Study On Enantioseparation Of Amino Acids And Enzyme Assay For D-amino Acid Oxidase Based On Optically Gated Capillary Electrophoresis-laser Induced Fluorescence Detection

With the speedy development of enantioseparation techniques,chiral separation has gained great research interest in the fields of analytical chemistry,pharmaceuticals,environmental and life sciences.It has long been presumed that amino acids(AAs)exist only in the l-form in nature,until 1970 s,the smaller amount of their D-enantiomers are found in mammalian tissues.Because D-amino acids(D-AAs)play an essential role in the regulation of many processes in living cells and the amount of D-AAs link D-amino acid oxidase(DAAO)to many diseases.Hence,detection of D-AAs and assay of DAAO activity are of vital importance in bioanalytical science.However,the fact that DAAO presents broad substrate activity and abundant L-AA enantiomers present in biological samples would interfere the reliability and accuracy of the DAAO assays.The key to solve this challenge is efficient enantioseparation of AAs.Among of many enatioseparation methods,capillary electrophoresis(CE)has been demonstrated as an efficient solution for AAs enantioseparation,due to its unique advantages of high separation efficiency,small sample requirement and economical equipment.To further reduce the analysis time in enantioseparation of AAs and improve the efficiency of DAAO assay,the mainly contents of this paper were listed on the following:(1)We present the application of an in-house constructed optically gated capillary electrophoresis with LIF detection(OGCE-LIF)system for efficient enantioseparation of AAs.To improve the enantioseparation of efficiency,various factors such as the concentration of HP-?-CD and borate buffer,the p H of buffer and the voltage for separation were investigated and optimized.Under the optimal OGCE-LIF conditions,five pairs of OPA/NAC labeled D/L-AAs are efficiently separated and low limit of detection of 1.3 ?M.The quantitative features of this proposed method were studied for exploring its potential application.High repeatability of the OGCE-LIF assay of amino acids(AAs)was achieved with relative standard deviation(RSD)(n = 15)less than 1.5% and 2.7% for migration time and peak height,respectively.The efficient enantioseparation of AA enantiomers can be accomplished with excellent repeatability and low limit of detection in OGCE-LIF system,indicating that the present method allows simultaneous quantification of the AA enantiomers.(2)Enzymatic assays of DAAO have been successfully achieved by detection the substrate consumption with OGCE-LIF,for either single or mixed AA substrates.Kinetic analysis of the parallel oxidation reactions of two different substrates has been performed,which is in good agreement with the experimental results.Our study indicates OGCE-LIF is a promising method for quantitatively assaying DAAO catalyzed reaction with the presence ofL-AA enantiomers in the sample.It is also proposed the feasibility of OGCE-LIF for assaying DAAO with multiple substrates,due to its rapid and efficient separation of mixed pairs of AA enantiomers,which would pave the way for accurate determination of D-AAs and DAAO enzymes in complicated biological samples.