A Sugaromics Method for Combined Targeted and Untargeted Sugar Profiling: Fit-for-Purpose Validation of a...

The composition of sugar compounds in human biofluids is affected by various factors, including diet, health, demographic background, and lifestyle. Accurate quantification of this profile enables identification and application of biomarkers linked to health and nutrition, offering valuable insights into the mechanistic background of sugar metabolism. However, existing methods typically quantify only a few sugar compounds simultaneously, constraining full assessment of the sugar profile. Hence, we propose a comprehensive two-dimensional gas chromatography coupled with a mass spectrometry-based detector sugaromics method applying a nonpolar-medium polar column setup. The method is a combination of a targeted and untargeted approach to absolutely quantify various sugars in urine (n = 40) and serum (n = 36) and simultaneously identify untargeted sugar compounds in urine (n = 35) and serum (n = 22). The method was evaluated through a fit-for-purpose validation using the guidelines of the U.S. Food and Drug Administration and of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use as guiding principles. The majority of sugars demonstrated satisfactory validity parameters in terms of linearity, lower and upper limit of quantification, precision, accuracy, and carryover. Furthermore, the validated method was applied to human urine and serum samples (n = 40), indicating quantifiable analyte concentrations within the expected range. In this respect, for healthy adults, absolute concentrations of seven sugars in urine and 14 in serum were reported for the first time. In conclusion, the newly developed method combining targeted and untargeted approaches demonstrates good performance and is promising for application in human studies investigating health and nutrition.