Nano-liquid chromatography in nutraceutical analysis: Determination of polyphenols in bee pollen

In this study, a nano-liquid chromatography based method for the simultaneous separation of 16 polyphenols employing UV–vis detection has been developed. A 100 μm I.D. capillary column packed with C18 core–shell particles (2.6 μm particle size, 100 Å) for 10 cm was employed. The separation of analytes was performed with a step gradient in less than 20 min, using 0.5% formic acid aqueous solution and acetonitrile as eluents. The optimized analytical method was validated and the resulting RSD% for intra-day and inter-day repeatability, related to retention time, retention factor and peak area, were below 4.68 and 5.57%, respectively. LOD and LOQ values were as low as 0.78 and 3.12 μg/mL, while linearity, assessed in the concentration range of interest for all analytes, gave R2 ≥ 0.990. The method was finally applied to the analysis of polyphenols extracted from a collected bee pollen. Nine polyphenols, namely o-, p-coumaric acid, ferulic acid, myricetin, cinnamic acid, quercetin, naringenin, hesperitin and kaempferol, were identified. All analytes, with the exception of p-coumaric acid and myricetin, which partially co-eluted with other pollen components, were also quantified in the sample.