A new approach to the rapid separation of isomeric compounds in a Silybum marianum extract using UHPLC core-shell column with F5 stationary phase

Abstract

In this paper, a new ultra-high performance liquid chromatography (UHPLC) method using a core–shell column with a pentafluorophenyl stationary phase for separation of seven active compounds of a Silybum marianum extract was developed and validated. Silymarin, an extract of Silybum marianum, is known for its abilities to protect the liver from toxic substances, hepatitis therapy, and anti-tumour activity. Silymarin is currently being widely used in commercial preparations and herbal teas. Separation of seven compounds contained in the Silybum marianum extract (taxifolin, silychristin, silydianin, silybin A, silybin B, isosilybin A, isosilybin B) and other substances occurring in real samples was performed on the Kinetex 1.7μ F5 100A (150×2.1mm), 1.7μm particle size core–shell column, with a mobile phase methanol/100mM phosphate buffer pH 2.0 according to the gradient program. A mobile phase 0.35mLmin−1 flow rate and 50°C temperature was used for the separation. The detection wavelength was set at 288nm. Under optimal chromatographic conditions, good linearity with a correlation coefficient of R2 >0.999 for all compounds was achieved. The available commercial samples of herbal teas and food supplements were extracted with methanol using an ultrasonic bath. After dilution with water and centrifugation, a 2μL sample of the filtered supernatant was directly injected into the UHPLC system. The use of a pentafluorophenyl stationary phase with methanol as the organic component of the mobile phase showed new ways to effectively separate isomeric compounds in herbal extracts, which could not be done with the conventional C18 stationary phase.