In this study, a low-cost efficient online derivatization system was developed which allows for the detection of various types of mono- and oligo-saccharides only utilizing high-performance liquid chromatography (HPLC)-ultraviolet detector (UV) system. In the proposed method, phenylhydrazine was used as the derivatization reagent and directly spiked in the mobile phase, allowing for the separation and detection of mono- and oligosaccharides in an accessible instrument system (HPLC-UV). And the online derivatization design of the proposed method has significantly reduced the potential harm of derivatization reagents to the analysts. Furthermore, critical chromatographic parameters were optimized via the Box-Behnken design strategy, culminating in the ideal response for saccharides. Finally, the methodology validation of the proposed method was conducted. The proposed method showed satisfactory linear ranges with acceptable correlation coefficients (R > 0.99), outstanding accuracy (Recovery: 95.3%-105.6%), high intra-day precision (relative standard deviation [RSD]: 1.4%-7.1%) and inter-day precision (RSD: 2.0%-7.4%). The robustness and ruggedness of the proposed method were proved as the recovery values in the range of 95.0%-104.6% and 95.1%-104.8% for robustness and ruggedness, respectively. These satisfactory validation results confirm the applicability and reliability of the proposed method for the analysis of saccharides in various complex real-world samples.
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