The biological properties (bioactivity) of the different origins and production methods are frequently debated, although on the effect of betaine numerous publications are available (Eklund et al. 2005, Ratriyanto et al. 2009). In particular, the osmoregulatory properties of chemically synthesized betaine hydrochloride compared with those produced from natural betaine sources i.e. sugar beet are in question. On the other hand also betaine monohydrate and anhydrous are criticized for being very hygroscopic, and thus negatively influencing the stability of feed.
Biological value of various Betaine sources
The aim of the first study was to evaluate the biological value of various betaine sources (hydrochloride, monohydrate and anhydrous), because in this respect the question is often asked whether osmoregulatory properties of betaine hydrochloride are equal betaine anhydrous. To investigate this question, an in vitro study was carried out in which a total of 4 samples were studied by simulating stomach conditions (betaine monohydrate or betaine anhydrous from sugar extraction (MonoEx or AnhydEx respectively), betaine hydrochloride or betaine anhydrous from chemical synthesis (HydroSyn or AnhydSyn respectively)). The samples (10 mg) were dissolved in 10 ml H2O, adjusted with a 37% HCl solution to pH 2.3 and pH 4.5 and subsequently analyzed by LC-MS and direct MS. The mass spectrometry was selected as a well defined chemical analysis because it is ideal for obtaining an insight into the chemical structure of a molecule. The mass spectra were determined on the one hand on an Agilent 1100 system with a Phenomenex Luna C18 column and DAD, in this case, the flow rate of 1 ml/min with 5 mM NH4OAc solution in water-acetonitrile was adjusted as the mobile phase. For direct MS an Agilent 1100 system was used with direct injection.
At pH 2.3 betaine was measured in the LC-DAD (220 nm) for all four samples after about 2.7 min. In the positive electro spray ionization mode of the total ion chromatogram (TIC) the protonated betaine molecules [M+H]+ at m/z 118 (m/z retention time of approximately 2.7 min, figure 1) and also the cluster [2M+H]+ at m/z 235 showed for all four samples a high intensity. In the negative electro spray ionization mode of the total ion chromatogram the ions [M+Cl]- at m/z 152/154 showed, on the other hand, only a low intensity. Similar results were obtained in the direct MS analyzes (table 1). Based on these data it can be concluded that betaine dissolved in acidic solutions (pH 2.3) leads to similar quantitative results by LC-MS analysis.