Study On Photodegradation Of Vitamin B₂ And Fluorescent Modification Of Microcystins Based On PCEC-LIF And UPLC-MS/MS

Pressurized capillary electrochromatography（pCEC）has the advantages of high efficiency,high resolution,high selectivity,and high speed.Coupled with the highly sensitive laser induced fluorescence detector（LIF）,pCEC has obvious advantages in trace separation and analysis.UPLC-MS-MS can provide molecular weight and structural information,which can further enhance the identification.Based on the above two kinds of technologies,we studied the photostability of vitamin B₂,the salt effect on the photodegradation of vitamin B₂,and deduced the structure of photodegradation product.At the same time,fluorescence modification was carried out on the microcystin molecules,and the modified molecules were characterized by pCEC-LIF assisted further by UPLC-MS-MS.The thesis contains five chapters as follows:The first chapter introduced the background of the subject,including the basic theory of ultra performance liquid chromatography,mass spectrometry,pressurized capillary electrochromatography,laser-induced fluorescence detector;fluorescence and fluorescence modification;drug stability and water quality monitoring,etc.The second chapter described a new method for the separation and analysis of vitamin B₂ and its fluorescent photolysis products based on pressurized capillary electrochromatography（pCEC）coupled with laser induced fluorescence detection（LIF）.The relationship between the photodegradation reaction rate of vitamin B₂ and the ionic strength in the aqueous solution and the phosphate buffered solution（PBS）was studied.The analysis was carried out by using a C18 capillary column.The mobile phase was acetonitrile-water with 0.1%trifluoroacetic acid with gradient elution.The excitation wavelength was 488 nm and the emission wavelength was 520 nm.Vitamin B₂ and nearly 10 kinds of fluorescent photolysis products were well separated.Based on the above,we studied how the photodegradation rate of vitamin B₂ was influenced by ionic strength.The apparent rate constant of photolysis was calculated by kinetic calculation.The results showed that the ionic strength had a significant effect on the photolysis of vitamin B₂ solution.The photodegradation rate of vitamin B₂ was positively related to ionic strength.The third chapter is based on the results of the previous chapter.The photodegradation products and the photodegradation mechanism of vitamin B₂ were explored.The data were analyzed based on the molecular ion peaks and fragment ion peaks of mass spectrogram.While lumiflavin and lumichrome were successively confirmed,the structures of seven kinds of photodegradation products of vitamin B₂ were speculated.The photodegradation reaction pathway we designed could well explained the experimental phenomena.The fourth chapter is to detect microcystins using pCEC-LIF.Three different fluorescence modification methods were designed:the direct reaction between microcystins and FITC;the two-step fluorescence modification based on"thiol-ene"click chemistry and the one-step fluorescence modification.The target compound was obtained after optimization and was confirmed by UPLC-MS-MS.At the same time,the method for the detection of trace amounts of microcystins by pCEC-LIF was established.The detection conditions were determined as follows:a C18 capillary column,pH 9.1 borate buffer and acetonitrile as mobile phase,gradient elution.The excitation wavelength was 488 nm and the emission wavelength was 520 nm.The limits of detection（LOD）（S/N=3）of MC-LR and MC-RR subtypes was 1.3×10^-12 mol/L and 1.2×10^-12 mol/L,respectively.In the fifth chapter,we summarized the experimental results of vitamin B₂ and microcystins,and look into the future of the application and the developmental direction.