Study On Extraction And Analysis Of Fatty Acids In Seaweeds

The algae seaweeds have the nutrition and medicine values due to containing unsaturated fatty acids such as DHA and EPA which is essential to human body.Establishing a rapid,sensitive and accurate method for analysis of fatty acids in algae is of great significance for the study of the relationship between fatty acid and seaweed biological activity.The aims of this thesis are(i)to optimize the derivatization reagent by comparing the derivative reaction of different derivatization reagents with fatty acids;(ii)to improve the MS response signal by modifying the structure of fatty acids;(iii)to develop the high performance liquid chromatography-tandem mass spectrometry methods for determination of fatty acids in algae.In the first part,the extraction methods of total lipids in seaweeds were established.After comparing a variety of different methods for extraction of total lipid in seaweeds such as solvent leaching extraction,ultrasonic extraction,soxhlet extraction,and improved Folch method,the improved Folch method was choose to extract total lipid in seaweeds finally.The optimum liquid ratio,extraction power,extraction temperature and extraction time conditions were as follows:methanol-dichloromethane was used as extraction solvent,extraction time was 90 min and the optimum temperature was 50 ?,the optimum power was 80 W.The extraction rate of total lipids was 5.7 % in seaweed.In second part,the method for fatty acid derivatization was established.N-(2-hydroxyethyl)-pyrrolidine,N,N-dimethylethylenediamine and N,N-dimethylethanolamine were used as derivative reagents to derivatize fatty acids and the conditions of derivatization were optimized.The N,N-dimethylethylenediamine was used as the best derivatization reagent after comparing the derivatization conditions and price of derivative agent.Due to containing negatively charged nitrogen atom of N,N-dimethylethylenediamine,the formation of quaternary ammonium salt after reaction with methyl iodide can greatly improve the ionization efficiency of mass spectrometry.The result shows that the detection sensitivity of three kinds of saturated fatty acid and five kinds of unsaturated fatty acid derivatives in mass spectrometric improved above100 times after derivatization.Then N,N-dimethyl functional group is water soluble group,so it will increase the water solubility.It is easier to shorten the retention time in the liquid phase column and improve the detection.In the third part,the content of fatty acids in seaweeds was determined by high performance liquid chromatography tandem mass spectrometry.The result shows that docosahexaenoic acid can not be detected in three species of marine algae.The eicosapentaenoic acid(20:5)and ?-linolenic acid are the most abundant fatty acids in seaweeds.Some fatty acids exist as isomerism and the content of palmitic acid is abundant in seaweed.Even if fatty acids may not be separated completely,and it can be detected accurately for quantitative analysis of fatty acids by using the method of tandem mass spectrometry.The method is a rapid,sensitive and accurate analysis method for the determination of fatty acids in seaweed.