Studies On Extraction And Analysis Of Phytosterols In Seaweeds

Phytosterols, a group of naturally occurring life-activated substances, have a series of physiological functions such as lowering cholesterol, anti-atherosclerosis and antioxidation et al. Therefore,they are widely used in the pharmaceutical, healthcare and other fields.This study aims to compare the derivative reaction of different derivatization reagents with phytosterols and select the best derivatization reagent for increasing of the mass spectral response signal. We established a rapid, sensitive and accurate derivatization high performance liquid chromatography-electrospray ionization -tandem mass spectrometry(HPLC-ESI-MS/MS) method to be used in determining phytosterols in seaweed. The main contents are as following:In the first part, we established the method for extraction of phytosterols from Sargassum, kelp and nori. The extraction efficiency of different extraction methods such as ultrasonic extraction, Soxhlet extraction, solvent extraction and solvent refluxing were compared. As a result, ultrasonic extraction was selected as the best extraction method.Extraction conditions such as extraction solvent, extraction time,extraction temperature, feed ratio were optimized. The optimum extraction conditions were as follows: ethanol as extraction solvent, ratio of solid to liquid (1:30), extraction time of 120 min, extraction temperature of 50 ?, extraction power of 80W. The extraction rate of phytosterols crude extract was 8.9%.In the second part, a method for the determination of phytosterols by pre-column derivatization HPLC-ESI-MS/MS was established. the derivatization reagents including of Acid chloride, hydroxylamine hydrochloride, dansyl hydrazide were used to react with phytosterols.By comparing the ionization efficiency of the derivative reaction, it was finally determined that dansyl hydrazide was used as the derivative reagents. The optimal derivatization reaction conditions were determined by optimizing the time of derivatization reaction, reaction temperature and derivatization reagent dosage. The proposed method was 2.3-75 times higher than that of underivatization high performance liquid chromatography- atmospheric pressure chemical ionization- tandem mass spectrometry(HPLC-APCI-MS/MS).In the third part, the phytosterols in seaweed were determined by derivatization HPLC-ESI-MS/MS. The content of phytosterols in three kinds of seaweed were also compared. Of those, the most abundant component is fucosterol. Meanwhile, the stigmasterol is not found. The main reason is that stigmasterol is widely present in cereals and vegetable oils. The Sargassum fusiforme and kelp have a similar phytosterols content and species. The phytosterols in Sargassum contains the most abundant species, so it has a high consumption and medicinal value.