Optimization and validation of reverse phase HPLC method for qualitative and quantitative assessment of polyphenols...

A simple reverse phase-high performance liquid chromatography (RP-HPLC) coupled to a diode array detector (DAD) and negative ion electrospray mass spectrometer (ESI-MS) method was developed for simultaneous identification and quantification of phenolic antioxidants in seaweed. The proposed method was validated in terms of linearity, limits of detection (LOD), limits of quantification (LOQ), recovery and intermediate precision. The calibration curves were linear with correlation coefficient ranging from 0.9909 to 0.9997 while the values of LOD (0.26-0.82mg/L), LOQ (0.77-2.50mg/L), recovery (≥97.2%) and precision in terms of retention time (%RSD ≤2.27) and peak area (% RSD ≤5.11) were satisfactory. Brown seaweed Himanthalia elongata used in this study was extracted with 60% methanol and the crude extract was cleaned with SPE (Solid Phase Extraction) cartridge. HPLC-DAD-MS/MS analysis of the SPE fraction allowed the identification of 7 phenolic compounds comprising phlorotannins, hydroxybenzoic acid, hydroxycinnamic acid and flavonols subclasses of polyphenols. Quantitative analysis of these compounds revealed the presence of phloroglucinol (394.1±4.33μg/g), gallic acid (96.3±3.12μg/g), chlorogenic acid (38.8±1.94μg/g), caffeic acid (44.4±2.72μg/g), ferulic acid (17.6±0.85μg/g), myricetin (8.6±0.85μg/g) and quercetin (4.2±0.15μg/g), in the extract. The SPE fraction were tested for antioxidant capacity which were significantly (P <0.05) higher (EC₅₀; 14.5±0.57mg/g) than the ascorbic acid (EC₅₀; 35.8±0.59mg/g) and the crude extract (EC₅₀; 46.3±0.48mg/g). The occurrence of all these phenolic antioxidant compounds in H. elongata extract suggested that the developed method is sensitive enough and reproducible and could be used for qualitative and quantitative assessment of polyphenols in seaweed.