Simultaneous determination of glucose, 1,5‐anhydrod‐glucitol and related sugar alcohols in serum by high‐performance liquid chromatography with benzoic acid derivatization

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Abstract
A new, simple and sensitive pre‐column high‐performance chromatographic method for the determination of diabetes marker d‐glucose, 1,5‐anhydro‐d‐glucitol and related compounds is reported. Sugars (d‐glucose, d‐galactose, d‐mannose, sucrose and arabinose) were derivatized with benzoic acid (BA) at 80°C for 60 min. l‐Fucose, fructose, d‐lactose, l‐rhamnose, arabinose and ascorbic acid were not reacted. Sugar alcohols (xylitol, erythritol, mannitol, sorbitol myo‐inositol) were also derivatized with BA at 80°C for 60 min. The fluorescence derivatives were separated on a TSK amide 80 column (4.6 mm i.d. × 250 mm, 5 µm) with acetonitrile–50 mm acetate buffer (pH 5.6; 4:96, v/v) as the mobile phase. The detection wavelength of beizoic acid derivatives was λex 275 nm and λem 315 nm. The detection limits of sugars were 10–80 µg/mL. The calibration graphs were linear up to 10 mg/mL. The relative standard deviations of 500 µg/mL sugars were 7.0–7.3%. The proposed method was compared with the enzymatic photometric glucose analysis method (Glucose B‐Test II Wako). The correlation coefficient was 0.83 (n = 20) and y = 0.82x + 5.91, where y and x are concentrations in µg/mL obtained by the proposed pre‐column HPLC and enzyme‐photometric method, respectively. The detection limits of sugar alcohols were 100–1000 ng/mL. The calibration graphs were linear to 50 µg/mL and relative standard deviations of 10 µg/mL were 7.2–8.2%. The 1,5‐AG data by the proposed method was also compared with the enzymatic photometric 1,5‐AG analysis method (Rana AG 1,5‐AG determination kit, Nihon Kayaku) and good correlation (r = 0.91, n = 20) was also obtained. The proposed method was applied to the simultaneous determination of d‐glucose, 1,5‐AG and related sugar alcohols in serum from healthy males. Copyright © 2005 John Wiley & Sons, Ltd.

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