Multiplex Isotope Labeling Coupled With LC-MS For The Quantitative Analysis Of Phenols In Environment

Phenolic compounds are an important class of substances in nature and are widely used in many fields such as industry and agriculture.Due to its wide range of applications,it is inevitably discharged into the environment,such as the atmosphere,water and soil.Since most phenolic compounds are toxic and difficult to degrade,their residues in environmental media will inevitably cause certain toxic effects on animals,plants and humans.Therefore,it is necessary to develop an analytical method for the detection of phenolic compounds.In general,the content of phenolic compounds in the environment is low,so efficient,sensitive and reliable analytical methods are necessary for detection.Among them,liquid chromatography-mass spectrometry(LC-MS)is one of the most effective methods at present.In order to achieve rapid,accurate and stable large-scale quantitative analysis of phenolic compounds in complex samples,we developed a multiplex isotope labeling method for the quantitative analysis of phenolic compounds in environmental samples by liquid chromatography-high resolution mass spectrometry(HPLC-HRMS).The method can simultaneously qualitatively and quantitatively analyse phenolic compounds in multiple environmental samples.In this experiment,two routes were first developed for the synthesis of quaternary ammonium salt isotope labeling reagents.After the synthesis,the high efficiency of labeling of phenolic compounds can be achieved by optimizing the reaction conditions.After the labeling is completed,the phenolic compounds originally detected in the negative ion mode were converted into positive ion mode detection by labeling reaction,thereby significantly improving the ionization efficiency of the electrospray ionization source(ESI)and improving the detection sensitivity of the mass spectrometry.In the LC-MS analysis,the retention time of the four-plex isotope labeling products are close(<0.05min),which can overcome the matrix effect existing in the detection of complex samples and improve the accuracy of quantitative analysis.The specific synthetic steps of the labeling reagent are as follows:(1)The 4-aminobutyric acid was reacted with deuterated methyl iodide or deuterated formaldehyde to form a quaternary ammonium salt,thereby introducing a corresponding isotope;(2)The obtained quaternary ammonium salt product was reacted with N-hydroxysuccinimide(NHS)to form the final labeling reagent-trimethylammo-nium butyric acid chloride N-hydroxysuccimide ester(TMABS),which can be used to label amino and phenolic compounds.By optimizing the synthesis conditions,the reaction efficiency of every step is above 99%.After the synthesis of the labeling reagent,15 kinds of representative phenolic compounds were selected,and the labeling conditions,Solid Phase Extraction(SPE)process and LC-MS detection conditions were systematically optimized.In order to verify the anti-interference ability of the method to the complex environmental matrix,we selected river water(samples from the compus river)to verify the method.By adding five different concentrations(0.04~5 ppb)of phenolic compounds to the school river,three sets of parallels were set.The test results indicated the linearity(R~2>0.99)and the reproducibility(RSD<10%)were good and the accuracy was high(0.81-1.03)in the test concentration range.The recoveries measured in this method were about 80.8~101.5%,and the LODs for 15 phenolic compounds were about 0.01~0.03 ppb.Subsequently,the synthesized four isotopic labeling reagents can be simultaneously used for the analysis of three different environmental samples.The three sets of samples were labeled with different isotope reagents and mixed with the internal standard.Quantitative analysis of three sets of samples can be achieved simultaneously by a single LC-MS.