Analysis of flavonoids with unified chromatography-electrospray ionization mass spectrometry-method development and application to compounds of pharmaceutical and cosmetic interest.

Abstract

In this project, we aimed at analysing flavonoid-type compounds with unified chromatography (joining supercritical fluid chromatography and enhanced fluidity liquid chromatography with carbon dioxide-methanol mobile phases covering a wide range of compositions) and diode-array and electrospray ionization mass spectrometric detection (UC-DAD-ESI-MS). First, the chromatographic method was developed for 9 standard flavonoid molecules from three different families (flavanols, flavanones and flavonols, glycosylated or not), with a strong focus on mobile phase composition to achieve the elution of a wide range of flavonoids with good chromatographic quality (efficiency and resolution). For this purpose, two stationary phases were selected (ACQUITY UPC2 DEA and Diol), and five different additives (formic acid, citric acid, phosphoric acid, methanesulfonic acid and ammonium hydroxide) were successively introduced in the methanol co-solvent. The composition containing 0.1% methanesulfonic acid in methanol was retained as it provided the best chromatographic quality together with the possibility of hyphenating the chromatography to mass spectrometry. The DEA column appeared to provide the best efficiency and was retained for further method development. The gradient method was then optimized to achieve a fast analysis, which involved elution with a wide range of mobile phase composition (from 20 to 100% co-solvent in methanol) together with reversed flow rate and reversed pressure gradients at fixed temperature. The final gradient lasted 10min, followed by 2.5min of re-equilibration. Then, ESI-MS detection was optimized. Because the single-quadrupole mass spectrometer employed (ACQUITY UPC2 QDa) allowed the variation of only a few parameters, a design of experiments was used to define the best compromise for three parameters (probe temperature, cone voltage and capillary voltage). The make-up fluid introduced before entering the MS was also varied: different compositions of methanol-water containing either formic acid, ammonium hydroxide or sodium chloride were tested. The best results in terms of signal-to-noise ratio were obtained with methanol containing 20mM ammonium hydroxide and 2% water. The optimal UC-DAD-ESI-MS method was then applied to two different flavonoid formulation ingredients. The first one, hidrosmin (5-O-(β-hydroxyethyl)diosmin), is known for its vasoprotective properties and therefore employed in pharmaceutical formulations. The second one, α-glucosyl-hesperidin (sometimes referred to as vitamin P), is employed in cosmetic formulations. Identification of the major compounds in each sample was achieved with the help of MS detection. Graphical abstract.