Application Investgation Of The Chromatographic Analysis Of Carboxyl Compounds Based On Derivatization

Carboxylic compounds are a class of organic compounds with carboxyl groups and carbon chains.Carboxylic compounds are widely present in the nature with the form of salts,esters and free acids.It is not only an intermediate metabolite of cell constituents of lipids,carbohydrates and amino acids,but also a substrate or product of biochemical reactions in the physiological activities of living bodies.Their concentration in life is closely related to the growth and development of the organism,aging and certain diseases?diabetes,inflammation?.In addition,as people abused carboxyl compounds for natural transformation,carboxyl compounds become a common environmental pollutant.Therefore,it is extremely important to establish a fast,high-sensitivity,high-accuracy analysis method for carboxyl compounds.In this paper,high performance liquid chromatography-fluorescence detection?HPLC-FLD?,capillary electrophoresis-laser induced fluorescence detection?CE-LIF?and ultra high performance liquid chromatography-mass spectrometry/mass spectrometry?UPLC-MS/MS?are combined Chemical derivatization,solid phase extraction and liquid-liquid extraction techniques to determine carboxyl compounds in plant and human blood samples.The research content mainly consists of the following four parts:?1?In this experiment,4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Propionyl Ethylenediamine?BODIPY?FL EDA,BODIPY fluorescent dye?was used as a pre-column derivatization reagent.Gibberellin 3?GA3?,indoleacetic acid?IAA?,abscisic acid?ABA?,indolebutyric acid?IBA?,4-dichlorobenzyloxyacetic acid?2,4-D?and naphthaleneacetic acid?NAA?of six carboxyl plant hormones were separated and quantified using HPLC-FLD analytical method.The chromatographic separation conditions and derivatization reaction conditions were discussed in detail.The derivatization reaction was completed at 20?within 20min.At the wavelength of?ex/?em=490 nm/510 nm,the six carboxyl phytohormones reached baseline separation at 17 min.The detection limits of GA3,IAA,ABA,IBA,NAA,2,4-D are respectively 0.75,2.00,0.55,3.00,0.05,0.50 nmol/L.The relative standard deviations?RSD?of the migration time and peak area of the derivatives were between 1.80-3.73%and 3.46-5.01%,respectively.This method has been successfully applied to the detection of phytohormone residues in five kinds of vegetables and fruits,and the recovery rate is within the range of94.12-106.75%.?2?CE-LIF was used to separate and detect of gibberellin 3,indoleacetic acid,abscisic acid,indolebutyric acid,naphthaleneacetic acid,2,4-dichlorobenzyloxyacetic acid of six carboxyl plant hormones,conbined liquid-liquid extraction methods.BODIPY?FL EDA was used as a fluorescent labeling reagent.A buffer solution of 28 mmol/L H₃BO₃-Na₂B₄O₇?containing 5mmol/L SDS,4%isopropanol?,p H=9.0 was used as the background buffer solution.At the wavelength of?ex=490 nm,the six carboxylic phytohormones reached baseline separation within 10 min.The detection limits of these six analytes were 0.05,0.05,0.05,0.075,0.0125,and 0.05 nmol/L.The intra-day and inter-day accuracy are less than 5.68 and 6.96%,respectively,and the relative standard deviation?RSD??n=5?ranges from 0.03-1.23%?migration time?and1.14-5.23%?peak area?.This method has been successfully applied to the determination of various endogenous phytohormones in a single flower of Arabidopsis thaliana,and is a powerful auxiliary tool for studying the function and regulation network of phytohormones.The method has also been successfully applied to the detection of phytohormone residues in five vegetables and fruits,and can be used as a analysis method for phytohormone residues in food.?3?A UPLC-MS/MS twin derivative method for the separation and detection of four branched fatty acid esters?FAHFAs?of 12-POHSA,12-PAHSA,12-OAHSA and 12-SAHSA was established.Solid phase extraction?SPE?was used to selectively enrich and purify FAHFAs from biological samples.In this method,a method of twin derivatization reagents was adopted,namely?2-aminoethyl?trimethylammonium?Cholamine?was used as the pre-column derivatization reagent,and 2-dimethylaminoethylamine?2-dimethylaminoethylamine,DMED?-derived FAHFAs are used as internal standards and as a substitute for stable isotopically labeled?SIL?.The derivatization reaction was completed within 1 min at room temperature.After Cholamine labeling,the results showed that the LOD and LOQ of FAHFAs were 0.02-0.07pg/m L and 0.06-0.12 pg/m L,respectively,and the detection sensitivity was increased by 50-126times.In addition,in the UPLC system,FAHFAs can be separated well within 15 min on the chromatographic column,and has a sharp peak shape.Using this method,we successfully quantified the content of FAHFAs in human serum samples,rat white fat,lung,kidney,liver and heart tissues.The results showed that three kinds of FAHFAs?12-PAHSA,12-SAHSA and12-OAHSA?were observed in different tissues of rats.In addition,we successfully detected the above three FAHFAs in human serum samples.?4?Preliminary exploration of UPLC-MS/MS twin derivative method for the determinated the13-PAHSA,12-PAHSA,10-PAHSA,9-PAHSA and 5-PAHSA five PAHSAs isomers.Adopted the derivation conditions in the previous section,the five isomers successfully derivatized with Cholamine and DMED,and PAHSAs derived from DMED were used as internal standards and as a substitute for SIL.The optimal conditions of MRM mode and the separation conditions of five isomers in UPLC system were preliminarily explored.Under the optimal separation conditions,the five isomers were successfully separated within 20 min.The LOD and LOQ of the five PAHSAs were 0.04-0.07 pg/m L and 0.10-0.20 pg/m L,respectively.The detection sensitivity has increased by 73-105 times,and this fast,highly sensitive and accurate quantitative analysis method is applied to the separation and quantitative determination of PAHSAs in human blood and mouse tissues.The results showed that 5 PAHSAs isomers were observed in different tissues of rats.Moreover,we successfully detected the above five PAHSAs isomers in human serum samples.