Determination of Aldoses, Deoxy-aldoses and Uronic Acids Content in a Pectin-Rich Extract by RP-HPLC-FLD after p-AMBA Derivatization

Abstract

A reversed-phase high-performance liquid chromatographic method with fluorometric detection is proposed for the simultaneous determination of different classes of neutral sugars, such as hexoses (galactose, glucose and mannose), pentoses (arabinose and xylose), deoxy-hexoses (fucose and rhamnose), as well as acidic sugars (galacturonic and glucuronic acids). The separation is carried out on a hydrophilic end capped C18 column following a pre-column derivatization with p-aminobenzoic acid. The fluorometric detection of the derivatives has shown a strong dependency with the mobile phase pH. The performance of the proposed methodology was evaluated and the prerequisites of linearity (r-value > 0.999), precision (intra-day CV < 6 % and inter-day CV < 11 %) and recovery (between 77 ± 7 and 103 ± 3 %) were satisfied. To our knowledge, the obtained values of limit of detection for neutral sugars (within the range 6.1–28 μg L−1) are the lowest reported using this derivatizing agent. In order to better judge the methodology presented herein, neutral sugars of a pectin-rich orange extract were also analysed by the conventionally used GC-FID (gas-chromatography with flame ionization detector) method of alditol acetate derivatives. A statistical test (paired t test) has proved that no significant differences (α = 0.05) were observed between these two methods.