Precolumn derivatization of reducing carbohydrates with 4-(3-Methyl-5-oxo-2-pyrazolin-1-yl) benzoic acid. Study of reaction, high-performance liquid chromatographic separation and quantitative performance of method

Summary A simple method for quantitative determination of carbohydrates by reversed-phase, high-performance liquid chromatography after pre-column derivatization and UV detection has been developed. Reducing sugars are condensed with 4-(3-methyl-5-oxo-2-pyrazolin-l-yl) benzoic acid (PMPA) to yield UV adducts absorbing at 271 nm, which are resolved under typical reversed-phase conditions. After derivatization, excess PMPA is easily removed from the reaction mixture by precipitation with mineral acids at pH<4. The influence of experimental conditions on reaction yield, as well as chromatographic separation of derivatives, were investigated. The quantitative performance was evaluated by means of a protocol comprising replicate measurements at several analyte levels. The calibration curves obtained for 8 sugars showed excellent linearity over 10–5000 pmol. Limits of detection and quantification for several monosaccharides were ca. 10 and 50 picomoles, respectively. Optimized conditions were successfully used for quantitative determination of monosaccharides released after hydrolysis from fetuin, mucin, α₁-acid glycoprotein, ovalbumin and transferrin.