Quantification of sugars from malt by liquid chromatography and refractive index detection

Malt plays an important role for the brewing industry since it is the main ingredient of beer. The germination of barley grains during malting increases the content of reducing sugars and amino acids when compared with barley composition. During thermal treatment of green malt, Maillard reactions occur and influence the levels of carbohydrates and amino acids. This undoubtedly affects the fermentation process and consequently beer composition [1]. In this study an extraction procedure for the HPLC analysis of free sugars in malt was optimized. Different solvents were tested for sugar extraction from malt and two different chromatographic methods with refractive index detection were compared for separation and quantification of sugars in malt samples. Fructose, glucose, sucrose, and maltose were separated by liquid chromatography on a NH2-column using isocratic elution with acetonitrile: water, 75:25 (v/v) and were also analyzed by a cationic resin-based column (SupelcogelCa column) using ultra-pure water as mobile phase. Pilsner malt samples were milled. Sugars were extracted using water, 50%, and 80% (v/v) ethanol. Extraction temperatures of 25 ºC and 50 °C, as well as simple or multiple extractions were combined with the different solvents. The regression equations revealed a good linear relationship between the amounts of each sugar and the IR response with r values that exceeded 0.99 in both columns and for all sugars. The intra-day and inter-day variations either for retention time and also areas were lower than 1.5 % for both columns. Limits of detection and quantification were 0.150 mg/g and 0.5 mg/g for NH2 column and 0.04 mg/g and 0.1 mg/g for SupelcogelCa column. This last column presents higher sensibility and good resolution for monosaccharides but disaccharides are co-eluted, so the separation of sucrose and maltose was not possible into this column. The run time was 15 minutes. Considering the NH2 column good separation of disaccharides, sucrose and maltose, was observed however the separation of glucose and fructose was not completely resolved. In 10 minutes all sugars were eluted. The optimum extraction protocol for the determination of soluble sugars from malt includes the use of 50% ethanol as solvent. Room temperature and simple extraction provided similar results as 50ºC or multiple extractions presenting yields higher than 80%. Lower yields were seen when higher alcohol percentage was used for extraction, namely for maltose, probably due to the lower solubility in ethanol. Whereas using only water as extraction solvent is not recommended because it does not inactivate malt enzymes and reproducibility difficult to achieve. In conclusion both HPLC columns presented advantages and limitations; however the NH2 allows the separation of the four main sugars in shorter time. Malt is a difficult matrix due to the high enzyme activity, however the optimal extraction conditions for sugars determination was achieved through the use of ethanol 50% as extraction solvent that prevents enzyme activity while maintaining good solubility of sugars.