Identification and LC-MS-MS Determination of Acteoside, the Main Antioxidant Compound of Euphrasia Rostkoviana, Using the Isolated Target Analyte as External Standard

The main compound of the traditionally known herbal eye-remedy, Euphrasia rostkoviana Hayne (eyebright) is isolated in high purity, identified and quantified in multiple reaction monitoring (MRM) mode with external calibration method using the isolated compound as standard. For structure elucidation high-performance liquid chromatography (HPLC)-electrospray ionization source (ESI)-tandem mass spectrometry, HPLC-ESI-time-of-flight, and nuclear magnetic resonance spectroscopy are used. The main compound is identified as acteoside, a phenyletanoid glycoside, exhibiting strong antioxidant activity. Purity of the isolated compound is over 97%, hence it is amenable to be used as standard for quantitation. Acteoside content is 2.56 ± 0.19 g / 100 g dry plant sample. According to the method performance test results the concentration-response plot is linear (r 2 = 0.997), precision and accuracy are all within acceptable range (highest RSD: 6.27%). SPE and method recovery are good, 98.6% (RSD 1.56%, n = 3) and 91.0% (RSD 2.1 %, n = 3), respectively. Determination is carried out with a reliable, fast, characterized method, using a self produced standard.