Determination of the betaine content of feed ingredients using high‐performance liquid chromatography

A series of studies was conducted to establish a methodology for the accurate and efficient determination of betaine in different feed ingredients. The final methodology involves an extraction step in which the feed sample is heated for 3 h in a methanolic KOH solution using a Goldfisch apparatus. Impurities are removed by the addition of activated charcoal and concentrated (36%) HCl. After centrifugation the extractant is passed through a strong cation exchange resin (Dowex 50W‐X12, H + ). The betaine retained in the column is eluted with 1.5  N HCl. A 2 ml aliquot of the elute is air dried and reconstituted with 1 ml of deionised water. HPLC separation with a cation exchange column (Partisil SCX‐10) is used for the separation of betaine from other compounds. The mobile phase is kept constant at 50 m M KH 2 PO 4 in water, and eluted compounds are detected by UV absorbance (200 nm). The flow rate is maintained at 1.5 ml min −1 . This assay is very accurate over the range of betaine concentrations from 15 to 650 µg ml −1 , with a lower detection limit in feeds of approximately 500 µg g −1 when 4 g of sample is extracted. Recovery assays done with standard betaine hydrochloride and hard red wheat resulted in a consistent recovery of 80%. Betaine content was quantified in several feed ingredients, including alfalfa (1.77 mg kg −1 ), wheat (3.96 mg kg −1 ), wheat middlings (4.98 mg kg −1 ) and poultry meal (0.77 mg kg −1 ). Betaine in corn and soybean meal was not detectable by this method, even when 16 g of sample was used (<125 mg kg −1 ). Betaine present in several feed ingredients should influence choline supplementation to animal feeds and may have implications for human health. © 2002 Society of Chemical Industry