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Objective: The impact of increased fructose consumption on carbohydrate metabolism is a topic of current interest, but determination of serum level has been hindered due to low concentration and interference from serum glucose. We are reporting a method for the quantification of glucose and fructose in clinical samples using gas chromatography/mass spectrometry (GC/MS). The accuracy and precision of GC/MS and an enzymatic assay were compared., Design and Methods: Mass spectrometry fragmentation patterns of methyloxime peracetate derivatized aldose and ketose were determined. Unique fragments for glucose and fructose were used for quantitative analysis using isotope labeled recovery standards., Results: Methyloxime peracetate derivatives of glucose and fructose showed characteristic loss of acetate (M-60) or ketene (M-42) under chemical ionization (CI). Under electron impact (EI) ionization, a unique C1-C2 fragment of glucose was formed, while a C1-C3 fragment was formed from keto-hexoses. These unique fragments were used in the quantitative assay of glucose and fructose in clinical samples. In clinical samples, the GC/MS assay has a lower limit of detection than that of the enzymatic assay. In plasma samples from patients evaluated for diabetes the average serum glucose and fructose were 6.19+/-2.72 mM and 46+/- 25.22 microM. Fructose concentrations in many of these samples were below the limit of detection of the enzymatic method., Conclusion: Derivatization of aldose and ketose monosaccharides to their respective O-methyloxime acetates for GC/MS analysis is a facile method for determination of serum/plasma glucose and fructose samples., (Copyright 2009 The Canadian Society of Clinical Chemists. All rights reserved.)