Application Of Isotope Labeling Chemical Derivatization-mass Spectrometry In The Analysis Of Sulfhydryl Compounds

Thiol-containing compounds are widely presented in living organisms and play an important role in maintaining redox balance and anti-oxidative stress in vivo.At the same time,thiol-containing compounds are also a widely used food flavor,naturally occurring in various food including meats,fruits,coffee and so on,with unique aroma characteristics and low odor threshold.As a class of important organic synthesis intermediates,thiol-containing compounds are also widely used in medicine,pesticides and other fields.Therefore,the highly sensitive and accurate detection of thiol-containing compounds is of great significance in the fields of biochemical research,disease diagnosis,food quality assessment and nutrition research,and environmental safety.Thiol-containing compounds are unstable,and it is easy to occur redox reaction.Meanwhile,its ultraviolet,fluorescence and mass spectrometry signals are relatively poor.The classical strategy is to use chemical derivatization reagent to derivatize it and then detect it.On the one hand,it can play the role of fixing thiol-functional groups.On the other hand,by the introduction of strong ultraviolet,fluorescence or mass spectrometry enhancement group,it can improve the detection sensitivity of the targeted thiol-containing compounds and the selectivity of the analytical method.Compared with the most common ultraviolet and fluorescence detection,the multiple reaction monitoring scanning mode（MRM）of mass spectrometry can simultaneously detect the parent ion and the product ion of the compound,which can effectively reduce the interference of noise,and improve anti-interference ability and sensitivity of the targeted thiol-containing compounds.Especially,it can obtain a pair of light/heavy isotope-labeled derivatives with the same functional groups after chemical derivatization of the tested sample and standard references using a pair of light/heavy isotope-labeled chemical derivatization（ILCD）reagents.It can improve the detection selectivity while the heavily-labeled derivatives are used as the internal standards（ISs）for quantitation analysis.This paper focuses on the design and synthesis of novel ILCD reagents in combination with high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）technique for the detection of thiol-containing compounds by the MRM-based detection mode.The specific work is as follows:Chapter one:Brief introduction of the general situation of thiol-containing compounds,the quantitative analysis methods for thiol-containing compounds,the basic theoretical knowledge of high performance liquid chromatography-tandem mass spectrometry-isotope labeling chemical derivatization technique,and the major researching contents of this paper.Chapter two:Two novel pairs of isotope-labeled chemical derivatization reagents were designed and synthesized and characterized in the presented work:N-（4-（acridone-10-yl）-phenyl）-N-d₀maleimide（NAPM-d₀）and its heavy form,and N-（4-（carbazole-9-yl）-phenyl）-N-d₀maleimide（NCPM-d₀）and its heavy form.Chapter three:The pair of isotope-labeled chemical derivatization reagents N-（4-（acridone-10-yl）-phenyl）-N-d₀/d₂maleimide have been used in the HPLC-MS/MS analysis for the determination of seven aroma-active thiol-containing compounds in the roasted sesame seeds.By combining gas purging micro-extraction with in situ isotope-labeled chemical derivatization method,the high-efficiency extraction and derivatization of aroma-active thiol-containing compounds in the roasted sesame seeds is carried out.The obtained heavy isotope-labeled derivatives were used as the internal standards for the absolute quantification analysis of thiol-containing compounds.This method not only greatly eliminated the matrix effect,but also improved the detection sensitivity,and also solved the problem of the lack of various isotope internal standard isotope of thiol-containing compounds.Chapter four:The pair of isotope-labeled chemical derivatization reagents N-（4-（carbazole-9-yl）-phenyl）-N-d₀/d₂maleimide have been used in the HPLC-MS/MS analysis for the determination of seven organophosphorus thioester pesticides.Firstly,organophosphorus thioester pesticides hydrolyzed to release cleavage products of thiol-containing compounds using silver ion exchanged zeolite based its catalysis action.Then,the cleavage products of thiol-containing compounds were derivatized using isotope-labeled chemical derivatization reagents.The obtained heavily-labeled derivatives were used as the internal standards for the precise quantitation of the cleavage products of thiol-containing compounds for the quantitation of the corresponding organophosphorus thioester pesticides.The established method was successfully applied to the determination of the residual organophosphorus thioester pesticides in agricultural products with satisfactory results.The method provides a new idea for the detection of residual organophosphorus pesticides:by in combination of cleavage products using chemical derivatization with HPLC analysis to achieve the quantitation analysis of residual organophosphorus pesticides in samples.Chapter five:The pair of isotope-labeled chemical derivatization reagents N-（4-（carbazole-9-yl）-phenyl）-N-d₀/d₂maleimide have been used in the HPLC-MS/MS analysis for the determination of four thiol-containing drugs.The established pre-column derivatization using isotope-labeled chemical derivatization reagents followed by HPLC-MS/MS analysis has been successfully applied for the analysis of four thiol-containing drugs with highly sensitivity.