Separation of polyphenols by HILIC methods with diode array detection, charged aerosol detection and mass...

The characterization of plant extracts is usually accomplished by reverse-phase liquid chromatography, but the development of new complementary approaches, such as HILIC, offers an orthogonal method. In this study, five HILIC stationary phases were evaluated to assess their ability to retain polyphenols. They were selected to cover the main different HILIC mechanisms: bare silica; silica with ethylene bridge; neutral amide; amino; zwitterionic. A total of 31 polyphenol standards were used for the screening, including 9 stilbenes, 8 flavonoids, 6 anthocyanins, and 8 phenolic acids. Three different detections were tested: diode array detector, charged aerosol detector and mass spectrometry. Results indicated that silica supports were not suitable for retaining polyphenols, with no or low retention observed except for anthocyanins. The effectiveness of stationary phases in retention of phenolics following the order related to increased retention: zwitterionic, amide, and amino. The choice of mobile phase also influenced retention. Mobile phases containing TFA as pH modifier limited retention, while formic acid was found to be more effective for polyphenol retention. Ammonium buffers also improved retention but often compromised peak shape. pH changes mainly impacted ionizable compounds, such as phenolic acids, by increasing their retention when they were ionized. DAD was wellsuited for detecting polyphenols that possess aromatic rings, though peak wavelengths depend on the structures of the polyphenols. CAD, while less sensitive than DAD and MS, provided an almost similar response for structurally related compounds, even with gradient elution. MS was the preferred detector for quantification when resolution between compounds was challenging, as it is often the case with natural extracts. The study successfully demonstrated that best HILIC conditions were obtained using an amino stationary phase composed of a polyethylenimine and formic acid-based mobile phase. These conditions were successfully applied to the analysis of stilbenoid-rich extracts from different parts of the vine. The elution order of stilbenoids followed the degree of polymerization. With CAD, the chromatographic profile was more representative of sample composition. It was demonstrated for the first time the interest of a combination of HILIC and CAD for analyzing stilbenes, offering a complementary approach to the classic RP analysis.