Determination of Neutral Monosaccharides as Per-O-methylated Derivatives Directly from a Drop of Whole Blood by Gas Chromatography-Mass Spectrometry Ionel Ciucanu,* ,† Luminit ̧ a Pilat, † Cristian Ionut ̧ Ciucanu, ‡ and Eugen Şis ̧ u* ,‡ † Department of Chemistry, West University of Timisoara, Strada Pestalozzi 16, RO-300115 Timisoara, Romania ‡ Faculty of Medicine, “Victor Babes” University of Medicine and Pharmacy of Timisoara, Piat ̧ a Eftimie Murgu 2, RO-300041 Timisoara, Romania ABSTRACT: A new analytical procedure was developed for the simultaneous quantification of neutral monosaccharides from a drop of whole blood using gas chromatography-mass spectrometry analysis (GC-MS) of their per-O-methylated derivatives. The per- O-methylation reaction with methyl iodide and solid sodium hydroxide in methyl sulfoxide was used for the first time for analysis of blood monosaccharides. A blood drop volume of 0.6 μL was used without special purification. The elimination of the undesirable components was carried out during methylation in the presence of a strong base and by liquid extraction of the per-O-methylated monosaccharides. The neutral monosaccharides with an anomeric center gave four per-O-methylated isomers, which were well- separated using a capillary column. Identification was done by electron impact mass spectrometry fragmentation, retention times, and library searching. The limits of detection were determined for standards and varied from 2.0 to 2.3 ng mL -1 . Recoveries for human blood samples varied from 99.22% to 99.65%. The RSD values ranged from 1.92 to 2.37. The method is fast, sensitive, reproducible, and an alternative to current methods for quantitative analysis of blood monosaccharides. T he human body regulates blood sugar concentration, maintaining a condition of equilibrium that depends on every individual alone. The changes in plasma monosaccharide concentrations above or below the normal range are the echo of the changes in cellular sugar and indicate the presence of some diseases. 1 Elevated glucose levels are present in diabetes mellitus, Cushing’s syndrome, liver disease, and hyperthyroid- ism, while decreased glucose levels are present in Addison’s disease, hyperinsulinism, and hypothyroidism. 2 The most prevalent of these diseases is diabetes mellitus. Left untreated, the diabetes can lead to complications affecting heart, kidney, teeth, eyes, and nerves. Mannose is a metabolic product in candidasis caused by infection with species of the genus Candida. A high concentration of mannose in body fluids is an indicator of the disseminated form of the Candida infection. 3 Galactose is recognized for its effect in galactosemia, which is a severe, hereditary disease resulting from the inability to metabolize galactose. 4 1,5-Anhydroglucitol (1,5-AG) levels could allow differentiation between subtypes of diabetes. 5 1,5- AG is a marker that responds to changes in glycemia over the course of weeks that may aid in the modification of therapy. 6 Fructose is a blood monosaccharide that is connected with obesity. 7 Current analysis of blood sugars involves a chemical or enzymatic reaction between blood sugars and a specific reagent followed by spectroscopic, electrochemical, or chromatographic measurement. With an enzymatic reagent, the method is specific for one sugar, and it cannot give information about the concentration of the other sugars. The chromatographic analysis can give information about all the monosaccharides from blood. The most often used methods are gas chromatography (GC) and liquid chromatography (LC). High-performance liquid chromatography (HPLC) can be done with a great variety of columns and detectors, with their advantages and disadvantages. GC is a good alternative for analysis of sugars because the separation power is good and the retention time is relatively short. Due to their high polarity and low volatility, all sugars need to be converted into volatile and stable derivatives for GC-MS analysis. Classical derivatization methods consist of substitution of the hydroxyl groups of neutral carbohydrates in order to increase their volatility. Methyl ethers, acetates, trifluoroacetates, and trimethylsilyl ethers are the most common derivatives used for the determination of carbohydrates. 8,9 Trimethylsilyl (TMS) ethers are the most widely used derivatives 10 because they are appropriate for a wide range of functional groups and have a good volatility. However, the TMS ethers are sensitive to decomposition even in the presence of air moisture and some Received: June 15, 2015 Accepted: October 7, 2015 Published: October 7, 2015 Article pubs.acs.org/ac © 2015 American Chemical Society 10856 DOI: 10.1021/acs.analchem.5b02252 Anal. Chem. 2015, 87, 10856-10861