acid.(c)Lactobacillus plantarum.—ATCC No.8014.For use in assay of niacin and pantothenic acid.(d)Lactobacillus casei subspecies rhamnosus.—ATCC No. 7469.For use in assay of riboflavin.D.Assay TubesMeticulously cleanse by suitable means(sodium lauryl sulfate USP has been found satisfactory as detergent),hard-glass test tubes, ca20×150mm,and other necessary glassware.(Test organisms are highly sensitive to minute amounts of growth factors and to many cleansing agents.Therefore,it may be preferred to follow cleansing by heating1–2h at ca250°C.This is of particular importance in cobalamin assay.)Prepare tubes containing appropriate standard solution as fol-lows:To test tubes add,in duplicate(or replicate),0.0(forTable960.46A Basal media stock solutions for250mL(proportionate amounts may be prepared)aIngredients(stock solutions,A)(a)Cobalamin(vitamin B12activity)(b)Folic acid(pteroylglutamic acid)(c)Niacin andniacinamide(d)Pantothenicacid(e)Riboflavin(vitamin B2)mL(a)Acid-hydrolyzed casein solution25252525(b)Adenine–guanine–uracil solution5 2.555(c)Asparagine solution515(d)Cystine solution25(e)Cystine–tryptophan solution2525(f)Manganese sulfate solution5(g)Photolyzed peptone solution50(h)Polysorbate80solution50.250.25(i)Salt solution A5555 (j)Salt solution B55555 (k)Tryptophan solution25(l)Vitamin solution I10(m)Vitamin solution II10(n)Vitamin solution III50(o)Vitamin solution IV55(p)Vitamin solution V5(q)Vitamin solution VI5(r)Xanthine solution55(s)Yeast supplement solution5 Solids gAscorbic acid1L-Cysteine⋅HCl⋅H2O0.19L-Cystine0.1Glucose,anhydrous1010101015 Glutathione0.0013K2HPO4,anhydrous 1.6NaCH3COO⋅3H2O8.38.38.3Na citrate,2H2O13D,L-Tryptophan0.1a Some commerical sources of basal media have been found satisfactory.uninoculated blanks),0.0(for inoculated blanks),1.0,2.0,3.0,4.0, and5.0mL,respectively,of standard solution.Prepare tubes containing appropriate test solution as follows:To similar test tubes add,in duplicate(or replicate),1.0,2.0,3.0,and 4.0mL,respectively,of test solution.To each tube of standard solution and test solution add H2O to make5.0mL.Then add5.0mL appropriate basal medium stock so-lution,Table960.46A,and mix.Cover tubes suitably to prevent bac-terial contamination,and sterilize(10min for titrimetric method,E; or5min for turbidimetric method,G)in autoclave at121–123°C, reaching this temperature in≤10min.Cool as rapidly as practicable to keep color formation at minimum.Take precautions to keep steril-izing and cooling conditions uniform throughout assay.Too close packing of tubes in autoclave,or overloading of it,may cause varia-tion in heating rate.Aseptically inoculate each tube,except1set of duplicate(or repli-cate)tubes containing0.0mL standard solution(uninoculated blanks),with1drop appropriate inoculum.Incubate for time perioddesignated in titrimetric method,E,or turbidimetric method,G,at any selected temperature between30and40°C held constant to within±0.5°C.Contamination of assay tubes with any foreign or-ganism invalidates assay.Titrimetric MethodE.DeterminationIncubate tubes72h and then titrate contents of each tube with0.1M NaOH,using bromothymol blue indicator,or to pH6.8measured potentiometrically.Disregard results of assay if response at inoculated blank level is equivalent to titration of>1.5mL greater than that at uninoculated blank level.Response at5.0mL level of standard solution should be equiva-lent to titration of ca8–12mL.Prepare standard concentration-response curve by plotting titration values,expressed in mL0.1M NaOH for each level of standard solution used,againstamount of reference standard contained in respective tubes. Determine amount of vitamin for each level of test solution by inter-polation from standard curve.Discard any observed titration values equivalent to<0.5mL or>4.5mL,respectively,of standard solution. Proceed as in H.Turbidimetric Method(Not applicable in presence of extraneous turbidity or color in amount that interferes with turbidimetric measurements.)F.Calibration of PhotometerUsing inoculum and standard stock solution as prescribed for ap-propriate vitamin in following table,and using suspension medium B(c),proceed as directed in Table960.46B.Aseptically add1mL inoculum to ca300mL sterile suspension medium containing1.0mL standard stock solution,and incubate mixture for same period and at same temperature to be employed in determination,G.After incubating,centrifuge and wash cells 3times with ca50mL portions0.9%NaCl solution;then resuspend cells in the NaCl solution to make25mL.Evaporate10mL aliquot of cell suspension on steam bath,and dry to constant weight at110°C in vacuum oven.Correcting for weight of NaCl,calculate dry weight of cells in mg/mL of suspension. Dilute second measured aliquot of cell suspension with0.9% NaCl solution so that each mL is equivalent to0.5mg dry cells.To test tubes add,in triplicate,0.0(for blanks),0.5,1.0,1.5,2.0,2.5, 3.0,4.0,and5.0mL,respectively,of this diluted cell suspension.To each tube add0.9%NaCl solution to make5.0mL.Then add5.0mL appropriate basal medium stock solution,Table960.46A,mix (1drop of suitable antifoam agent may be added;1–2%solution of Dow Corning Antifoam AF Emulsion or Antifoam B has been found satisfactory),and transfer to optical cell.With blanks set at100%T, measure%T of each tube under same conditions to be used in re-spective assay.Prepare curve by plotting%T readings for each level of diluted cell suspension used against cell content(mg dry weight) of respective tubes.Repeat appropriate calibration step at least twice more for pho-tometer to be used in respective assay.Draw composite curve,best representing3or more individual curves,relating%T to mg dried cell weight for photometer under conditions of respective assay. Once appropriate curve for particular instrument is established,all subsequent relationships between%T and cell weight are deter-mined directly from this curve.Respective assay limits expressed as mg dried cell weight/tube are so determined.G.DeterminationIncubate tubes16–24h until maximum turbidity is obtained,as dem-onstrated by lack of significant change during2h additional incubation period in tubes containing highest level of standard solution. Determine T of tubes as follows:Thoroughly mix contents of each tube(1drop of suitable antifoam agent solution may be added;1–2% solution of Dow Corning Antifoam AF Emulsion or Antifoam B has been found satisfactory),and transfer to optical cell.Agitate con-tents,place cell in photometer set at any specific wavelength be-tween540and660nm,and read%T when steady state is reached. Steady state is observed few s after agitation when galvanometer reading remains constant≥30s.Allow ca same time interval for reading on each tube.With T set at100%for uninoculated blank level,read%T of inoc-ulated blank level.If this reading corresponds to dried cell weight >0.6mg/tube,disregard results of assay.Then with T reset at100% for inoculated blank level,read%T for each of remaining tubes.Dis-regard results of assay if%T observed at5.0mL level of standard so-Table960.46B Procedures for inoculum and standard stocksolution for different vitaminsVitamin Inoculum Standard stock solution Cobalamin952.20C a(see45.2.02)952.20B(a)(see45.2.02)Folic acid944.12C(see45.2.03)944.12B(b)(see45.2.03) Niacin944.13G(see45.2.04)944.13B(a)(see45.2.03) Pantothenic acid945.74G(see45.2.05)945.74B(a)(see45.2.05) Riboflavin940.33G(see45.2.06)940.33B(a)(see45.2.06)a Proceed as in952.20C(see45.2.02),except replace fifth sentence with thefollowing:“Dilute0.2–1.0mL aliquot of this suspension with10mL sterile suspension medium.”lution(against inoculated blank)is equivalent to that for dried cell weight of<1.25mg/tube.Prepare standard concentration-response curve by plotting%T readings for each level of standard solution used against amount of reference standard contained in respective tubes.Determine amount of vitamin for each level of test solution by in-terpolation from standard curve.Discard any observed T values equivalent to<0.5mL or>4.5mL,respectively,of standard solu-tion.Proceed as in H.H.Calculation for Both Titrimetric and Turbidimetric Methods For each level of test solution used,calculate vitamin content/mL of test solution.Calculate average of values obtained from tubes that do not vary by>±10%from this average.If number of acceptable values remaining isof original number of tubes used in the 4levels of test data are insufficient for calculating po-tency of product.If number of acceptable values remaining isof original number of tubes,calculate potency of product from aver-age of them.CAS-68-19-9(cobalamin;vitamin B12)CAS-59-30-3(folic acid)CAS-59-67-6(niacin)CAS-79-83-4(pantothenic acid)CAS-83-88-5(riboflavin)。