RECOMMENDED METHODS OF ANALYSIS AND SAMPLING CODEX STAN 234-19991 1 The most updated version of the method should be used, in application of ISO/IEC 17025: 1999. The present list of methods reflects the amendments adopted by the 37th Session of the Codex Alimentarius Commission in 2014. CODEX STAN 234-19992 Table of Contents 1.PART A METHODS OF ANALYSIS BY COMMODITY CATEGORIES AND NAMES All Foods Cereals, Pulses and Legumes and Derived Products Cocoa Products and Chocolate Fats and Oils and Related Products Fish and Fishery Products Foods for Special Dietary Uses Fruit Juices Milk and Milk Products Natural Mineral Waters Processed Fruits and Vegetables Processed Meat and Poultry Products and Soups and Broths Quick Frozen Fruits and Vegetables Sugars and Honey Miscellaneous Products 2. PART B METHODS OF SAMPLING BY COMMODITY CATEGORIES AND NAMES CODEX STAN 234-19993 PART A METHODS OF ANALYSIS BY COMMODITY CATEGORIES AND NAMES CommodityProvisionMethodPrincipleType All Foods All foodsAcesulfame K, Aspartame EN 12856High performance liquid chromatography II All foodsCyclamateEN 12857High performance liquid chromatography II All foodsCyclamateNMKL 123SpectrophotometryIII All foods SaccharinEN 12856High performance liquid chromatography III All Foods (see also meat products)Nitrates and/or NitritesEN 12014-1Part 1- General considerationsN/A Individual Foods2Sulphites EN 1988-1 AOAC 990.28 Part 1: Optimized Monier-Williams method III Individual Foods3Sulphites EN 1988-2 NMKL 135Part 2: Enzymatic method III Cereals, Pulses and Legumes and Derived Products Certain pulsesMoistureISO 665GravimetryI Degermed maize (corn) meal and maize (corn) grits AshAOAC 923.03 ISO 2171 ICC Method No 104/1GravimetryI Degermed maize (corn) meal and maize (corn) grits Fat, crudeAOAC 945.38F; 920.39CGravimetry (ether extraction)I Degermed maize (corn) meal and maize (corn) grits MoistureISO 712 ICC Method No 110/1GravimetryI Degermed maize (corn) meal and maize (corn) grits Particle size (granularity)AOAC 965.22SievingI 2 Hominy, fruit juice, sea food3 Wine, dried apples, lemon juice, potato flakes, sultanas, beer CODEX STAN 234-19994 CommodityProvisionMethodPrincipleType Degermed maize (corn) meal and maize (corn) grits ProteinICC Method No 105/1 Titrimetry, Kjeldahl digestionI Durum wheat semolina and durum wheat flour Ash (semolina)AOAC 923.03 ISO 2171 GravimetryI Durum wheat semolina and durum wheat flour MoistureISO 712ICC 110/1GravimetryI Durum wheat semolina and durum wheat flour Protein (N x 5.7)ICC 105/1Titrimetry, Kjeldahl digestionI Instant Noodles Extraction of oil from instant noodles described in the standardGravimetryI Instant Noodles Acid Valuedescribed in the standardTitrimetryI Instant NoodlesMoisturedescribed in the standardGravimetryI Maize (corn)MoistureISO 6540GravimetryI Peanuts (raw)Aflatoxins, totalAOAC 991.31Immunoaffinity column (Aflatest)II Peanuts (raw)Aflatoxins, totalAOAC 993.17Thin layer chromatographyIII Peanuts (intended for further processing) Aflatoxins, totalAOAC 975.36Romer minicolmnIII Peanuts(Cereals, shell-fruits and derived products ( including peanuts)) Sum of aflatoxins B1, B2, G1 and G2 EN 12955ISO 16050:HPLC with post column derivatization and immunoaffinity column clean up III Peanuts (intended for further processing) Aflatoxins, totalAOAC 979.18Holaday-Velasco minicolumnIII Pearl millet flourAshAOAC 923.03GravimetryI Pearl millet flourColourModern Cereal Chemistry, 6th Ed., D.W. Kent-Jones and A.J. Amos (Ed.), pp. 605-612, Food Trade Press Ltd, London, 1969. Colorimetry using specific colour grader IV Pearl millet flourFat, crudeAOAC 945.38F; 920.39CGravimetry (ether extraction)I Pearl millet flourFibre, crudeISO 5498: (B.5 Separation)GravimetryI Pearl millet flourMoistureISO 712: ICC 110/1 GravimetryI CODEX STAN 234-19995 CommodityProvisionMethodPrincipleType Pearl millet flourProteinAOAC 920.87Titrimetry, Kjeldahl digestionI Sorghum flourAshAOAC 923.03 ISO 2171ICC 104/1GravimetryI Sorghum flourColourModern Cereal Chemistry, 6th Ed., D.W. Kent-Jones and A.J. Amos (Ed.), pp. 605-612, Food Trade Press Ltd, London, 1969. Colorimetry using specific colour grader IV Sorghum flourFat, crudeAOAC 945.38F; 920.39CGravimetry (ether extraction)I Sorghum flourFibre, crudeICC 113ISO 6541:1981GravimetryI Sorghum flourMoistureISO 712:1998 ICC 110/1GravimetryI Sorghum flourParticle size (granularity)AOAC 965.22SievingI Sorghum flourProteinICC 105/1 Titrimetry, Kjeldahl digestionI Sorghum flourTanninsISO 9648SpectrophotometryI Sorghum grainsAshAOAC 923.03 ISO 2171:1993 ICC 104/1GravimetryI Sorghum grainsFat, crudeAOAC 945.38F, 920.39CGravimetry (ether extraction)I Sorghum grainsMoistureISO 6540GravimetryI Sorghum grainsProteinICC 105/1 Titrimetry, Kjeldahl digestionI Sorghum grainsTanninsISO 9648SpectrophotometryI Soy protein productsAshAOAC 923.03 ISO 2171: (Method B) GravimetryI Soy protein productsFatCAC/RM 55 -Method 1Gravimetry (extraction)I Soy protein productsFibre, crudeISO 5498GravimetryI Soy protein productsMoistureAOAC 925.09Gravimetry (vacuum oven)I CODEX STAN 234-19996 CommodityProvisionMethodPrincipleType Soy protein productsProteinAOAC 955.04D (using factor 6.25)Titrimetry , Kjeldahl digestionII Vegetable protein productsAshAOAC 923.03 ISO 2171 (Method B) Gravimetry, DirectI Vegetable protein productsFatCAC/RM 55 - Method 1Gravimetry (extraction)I Vegetable protein productsFibre, crudeAACC 32-17Ceramic fiber filterationI Vegetable protein productsMoistureAOAC 925.09Gravimetry (vacuum oven)I Vegetable protein productsProteinAOAC 955.04D (using factor 6.25)Titrimetry, Kjeldahl digestionII Wheat flourAshAOAC 923.03 ISO 2171:ICC 104/1GravimetryI Wheat flourFat acidityAOAC 939.05TitrimetryI Wheat flourMoistureISO 712:ICC 110/1GravimetryI Wheat flourParticle size (granularity)AOAC 965.22SievingI Wheat flourProteinICC105/1 Titrimetry, Kjeldahl digestionI Wheat protein products including wheat gluten ProteinVital wheat gluten and devitalized wheat glutenAOAC 979.09 (wheat protein in grain Nx5.7) KjeldahlI Solubilized wheat protein AOAC 920.87(wheat protein in flour Nx5.7) KjeldahlI Wheat protein products including Wheat gluten Fibre, crudeAOAC 962.09Ceramic fiber filterationI Wheat protein products including Wheat gluten AshAOAC 923.03 ISO 2171: method B GravimetryI Whole and decorticated pearl millet grains AshAOAC 923.03GravimetryI CODEX STAN 234-19997 CommodityProvisionMethodPrincipleType Whole and decorticated pearl millet grains Fat, crudeAOAC 945.38F; 920.39CGravimetry (ether extraction)I Whole and decorticated pearl millet grains Fibre, crudeISO 5498 (B.5 Separation)GravimetryI Whole and decorticated pearl millet grains MoistureISO 712 ICC 110/1GravimetryI Whole and decorticated pearl millet grains ProteinAOAC 920.87Titrimetry, Kjeldahl digestionI Whole maize (corn) mealAshAOAC 923.03 ISO 2171ICC 104/1GravimetryI Whole maize (corn) mealFat, crudeAOAC 945.38F; 920.39CGravimetry (ether extraction)I Whole maize (corn) mealMoistureISO 712ICC 110/1GravimetryI Whole maize (corn) mealParticle size (granularity)AOAC 965.22SievingI Whole maize (corn) mealProteinICC 105/1 Titrimetry, Kjeldahl digestionI Cocoa Products and Chocolate Chocolate and chocolate productsCocoa butterAOAC 963.15 IOCCC 14 Gravimetry (Soxhlet extraction)I Chocolate and chocolate productsFat-free cocoa solidsAOAC 931.05Oven evaporation and factorI Chocolate and chocolate productsFat-free milk solidsIOCCC 17 or AOAC 939.02Titrimetry, Kjeldahl digestion; after extraction of milk proteins II Chocolate and chocolate productsFat, totalAOAC 963.15Gravimetry (Soxhlet extraction)I Chocolate and chocolate productsMilkfatIOCCC 5 AOAC 945.34; 925.41B; 920.80 Titrimetry/DistillationI Chocolate and chocolate productsMoistureIOCCC 26 or AOAC 977.10 (Karl Fischer method); or AOAC 931.04 or IOCCC 1 GravimetryI CODEX STAN 234-19998 CommodityProvisionMethodPrincipleType Chocolate and chocolate productsNon-cocoa butter vegetable fat AOCS Ce 10/02 and described in the Standard Described in the StandardI Cocoa (Cacao) Mass or Cocoa/ Chocolate Liquor, and Cocoa Cake Cocoa shellAOAC 968.10 and 970.23Spiral vessel count, Stone cell countI Cocoa (Cacao) Mass or Cocoa/ Chocolate Liquor, and Cocoa Cake FatAOAC 963.15 or IOCCC 14 Gravimetry (Soxhlet extraction)I Cocoa butterFree fatty acids ISO 660 or AOCS Cd 3d-63 TitrimetryI Cocoa butterUnsaponifiable matter ISO 3596 or ISO 18609 or AOCS Ca 6b-53 Titrimetry after extraction with diethyl ether I Cocoa powders (cocoa) and dry cocoa-sugar mixtures Moisture IOCCC 26 or AOAC 977.10 (Karl Fischer method)GravimetryI Fats and Oils and Related Products Fats and Oils (all)ArsenicAOAC 952.13 (Codex general method) Colorimetry (diethyldithiocarbamate)II Fats and Oils (all)ArsenicAOAC 942.17 (Codex general method)Colorimetry (molybdenum blue)III Fats and Oils (all)ArsenicAOAC 986.15 (Codex general method)Atomic absorption spectrophotometryIII Fats and oils Butylhydroxyanisole, butylhydroxytoluene, tert-butylhydroquinone, & propyl gallate AOAC 983.15; or AOCS Ce-6-86 Liquid chromatographyII Fats and Oils (all)Insoluble impurities ISO 663 GravimetryI Fats and Oils (all)LeadAOAC 994.02 ISO 12193 (Codex general method) or AOCS Ca 18c-91Atomic absorption spectrophotometry (direct graphite furnace) II Fats and Oils (all)Matter volatile at 105CISO 662Gravimetry (open-drying)I Fats and Oils (all)Soap contentBS 684 Section 2.5; or AOCS Cc 17-95 GravimetryI Fats and oils not covered by individual standards Acid ValueISO 660; or AOCS Cd 3d-63 TitrimetryI CODEX STAN 234-19999 CommodityProvisionMethodPrincipleType Fats and oils not covered by individual standards Copper and IronAOAC 990.05 ISO 8294or AOCS Ca 18b-91(Codex general method) Atomic absorption Spectrophotometry (direct graphite furnace) II Fats and oils not covered by individual standards Peroxide value AOCS Cd 8b-90ISO 3960 Titrimetry using iso-octaneI Fat spreads and blended spreadsFat contentISO 17189 | IDF 194GravimetryI Named Animal FatsAcidityISO 660; or AOCS Cd 3d-63 TitrimetryI Named Animal FatsCopper and IronAOAC 990.05 ISO 8294; or AOCS Ca 18b-91(Codex general method) Atomic absorption Spectrophotometry (direct graphite furnace) II Named Animal FatsGLC ranges of fatty acid composition ISO 5508 and ISO 12966-2:or AOCS Ce 2-66 and Ce 1e-91or Ce 1f-96Gas chromatography of methyl estersII Named Animal Fats Iodine value (IV)ISO 3961; or AOAC 993.20; or AOCS Cd 1d-92 Wijs-Titrimetry I Named Animal FatsPeroxide value AOCS Cd 8b-90ISO 3960 Titrimetry using iso-octaneI Named Animal FatsRelative densityISO/AOCS method for apparent density to be inserted PycnometryII Named Animal FatsRefractive indexISO 6320; or AOCS Cc 7-25 RefractometryII Named Animal FatsSaponification valueISO 3657; or AOCS Cd 3-25 TitrimetryI Named Animal FatsUnsaponifiable matterISO 3596 or ISO 18609; or AOCS Ca 6b-53 Titrimetry after extraction with diethyl ether I Named Animal FatsTitreISO 935; or AOCS Cc 12-59 ThermometryI Named Vegetable Oils AcidityISO 660; or AOCS Cd 3d-63 TitrimetryI Named Vegetable OilsApparent densityISO 6883, with the appropriate conversion factor; or AOCS Cc 10c-95PycnometryI CODEX STAN 234-199910 CommodityProvisionMethodPrincipleType Named Vegetable OilsBaudouin test (modified Villavecchia or sesameseed oil test) AOCS Cb 2-40Colour reactionI Named Vegetable Oils Carotenoids, total BS 684 Section 2.20 SpectrophotometryII Named Vegetable Oils Copper and ironISO 8294; or AOAC 990.05; or AOCS Ca 18b-91AAS II Named Vegetable Oils Crismer valueAOCS Cb 4-35 and AOCS Ca 5a-40 TurbidityI Named Vegetable Oils GLC ranges of fatty acid composition ISO 5508 and ISO 12966-2; orAOCS Ce 2-66 and Ce 1--62 or Ce 1h-05Gas chromatography of methyl estersII Named Vegetable OilsHalphen test AOCS Cb 1-25ColorimetryI Named Vegetable Oils Insoluble impuritiesISO 663 GravimetryI Named Vegetable Oils Iodine value (IV)Wijs - ISO 3961; or AOAC 993.20; or AOCS Cd 1d-92; or NMKL 39 Wijs-Titrimetry 4I Named Vegetable Oils LeadAOAC 994.02; or ISO 12193; orAOCS Ca 18c-91 Atomic AbsorptionII Named Vegetable Oils Moisture & volatile matter at 105C ISO 662Gravimetry I Named Vegetable Oils Peroxide value (PV)AOCS Cd 8b-90 or ISO 3960 Titrimetry I Named Vegetable Oils Refractive indexISO 6320 or AOCS Cc 7-25 RefractometryII Named Vegetable Oils Reichert value and Polenske value AOCS Cd 5-40 TitrimetryI Named Vegetable OilsRelative densityIUPAC 2.101 with the appropriate conversion factorSee comment above (Named Animal Fats) 5 PycnometryI Named Vegetable Oils Saponification value (SV)ISO 3657 or AOCS Cd 3-25 TitrimetryI 4It is possible to calculate the Iodine Value from fatty acid composition data obtained by gas chromatographye.g. usingAOCS Cd 1b-875 The method is no longer available. CODEX STAN 234-199911 CommodityProvisionMethodPrincipleType Named Vegetable Oils Slip pointISO 6321 for all oils; AOCS Cc 3b-92 for all oils except palm oils; AOCS Cc 3-25 for palm oils only Open ended capillary tubeI Named Vegetable Oils Soap contentBS 684 Section 2.5; or AOCS Cc 17-95 GravimetryI Named Vegetable Oils Sterol contentISO 12228; or AOCS Ch 6-91 Gas chromatographyII Named Vegetable Oils Tocopherol contentISO 9936or AOCS Ce 8-89HPLCII Named Vegetable Oils Unsaponifiable matterISO 3596; or ISO 18609; or AOCS Ca 6b-53 Gravimetry I Olive Oils and Olive Pomace Oils Absorbency in ultra-violetCOI/T.20/Doc. No. 19 or ISO 3656 or AOCS Ch 5-91Absorption in ultra violetII Olive Oils and Olive Pomace Oils Acidity, free (acid value)ISO 660 or AOCS Cd 3d-63TitrimetryI Olive Oils and Olive Pomace Oils Alpha-tocopherolISO 9936HPLCII Olive Oils and Olive Pomace Oils Difference between the actual and theoretical ECN 42 triglyceride content COI/T.20/Doc. no. 20 or AOCS Ce 5b-89 Analysis of triglycerides of HPLC and calculation I Olive Oils and Olive Pomace OilsErythrodiol + uvaolCOI/T.20/Doc.no. 30Gas chromatography II Olive Oils and Olive Pomace Oils Halogenated solvents, tracesCOI/T.20/Doc. no. 8Gas chromatographyII Olive Oils and Olive Pomace Oils Insoluble impurities in light petroleum ISO 663GravimetryI Olive Oils and Olive Pomace Oils Iodine valueISO 3961 or AOAC 993.20 or AOCS Cd 1d-92 or NMKL 39Wijs-TitrimetryI Olive Oils and Olive Pomace Oils Iron and copperISO 8294 or AOAC 990.05 AAS II Olive Oils and Olive Pomace Oils LeadAOAC 994.02 or ISO 12193 or AOCS Ca 18c-91 AASII Olive Oils and Olive Pomace Oils Moisture and volatile matterISO 662GravimetryI Olive Oils and Olive Pomace Oils Organoleptic characteristicsCOI/T.20/Doc. no. 15Panel testI Olive Oils and Olive Pomace Oils Peroxide valueISO 3960 or AOCS Cd 8b-90 TitrimetryI Olive Oils and Olive Pomace Oils Relative densityIUPAC 2.101, with the appropriate conversion factor. See comment above PycnometryI CODEX STAN 234-199912 CommodityProvisionMethodPrincipleType Olive Oils and Olive Pomace Oils Refractive indexISO 6320 or AOCS Cc 7-25 RefractometryII Olive Oils and Olive Pomace Oils Saponification valueISO 3657 or AOCS Cd 3-25 TitrimetryI Olive Oils and Olive Pomace Oils Sterol composition and total sterols COI/T.20/Doc. no. 10 or ISO 12228 or AOCS Ch 6-91Gas chromatographyII Olive Oils and Olive Pomace Oils StigmastadienesCOl/T.20/Doc. no. 11 or ISO 15788-1 or AOCS Cd 26-96 Gas chromatographyII Olive Oils and Olive Pomace Oils StigmastadienesISO 15788-2HPLCIII Olive Oils and Olive Pomace Oils Trans fatty acids contentCOI/T.20/Doc no. 17 or ISO 15304 or AOCS Ch 2a-94 Gas chromatography of methyl esters II Olive Oils and Olive Pomace Oils Unsaponifiable matterISO 3596 or ISO 18609 or AOCS Ca 6b-53 GravimetryI Olive Oils and Olive Pomace Oils Wax contentCOI/T.20/Doc. no. 18 or AOCS Ch 8-02 Gas chromatographyII MargarineFatIUPAC 2.801GravimetryI MargarineMilkfatCAC/RM 15TitrimetryI MargarineSodium chlorideAOAC 971.27 (Codex general method)PotentiometryII MargarineVitamin AAOAC 960.45SpectrophotometryII MargarineVitamin DAOAC 936.14BioassayII MargarineVitamin EIUPAC 2.411TLC followed by spectrophotometry or GLC II MargarineWaterCAC/RM 17 (described in the Standard)GravimetryI MinarineFatIUPAC 2.801GravimetryI MinarineMilkfatCAC/RM 15 (described in the Standard)TitrimetryI MinarineSodium chlorideAOAC 971.27 (Codex general method)PotentiometryII MinarineVitamin AAOAC 960.45SpectrophotometryII MinarineVitamin DAOAC 936.14BioassayII MinarineVitamin EIUPAC 2.411TLC followed by spectrophotometry or GLC II CODEX STAN 234-199913 CommodityProvisionMethodPrincipleType MinarineWaterCAC/RM 17GravimetryI Fish and Fishery Products Fish and fishery productsHistamineAOAC 977.13FluorimetryII Fish and fishery productsMercuryAOAC 977.15Flameless atomic absorption spectrophotometry III Fish and fishery products: canned productsDrained weightDescribed in the StandardWeighingI Fish and fishery products: canned products Net weightDescribed in the StandardWeighingI Boiled Dried Salted Anchovies Sodium Chloride(chloride expressed as sodium chloride) AOAC 937.09 TitrimetryII Canned shrimps or prawnsSize, determination of Described in the StandardNumber per 100 gI Fish Saucetotal nitrogenAOAC 940.25digestionI Fish Sauceamino acid nitrogenAOAC 920.04 and AOAC 920.03determining formaldehyde titration method subtracting by ammoniacal nitrogen (magnesium oxide method) I Fish SaucepHAOAC 981.12 The pH shall be measured in a sample of fish sauce diluted with water to 1:10 using a pH meter. The dilution of fish sauce is necessary because of the high ionic strength in the undiluted sauce. electrometryIII Fish Saucesodium chlorideAOAC 976.18potentiometryII Fish Saucesodium chlorideAOAC 937.09titrimetryIV Fish SaucehistamineAOAC 977.13FluorimetryII Frozen abalone (covered by glaze)Net weight AOAC 963.18GravimetryI Frozen fish and fishery productsThawing and cooking procedures Described in the StandardsThawing and heatingI CODEX STAN 234-199914 CommodityProvisionMethodPrincipleType Quick frozen blocks of fish fillet, minced fish flesh and mixtures of fillets and minced fish flesh Proportion of fish fillet and minced fish AOAC 988.09Physical separationI Quick frozen blocks of fish fillet, minced fish flesh and mixtures of fillets and minced fish flesh Net content of frozen fish blocks covered by glaze Described in the StandardGravimetryI Quick frozen blocks of fish fillet, minced fish flesh and mixtures of fillets and minced fish flesh Sodium chlorideAOAC 971.21 (Codex general method)PotentiometryII Quick frozen fish filletsNet weight of products covered by glaze Described in the StandardWater spraying and sievingI Quick Frozen Fish sticks (fish fingers) and fish portions - breaded or in batterFish content (declaration)AOAC 996.15 and calculation (described in the standard) GravimetryI Quick frozen fish sticks (fish fingers) and fish portions - breaded or in batter Net weightDescribed in the StandardWeighingI Quick Frozen Fish Sticks (fish fingers) and Fish Portions-Breaded and in Batter (except for certain fish species with soft flesh) Proportion of fish fillet and minced fish WEFTA Method (described in the Stnadard) Gravimetry I Quick frozen fish sticks (fish fingers) and fish portions - breaded or in batter Sodium chlorideAOAC 971.27 (Codex general method)PotentiometryII Salted Atlantic Herring and Salted Sprat Water contentAOAC 950.46Bair dryingI Salted Fish of the Gadidae FamilySalt Described in CODEX STAN 167-1989Titrimetry (Mohr) Salt determined as chloride expressed as sodium chloride I Salted Fish and Dried Salted Fish of the Gadidae Family of Fishes Salt Content Water content Sampling and method described in the Standard Gravimetry I Smoked Fish, Smoke-Flavoured fish and Smoke-dried fish Water phase saltAOAC 952.08 AOAC 937.09 Described in standard6 CalculationI 6 % salt 100 / (%water + %salt) CODEX STAN 234-199915 CommodityProvisionMethodPrincipleType Smoked Fish, Smoke-Flavoured fish and Smoke-dried fish Water activityNMKL 168 | ISO 21807Electrometry III Sturgeon CaviarSalt content Described in CODEX STAN 167-1989Titrimetry (Mohr) Salt determined as chloride expressed as sodium chloride I Method Performance Criteria for histamine in smoked fish, smoke-flavoured fish and smoke-dried fish ProvisionML(mg/100 g) Minimum applicable range (mg/100 g) LOD (mg/100 g) LOQ (mg/100 g) RSDR(%)RecoveryApplicable methods that meet the criteria Principle Histamine10 (average) 8 121216.090 107AOAC 977.13 | NMKL 99,NMKL 196,Fluorometric HPLC Histamine20(each unit) 16 242414.490 107AOAC 977.13 | NMKL 99,NMKL 196,Fluorometric HPLC Determination of Biotoxins in live and raw bivalve molluscs The method selected should be chosen on the basis of practicability and preference should be given to methods which have applicability for routine use. Criteria for determination of Toxin Analogues by chemical methods Methods shall meet the numerical criteria listed in Table 1 and may either meet the minimum applicable range, or LOD and LOQ criteria listed. Table 1. Criteria for determination of Toxin Analogues by Chemical Methods Toxin GroupToxinMinimum applicable range (mg/kg) LOD (mg/kg) LOQ (mg/kg) Precision (RSDR) (%) No more than Recovery percentApplicable methods that meet the criteria STX GroupSaxitoxin (STX) 0.05 0.20.010.0244%50 130AOAC 2005.06NMKL 182, EN 14526AOAC 2011.02 NMKL 197 NEO0.05 0.20.010.0244%50 130 dcSTX0.05 0.20.010.0244%50 130 GTX10.05 0.20.010.0244%50 130 GTX20.1 0.50.030.0638%50 130 GTX30.1 0.50.030.0638%50 130 GTX40.05 0.20.010.0244%50 130 GTX50.1 0.50.030.0638%50 130 GTX60.1 0.50.030.0638%50 130 dcGTX20.1 0.50.030.0638%50 130 CODEX STAN 234-199916 Toxin GroupToxinMinimum applicable range (mg/kg) LOD (mg/kg) LOQ (mg/kg) Precision (RSDR) (%) No more than Recovery percentApplicable methods that meet the criteria dcGTX30.1 0.50.030.0638%50 130 C10.1 0.50.030.0638%50 130 C20.1 0.50.030.0638%50 130 C30.5 1.50.10.232%50 130 C40.5 1.50.10.232%50 130 OA GroupOA0.03 0.20.010.0244%60-115See reference below DTX10.03 0.20.010.0244%60-115 DTX20.1 0.50.030.0638%60-115 Domoic AcidDA14 262420%80-110 AZA GroupAZA10.03 0.20.010.0244%40 - 120See reference below AZA20.03 0.20.010.0244%40 - 120 AZA30.03 0.20.010.0244%40 - 120 Reference: http://aesan.msssi.gob.es/en/CRLMB/web/procedimientos_crlmb/crlmb_standard_operating_procedures.shtml Harmonised-SOP-LCMS-OA-Version4.pdf Totaltoxicityisestimatedasthesumofthemolarconcentrationsofdetectedanalogsmultipliedbytherelevantspecifictoxicityequivalencyfactors(TEFs). InternationallyscientificallyvalidatedTEFsmustbeused.ThesciencebehindTEFsisdeveloping.CurrentinternationallyvalidatedTEFswillbefoundontheFAO website. Information on TEFs could be incorporated in this standard at a future date. Methods should be validated and used for the relevant toxin analogues that may contribute to total toxicity. Currently known toxin analogues to consider are listed in Table 1. WheretoxinanaloguesthatarenotlistedinTable1aredeterminedthecompetentauthoritymustassessthecontributionoftheseanalogstototaltoxicitywhilst conducting further investigations.Foods for Special Dietary Uses Special foodsAshAOAC 942.05GravimetryI Special foodsCalciumAOAC 984.27ICP emission spectrometryIII Special foodsCalories by calculationMethod described in CAC/VOL IX-Ed.1, Part III Calculation methodIII Special foodsCarbohydratesMethod described in CAC/VOL IX-Ed.1,Part III CalculationIII Special foodsChlorideAOAC 971.27 (Codex general method)PotentiometryII Special foodsDietary fibre, totalAOAC 985.29Gravimetry (enzymatic digestion)I CODEX STAN 234-199917 Foods for Special Dietary Uses Special foodsFatCAC/RM 55Gravimetry (extraction)I Special foodsFat in foods not containing starch, meat or vegetable products CAC/RM 1, B-2GravimetryI Special foodsFill of containersCAC/RM 46WeighingI Special foodsFolic acidAOAC 944.12MicrobioassayII Special foodsLinoleate (in the form of glycerides)AOAC 922.06; 969.33; 963.22Acid hydrolysis, preparation of methyl esters and gas chromatography II Special foodsLinoleate (in the form of glycerides)AOAC 922.06; 979.19Acid hydrolysis and spectrophotometry III Special foodsLoss on drying (milk based)AOAC 925.23 ISO 6731|IDF 21 Gravimetry I Special foodsNicotinamide for foods not based on milkAOAC 961.14ColorimetryII Special foodsNicotinamide for milk-based foodsAOAC 944.13MicrobioassayII Special foodsPantothenic acid/enriched foodsAOAC 945.74MicrobioassayII Special foodsPantothenic acid/non-enriched foodsThe Analyst 89 (1964):1, 3-6, ibid. 232 US Dept Agr., Agr. Handbook 97 (1965) MicrobioassayIV Special foodsPhosphorousAOAC 986.24Colorimetry (molybdovanadate)II Special foodsProtein efficiency ratio (PER)AOAC 960.48Rat bioassayI Special foodsProtein, crudeMethod described in CAC/VOL IX-Ed. 1,Part IIITitrimetry, Kjeldahl digestionI Special foodsRiboflavinAOAC 970.65FluorometryII Special foodsSodium and PotassiumISO 8070|IDF 119Flame atomic absorption spectrometry II Special foodsSodium and potassiumAOAC 984.27ICP emission spectrometryIII Special foodsVitamin AAOAC 974.29ColorimetryIV Special foodsVitamin A in foods in which carotenes have been added as a source of vitamin A AOAC 941.15SpectrophotometryIII CODEX STAN 234-199918 Foods for Special Dietary Uses Special foodsVitamin B12AOAC 952.20MicrobioassayII Special foodsVitamin B6AOAC 961.15MicrobioassayII Special foodsVitamin CAOAC 967.22MicrofluorometryII Special foodsVitamin CAOAC 967.21Colorimetry (dichloroindophenol)III Special foodsVitamin DAOAC 936.14Rat bioassayIV Special foodsVitamin D (D3, milk based infant formula)AOAC 992.26Liquid chromatographyII Special foodsVitamin EAOAC 971.30ColorimetryIV Special foodsVitamin E (milk based infant formula)AOAC 992.03Liquid chromatographyII Special foodsSodium and PotassiumISO 8070|IDF 119Flame atomic absorption spectrometryII Follow-up formulaDietary fibre, totalAOAC 991.43Gravimetry (enzymatic digestion)I Follow-up formulaIodine (milk based formula)AOAC 992.24Ion-selective potentiometryII Follow-up formulaPantothenic acidAOAC 992.07 Measures total pantothenate(free pantothenic acid + CoA- + ACP-bound) and measured as D-pantothenic acid (or calcium D-pantothenate) MicrobioassayII Follow-up formulaVitamin AAOAC 974.29ColorimetryIV Follow-up formulaVitamin A (retinol isomers)AOAC 992.04HPLCII Follow-up formulaVitamin A (retinol) (above 500 IU/l milk after reconstitution) AOAC 992.06HPLCIII Follow-up formulaVitamin K AOAC 999.15 EN 14148 (vitamin K1) (Measures either aggregated cis + trans K1 or can measure individual cis and trans forms depending on LC column.) HPLC with C30 column to separate the cis- and the trans- K vitamins II Foods with low-sodium content (including salt substitutes) IodineAOAC 925.56TitrimetryII CODEX STAN 234-199919 Foods for Special Dietary Uses Foods with low-sodium content (including salt substitutes) Silica (colloidal, calcium silicate) AOAC 950.85NGravimetryIV Gluten-free foodsGluten Enzyme-Linked Immunoassay R5 Mendez (ELISA) Method Eur J Gastroenterol Hepatol 2003; 15: 465-474 ImmunoassayI Infant formula Biotin EN 15607 (d-biotin) (Measures total D-biotin (free + D-biocytin) HPLCII Infant formula Calories (by calculation)Method described inCAC/Vol IX-Ed.1,Part III7 Calculation I Infant formula CalciumISO 8070 IDF 119Flame atomic absorption spectrophotometry II Infant formula CalciumAOAC 985.35Flame atomic absorption spectroscopy III Infant formula CalciumAOAC 984.27ICP emission spectroscopy III Infant formula ChlorideAOAC 986.26Potentiometry III Infant formula CholineAOAC 999.14 Enzymatic Colorimetric Methodwith limitations on applicability due to choline and ascorbate concentration. II Infant formula CopperAOAC 985.35Flame atomic absorption spectroscopy II Infant formula CopperAOAC 984.27ICP emission spectroscopy III 7 Section 9. Calories by calculation Section 9.2 Conversion Factors(a) protein 4 kcal per g(b) carbohydrate 4 kcal per g (c) fat9 kcal per g (d) monosaccharides 3.75 kcal per g (e) specific food ingredients See Energy and Protein Requirements(FAO Nutrition Meeting Report Series No. 52 or WHO Technical Report Series No. 522) (f)otherspecificcalorieconversionfactorsmaybeusedwheretheformulationofthefoodandthenutrientcontentareknownandwheresuchspecificconversionfactorsare physiologically more meaningful than the factors listed above CODEX STAN 234-199920 Foods for Special Dietary Uses Infant formula Chromium (Section B of STAN 72 only) EN 14082Graphite furnace atomic absorption after dry ashing II Infant formula Chromium (Section B of STAN 72 only) EN 14083 Graphite furnace AAS after pressure digestion III Infant formula Chromium (Section B of STAN 72 only) AOAC 2006.03ICP emission spectroscopy III Infant formula Crude protein8AOAC 991.20 ISO 8968-1 | IDF 20-1 Titrimetry (Kjeldahl)I Infant formula Fatty acids (including trans fatty acid) AOAC 996.06 Gas chromatography II Infant formula Fatty acids (including trans fatty acid) AOCS Ce 1h-05 Gas chromatography III Infant formula Folic acidAOAC 992.05 (Measures free folic acid + free, unbound natural folates, aggregated and measured as folic acid)EN 14131 (Total folate (free + bound), aggregated and measured as folic acid) Microbioassay II Infant formula Folic acidJ AOAC Int. 2000:83; 1141-1148 (Measures free folic acid + proportion of free, natural folate) Optical Biosensor Immunoassay IV Infant formula Folic acidJ Chromatogr. A., 928, 77-90, 2001 (Measures total folates after conversion to, and measurement as 5-Me-H4PteGlu) HPLC,incorporating immunoaffinity clean-up and conversion to 5-methyltetrahydrofolate IV Infant formula Iodine (for milk-based formula) AOAC 992.24Ion-selective potentiometry II 8 Determination of Crude Protein The calculation of the protein content of infant formulas prepared ready for consumption may be based on N x 6.25, unless a scientific justification is provided for the use of a different conversion factor for a particular product. The value of 6.38 is generally established as a specific factor appropriate for conversion of nitrogen to protein in other milk products, and the value of 5.71 as a specific factor for conversion of nitrogen to protein in other soy products CODEX STAN 234-199921 Foods for Special Dietary Uses Infant formula Iron9AOAC 985.35Flame atomic absorption spectrophotometry III Infant formula IronAOAC 984.27ICP emission spectroscopy III Infant formula IronAOAC 999.11NMKL139AAS after dry ashing II Infant formula MagnesiumISO 8070 IDF 119Flame atomic absorption spectrophotometry II Infant formula MagnesiumAOAC 985.35Flame atomic absorption spectroscopy III Infant formula MagnesiumAOAC 984.27ICP emission spectroscopy III Infant formula ManganeseAOAC 985.35Flame atomic absorption spectrophotometry II Infant formula ManganeseAOAC 984.27ICP emission spectroscopy III Infant formula Molybdenum (Section B of STAN 72 only) EN 14083Graphite furnace AAS after pressure digestion II Infant formula MelamineISO/TS 15495 | IDF/RM 230LC-MS/MS IV Infant formula Molybdenum (Section B of STAN 72 only) AOAC 2006.03ICP emission spectroscopy III Infant formula NiacinAOAC 985.34 (niacin (preformed) and nicotinamide) Microbioassay and turbidimetry III Infant formulaNiacinEN 15652 (Free and bound and phosphorylated forms measured either as aggregate of nicotinic acid + nicotinamide, or as individual forms) HPLC II10 Infant formula PhosphorusAOAC 986.24Spectrophotometry (molybdovanadate) II Infant formula PhosphorusAOAC 984.27ICP emission spectroscopy III Infant formula RiboflavinAOAC 985.3111Fluorimetry III 9General Codex methods are also available 10 When published as EN method 11 Care should be taken in the application of the method due to spectral interference CODEX STAN 234-199922 Foods for Special Dietary Uses Infant formula RiboflavinEN 14152 (Measures natural and supplemental forms, free, bound and phosphorylated (FMN and FAD) aggregated and measured as riboflavin.) HPLC II Infant formula SeleniumAOAC 996.16 or AOAC 996. 17Continuous hydride generation Flame atomic absorption spectrometry (HGAAS) III Infant formula SeleniumEN 14627Hydride generation atomic absorption spectrometry (HGAAS) II Infant formula SeleniumAOAC 2006.03ICP emission spectroscopy III Infant formula Sodium and potassiumAOAC 984.27 ICP emission spectrometryIII Infant formula Sodium and potassiumISO 8070 | IDF 119 Flame atomic absorption spectrophotometry II Infant formula ThiamineAOAC 986.2712Fluorimetry III Infant formula ThiamineEN 14122 (Measures all vitamin B1 forms (natural and added free, bound and phosphorylated) following extraction and conversion to thiamine) HPLC with pre-or post column derivatization to thiochrom II Infant formula Total carbohydrates Moisture/Total Solids Ash AOAC 986.25 AOAC 990.19orAOAC 990.20 ISO 6731|IDF 21 AOAC 942.05 Determination by difference Gravimetry GravimetryI Infant formula Total fatAOAC 989.05 ISO 8381|IDF 123 Gravimetry (Rse-Gottlieb) I 12 Care should be taken in the application of the method due to spectral interference CODEX STAN 234-199923 Foods for Special Dietary Uses Infant formula Total fat for milk-based infant formula (Products not completely soluble in ammonia) ISO 8262-1 |IDF 124-1Gravimetry (Weibull-Berntrop)I Infant formula Total phospholipidsAOCS Ja7b-91Gas chromatographywith suitable extraction and preparation procedures III Infant formula Vitamin A EN 12823-1 (all-trans-retinol and 13-cis-retinol) Vitamin A (both natural + supplemental ester forms) aggregated and quantified as individual retinol isomers (13 - cis and all-trans) HPLC III Infant formula Vitamin D AOAC 992.26 D3 measured HPLC III Infant formula Vitamin D EN 12821 (D2 and/or D3 measured as single components. Hydroxylated forms not measured.) NMKL 167 HPLC II Infant formula Vitamin D AOAC 995.05 D2 and D3 measured HPLC III Infant formula Vitamin EAOAC 992.03 Measures all rac-vitamin E (both natural + supplemental ester forms) aggregated and quantified as -congeners HPLCIII Infant formula Vitamin E EN 12822 (Measures Vitamin E (both natural + supplemental ester forms) aggregated and quantified as individual tocopherol congeners (, , , ). HPLC II Infant formula Vitamin B6AOAC 985.32Microbioassay III CODEX STAN 234-199924 Foods for Special Dietary Uses Infant formula Vitamin B6 EN 14166(Aggregates free and bound pyridoxal, pyridoxine and pyridoxamine and measures as pyridoxine) MicrobioassayIII Infant formula Vitamin B6 AOAC 2004.07 EN 14164 (Free and bound phosphorylated forms (pyridoxal, pyridoxine and pyridoxamine) converted and measured as pyridoxine) HPLCII Infant formula Vitamin B6 EN 14663 (includes glycosylated forms) (Free and bound phosphorylated and glycosylatedforms measured as the individual forms pyridoxal, pyridoxine and pyridoxamine) HPLC III Infant formula Vitamin B12 AOAC986.23 (Measures total vitamin B12 as cyanocobalamin) Turbidimetric MethodII Infant formula ZincAOAC 985.35Flame atomic absorption spectroscopy II Infant formula ZincAOAC 984.27ICP emission spectroscopy III Methods of analysis for dietary fibre: Guidelines for Use of Nutrition and Health Claims: Table of Conditions for Claims StandardProvisionsMethodPrincipleType General methods that do not measure the lower molecular weight fraction (i.e. monomeric units 9) and the lower molecular weight fraction (monomeric units 0.1 mg/L< 4171-102ISO 9377-2GC PCB->15 ng/L 15 ng/ L