The paper presents development and validation of a RP-HPLC-PDA method for quantification of 30 phenolic constituents of the blackthorn (Prunus spinosa L.) flower, a traditional European herbal medicine with a unique and complex composition. The target analytes were selected from over 50 active compounds present in the investigated plant material, and their separation was optimized on a C18 Ascentis Express fused-core column (2.7 μm, 150 mm × 4.6 mm), in a step-by-step process, in terms of elution solvents, gradient profile, temperature, and flow rate. The final procedure was carried out with an acetonitrile-tetrahydrofuran gradient at a flow rate of 1.09 mL/min and column temperature of 28°C. Under those conditions, the matrix peaks were satisfactorily separated within 35 min. The validation showed good precision (RSD < 5 %), accuracy (93.5-102.1 %), linearity (r > 0.9998), and sensitivity (LODs 0.51-2.05 ng) of the method. The real sample analysis demonstrated its applicability for quantification of the phenolics both in commercial samples of P. spinosa flowers (different manufacturers and years of collection), as well as in the extracts (of different polarity) prepared thereof. Thus, the developed procedure proved to be a useful tool in quality control, and the optimization approach might serve as a practical guideline for LC-method development in complex matrices.
Keywords: High performance liquid chromatography; Method development; Polyphenols; Prunus spinosa; Quality control; Tertiary gradient.
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