The Studies of a Pre-column Derivatization-HPLC Method for the Determination of α-dicarbonyl Compounds

Author:Wang Zuo Jie

Supervisor:gao wen yun


Degree Year:2019





α-Dicarbonyl compounds(α-DCs) have been widely studied because they are the main precursors of the advanced glycation end products(AGEs).Up to date,plenty of investigations have confirmed the existence of this kind of compounds in different food samples,especially in fermented foods.Furthermore,the presence of variousα-DCs in vivo has been associated with the development of complications of diabetes mellitus(DM) and also with other chronic diseases,such as Alzheimer’s disease,Parkinson and so on.Therefore,quantitative analysis ofα-DCs in different samples is crucial to understand their roles both in vivo and vitro.Presently,the pre-column derivatization-HPLC methods have been widely employed for detectingα-DCs in various samples.The mostly used derivatizing reagents are o-phenyldiamine and its derivatives and analogues because they can form quinoxaline substructure withα-DCs which is stable and has excellent UV absorption.These advantages largely facilitate the HPLC separation and UV detection of these compounds.All these derivatizaing reagents,however,share a common drawback,namely their derivatizing reactions withα-DCs need prolonged time at room terperature or to be heated to reach optimum result,which undoubtedly brings more challenges to the precise determination of theα-DCs.In order to improve the research onα-DCs detection,we synthesized in this thesis a new derivatizing reagent and destablished a novel analytical procedure for the determination of these compounds by pre-column derivatization HPLC-UV.The advantages of this method are:i)the derivatization reaction could be carried out quickly under mild conditions;ii)the HPLC-UV process displays efficient chromatographic separation,approving linearity,and satisfactory analyses of various samples.Firstly,the new derivatization reagent 4-(2,3-dimethyl-6-quinoxalinyl)-1,2-o-phenylene-diamine(DQB) was synthesized and used to determine theα-DCs including3-deoxyglucosone(3-DG),glyoxal(Gly),methylglyoxal(MGly),diacetyl(DA),and pentane-2,3-dione(PD).The optimum derivatization conditions were determined(reaction pH 9.0,room temperature,30 min).The fiveα-DCs showed wide linear ranges and good linear correlation coefficients(R~2>0.99);the limits of detection(LOD) of the five compounds are 3-DG,0.05μM;Gly,0.05μM;MGly,0.05μM;DA,0.02μM;PD,0.025μM,respectively.The intra-and inter-day relative standard deviation(RSD) of the five compounds are 0.34-2.70% and 0.34-2.06%,respectively;the reaction products of DQB and theα-DCs are stable for at least 24 h at room temperature.Secondly,the established detection method was applied to measure theα-DCs in different food samples after its reliability in these kind of samples has been evaluated.The results displayed that the recoveries of the five compounds ranged from 85.35 to 105.38%,and the RSD ranged from 0.43 to 4.07%.These data indicate that the method is suitable for determining theα-DCs in food samples.After that,theα-DCs in seven types of foods were quantified,including beer,alcohol,carbonated drinks,dairy products,fruit drinks,coffee samples,and teas.These tests provide a new approach for food quality assessment and also provide a dietary reference for the patients of chronic diseases like diabetes.Thirdly,the detection method was also used to quantitatively analyze and evaluate theα-DCs in human plasma after its reliability in this kind of sample has been evaluated.The recovery rates of the fiveα-DCs are between 84.38%and 103.99%,the intra-and inter-day RSDs are in the range of 1.28-5.69% and 2.26-6.34%,respectively,and the derivatives are stable for at least 24 h at 4~oC.Based on the evaluation,the concentrations of thea-DCs in the blood plasma of 47 healthy human subjects and 50 type 2 diabetes mellitus(T2DM) patients(including 15 diabetic nephropathy patients) under hypoglycemic treatment were determined.The data showed that the levels of 3-DG,Gly,and MGly in the plasma of diabetic patients were significantly higher than those in healthy subjects and the levels of Gly and MGly in patients with diabetic nephropathy(DN) were higher than those in patients with T2DM alone.Additionally,Pearson’s correlations were calculated and correlation tests between HbA1c,plasma glucose and thea-DCs were conducted,respectively by using the quantification results ofa-DCs in the plasma of T2DM patients.The results indicate that the levels of 3-DG,Gly,and MGly positively correlate to glycosylated hemoglobin(HbA1c) and fasting-blood glucose.We also evaluated the correlation between microalbuminuria and the levels of Gly and MGly in the DN patients.The results display positive correlations between Gly or MGly with microalbuminuria.The data obtained in this study exhibit for the fist time that the blood level of Gly might play a role in the development of DN as well.Fourthly,we detected the levels of theα-DCs in human saliva for the first time by employing the established DQB approach.The recovery rates of the fiveα-DCs in saliva ranged from 80.26% to 100.63%,and the RSD ranged from 0.34% to 4.64%.Using this method,the contents of fiveα-DCs in saliva samples of 23 T2DM patients and 15 healthy subjects were determined,and the levels of Gly and MGly in saliva of diabetic patients were found to be higher than those in healthy subjects.In addition,the levels of glucose in the saliva of the two groups of subjects were evaluated.The results showed that the salivary glucose level of T2DM patients was significantly higher than that of the healthy control group,and salivary glucose was found to be positively correlated with fasting blood glucose and HbA1c.Finally,anotherα-DC,namely D-glucosone(GS) was detected by using DQB as a derivatization reagent.The optimal reaction conditions for this compound were pH 5.0,80 ℃ for 50 min.The results showed that GS had a good linearity(R~2=0.993)over a range of1-150μM with an LOD of 0.01μM and RSD<5%.The detection method was then utilized to quantitatively analyze the GS level in human plasma,including 50 patients with type 2diabetes and 47 healthy subjects.The data exhibited the average level of GS in plasma of T2DM patients was higher than that of healthy controls.Moreover,its level also had a positive correlation with HbA1c and fasting blood glucose.In this thesis we first prepared a new reagent DQB that can react withα-DCs to form quinoxaline derivatives.Then we set up and evaluated a pre-column derivatization-HPLC method to quantify theα-DCs in various samples by using this reagent.Based on the method,the concentrations of fiveα-DCs in different foods and the plasma and saliva of diabetic patients were determined and informative results were obtained.The importance of this research lies in:i)the determination of theα-DCs in various foods could present reference for the patients of the chronic diseases like diabetes in their daily diet;ii)the quantification of theα-DCs in the plasma and saliva of diabetic patients and the comparison of these data with those of the healthy controls could provide significant information for us to explore the relationship between the in vivo α-DCs levels and the occurrence and development of diabetes and its related complications.In addition,these data could furnish possible solutions as well for the screening and monitoring of this chronic disease.