| There are four parts in this paper. The first part was establishing the method for analyzing sphingomyelin (SM) and sulfatide (ST) by direct injection electrospray ionization tandem mass spectrometry (DI-ESI-MS/MS). The second part of the thesis was establishing the method for analyzing SM and ST by flow injection electrospray ionization tandem mass spectrometry (FI-ESI-MS/MS). In the third part of the paper, the two methods established in the previous chapters were compared. In the last part, the content of brain from chicken, duck and pig was analyzed, SM and ST profiling from pig brain during storage at different temperatures was tracked by DI-ESI-MS/MS. Also the effect of selenium (Se) to SM and ST profiling from mouse plasma was investigated by FI-ESI-MS/MS.Firstly, the method for analyzing SM and ST by DI-ESI-MS/MS was established. The extract method, injection speed, scanning mode of SM and ST, and tandem mass spectrum parameters were optimized in the experiment. The linear relation, extraction recovery, within-day precision, inter-day precision were investigated finally. Sphingolipids (SPs) were extracted with a modified Bligh&Dyer method combine with NaOH-CH3OH, and then the analysis was taken directly into the electrospray ionization source at the rate of5μL/min by injection pump. The MS/MS experiments combining precursor ion scan (PIS) was performed to monitor SM and ST by tandem triple quadrupole mass spectrometry. Methodological evaluation showed that the linear range was5-2000ng/mL, in this concentration the linear relation (0.9967,0.9913. respectively) were good. The recovery (88.7-107.8%) and precision (less then8.72%) of the method met the requirements for analyzing biological samples. The method for analyzing SM and ST by DI-ESI-MS/MS was simple, fast and accurate for qualitative and quantitative, with excellent stability and reproducibility, thus making it suitable to analyze SMand ST from various biological samples.Subsequently, the method for analyzing SM and ST by FI-ESI-MS/MS was established. The injection speed, sample size, mobile phase and tandem mass spectrum parameters were optimized in the experiment. The linear relation, extraction recovery, within-day precision, inter-day precision were investigated finally. SPs were extracted with a modified Bligh&Dyer method combine with NaOH-CH3OH, and then on the base of qualitative test by FI-ESI-MS/MS, the analysis was taken directly into the electrospray ionization source at the rate of0.15mL/min by flow injection system The MS/MS experiments combining Multiple Reaction Monitoring (MRM) was performed to monitor SM and ST by tandem triple quadrupole mass spectrometry. Methodological evaluation showed that the linear range was5-2000ng/mL, in this concentration the linear relation (0.9954,0.9972. respectively) were good. The recovery (87.3-103.2%) and precision (less then8.21%) of the method met the requirements for analyzing biological samples. The method for analyzing SM and ST by FI-ESI-MS/MS was confirmed to have a good pretreatment, degree of automation, reproducibility, specifity and sensitivity, thus making it suitable to analyze SM and ST from various biological samples.Then we compared the two methods established in the previous chapters. The two methods have a little bit difference, but they all have excellent stability and reproducibility, and would be suitable for analysis SM and ST from various biological samples.Finally, we analyzed the content of brain from chicken, duck and pig, tracked SM and ST profiling from pig brain during storage at different temperatures by DI-ESI-MS/MS. Also investigated the effect of Se to SM profiling from mouse plasma by FI-ESI-MS/MS. In the application of the DI-ESI-MS/MS method, we found that the molecular species were similar in the brain from chicken, duck and pig and the content of SM and ST among them was different, these differences are expected to used for different animal tissue source identification. And during storage at+20℃, the content of SM and ST increased and then decreased gradually, at last kept steady. The oxidation and hydrolysis of SM and ST storage at+20℃was much faster than that at+4℃. The molecular content of SM and ST storage at-20℃for60day did not have significantly change. It indicated that the SM and ST could stay stability during low temperature storage for a long time. In the application of the FI-ESI-MS/MS method, the effect of Se profiling from mouse plasma was discussed. The results showed that the content of SM increased along with the increase of Se. |
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