Quality Control Of Polyphenolic Components In Hawthorn By HPLC-MS

Hawthorn (Crataegus oxyacantha), one of the most common Chinese medicine, is used for the treatment of digestive ailments, dyspnea, kidney stones, and cardiovascular disorders. It is rich in polyphenolic components such as flavonoids and phenolic acids. A HPLC-MS method was developed to determine polyphenolic components in Hawthorn and it provided a quality controlling method for Hawthorn and its processed products. The main work in this dissertation was focused on the following aspects.1. A method for detection of total flavonoids by UV was developed and it was shown that the method was reeligible, accurate and reliable. The optimal conditions for the ultrasonic extraction of total flavonoids were obtained by Response Surface Methodology (RSM). The results were as follows: ethanol concentration 66.3%, extraction time 47min, ethanol volume/hawthorn powder 70mL/g, and the predicted content of total flavonoids was 75.87mg/g. The relative error was 0.34% between predicted and experimental results with validation tests, which showed reasonable.2. The HPLC-ESI-IT/MS/MS method to simultaneously qualify 12 polyphonelic components in Hawthorn was developed. The analysis was performed by using a Waters Atlantis-T3 column (4.6mm×250mm, 5μm) at gradient elution of water (A) and acetonitrile (B) both containing 0.1% formic acid. 12 peaks were identified on the basis of MS2 data and some interrelated literature. They were citric acid, chlorogenic acid, anthocyanin B2, epicatechin, anthocyanin B3, rutin-glucoside, vitexin-glucoside, maslinic acid, vitexin-rhamnoside, rutin, isoquercetin and hyperoside.3. The HPLC-ESI-IT/MS internal standard method with additional in-source collision induced dissociation (SCID) and selected ion monitor (SIM) scan was established to determine the contents of 7 polyphenolic components identified before. Daidzein was chosen as the internal standard. The detection limits was 13 pg/mL and the relative standard deviations (RSDs) of retention time (tR) and relative peak area (RPA) were 0.47~3.65% and 1.28~6.03% respectively for interday precision; 0.36~3.09% and 0.16~6.17% for intraday precision. The recoveries were from 84.03% to 101.14%.4. The HPLC-ESI-IT/MS internal standard method developed above was applied in determination of 7 polyphenolic components in Hawthons from 10 batches and different processed Hawthorn products. It was shown that in Hawthorn citric acid with the highest content was about 570~3300 times as much as hyperoside with the lowest content. Based on the content differences of 7 polyphenolic components, 10 Hawthorns were divided into 5 categories. The contents of all 7 polyphenolic components including organic acids and flavonoids decreased in processed products.