Stable Isotope Labeling Combined With LC-MS For The Quantification Of Benzoic Acids In Environment

Benzoic acids?BAs?are an important class of chemical compounds and widely used in many fields such as industry,agriculture and food.Thus they will inevitably enter the environment,such as soil,water and food.Because of the toxicity,they may cause potential harmful effect to animals or humans after entering the environment.Therefore,it is necessary to monitor the BAs in the environment.Because BAs in environmental media were at trace levels,highly sensitive analytical methods were needed for termination,and liquid chromatography-mass spectrometry?LC-MS?is one of the most effective and widely used methods.In order to realize more rapid and accurate quantitative analysis of BAs,overcome matrix interference effect and improve detection capability,a stable isotope labeling method combined with HPLC-HRMS and solid phase extraction?SPE?was developed to determine trace BAs pollutants in the environment.Firstly,a pair of quaternary ammonium isotope labeling reagents was synthesized.The BAs,conventionaly were detected in negative mode,could be converted to positive mode after labeling.After derivitzation,the ionization efficiency of the BAs in ESI was dramatically improved,and the signal response was also enhanced,which led to a better detection capability of the instrument.In LC-MS,the retention time of the analytes labeled with light and heavy labeling reagent were identical,and the matrix effect could be well corrected,resulting in better accuracy in quantitative analysis.The synthesis steps of the labeling reagents were:?1?N,N-dimethylene-diamine reacted with an amino protecting reagent Di-tert-butyl dicarbonate??BOC?₂O?under basic condition;?2?The obtained intermediate product protected by BOC was reacted with methyl iodide or deuterium substituted methyl iodide under alkaline conditions to turn the tertiary amine to quaternary ammonium and introduce the corresponding isotopes;?3?The BOC protecting group was dissociated in HCl ethyl acetate solution to recover the primary amine group which was used for labeling pollutants with carboxyl group.After optimizing the synthesis conditions,the reaction efficiency of over 98%for each reaction step was achieved.After synthesis of the labeling reagents,seven different BAs and six benzoic acid pesticides or pesticide metabolites were selected as analytes.and the experimental conditions for labeling,SPE,and HPLC-HRMS were systematically optimized.In order to verify the applicability of the method,it was applied to the analysis of BAs in real environmental water samples collected from Liao River estuary.Six BA pesticide or pesticide metabolite standards with different concentrations?0.02-5?g/L?were spiked to the samples,and good linearities?R²?0.99?and rebpoducibility?relative standard deviation,RSD?10%,n=3?were obtained.The accuracy of 84%¹02%was achieved by comparing the experimental and theoretical values of spiked samples?1?g/L,5?g/L and 10?g/L?.The recoveries of the SPE for all tested compounds were 82.3%¹00.2%.LODs of the tested BAs were all in the range of 0.01-0.2?g/L.All these results indicated that this newly developed methodexhibitedexcellentanti-matrixinterferencecapability,realizingaccurate quantification of BA pollutants in the environment,which could be further applied to the analysis of BAs in other complex samples.