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

Abstract

AbstractA 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 mM KH2PO4 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