| The chiral selection rule is one of the natural attributes of the living system.Many living substances such as proteins and nucleic acids are chiral substances.After chiral drugs and pesticides enter the human body,the enantiomers are different in efficacy,toxicology,and metabolic pathways,which are closely related to people's health.The development of highly efficient chiral separation materials and the establishment of a new LC-MS/MS method for rapid,sensitive and accurate determination of enantiomer content are conducive to a more scientific evaluation of enantiomer safety.In this dissertation,based on the“thiol-ene”addition Click Chemistry reaction,the convienent preparation methods for the new chiral stationary phases including derivatized cyclodextrin,teicoplanin and cellulose-bonded phases were developed,respectively.On the basis of characterizing the structures of the stationary phases,the chiral chromatographic performance of the new stationary phases were systematically evaluated and applied to the analysis of actual samples.The new LC-MS/MS methods for enantiomer determination of related chiral markers,chiral pesticides and illegal chiral additives in human urine and food were established respectively,which has important research significance and application prospect for ensuring food and drug safety.The main research work includes the following aspects:1.Firstly,the various types of chiral separation methods developed by our predecessors and their basic principles were reviewed,focusing on the development process and characteristics of chiral stationary phases that are widely used for high-performance liquid chromatography,and related to ordered mesoporous materials as chromatographic bonding matrix.This is used as the theoretical basis and starting point of the research work in this dissertation.2.A benzylureido-?-cyclodextrin was synthesized by the reaction of6-amino-?-cyclodextrin with an active benzyl isocyanate following by a double bond was introduced.Then,it was bonded onto silica gel based on a“thiol-ene”addition reaction,obtaining a new benzylureido-?-cyclodextrin-based chiral stationary phase ?BzCDP?.After structural characterization,BzCDP was successfully used to the simultaneous chiral resolution and quantitative analysis of phenylmercapturic acid?PMA?and benzylmercapturic acid?BMA?enantiomers,which were confirmed as biomarkers of exposure to benzene and toluene in human urine.It was firstly confirmed that biomarkers in human metabolism exist in the form of two enantiomers.Bz CDP was able to rapidly separate the PMA and BMA enantiomers with resolutions up to 2.25 and 2.14 within 30 min,respectively.A new LC-MS/MS quantitative method for the PMA and BMA enantiomers in human urine was established by negative ion multiple reaction monitoring?MRM?and an isotope-labeled PMA?d2-PMA?as an internal standard.The linear ranges of all enantiomers were from 0.5?g L-11 to 250?g L-1,the recoveries were more than 82%,the detection limits?LODs?were less than 0.17?g L-1,and the intra-and inter-day average relative standard deviations?RSDs?were less than 13.1%.The method was successfully applied to urine testing of 60 painting and printing workers.The results showed that both biomarkers in positive urine were present in different enantiomeric forms,such as L-PMA(27.5-106?g L-1)and D-PMA(19.9-82.8?g L-1),indicating that benzene pollution was more serious,which should be paid more attention to the occupational health of this group.This will help to more scientifically assess the harmful effects of benzene and benzene series on humans.3.An S-?-?-2-benzylamino-1-phenylethanol mono-derivative-?-cyclodextrin bonded stationary phase?BzCSP?was prepared based on the"thiol-ene"addition reaction,and its basic structurethe was characterized.By introducing an aryl group and a chiral center,the chiral separation ability of the cyclodextrin stationary phase was further improved,the chiral separation range was broadened,and the practicability of the stationary phase was enhanced.The chiral compounds including four types of chiral compounds,such as cyclic ketone drugs,triazole pesticides,amine-containing drugs,and amino alcohols drugs were used as chiral probes,and their chiral chromatographic properties were evaluated.The results showed that the novel stationary phase was suitable for a variety of chromatographic modes?normal phase,reversed-phase,polar organic modes?.Most drugs could be separated in reversed-phase mode.Among them,the resolutions?Rs?of the cyclic ketones and the triazole pesticides were up to 5.33 and 2.05 in shorter time,respectively.Some compounds could be resolved only in normal-phase mode.For example,the Rs of warfarin was 2.46,and the Rs of benalaxyl reached 8.7.Moreover,it was found that the separation ability of S-?-?-2-benzylamino-1-phenylethanol derivatized CD-based stationary phase was stronger than that of the corresponding R?+?-type stationary phase,probably because the former was more compatible with the"three-point"effect with the analytes than the latter,which was beneficial to chiral separations.In addition,the new stationary phase could also separate the commonly used cardiovascular chiral drugs such as propranolol with a high resolution of 1.53 in polar organic mode.It is indicated that the stationary phase prepared by the"thiol-ene"addition reaction is a new type of multi-mode stationary phase,which has a high application value.In order to verify the practicability of the new BzCSP,a new LC-MS/MS method for the determination of the enantiomeric residues of three chiral pesticides hexaconazole,tebuconazole and triticonazole in five kinds of fruits and vegetables was established.The established method had the characteristics of high selectivity,high sensitivity,strong resistance to matrix interference and good reproducibility.At present,there is still no systematic research on the enantiomeric analysis methods of chiral pesticides at home and abroad.4.A new preparation method of teicoplanin-bonded chiral stationary phase ?TCSP?by the“thiol-ene”addition reaction was prepared for the first time.After the methacrylation of teicoplanin,the methacryl and mercaptopropyl silica gel were subjected to the“thiol-ene”addition reaction to obtain a new TCSP.The new preparation method has mild reaction conditions,high bonding amount and low cost.The high resolution?Rs?2.72 for clenbuterol?CLEN?and 1.91 for salbutamol?SAL?enantiomers on homemade TCSP were simultaneously achieved by an optimal polar organic mobile phase?methanol/acetonitrile/ammonium formate/acetic acid,480/120/0.3/0.04,v/v/m/v?,with a flow rate of 0.5 mL min-1 at 35°C within 20 min.After the solid-phase extraction,a fast and sensitive LC-MS/MS method for the enantiomeric quantitation of the?2-agonists CLEN and SAL in 200 pieces of meats samples was established by multi-reactive positive ion monitoring?MRM?.The precision,accuracy,stability,linearity,limits of detection?LODs?and matrix effects of the method were investigated,separately.This new method has good linear relationship for all enantimers over ranges of 0.5-50?g L-1?r?0.995?,high recoveries?87-103%for CLEN,93-103%for SAL?and high reproducibilities?RSDs,2.65-7.98%for intra-day,4.23-9.29%for inter-day?.The enantiomeric LODs were below 0.018 for CLEN and 0.076?g kg-1 for SAL.The results showed that all positive samples contained unequal enantiomers,especially the R/S isomerization ratio in pork livers was as high as 1.3,which revealed that the?2-agonists were enantioselectively metabolized in animal bodies.Therefore,the scientific assessment of the toxicity of?2-agonists to humans requires precise determination of the enantiomeric content.5.A new 3,5-dichlorophenylcarbamated cellulose-bonded silica gel stationary phase?CELCSP?was prepared by a“thiol-ene”addition reaction.The alkenyl group was first introduced onto the cellulose,and then the cellulose was completely isocyanated with 3,5-dichlorophenyl isocyanate.Finally,the alkenyl group was reacted with the 3-mercaptopropyl silica gel to obtain the cellulose-bonded stationary phase.The structures of the ligand and the stationary phase were characterized by infrared spectroscopy,nuclear magnetic resonance spectroscopy and elemental analysis.The newly prepared cellulose-bonded phase was solvent resistant and could be used in reversed phase chromatography and was compatible with ESI-MS,which was successfully used for the enantio-resolution of six common chiral fungicides including triticonazole,hexaconazole,tebuconazole,triadimefon,metalaxyl and benalaxyl,respectively.The resolution?Rs?and selectivity factor???of the above fungicide enantiomers on CELCSP could reach 3.46 and 1.27,respectiviely,by using the common 0.1%formic acid-acetonitrile as the mobile phase.Base on the CELCSP column,a new LC-MS/MS method for the quantitative determinations of all six chiral fungicide enantiomers in ten kinds of fruits and vegetables such as cucumber,grape,etc.was established within 30 min.After the rapid sample pretreatment with Fe3O4magnetic particles,the enantiomers were separated by LC and determined in positive ion multi-reaction monitoring?MRM?by mass spectrometry.The good linear relationship between the response and the concentration of the enantiomers were observed in a range from 0.10?g L-1 to 100?g L-1 with the correlation coefficient?0.9965–0.9982?.The averaged recoveries from fruits and vegetables were between65%and 110%?n=3?.The limits of detection?LODs?and limits of quantitation?LOQs?for the enantiomers were 0.05-0.61?g kg-1 and 0.18-2.01?g kg-1,respectively.The RSDs of the repeated determination in the samples were 1.2%–6.0%?intra-day,n=5?and 2.5%–13.0%?inter-day,n=10?,respectively.The mild condition of the thiol-ene addition reaction was beneficial to maintain the order stereostructure of cellulose,while its high yield of the oriented synthesis could also provide sufficient binding amount of chiral ligand,which provided a guarantee for establishing a reliable food safety analysis method for monitoring pesticide enantiomer residues by LC-MS/MS. |
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