Novel GC–MS/MS Strategy for Fructose Quantification and Stable Isotope Tracing: Development, Validation, and SIM vs MRM Comparison

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Abstract High dietary fructose consumption is a major contributor to the development of obesity and related cardiometabolic diseases, highlighting the need for accurate assessment of fructose metabolism in humans. Stable isotope tracer approaches, such as 13 C 6 -fructose, require highly sensitive and specific analytical methods to quantify both concentrations and isotopic enrichments. In this study, we developed and validated a robust gas chromatography–triple quadrupole mass spectrometry (GC– QQQ)-based method for the simultaneous measurement of unlabeled and 13 C 6 -fructose in human plasma. The method employs oximation and per-acetate derivatization, and demonstrates high specificity and accuracy. Intra- and inter-assay precision were below 10%, with no detectable carry-over, and a lower limit of quantification (LLOQ) of 0.1 nmol/mL for concentration and 0.02 molar percent excess (MPE%) for enrichment and no interference from glucose. We further compared data acquisition using multiple reaction monitoring (MRM) and selected ion monitoring (SIM). MRM showed superior performance at the low concentrations and enrichment levels characteristic of clinical plasma samples, resulting in improved sensitivity and lower LLOQs compared to SIM. Overall, this validated method provides a sensitive and reliable approach for fructose tracer studies in humans. Its application will facilitate robust investigations into fructose metabolism, and its role in metabolic dysregulation and obesity-related disease.

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