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BACKGROUND:The increasing importance of plant-based proteins in the food sector makes a reliable compositional analysis of plant-based high-protein ingredients a necessity. Specifically, the quantification of short-chain carbohydrates is relevant for multiple areas, including food product development, food labelling and fundamental food chemistry and food technology research. Commonly used extraction procedures for subsequent high-performance liquid chromatographic separation and quantification of short-chain carbohydrates have been discussed controversially regarding a range of complications that can potentially lead to inaccurate sugar determination. The present study compares the sugar levels in wheat flour and wholemeal wheat flour determined with different aqueous and ethanolic extraction procedures. These procedures included measures to prevent enzyme activity and microbial growth, which represent two of the most relevant challenges in sugar extraction from food samples. RESULTS: Differences in sugar levels (sum of sucrose/maltose, glucose and fructose) as high as 1.8% dry matter (wheat flour) were observed between the employed extraction procedures. Ethanolic extraction (80% ethanol in ultrapure water) with the use of the antimicrobial agent sodium azide but without Carrez clarification was identified as most promising for sugar determination in plant-based high-protein ingredients.CONCLUSION: A screening of high-protein ingredients derived from cereals (wheat gluten), pseudocereals (quinoa, amaranth, buckwheat) and legumes (soy, pea, lupin, lentil, carob, chickpea, faba bean) concerning their levels of sucrose, maltose, glucose and fructose confirmed the applicability of the chosen extraction procedure.