Determination of salicinoids by micro-high-performance liquid chromatography and photodiode array detection

Introduction Plant-derived salicinoids (conjugates of glucose and salicylate phenolic moieties) are potent modulators of plant-herbivore interactions. We demonstrate the use of micro-high-performance liquid chromatography (μHPLC) with photodiode-array detection (DAD) for quantification of four salicinoids (salicin, salicortin, hydroxycyclohexen-on- oyl salicortin and tremulacin) in methanolic extracts of Populus. Objective To develop and implement a solvent-conserving μHPLC method to quantify salicinoids in methanolic extracts of Populus tissue. Methods Salicinoids were extracted from Populus tissue into methanol, filtered, and introduced to μHPLC. Extracted analytes were separated on a Zorbax SB C₁₈-column with a binary gradient of methanol and water (with 2% tetrahydrofuran; 20 μL/min), and quantified by DAD (274 nm). We confirmed measurement reliability through standard addition, comparison with an accepted method, and assessment of chromatographic peak purity by ultraviolet absorbance spectra. Results Method detection and quantification limits for the salicinoids as a percentage of dry leaf weight were as follows: salicin (0.1%, 0.2%), salicortin (0.001%, 0.02%), hydroxycyclohexen-on-oyl salicortin (0.02%, 0.06%) and tremulacin (0.0006%, 0.002%). Calibrations by external standardisation were linear over 1.5 orders of magnitude with acceptable accuracy and reproducibility. Conclusion Micro-HPLC can serve as a solvent-conserving alternative to conventional HPLC for quantification of salicinoids in Populus tissue.