Study On Screening And Analysis Of Toxicity Of Marine Harmful Algae By Liquid Chromatography - Mass Spectrometry

In recent years, harmful algal bloom(HAB) incidents are becoming more common due to the growing pollution of marine environment. Especially, some of marine biotoxins produced by the toxic algae have an impact on marine ecosystem, marine fishery resources and marine breeding industry. Moreover, algal toxins transferred into human beings through food chain can cause human toxicosis or even death. To date, over 200 marine algal toxins and derivatives have been discovered. They can be classified as lipophilic and hydrophilic algae toxins based on their water solubility. Since there are varieties of marine algal toxins and each type consists of dozens or event hundreds of toxin monomer, the authorities of marine environmental management has to determine whether the HAB produces toxin and what type of toxin was produced, a rapid and highly efficient method for the screening and identification of marine algal toxins was urgently needed. This study describes the use of liquid chromatography–mass spectrometry(LC–MS) for screening and identification of multipal classes of algal toxins in marine toxic algae. Firstly, the MS characteristic of eight typical hydrophilic toxins was investigated by electrospray ionization mass spectrometry in negative ion mode. Secondly, a method was developed for the screening and identification of over 20 hydrophilic toxins in marine toxic algae by hydrophilic interaction liquid chromatography–high resolution mass spectrometry(HILIC–HR–MS) combined with accurate mass database. Finally, we apply the revers phase liquid chromatography(RPLC) combined with HILIC and HR–MS establishes a new method for simultaneous screening and identification of over 20 hydrophilic algae toxins and 200 lipophilic algae toxins, the details are as follows:1. Paralytic shellfish poisoning(PSP) toxins are the most toxic and widely distributed hydrophilic algae toxins. The MS characteristic of eight commen sulfur containing PSP toxins(GTX1, GTX 4, GTX2, GTX 3, dc GTX2, dc GTX3, C1 and C2) were investigated by ESI–MSn in negative ion mode, the results show that the quasi–molecular ion peak [M–H]– of each PSP toxin was detected by ESI–MS. Stability characteristic fragment ions were observed by the neutral loss of –H2O and –NHCO for ESI–MS2 detection and the MS2 spectra of PSP toxin isomers were different, which could provide experimental basis for qualitative and quantitative determination of sulfur containing PSP toxins by ESI–MS2. ESI–MS3 detection was performed for the main characteristic fragment ions of the target toxins produced during ESI–MS2 analysis. The main MS3 characteristics of the fragment routes were also discussed, this study can help to offer the reference and basis for determination of sulfur containing PSP toxins by ESI–MS in negative mode.2. A new method was developed for rapid screening and identification of 25 PSP toxins in marine toxic algae by HILIC–HR–MS combined with accurate mass list. Toxic algae sample was extracted with water containing 0.1 mol/L acetic acid after cell–breaking, and then the crude extracts were analyzed by HILIC–HR–MS in positive and negative full scan modes. The accurate mass of quasi–molecular ion and main fragment ion peaks of PSP toxins in toxic algae were acquired and the PSP toxins in algae could be successfully qualitative identification by database screening and HR–MS data analysis using commercial software. The created database includes data not only the accurate relative molecular masses of 25 common PSP toxins but also the fragment ions. The results showed that the proposed HILIC–HR–ESI/MS method was sensitive and had the high separation efficiency. The limits of detection(LOD) of ten common PSP compounds were in the range of 10~80 nmol/L and it could meet the requirements for the actual screening of toxic algae samples. The developed method was applied to screen the PSP toxins in three kinds of toxic algae which demonstrated that the method was a useful tool for the rapid screening and qualitative identification of common PSP toxins in harmful algae.3. Serial coupling of RPLC and HILIC–HR–MS was investigated for the simultaneous screening and identification of multiple classes of hydrophilic and lipophilic algae toxins in marine toxic algae combined with accurate mass list. First of all, ultrasonic cell disruption method was applied to process the toxic algae. Then, toxins in algae were extracted by the mixed solution of methanol and 0.1 mol/L acetic acid(1:1, V/V). Finally, both the lipophilic and hydrophilic in the crude extracts were separated and determined by RPLC/HILIC–HR–ESI/MS in positive and negative full scan modes. Among them, RPLC and HILIC column were coupled in series via a zero dead volume T–piece and a mixer, the RPLC column with low flow and high water content(0.2 m L/min, 80% water), while HILIC column with high flow and high acetonitrile content(1 m L/min, 85% acetonitrile), which caused hydrophilic and lipophilic algae toxins be simultaneously separated in a single injection. Eleven lipophilic toxins and eight hydrophilic toxins were used to determine the reliability of the method. The precision results showed that the relative standard deviations(RSDs) of peak area of 19 toxins were all lower than 4.55%, and the RSDs of the retention time were all better than 0.96%. The RSD values for the intra–day and inter–day variation of the proposed method ranged from 4.38% to 7.92% and 8.37% to 14.87%, respectively. The instrumental limit of detection(LOD) for 19 algal toxins was in the range of 0.016~50.9 ng, it did fully satisfy the requirements for screening and identification of multiple classes of lipophilic and hydrophilic toxins in the real samples of algae.