Reducing sugars, particularly aldoses with cyclic structures, efficiently react with 1-phenyl-3-methyl-5-pyrazolone (PMP), forming derivatives that are detectable by high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) at 248 nm. However, a major challenge in this type of analysis is the co-elution of xylose and arabinose, making their individual identification and quantification difficult. This study aimed to optimize an HPLC-DAD method to separate and quantify seven monosaccharides (mannose, rhamnose, galacturonic acid, glucose, galactose, xylose, and arabinose) in complex matrices while minimizing total analysis time.

A C18 column was used, and various chromatographic conditions were evaluated, including pH and composition of the mobile phase, gradient profile, flow rate, and column temperature. Derivatization with PMP was carried out under alkaline conditions. Method validation included assessment of linearity, limits of detection (LOD) and quantification (LOQ), repeatability, interday precision, and recovery rates.

The optimized method reduced the total analysis time to 15 minutes and achieved baseline separation between xylose and arabinose. Final chromatographic conditions included a flow rate of 0.6 mL/min, a column temperature of 15°C, and mobile phase A consisting of 50 mM ammonium acetate adjusted to pH 7.5.

This method enables individual identification and quantification of monosaccharides in natural matrices, resolving the co-elution of xylose and arabinose and significantly reducing analysis time.