625 TOTAL CRUDE PROTEIN IN FEED MATERIALS - LECO METHOD
Protein
Protein by Nitrogen Gas Analyzer utilizing induction furnace and thermal conductivity (LECO FP-528).
Summary: This method quantitatively determines the amount of nitrogen in all forms (ammonium, nitrate, protein and heterocyclic nitrogen) in botanical materials using an induction furnace and a thermal conductivity detector. Samples are ignited in an induction furnace at approximately 900°C in helium and oxygen environment in a quartz combustion tube. An aliquot of the combustion gases is passed through a copper catalyst to remove oxygen and convert nitrous oxides to N2, scrubbed of moisture and carbon dioxide, and nitrogen content determined by thermal conductivity. Total crude protein is calculated from the nitrogen content of the feed material, based on sample type. The method has a detection limit of 0.1% protein (dry basis) and is generally reproducible within 5%.
AOAC Official Method 990.03, in Official Methods of Analysis of AOAC International, 16th edition, Volume I Chapter 4, pp 18-19.
640 ACID DETERGENT FIBER (ADF), LIGNIN (Ash Free), ACID DETERGENT INSOLUBLE NITROGEN (ADIN), TOTAL DIGESTIBLE NUTRIENTS (TDN) AND CELLULOSE - REFLUX METHOD
ADF, ADF (Ash Free), Lignin (Ash Free), ADIN, TDN, Cellulose
Determination of the amount of acid detergent fiber and lignin in feed materials using acid detergent, sulfuric acid and heat.
Summary: This procedure is used for the determination of acid detergent fiber (ADF) and ADF-derived tests including lignin, cellulose, TDN (total digestible nutrients) and ADIN (acid detergent insoluble nitrogen) in all types of forages. A hot, acidified detergent solution is used to dissolve cell solubles, hemicellulose and soluble minerals leaving a residue of cellulose, lignin, and heat damaged protein and a portion of cell wall protein and minerals (ash). The analyses require approximately 10 g, but a minimum of 20 g is requested to allow for quality control samples.
ADF is determined gravimetrically as the residue remaining after acid detergent extraction. Lignin is determined gravimetrically after the ADF residue is extracted with 72% H2SO4 and ashed. Cellulose is determined by subtracting the pre-ash lignin value from the ADF value. TDN is a calculation based on the ADF value and is reported based on the formula for alfalfa. The TDN result is standardized to a 90% dry matter value. ADIN is determined by nitrogen analysis (combustion) of a sub-sample of the ADF residue. (See SOP 525 for information on nitrogen analysis.)
| Analyte | Typical MDL |
| ADF | 0.5 % |
| Lignin | 1.0 % |
| Cellulose | 1.0 % |
| TDN (@90%DM) | 0.5 % |
| ADIN | 0.05 % |
AOAC Official Method 973.18, Fiber (Acid Detergent) and Lignin in Animal Feed, in Official Methods of Analysis of AOAC International, 16th edition (1997), Chapter 4, pp. 28-29, AOAC International, Arlington, VA.
650 NEUTRAL DETERGENT FIBER (NDF) and HEMICELLULOSE - REFLUX METHOD
NDF
Determination of the amount of neutral soluble fiber material and lignin in feed materials using a detergent and heat.
Summary: This procedure is used for the determination of amylase-treated neutral detergent fiber (NDF) in feed materials using a neutral detergent solution and heat. Sodium sulfite is used in the procedure to aid in the removal of some nitrogenous matter. Heat-stable amylase is used in the procedure to allow for the removal of starch and to inactivate potential contaminating enzymes that might degrade fibrous constituents. Hemicellulose is determined by performing the NDF procedure followed by the ADF procedure (see SOP 640 for ADF details). The detection limit for NDF and hemicellulose are approximately 0.5 %, and 1.0 % respectively. On homogenous sample material, the method is generally reproducible within 8% (relative). The analysis requires approximately 1 g, but 10 g is requested to allow for quality control. Results for NDF can be reported on an ash-free basis upon client request.
AOAC Official Method 2002.04, Amylase-Treated Neutral Detergent Fiber in Feeds, Using Refluxing in Beakers or Crucibles, in Official Methods of Analysis of AOAC International, (2006), Chapter 4, pp. 48-55, AOAC International, Arlington, VA.
660 ACID DETERGENT FIBER (ADF), LIGNIN AND TOTAL DIGESTIBLE NUTRIENTS (TDN) - ANKOM METHOD
ADF @ 3%, ADF @ 8%, Lignin, TDN
Determination of the amount of acid soluble fiber material and lignin in feed materials using the Ankom fiber analyzer.
Summary: This method semi-quantitatively determines the amount of acid soluble fiber material and lignin in feed materials using sulfuric acid and heat. It is based on the Ankom Technology method for determining acid detergent fiber and lignin. It is a relatively simple procedure when compared to crude fiber determination. The method has a detection limit of 0.1% ADF percentage (dry basis) and is generally reproducible within 8% (relative). Total digestible nutrients (TDN) is a calculation based on the ADF value.
Ankom Technology, 1998. Method for Determining Acid Detergent Fiber.
Ankom Technology, 1998. Method for Determining Acid Detergent Lignin in Beakers.
665 NEUTRAL DETERGENT FIBER (NDF) - ANKOM METHOD
NDF
Determination of the amount of neutral soluble fiber material and lignin in feed materials using the Ankom fiber analyzer.
Summary: This method semi-quantitatively determines the amount of neutral detergent soluble fiber material feed materials using the Ankom Fiber Analyzer. It is based on the Ankom Technology method for determining neutral detergent fiber. It is a relatively simple procedure when compared to crude fiber determination. The method has a detection limit of 0.1% NDF percentage (dry basis) and is generally reproducible within 8% (relative).
Ankom Technology, 1998. Method for Determining Neutral Detergent Fiber.
670 ASH CONTENT IN BOTANICAL MATERIALS - GRAVIMETRIC METHOD
ASH
Gravimetric determination using high temperature.
Summary: This method quantitatively determines the amount of ash in feed materials based on the gravimetric loss by heating to 550°C for a period of at least eight (8) hours. The method has a detection limit of 0.01% is generally reproducible within 7% (relative).
J. Assoc. Off. Anal. Chem. 25:857; 26:220.
680 SOLUBLE CARBOHYDRATES - HPLC METHOD
Soluble Carbohydrates
Determination of the free sugars glucose, fructose and sucrose by HPLC.
Summary: This method quantitatively determines the amount of the free sugars fructose, glucose and sucrose in botanical materials. Sorbitol content can also be determined upon request. Samples are extracted by hot deionized water and analyzed by HPLC with mass selective detection. The analysis uses a Phenomenex Luna NH2 (250 mm x 4.6 mm) HPLC column at a flow rate of 2.75 mL min-1 acetonitrile:water (78:22). The method has a detection limit of 0.2% and is reproducible within 10% (relative).
Johansen, H. N., Glitso, V. and Knudsen, K. E. B. 1996. Influence of Extraction Solvent and Temperature on the Quantitative Determination of Oligosaccharides from Plant Materials by High-Performance Liquid Chromatography. J. Agric. Food Chem. 44:1470-1474.
690 TOTAL GLUCOSE FOR TOTAL NONSTRUCTURAL CARBOHYDRATES (TNC) AND STARCH
TNC, Starch
Determination of total non-structural carbohydrates and starch by enzymatic hydrolysis and HPLC determination of the glucose content.
Summary: Nonstructural carbohydrates are those that can be accumulated and then readily mobilized in order to be metabolized or translocated to other plant parts. This method quantitatively determines the amount of the total glucose following enzymatic hydrolysis with hydrolysis. Total non-structural carbohydrates (TNC) is the sum of total glucose, free fructose and free sucrose. Starch is the total glucose minus the free glucose multiplied by 0.9. The free carbohydrates are determined by a separate analysis. The samples for total glucose are enzymatically hydrolyzed at 55°C with amyloglucosidase for 12 hours and analyzed by HPLC with mass selective detection. The analysis uses a Phenomenex Luna NH2 (250 mm x 4.6 mm) HPLC column at a flow rate of 2.75 mL min-1 acetonitrile:water (78:22). The method has a detection limit of 0.5% and is reproducible to within 10% (relative).
Smith, Dale. Removing and Analyzing Total Nonstructural Carbohydrates from Plant Tissue. Wisconsin Agric. Exp. Sta. Res. Report 41. 1969.
By Special Request
Quantitation of resveratrol by HPLC with tandem mass spectrometry detection.
Summary:This method quantitatively determines the amount of the resveratrol in botanical materials. Freeze dried samples are extracted by homogenization with methanol, followed by heating at 60 degrees for 30 minutes and analyzed by HPLC with tandem mass selective detection. The analysis uses a Phenomenex SynerGi Hydro-RP (150 mm x 4.6 mm) HPLC column at a flow rate of 0.75 mL min-1 and a gradient elution using methanol:water. The method has a detection limit of 0.2 ppm.
Romero-Perez, A. I., Lamuela-Ravento, R. M., Andres-Lacueva, C. and de la Torre-Boronat, M. C. Method for the Quantitative Extraction of Resveratrol and Piceid Isomers in Grape Berry Skins. Effect of Powdery Mildew on the Stilbene Content. J. Agric. Food Chem. 2001, 49, 210-215.
Wang, Y., Catana, F., Yang, Y., Roderick, R. and van Breeman, R. An LC-MS Method for Analyzing Total Resveratrol in Grape Juice, Cranberry Juice, and in Wine. J. Agric. Food Chem. 2002, 50, 431-435.