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

Abstract

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 þ ). 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 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 À1 . This assay is very accurate over the range of betaine concentrations from 15 to 650 mg ml À1 , with a lower detection limit in feeds of approximately 500 mg g À1 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 À1 ), wheat (3.96 mg kg À1 ), wheat middlings (4.98 mg kg À1 ) and poultry meal (0.77 mg kg À1 ). Betaine in corn and soybean meal was not detectable by this method, even when 16 g of sample was used (<125 mg kg À1 ). Betaine present in several feed ingredients should influence choline supplementation to animal feeds and may have implications for human health.

Recovery from adverse effects of heat stress on slow-growing chicks in the tropics 1: Effect of ascorbic acid and different levels of betaine

“…The Bet in corn and soybean meal were reported to be below the detectable level (Chendrimada et al 2002), and was not detected in corn-soybean meal diet (Waldroup and Fritts 2005). On the other hand, data related to AA in animal feedstuffs are rare.…”

A Review of Extraction and Analysis: Methods for Studying Osmoregulants in Plants

“…As betaine is charged at low pH, ion exchange columns [58,62,108] are commonly used for its separation. However, the use of an RP column [36] has also been reported where retention is increased by derivatization or the addition of an IPR to the mobile phase.…”

“…However, the use of an RP column [36] has also been reported where retention is increased by derivatization or the addition of an IPR to the mobile phase. For detection, UV is most commonly used [62,[108][109][110][111] which is most likely due to ease of access to this detector. However, use of RI [36] and MS [112] has also been described for betaine analysis.…”

“…However, use of RI [36] and MS [112] has also been described for betaine analysis. As betaine lacks a choromophore, UV detection is only possible at low wavelengths [58,62,108] and with relatively high LOD (e.g., 15 µg/mL or 0.128 µM) [108], for sensitive detection derivatization is essential (Table 3) [109,111]. Betaine and its analogues were determined in vegetables after dertivatization with 2-naphthacyl trifluoromethane sulfonate for UV detection, and separation was performed using a RP column [111] achieving detection limits as low as 1 µg/g or 0.0085 µM.…”

Potentiometric Batch and Flow Injection Analysis of Betaine Hydrochloride

“…1 Several analytical techniques have been reported for the quantitative determination of Be.Cl in pure solutions and pharmaceutical preparations. Among these are HPLC, [2][3][4][5][6][7] TLC, 8 capillary electrophoresis, determination of ions in solution with high selectivity. Most ISE's are of low-cost, their use and maintenance being very simple, assay procedures involving such electrodes are generally simple and fast.…”