Method References
It is the policy of the University of Missouri Agricultural Experiment Station Chemical Laboratories to declare the primary references for all official methods that are employed for the major assays routinely performed and/or requested. This is not meant to be an exhaustive listing, especially for 'research' procedures where several methods may be available, depending on sample matrix.
- Amino Acid Analyses
- Proximates, Other Fiber Analyses
- Sugars and Carbohydrates
- Lipids, Fats and Oils
- Mineral Analyses
- Water and Wastewater Analyses
- Fertilizer and Limestone
- Mass Spectrometry
Standard Amino Acid Analyses
- Complete Amino Acid Profile (AAP)
- AOAC Official Method 982.30 E(a,b,c), chp. 45.3.05, 2006 ** see "Tryptophan (T)"
- Amino Acid Profile without Tryptophan (AAP-T)
- AOAC Official Method 982.30 E(a,b), chp. 45.3.05, 2006.
- NIST Standard Reference Material 2389a (click here)
- Cysteine, Methionine and Lysine +9 (CML+9)
- AOAC Official Method 994.12
- JAOAC 70:171-174, 1987.
- Standard Protein Hydrolysate (SPH)
- AOAC Official Method 982.30 E(a), chp. 45.3.05, 2006.
- Methionine, Lysine or Hydroxyproline (M, L or HP)
- AOAC Official Method 982.30 E(a), chp. 45.3.05, 2006, acid hydrolysis.
- Cysteine & Methionine (CM)
- AOAC Official Method 982.30 E(b), chp. 45.3.05, 2006; peformic acid oxidation/acid hydrolysis.
- Tryptophan (T)
- by Enzymatic hydrolysis - colorimetric determination.
- by Alkaline hydrolysis - AOAC Official Method 988.15 , chp. 45.4.04, 2006
- Free Supplemental Amino Acids (FM, FL or FML)
- Methionine or Lysine, JAOAC 65:798, 1982.
- Methionine and lysine, JAOAC 65:798, 1982.
- AOAC Official Method 999.13
- Free Amino Acid Profile - Foods/Feeds (FAAP)
- AOAC Official Method 999.13
- Available Lysine (AL)
- AOAC Official Method 975.44, chp. 45.4.03, 2006.
- Available Lysine and Complete Amino Acid Profile (AL+AAP)
- AOAC Official Method 975.44 ; 982.30 E(a,b,c), chp. 45.3.05, 2006.
- Glutamine (peptides and proteins)
- J. Agric. Food Chem. (1996) 44:1808-1811
- HMB (also known as MHA and others) - Amino Acid Analogue for Methionine
- R.R.Ontiveros, W.S.Shermer, and R.A.Berner, An HPLC method for the determination of 2-hydroxy-4-
- (methylthio)butanoic acid (HMB) in supplemental animal feeds, J.Agric. Food Chem., 35(5), 692, 1987.
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Physiological Analyses of Biological Fluids
- Physiological Analyses
- Free amino acids in Plasma, Serum, Urine,Cerebrospinal Fluid and other biological fluids. (0.5 mL - 1.0 mL is required)
- Clinical Chemistry 43(8) 1421-1428 (1997) (click here for pdf); Beckman 121-M Application Notes. 121-M-TB-004A, March 1975; and 121 MB-TB-018, January, 1980. Deyl, Z., Hyanek, J. and Horak ova, M. (1986) Profiling of amino acids in body fluids and tissues by means of liquid chromatography. J. Chromatogr. 379, 177-250.; Fekkes, D. (1996) State of the art-of-the-art high-performance liquid chromatographic analysis of amino acids in physiological samples. J. Chromatog. B. Biomed. Appli. 682, 3-22.
- EU BIOMED 2-Project SOP: (click here for pdf)
Microanalysis of Amino Acids
- Amino Acid Profile (Tryptophan not included, inquire)
- Hyp, Asp, Thr, Ser, Glu, Pro, Lys, Ala, Cys, Val, Met, Ile, Tyr, Phe, Hyl, His, Lys, Arg
- Standard Protein and Peptide Hydrolysis
- Hyp, Asp, Thr, Ser, Glu, Pro, Lys, Ala, Cys, Val, Met, Ile, Tyr, Phe, Hyl, His, Lys, Arg
- Sample in stained/unstained polyacrylamide gel.
- Lyophilized sample or in solution.
- Sample on PVDF, preferred, nitrocellulose, Immobilon (or similar membrane), resin, etc.
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Proximate Analyses
- Total Nitrogen or Crude Protein (CP)
- Combustion Analysis (LECO) AOAC Official Method 990.03, 2006
- Kjeldahl, AOAC Official Method 984.13 (A-D), 2006
- Standard report of 'crude protein' in general matricies utilizes the calculation: 6.25 X Nitrogen value
- For other specific factors for the conversion of nitrogen content to protein content. (click here)
- Examples of other factors: Wheat (5.70); Dairy (6.38); Peanuts (5.46); Tree Nuts (5.30).
- Ash Determination (Ash)
- AOAC Official Method 942.05
- Acid Insoluble Ash
- AOCS Official Method Ba 5b-68
- Crude Fat (CF)
- By Ether Extraction, AOAC Official Method 920.39 (A)
- Acid Hydrolysis, baked goods & pet food, AOAC Official Method 954.02, 2006.
- For biological tissues and liquids: J. Folch, et al., J. Biol. Chem. 1957, 226, 497-509 (click here)
- Crude Fiber (CFiber)
- AOCS Approved Procedure Ba 6a-05
- AOAC Official Method 978.10, 2006
- Moisture (M)
- AOCS Am 5-04, Filter Bag (ANKOM).
- AOAC Official Method 934.01, 2006, vacuum oven.
- Freeze-drying - General Reference: Eur J Pharm and Biopharm 45:221-229(1998) Online
- Total Carbohydrates, crude 'by difference'
- Calculation, 100% - %(CP+ Ash + Crude Fat + M).
- Food Energy - methods of analysis and conversion factors. Report of a technical Workshop, Rome, 3-6 December 2002, Food and Agriculture Association of the United Nations, Room 2003. FAO Food and Nutrition Paper 77. FAO Corporate Document Repository
- Available Carbohydrates
- Calculation, 100% - %(CP+ Ash + Crude Fat + M + Crude Fiber).
- Non-structural Carbohydrates
- Calculation, 100% - %( NDF + CP + ether extract + ash)
- Gross Calories
- Calculation: Protein = 4 calories/gram, Carbohydrate = 4 calories/gram, Fat = 9 calories/gram.
- Merrill, A.L. and Watt, B.K. 1973. Energy Value of Foods...Basis and Derivation. Agriculture Handbook No. 74. U.S. Government Printing Office. Washington, DC. 105p (out-of-print article site). Also: Food Labeling - Requirements for FDA Regulated Products, by James L. Vetter, E.M. Meloan, Ed., AIB International, Manhattan, KS, 2007.
- Acid Detergent Fiber (ADF) AOAC Official Method 973.18 (A-D), 2006.
- ADF = cellulose & lignin.
- Neutral Detergent Fiber (NDF)
- NDF = cellulose, lignin, hemicelluose.
- JAOAC 56, 1352-1356, 1973.
- General reference: J Dairy Science 74(10):3583-3957(1991) Online
- Cellulose (Cel) AOAC Official Method 973.18 (A-D), 2006.
- Cellulose = Acid Detergent Fiber (ADF)- Acid Detergent Lignin (ADL).
- See J. Dairy Sci. 89:2168-2176(2006) as an excellent research paper on method.
- Hemicellulose Calculation: NDF - ADF.
- Nitrogen-free Extract Calculation: 100 - (Moisture + Ash + Protein +Fiber + Fat).
- "Feeds and Feeding, Handbook for the Student and Stockman", 21st Ed., by Frank B. Morrison, The
- Morrison Publishing Co., Ithaca, NY, 1950.
- Lignin AOAC Official Method 973.18 (A-D), 2006. Requires ADF and Cellulose analyses.
- Total Dietary Fiber (TDF) in Foods AOAC Official Method 985.29, 2006.
- * Note: TDF requires NDF analyses. Some matricies may also require proximates.
- Total, Soluble and Insoluble Dietary Fiber (Foods) AOAC Official Method 991.43
- * Note: Requires NDF analyses. Some matricies may also require proximates.
- Pepsin Digestibility of Animal Protein AOAC Official Method 971.09, 2006.
- 0.2% pepsin as per AOAC - includes crude protein determination.
- Protein Dispersibility Index (PDI) AACC Official Method 46-24, 2006.
- Nitrogen Solubility Index (NSI) AOCS Official Method Ba 11-65, 2006.
- Protein Solubility, KOH method J Anim Sci, 69:2918-2924, 1991.
- Soluble Non-Protein Nitrogen (SNPN)
- Prigge, E.C., et al., "Soluble Nitrogen and Acid Production of High Moisture Corn", J Animal Sci 42:490-496(1976).
- Wohlt, J.E., et al., "Measurement of Protein Solubility in Common Feedstuffs", J Dairy Sci 56:1052-1057(1973).
- Burroughs, et al., "Preliminary Observations Upon Factors Influencing Cellulose Digestion By Rumen Microorganisms", J Nutrition 40:9-24(1949).
- Trypsin Inhibitor Activity -Soybean (TIU/g) AACC Official Method 22-40, 2006.
- per Stauffer,C., Cereal Chem. 67(3):296-302(1990), 1 TIU = 8.6 x 10-12 mol of inhibitor. Determination of
- the absolute inhibitor concentration (i.e., mg/g) depends on the molecular weight of the inhibitor, itself.
- (Ex., Kunitz TI (Soybean) = 21.5 kDa; Bowman-Birk TI = 8 kDa; Bovine Pancreatic TI = 6.5 kDa;
- Ovomucoid TI = 28 kDa; Lima Bean TI = 8-10 kDa)
- Urease Activity (soybean meals, flour, millfeeds) AACC Official Method 22-90
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Sugars and Carbohydrate Analyses
- Sugar Profile I (glucose, fructose, sucrose, lactose, maltose)
- Free sugar profile only.
- Base references: Churms, S.C., “Carbohydrates” in Handbook of Chromatography, by Gunter Zweig and Joseph Sherma, Eds-in-Chief, CRC Press, FL, 1982; Kakehi, K. and Honda, S., “Silyl ethers of carbohydrates”, Chpt. 4, in Analysis of Carbohydrates by GLC and MS, Biermann, CJ and McGinnis, Eds., CRC Press, 1989, p. 43-85.
- Sugar Profile II (xylose, arabinose, fucose, ribose, mannose, glucose, galactose)
- Free or hydrolysis sugar profile. (please specify)
- Base references: “Monosaccharide Composition Analysis: Alditol Acetates" contributed by David Oxley, Graeme Currie, and Antony Bacic, Chapter 25, in Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.” The base protocol can be found on the website at Cold Spring Harbor ; J. R. Hudson, S. L. Morgan, A. Fox (1982) High resolution glass capillary columns for the gas chromatographic analysis of alditol acetates of neutral and amino sugars. J. High Res. Chromatogr. Chromatogr. Commun., 5, 285. ; Kiho, T., Ukai, S., and Hara, C., (1986) Simultaneous determination of the alditol acetate derivatives of amino and neutral sugars by gas-liquid chromatography. J. Chromatogr. 369:415. Fox, A., Morgan, S.L. and Gilbert, J. “Preparation of alditol acetates and their analysis by gas chromatobraphy (GC) and mass spectrometry (MS)”, Chapter 5, in Analysis of Carbohydrates by GLC and MS, Biermann, CJ and McGinnis, Eds., CRC Press, 1989, p. 87-117.
- Sugar Profile III (N-acetylglucosamine and N-acetylgalactosamine)
- Free or hydrolysis sugar profile. (please specify)
- Phytate, in Foods (hexaphosphorylinositol)
- AOAC Official Method 986.11, 2006 -- may be modified to utilize ICP to analyze phosphorus.
- JAOAC 74:32-36, 1991.
- Reading reference: N.R. Reddy, et al., Phytates in Cereals and Legumes, CRC Press (1989)
- Sucrose, Raffinose, Stachyose, Verbascose
- J. Chromatogr., 153:539(1978); Biochim. Biophys. Acta, 222:339(1970); Handbook of Chromatography, 1982, Gunter Zweig and Joseph Sherma, Eds. In Chief, Shirley C. Churms, Ed., CRC Press, Boca Raton, FL.
- Total Reducing Sugars (colorimetric)
- Anal. Chem.. 28:350(1956) (colorimetric).
- Quantitative estimation of simple sugars, oligo- and polysaccharides in solution.
- Not applicable with amino sugars and sugar alcohols.
- Uronic acid (as glucuronic acid)
- Analytical Biochemistry 4, 330-334 (1962) (Colorimetric; Carbazole).
- Anal. Biochem. 57:268-273 (1974) (Colorimetric; Harmine alternative to carbazole)
- Glycerol (also referred to as glycerin)
- Am. J. Enol. Vitic. 21:4:205-212 (1970) (gas-liquid chromatography, trimethylsilyl derivative).
- Lactose
- JAOAC 62(2):262-265(1979)(gas-liquid chromatography, capillary column, trimethylsilyl derivative).
- see also Biotechnology and Bioengineering 28:1119-1121 (uses trimethylsilylimidizole for silylation).
- Starch, Total (amylose/amylopectin)
- Base method: Amer. Assoc. Cereal Chemists, Approved Methods, no. 76-13.
- Modified: Sigma (St. Louis, MO) Starch Assay Kit, Product Code STA-20.
- Megazyme assay kit K-AMYL
- β-Glucan (Please note that this method is only by special request, at this time.)
- AOAC Official Method 995.16; AACC Method 32-23.
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Lipids, Fats and Oils
- Fatty Acid Profile - saturated, mono- and polyunsaturates *
- AOAC Official Method 996.06 [Analysis of methyl esters by Capillary GLC]
- AOCS Official Method Ce 2-66 [Preparation of Methyl Esters of Fatty Acids].
- AOAC Official Method 965.49 [Preparation of Methyl Esters of Fatty Acids].
- AOAC Official Method 969.33 [Oils and Fat, Boron Trifluoride method].
- Omega 3 Fatty Acid Profile *
- AOCS Official Method Ce 1b-89 [Marine Oil - Fatty Acid Composition by GLC]
- AOCS Official Method Ce 1d-91 [n–3 and n –6 Fatty Acids by Capillary GLC]
- Trans Fats - total * AOCS Official Method Ce 1f-96 [Capillary GLC]
- Total Fatty Acids (Crude) AOAC Official Method 996.06 & AOCS Official Method Ca 5b-71
- Free Fatty Acids AOCS Official Method Ca 5a-40
- Volatile Fatty Acids Cold Spring Harb Protoc, 2017(5). doi: 10.1101/pdb.prot085464
- Conjugated Linoleic Acids (CLA, reported as 'total')
- AOCS Official Method Ce 1h-05 (include c9,t11- and c10,t12- octadecadienoic acids)
- Fat by Acid Hydrolysis AOAC Official Method 954.02, 2006
- Folch Extraction for Total Lipids from Animal Tissues
- Folch, J. et al., J. Biol. Chem. 226:497, 1957. (chloroform : methanol : water, 8:4:3, v/v/v)
- Bligh, E.G. and Dyer, J., Can. J. Biochem. Physiol. 37:911, 1959. (Folch extraction, variable solvent volumes, larger scale preparations)
- Peroxide Value of Fats and Oils
- AOAC Official Method 965.33
- AOCS Official Method Cd 8-53, Acetic acid - chloroform method (requires 5 milliliters of fat or oil)
- 4-Hr. or 20-Hr. AOM Peroxide Value
- Active Oxygen Method, AOM; AOCS Method Cd. 12-57
- (Please note: Official method requires 2 x 20 milliliters of fat or oil (i.e.: 40 mL total)
- Iodine Value AOCS Recommended Practice Ja 14-91
- TBA (Thiobarbituric Acid) Rancidity
- AOCS Official Method Cd 19-90
- TBARS (2-Thiobarbituric Acid Reactive Substances, non-AOCS)
- Current Protocols in Food Analytical Chemistry (2001), D2.4.1-D2.4.18
- Color, FAC AOCS Official Method Cc 13a-43
- Cholesterol May require sample preparation depending on sample type (inquire).
- AOAC Official Method 994.10 [in Foods by GLC]
- AOAC Official Method 970.51 [in Vegetable Fat/Oils by GLC]
- Phospholipids, Total AOAC Official Method 15:949.07
- Choline, Total Glick, D., J. Biol. Chem. 156:643, 1944
- Total Carotenoids AOAC Official Method 938.04
- Xanthophylls and Carotene AOAC Official Method 970.64
- Antioxidants **
- Ethoxyquin, in eggs, tissue and fat.
- BHA, BHT, PG, DG, TBHQ (in fats and oils)
*methods require 'crude fat' analysis for feeds, foods, grains, etc., that are not fats and oils. ** estimated ranges of all supplements must be declared.
Other Lipid, Fat, Specific Fatty Acids and Oil Analyses
- Insoluble AOCS Official Method Ca 3a-46
- Unsaponifiable AOCS Official Method Ca 6a-40
- Moisture AOCS Official Method Ca 2c-25
- Saponification Value AOCS Official Method Cd 3-25
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Inorganic, Elemental, Mineral Analyses
- Inductively Coupled Plasma - Mass Spectrometry (ICP-MS)
- AOAC Official Method 993.14 Trace Elements in Waters and Wastewaters
- AOAC Official Method 2015.01 Heavy Metals in Food (Microwave assisted acid digestion).
- Inductively Coupled Plasma - Optical Emission Spectoscopy (ICP-OES)
- AOAC Official Method 985.01(A, B, D) Metals and Other Elements in Plants and Pet Foods.
- AOAC Official Method 990.08 Metals in Solid Wastes ICP Spectroscopic Method.
- Flame Atomic Absorption Spectroscopy (FAAS)
- AOAC Official Method 968.08, Minerals in Animal Feed and Pet Food.
- AOAC Official Method 975.03B(b) Metals in Plants and Pet Foods Atomic Absorption Spectrophotometric Method (applicable to Ca, Cu, K, Mg, Mn, and Zn) nitric-perchloric wet ash sample preparation with hydrochloric acid sample matrix
- Flame Emission Photometry - sodium, potassium
- AOAC Official Method 956.01
- Sulfur, gravimetric AOAC Official Method 956.01, 2006.
- Boron, volumetric AOAC Official Method 958.03, 2006.
- Chloride, manual titration AOAC Official Method 9.15.01, 943.01, 2006.
- Iodine, volumetric AOAC Official Method 935.14, 2006.
- Silicate, gravimetric AOAC Official Method 920.08, 2006.
- Titanium in Feeds & dried intestinal / feces
- Journal of Animal Science (2004) 82:179-183.
- Chromium in Feeds and dried Feces
- Nitric/Perchloric Wet Ash Sample Preparation: Official Method 968.08D(b) - see also 935.13
- AOAC Official Method 990.08, 2006, ICP method.
- Selenium AOAC Official Method 996.16(G): Selenium in Feeds and Premixes: ICP method, 2006.
- Ytterbium in Feeds and dried Feces J. Dairy Sci. 75:2176-2183 I1992)
- Phosphorus, gravimetric AOAC Official Method 966.01 (plant tissue)
- Other mineral analyses (please inquire)
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Fertilizer Analyses
- Nitrogen (Kjeldahl method)
- AOAC Official Method 978.02, See also 920.02A(a),(c),(f),(i),(j),(k); 970.02A (a),(b); 970.02B & 955.05C.
- Available Phosphorus AOAC Official Method 960.03B(a)(1) & 978.01 A to H
- Soluble Potassium AOAC Official Method 983.02B(a) or (b) & 983.02 A, C-J
- Moisture vacuum dessication, AOAC Official Method 965.08B
Secondary and Micronutrients
- Calcium, Magnesium, Zinc, Copper, Iron, Manganese, Sodium
- Silicates in plant tissue
- Sulfur AOAC Official Method 980.02
- Boron AOAC Official Method 982.01B(a); determination 982.01 A, C and D
- Chloride
Limestone Analyses
- Calcium Carbonate Equivalent (CCE) AOAC Official Method 955.01A-B
- Magnesium (Mg)
- CCE by Neutralization and Magnesium (Mg)
- CCE plus sieve analysis profile, ENM* per ton
- CCE, Magnesium and sieves profile, ENM and EMG** per ton
- Sieve analysis profile only
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