Precolumn Derivatization of Reducing Carbohydrates with 4-(3-methyl-5-oxo-2-pyrazolin-1-yl) Benzoic Acid. Study of the Reaction, High-Performance Liquid Chromatographic Separation and Quantitative Performance of the Method

C. B. CASTELLS *; ARIAS, V. C.; CASTELLS, R. C.

CHROMATOGRAPHIA

VIEWEG

Lugar: ALEMANIA; Año: 2002 vol. 56 p. 153 - 160

0009-5893

A simple method for the 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-1-yl) benzoic acid (PMPA) to yield UV adducts absorbing at 271 nm, which are resolved under typical reversed phase conditions. After the derivatization reaction, the PMPA excess is easily removed from the reaction mixture by precipitation with mineral acids at pH below 4. The influence of experimental conditions on the reaction yield, as well as the chromatographic separation of the derivatives, were investigated. The quantitative performance was evaluated by means of a protocol consisting in replicate measurements at several analyte levels. The calibration curves obtained for eight sugars showed excellent linearity over a range of 10-5000 pmol. Limits of detection and quantification for several monosaccharides were about 10 and 50 picomoles, respectively.Optimized conditions are successfully used for the quantitative determination of monosaccharide composition released from fetuin, mucin, a1-acid glycoprotein, ovalbumin and transferrin.