A new approach to the rapid separation of isomeric compounds in aSilybum marianumextract using UHPLC core-shell column with F5 stationary phase

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HIGHLIGHTSThe method for separation of diastereoisomeric flavonolignans was developed.Method shows short time (10 min) of analysis and reliability for routine quality control.Quality control of Silybum marianum herbal extracts showed variable results.The method shows unique selectivity of F5 stationary phase for separation of silymarin isomers.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 1.7 μ F5 100A (150 × 2.1 mm), 1.7 μm particle size core–shell column, with a mobile phase methanol/100 mM phosphate buffer pH 2.0 according to the gradient program. A mobile phase 0.35 mL min−1 flow rate and 50 °C temperature was used for the separation. The detection wavelength was set at 288 nm. 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 2 μL 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.

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