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1、4.1.11 AOAC Official Method 994.12 Amino Acids in Feeds Performic Acid Oxidation with Acid HydrolysisSodium Metabisulfite Method First Action 1994 Final Action 1997 (Applicable to determination of amino acids including methionine and cystine in feeds. Not applicable to determination of tyrosine and
2、tryptophan.) See Tables 994.12AE for the results of the interlaboratory study supporting acceptance of method. A. Principle Performic acid oxidation is performed prior to hydrolysis to oxi- dize cystine and methionine to cysteic acid and methionine sulfone, respectively. Sodium metabisulfite is adde
3、d to decompose performicacid.Aminoacidsareliberatedfromproteinbyhydrolysis with 6M HCl. Hydrolysates are diluted with sodium citrate bufferorneutralized,pHisadjustedto2.20,andindividualaminoacid componentsareseparatedonion-exchangechromatograph.Tyrosineis destroyedbyoxidation.Tryptophanisdestroyedby
4、hydrolysis,sothose amino acids cannot be determined. B. Apparatus (a) Amino acid analyzer.Ion-exchange resin with ninhydrin post-column derivatization. (b) Analytical balance.Accurate to 0.1 mg. (c) Balance.Top loading. (d) Bottle.50 mL; polyethylene. (e) Digestion tubes.Boiling flasks are suitable.
5、 (f) Digestion block.Heating mantle is suitable. (g) Filter units.0.22 m (Millex GS, Millipore are suitable). (h) Magnetic stirring plate. (i) pH meter.Calibrated with buffers of pH 2.0, 4.0, and 7.0. (j) Reflux condensers. (k) Rotary evaporator. (l) Vacuum flask.250 mL. (m) Glassware.Glass beakers,
6、 250 and 1000 mL; Erlenmeyer flask,150mL;round-bottomevaporatingflask,1000mL;graduated cylinders,100,500,and1000mL;volumetricflask,1000mL;volu- metric pipets, 10 and 20 mL. (n) Sintered glass filter.Porosity 1015 m. (o) Ice bath. (p) Syringes. C. Reagents (a) Formic acid.88%. (b) Hydrogen peroxide.3
7、0%. (c) Sodium metabisulfite. (d)DL-Norleucine.Crystals. (e) HCl.Concentrated. (f) NaOH.30% solution (30 g/100 mL). (g) Phenol.Crystals. (h) Thiodiglycol.98% solution. (i) Tri-sodium citrate dihydrate. (j) pH buffer.pH 2.0, 4.0, and 7.0. (k) Amino acid standard kit.To calibrate amino acid analyzer;
8、available from Aldrich Chemical Co., Inc., 1001 West Saint Paul Ave, Milwaukee, WI 53233, USA. D. Preparation of Solutions (a) Sodium citrate buffer, pH 2.20.Weigh 19.60 g tri-sodium citratedihydratein1000mLbeakeranddissolveinca800mLH2O. Whilestirring,add10mL98%thiodiglycolsolutionand15mLcon- centra
9、ted HCl. Transfer solution quantitatively into 1000 mL volu- 2000 AOAC INTERNATIONAL Table 994.12AResults of interlaboratory study for determination of amino acids in broiler finisher feed by sodium metabisulfite method Amino acidsNMeansrRSDr, %sRRSDR, %raRb Alanine441.170.0322.740.0998.460.0900.277
10、 Arginine441.280.0302.340.1108.590.0840.308 Aspartic acid461.680.0472.800.1217.200.1320.339 Cystine380.320.0103.130.03611.250.0280.101 Glutamic acid443.250.0521.600.2266.950.1460.632 Glycine461.270.0292.280.0856.690.0810.238 Histidine340.500.0204.000.09919.800.0560.277 Isoleucine420.760.0243.160.052
11、6.840.0670.146 Leucine461.660.0442.650.1056.330.1230.294 Lysine441.070.0373.460.0968.970.1040.269 Methionine380.530.0061.130.0407.550.0170.112 Phenylalanine440.870.0384.370.12714.600.1060.356 Proline361.390.0443.170.1248.920.1230.347 Serine440.940.0444.680.12713.510.1230.356 Threonine400.730.0202.74
12、0.0608.220.0560.168 Valine440.920.0353.800.11712.720.0980.328 a r = 2.8 sr. b R = 2.8 sR. metric flask and dilute to mark with H2O. Filter buffer solution through sintered glass filter, B(n). Adjust pH to 2.20 with concen- trated HCl or 2M NaOH. (b) 6M HClphenol solution.Weigh 1 g phenol crystals in
13、to tared1000mLbeaker.Dissolvecrystalsin500mLH2O.Whilestir- ring, slowly add 500 mL concentrated HCl. (c) HCl solutions.(1) 1M HCl.Pour ca 800 mL H2O into 1000mLvolumetricflask,andthenadd83.3mLconcentratedHCl, using pipet. Dilute to the mark with H2O and mix thoroughly. (2) 0.1M HCl.Pour ca 800 mL H2
14、O into 1000 mL volumetric flask, and then add 100 mL 1M HCl, using pipet. Dilute to the mark with H2O and mix thoroughly. 2000 AOAC INTERNATIONAL Table 994.12B Results of interlaboratory study for determination of amino acids in broiler starter feed by sodium metabisulfite method Amino acidsNMeansrR
15、SDr, %sRRSDR, %raRb Alanine461.280.0272.110.1007.810.0760.028 Arginine461.570.0422.680.1298.220.1180.361 Aspartic acid402.290.0351.530.1375.980.0980.384 Cystine380.350.0061.710.05616.000.0170.157 Glutamic acid464.040.0721.780.3398.390.2020.949 Glycine461.270.0342.680.0907.090.0950.252 Histidine400.6
16、50.0182.770.10015.380.0500.280 Isoleucine460.950.0192.000.09810.320.0530.274 Leucine461.970.0331.680.1246.290.0920.347 Lysine461.350.0322.370.1229.040.0900.342 Methionine420.620.0132.100.06310.160.0360.176 Phenylalanine421.120.0252.230.1019.020.0700.283 Proline361.470.0604.080.1288.710.1680.358 Seri
17、ne441.120.0282.500.15313.660.0780.428 Threonine441.120.0242.730.0879.890.0670.244 Valine401.110.0191.710.0988.830.0530.274 a r = 2.8 sr. b R = 2.8 sR. Table 994.12CResults of interlaboratory study for determination of amino acids in corn by sodium metabisulfite method Amino acidsNMeansrRSDr, %sRRSDR
18、, %raRb Alanine440.610.0091.480.0498.030.0250.137 Arginine440.400.0133.250.0389.500.0360.106 Aspartic acid440.540.0152.780.0458.330.0420.126 Cystine380.180.0073.890.02513.890.0200.070 Glutamic acid401.510.0362.380.0946.230.1010.263 Glycine460.330.0103.030.0309.090.0280.084 Histidine420.270.0197.040.
19、06323.330.0530.176 Isoleucine440.280.0155.380.04114.620.0420.115 Leucine440.990.0191.920.0696.970.0530.193 Lysine440.260.0083.080.03413.080.0220.095 Methionine420.180.0105.560.02111.670.0280.059 Phenylalanine400.380.0092.370.07319.210.0250.204 Proline340.730.0192.600.0446.030.0530.123 Serine400.390.
20、0071.790.04010.260.0200.112 Threonine440.290.0124.140.03411.720.0340.095 Valine460.380.0092.370.06116.050.0250.171 a r = 2.8 sr. b R = 2.8 sR. (d) NaOHsolutions.(1) 7.5MNaOH.Weigh300.0gNaOHin tared 1000 mL beaker. (2) 2M NaOH.Weigh 80.0 g NaOH in tared 1000 mL beaker. Slowly dissolve pellets in each
21、 beaker in ca 600 mL H2O. Cool solutions and transfer quantitatively to separate 1000 mL volumetric flasks. Dilute to mark with H2O and mix thoroughly. (e) Norleucine standard solution.Accurately weigh 195200 mgDL-norleucine crystals into tared 150 mL Erlenmeyer flask. Dissolve crystals with 100 mL
22、1M HCl. Trans- fer solution quantitatively into 1000 mL volumetric flask and dilute to mark with H2O. (f) Performicacidreagent.Prepareinhood.Weigh25mgphe- nol crystals in 25 mL test tube; then add 0.5 mL 30% H2O2, using micropipet, and 4.5 mL 88% formic acid solution. Cover test tube with stopper, a
23、nd let mixture stand 30 min at room temperature. Af- ter 30 min, place test tubes in ice bath and cool performic acid mix- ture for 15 min. Prepare reagent just before use. 2000 AOAC INTERNATIONAL Table 994.12DResults of interlaboratory study for determination of amino acids in fishmeal by sodium me
24、tabisulfite method Amino acidsNMeansrRSDr, %sRRSDR, %raRb Alanine423.500.0732.090.2236.370.2040.624 Arginine463.400.1173.000.2807.180.3280.784 Aspartic acid465.220.1082.070.3276.260.3020.916 Cystine380.480.0193.960.09118.960.0530.255 Glutamic acid467.370.0630.850.3474.710.1760.972 Glycine463.840.059
25、1.540.2155.600.2650.602 Histidine381.370.0332.410.17612.850.0920.493 Isoleucine462.320.0492.110.23810.260.1370.666 Leucine464.070.0791.940.2766.780.2210.773 Lysine444.220.1172.770.3357.940.3280.938 Methionine421.610.0301.860.1569.690.0840.437 Phenylalanine402.290.0371.620.1767.690.3280.938 Proline36
26、2.620.0793.020.32612.440.2210.913 Serine422.210.0482.170.24811.220.1340.694 Threonine442.280.0813.550.24410.700.2270.683 Valine442.780.0632.270.31111.190.1760.871 a r = 2.8 sr. b R = 2.8 sR. Table 994.12EResults of interlaboratory study for determination of amino acids in poultry meal by sodium meta
27、bisulfite method Amino acidsNMeansrRSDr, %sRRSDR, %raRb Alanine424.260.0872.040.2104.930.2440.588 Arginine464.350.1443.310.4209.660.4031.176 Aspartic acid444.920.1322.680.3767.640.3701.053 Cystine420.810.0374.570.14317.650.1040.400 Glutamic acid467.970.2162.710.7289.130.6052.038 Glycine386.900.0851.
28、230.2864.140.2380.801 Histidine381.310.0362.750.24218.470.1010.678 Isoleucine462.240.0602.680.26111.650.1680.731 Leucine464.090.1012.470.3107.580.2830.868 Lysine463.630.1123.090.3609.920.3141.008 Methionine401.170.0252.140.14011.970.0700.392 Phenylalanine442.330.0823.520.2159.230.2300.602 Proline344
29、.530.1022.250.2836.250.2860.792 Serine442.760.1164.200.34712.570.3250.972 Threonine442.320.0733.150.2129.140.2040.594 Valine442.820.0903.190.36112.800.2521.011 a r = 2.8 sr. b R = 2.8 sR. E. Performic Acid Oxidation Finely grind test sample to pass 0.25 mm sieve. Accurately weigh ca 1001000 mg test
30、portions to the nearest 0.1 mg (equivalent to ca 10 mg nitrogen content) into labeled digestion tubes. Calculate approximate amount of test portion to use as follows: Ws= 1000 Ns where Ns= nitrogen content of test portion, %; Ws= weight of test portion equivalent to 10 mg nitrogen content, mg. Put m
31、agnetic stirrer into each tube and place digestion tubes in ice bath (0C). Afterboththeperformicacidandtestportionhavecooledatleast 15min,add5mLperformicacidintodigestiontube,coveralltubes with glass stoppers and stir 15 min on magnetic stirring plate. Return digestion tubes to ice bath and let oxid
32、ize 16 h. Remove glass stoppers and add ca 0.84 g sodium metabisulfite to decompose performic acid. Stir for 15 min to liberate SO2. F. Hydrolysis Add 50 mL 6M HClphenol solution, D(b), to test solution and briefly stir. Remove stirring bar using magnetic stirring rod, and rinse with small volume of
33、 0.1M HCl into tube. Add 23 pieces of boiling chips to test solution. Hydrolyze under reflux for 24 h at 110120C using digestion block, B(f). (Caution: Perform this step inside fume hood with ade- quate ventilation.) Remove digestion tubes from heat and cool to room temperature. Add20mLnorleucinesta
34、ndardsolution,D(e),toeachhydrolysateus- ingvolumetricpipet.Mixsolutionsbyswirlingflasks.Proceedwith(a) or (b) below. Note: If low sodium concentration is required for chro- matography,evaporateHClcarefullyperformstep(a).Iflowsodium concentration is not required perform neutralization step, (b). (a)
35、Filter hydrolysates through sintered glass filter into labeled 1000 mL evaporating flasks. Connect flasks to rotary evaporators, andevaporateundervacuumat40Ctoca5.0mL.(Note:Donotlet solutionevaporatetodryness.)Removeflasksfromevaporator.Add 50 mL sodium citrate buffer, D(a), to evaporated test solut
36、ion, mix well,andtransferintolabeled50mLpolyethylenebottle,B(d).Pro- ceed to G, or freeze until measurement. (b) Filter hydrolysates into 250 mL vacuum flask, B(l), through sintered glass filter, then transfer filtrate to 250 mL beaker. Place beaker in ice bath. Partly neutralize hydrolysates with c
37、a 40 mL 7.5M NaOH, D(d)(1), while stirring. (Note: Temperature can not exceed40C.)AdjustpHto2.20using2MNaOH,D(d)(2).Proceed to G. G. Determination Dilute aliquot of evaporated hydrolysate F(a) with sodium citrate buffer,D(a),andadjustpHto2.20with2MNaOH.Whenneutralized hydrolysatesF(b)areused,dilutea
38、liquotwithH2O.Filterthroughfil- ter unit, B(g), into autosampler tube and inject into analyzer. (Note: Volume of aliquot and dilution depends on response of analyzer.) Calibrate amino acid analyzer with amino acid standard kit solu- tion, C(k), containing norleucine. Operate amino acid analyzer ac-
39、cording to manufacturers specifications. Adjust analyzer conditionstoensurebaselineseparationofpeaks.Minimumresolu- tion between 2 peaks should be 90%. H. Calculations Calculate response factor (RFaa) for each amino acid as follows: RFaa= PW PW naa aan where Paa= peak area of amino acid; Pn= peak ar
40、ea of norleucine; Waa= weight of amino acid, mg; Wn= weight of norleucine, mg. Calculate internal standard (IS) factor as follows: IS = Wn2 102 wheremgnorleucine=norleucinecontentin20mLnorleucinestan- dard, D(e). Calculateaminoacid(AA)contentofthetestsampleasfollows: AA, % = PRFIS PW aaaa ns 100 whe
41、rePaa=peakareaofaminoacid;Pn=peakareaofnorleucine;Ws =weightoftestportion,mg;RFaa=aminoacidresponsefactor;IS= internal standard factor. Performic Acid Oxidation with Acid HydrolysisHydrobromic Acid Method (Applicable to determination of amino acids including methionine and cystine in feeds. Not appl
42、icable to determination of phenylalanine, tyrosine, histidine, and tryptophan.) See Tables 994.12FJ for the results of the interlaboratory study supporting acceptance of method. A. Principle Performicacidoxidationisperformedpriortohydrolysistooxi- dize cystine and methionine to cysteic acid and meth
43、ionine sulfone, respectively. Hydrobromic acid is added to decompose performic acid. Amino acids are liberated from protein by hydroly- siswith6MHCl.Hydrolysatesaredilutedwithsodiumcitratebuffer and individual amino acid components are separated by ion-exchange chromatography. Tryptophan is destroye
44、d by hydro- lysis. Tyrosine, phenylalanine, and histidine are destroyed during oxidationprocessandbyreactionwithbromine,andcannotbeaccu- rately analyzed. B. Apparatus (a) Amino acid analyzer.Ion-exchange resin with ninhydrin post-column derivatization. (b) Analytical balance.Accurate to 0.1 mg. (c)
45、Balance.Top loading. (d) Bottle.50 mL; polyethylene. (e) Digestion tubes.Boiling flasks are suitable. (f) Digestion block.Heating mantle is suitable. (g) Filter units.0.22 m (Millex GS, Millipore are suitable). (h) Magnetic stirring plate. (i) pH meter.Calibrated with buffers of pH 2.0, 4.0, and 7.0
46、. (j) Reflux condensers. (k) Rotary evaporator. (l) Glassware.Glass beakers, 250 and 1000 mL; Erlenmeyer flask,150mL;round-bottomevaporatingflask,1000mL;graduated 2000 AOAC INTERNATIONAL cylinders,100,500,and1000mL;volumetricflask,1000mL;volu- metric pipets, 10 and 20 mL. (m) Sintered glass filter.P
47、orosity 1015 m. (n) Ice bath. (o) Syringes. C. Reagents (a) Formic acid.88%. (b) Hydrobromic acid.48%. (c) Hydrogen peroxide.30%. (d)DL-Norleucine.Crystals. (e) HCl.Concentrated. (f) NaOH.30% solution (30 g/100 mL). (g) Phenol.Crystals. (h) Thiodiglycol.98% solution. (i) Tri-sodium citrate dihydrate
48、. (j) pH buffer.pH 2.0, 4.0, and 7.0. (k) Amino acid standard kit.To calibrate amino acid analyzer; available from Aldrich Chemical Co., Inc., 1001 West Saint Paul Ave, Milwaukee, WI 53233, USA. D. Preparation of Solutions (a) Sodium citrate buffer, pH 2.20.Weigh 19.60 g tri-sodium citratedihydratei
49、n1000mLbeakeranddissolveinca800mLH2O. Whilestirring,add10mL98%thiodiglycolsolutionand15mLcon- centrated HCl. Transfer solution quantitatively into 1000 mL volu- 2000 AOAC INTERNATIONAL Table 994.12F Results of interlaboratory study for determination of amino acids in broiler fnisher feed by hydrobromic acid method Amino acidsNMeansrRSDr, %sRRSDR, %raRb Alanine281.200.0231.920.1038.580.0640.288 Arginine301.220.0322.620.17013.930.0900.476 Aspartic acid261.750.0321.830.0653.710.0900