Establishment And Application Of Analytical Methods For The Determination Of Several Representative Phosphonates With Liquid Chromatography In Seawater Matrix

Phosphorus(P)is an essential nutrient element for the growth of marine phytoplankton.As dissolved inorganic P is usually depleted in the euphotic zone of oligotrophic open ocean waters,the growth of marine phytoplankton is limited.Thus,the bioavailability of various forms of dissolved organic P in the ocean makes a significant contribution to global biogeochemical P cycling and primary production under P-limitation conditions.A number of biological incubation experiments have suggested that phosphonates are a significant P source in the oceans.For example,glyphosate(GLYP)and its main degradation product,aminomethylphosphonic acid(AMPA),2-aminoethylphosphonic acid(2-AEP),2-amino-3-phosphonopropionic acid(2-AP3)and some other phosphonates could serve as alternative P sources for environmental microorganisms.Due to the lack of appropriate analytical methods in seawater matrix,there are no available data on the phosphonates concentration variation during these incubation experiments.As a result,some important scientific theory lack the support of concentration data and the further studies of phosphonates bioavailability are limited.To meet these requirements,the present study aimed to develop simple and fast methods for the determination of several representative phosphonates in seawater matrix,which possessed the feature of high recoveries,good reproducibility and relatively high sensitivity.The contents and results are summarized as the follows:(1)A method for the determination of GLYP and AMPA in seawater matrix has been developed based on the derivatization with 9-fluorenylmethylchloroformate(FMOC-Cl),using high performance liquid chromatography(HPLC)equipped with fluorescence detector(FLD).The derivatization procedure was carefully optimized regarding concentration of FMOC-Cl,volume and pH of borate buffer,mixing and derivatization time.The derivatization.reaction could be completed within 30 min in seawater samples without any additional clean-up or desalting steps.Under the optimized conditions,the developed HPLC method showed a wide linear response with good linearity(R2>0.990).The limits of detection(LOD)were 0.60 ?g/L and 0.30 ?g/L for GLYP and AMPA in seawater matrix,respectively.The relative standard deviation(RSD)was 14.0%for GLYP and 3.1%for AMPA in saline samples with three different operators(n=24).This method was successfully applied to determine the concentration of GLYP and AMPA in seawater alga culture media.Spiked GLYP and AMPA were recovered 80-120%in more than 90%seawater culture media samples and good recoveries were also observed in other different water samples,indicating minimal matrix interference.(2)A method for the determination of 2-AEP in seawater matrix has been developed based on the derivatization with o-phthalaldehyde(OPA)and ethanethiol(ET),using HPLC-FLD.The derivatization procedure and HPLC conditions were carefully optimized regarding excitation and emission wavelength of FLD,concentration and pH of ammonium acetate in mobile phase,content and composition of OPA-ET reagent and derivatization time.Under the optimized conditions,the developed HPLC method showed a wide linear response with good linearity(R2>0.990)and high reproducibility(RSD<7.8%,n=7)in different matrix at three concentration levels.The LOD of 2-AEP was 15.0 ?g/L in seawater matrix.This method was successfully applied to determine the concentration of 2-AEP in seawater alga culture media and in different matrix during degradation process.Spiked 2-AEP was recovered 79.3-99.3%in seawater culture media samples and good recoveries were also observed in other different water samples,indicating minimal matrix interference.(3)A method for the determination of 2-AP3 and 2-AEP in seawater matrix has been developed based on the derivatization with OPA-ET,using HPLC-FLD.Under the optimized conditions,the developed HPLC method showed a wide linear response with good linearity(R2>0.999)and high reproducibility(RSD was less than 1.7%for 2-AP3 and 4.1%for 2-AEP,n=7)in saline samples at three concentration levels.The LOD were 12.0 ?.g/L for 2-AP3 and 2-AEP in seawater matrix.Spiked 2-AP3 was recovered 72.6-98.6%and 2-AEP was recovered 83.0-103.8%in different water samples including seawater culture media samples,indicating minimal matrix interference.In the study,the difference of derivatization rate and stability of derivatives between 2-AP3 and 2-AEP were analyzed,providing the reference information for simultaneous analysis of more phosphonates.