The Determination And Application Of Marine Phytoplankton Pigments By Using UHPLC-MS/MS

Phytoplankton are the most important primary producers in marine ecosystems,and their community and biomass could not only put impact on food web and biogeochemical cycles,but also influence climate change and mitigate the effects of global warming.As an important biological marker for phytoplankton community,phytoplankton pigment detection plays significant role in research of marine ecological technology.High performance liquid chromatography(HPLC),which is commonly used to analyze phytoplankton pigments,still has some limitations(e.g.,Low sensitivity and low accuracy).Concerning this issue,this study developed two ultrahigh performance liquid chromatography tandem triple quadrupole mass spectrometry(UHPLC-MS/MS)methods for multiple pigments analyzing with high sensitivity and accuracy,tested their feasibility of detecting phytoplankton pigments in seawater samples,and applied them for analyzing phytoplanktion size fraction.The main research contents and results include the following three aspects:(1)Development of a sensitive and specific UHPLC–MS/MS method for analyzing 14 marker pigments14 marker pigments were determined and validated using an internal standard(IS)method under electrospray ionization(ESI)in positive mode.Under the optimized conditions of chromatogram and mass spectrum,14 types of pigments were separated in less than 15 min and presented good linearity,with the coefficients ranging from 0.9915 to 0.9991.The limit of quantification(LOQ)attained for 14 pigments were 0.005-0.500 μg/L,which enabled quantification of the pigments in seawater at ppt level.Both intra-day and inter-day precisions were less than 15%.The method was validated using seawater samples and the recovery of the analytes ranged from 62.8% to 120.2% at two spiked concentrations.Compared with previous studies,UHPLC–MS/MS method takes the advantage of improved efficiency of qualitative analysis and significantly decreased detection LOQ on analyzing marker phytoplankton pigments at trace/ultra-trace levels in complex matrices.(2)Increase of the identifiable pigment types by UHPLC-MS/MS method based on diagnostic fragmentation patternsThe application of the UHPLC-MS/MS method is limited due to the lack of commercial standards.In this research,the transitions of 38 pigments and their formation patterns during electrospray ionisation(ESI)in positive mode were studied.Based on the diagnostic fragmentation patterns,the transitions and collision energy of more types of pigments were derived,and 64 pigments(including 26 types of pigment without pure standards)were determined from the mixed standards in one injection within 20 min.The method presented high sensitivity even when determined 64 pigments at one injection with the limit of quantitation(LOQ)of 36 types of pigments ranging from 0.2-0.005 μg/L.The developed method expanded the application of UHPLC-MS/MS to pigment without standards,and it can be used for analyzing more pigments with those of known structures.(3)Feasibility study of UHPLC-MS/MS method for size fraction analysingSeawater samples collected from 3 different water layers at 47 sites in the Yellow Sea in August 2015 were analyzed.Comparisons were carried in terms of pigment concentrations acquired by ultra-high performance liquid chromatography(UHPLC)and UHPLC-MS/MS.No significant differences were observed from the result comparisons for most cases although UHPLC-MS/MS were more sensitive,and two methods were both suitable for pigment detection.Based on the pigment results provided by UHPLC-MS/MS,the spatial distribution of chlorophyll a(Chl a)and size fraction were studied.The concentration of Chl a was much higher in the northeast of Yangtze River mouth and coastal waters of Shandong Peninsula,where micro phytoplankton was the dominant size fraction.The concentration of Chl a was much lower in the central Yellow Sea,and the contribution of nano-and pico-phytoplankton increased significantly in this region.Compared with previous studies,the absolute concentrations of Chl a and contribution of size fractions in our study showed some differences,which may be caused by the differences of investigation time,sites or analytical methods.Thus,more comparison study is still needed to further probe the reason behind these differences.