The Establishment And Application Of HPLC Method Determination On 11 Substances Related To Diabetic Complications

Background:In recent years,the incidence of diabetes gradually increased and the complications of diabetes were widely concerned.Diabetic patients are often accompanied by brain diseases?diabetic encephalopathy,DE?which clinical manifestations are mild to moderate cognitive impairment,decreased the ability of learning,memory,judgment,comprehension,and others with sluggish eyes and unresponsive.Whats`more,learning and memory disorders are the main clinical manifestations of DE.The pathogenesis of diabetic encephalopathy is multifactorial.The role of neurotransmitters in diabetic complications is being studied.Neurotransmitters and their related substances in animals play an important role in the transmission of neural pathways which can regulate the acquisition and storage of information and physical activities,such as learning and memory.It has been accepted that different parts of the brain have different effects on learning and memory and that the cerebral cortex and hippocampus are predominant.Neurotransmitters in the brain can be divided into excitatory and inhibitory neurotransmitters.Excitatory neurotransmitters act on receptors to promote learning and memory physiological processes.If they increase excessively,neurons are continuously depolarized,resulting in neurotoxicity and eventually cell death.Inhibitory neurotransmitters act on presynaptically to reduce the release of Glu while leaving postsynaptic neurons in a protective state.When the concentration of inhibitory neurotransmitters rises,neurons are in a state of hyper-suppression,affecting the process of neurotransmitter transmission,and making the learning and memory ability decline.Neurotransmitters in the brain play a key role in maintaining the normal physiological activities of the brain.Alteration of concentrations can induce cognitive defects,learning disabilities,Parkinson's and other neurological diseases.Quantitative determination of neurotransmitters in the brain plays an important role in the control of a clinical disease.Fewer methods were established to simultaneously determine amino acids and monoamine neurotransmitters with HPLC-FLD.Therefore,this article is divided into three parts,respectively,to explore the determination method of nine neurotransmitters by HPLC-FLD,and the determination method of homocysteine and cysteine by HPLC-ECD,and the concentration alteration of 11 neurotransmitters in brain and plasma of diabetes mellitus rat.Part I:The establishment of a method for the determination of 9substances in ratObjective:To establish an HPLC-FLD method for simultaneous determination of nine substances in rat brain and plasma.Method:The HPLC system?Agilent 1260?equipped with a fluorescence detector?FLD-G1321B?and UV detector was employed in our study.The separations were performed on a Hypersil BDS column?4.6 mm×250mm,5?m?fitted with a C18 security guard cartridge.The mobile phase A consisted of 35 mmol·L^-1 sodium acetate and 5 mmol·L^-1 citric acid,adjusting pH to 6.0.The mobile phase B was methanol.The gradient elution,at a flow rate of 1.0 mL.min^-1,was used.28%B from 0 to 23 min,then 60%B at 23.01 min till the end of the analysis.Fluorescence detection was performed with excitation at 340 nm and emission at 460nm.The column oven temperature was set at 30?.The injection volume was 20?L.The samples of brain tissue and plasma were pretreated as follows:ultrapure water was added at a ratio of 1:3?m:v?to cortex and hippocampus,the mixture was manually homogenized with a glass homogenizer,then 200?L of 0.4 mol·L^-1 perchloric acid was added to the100?L homogenate.After thoroughly mixed,the mixture was centrifuged at 15000×g,4°C for 15 min,the supernatant was diluted and injected.Results:Asp,Glu,Gly,Tau,GABA,Trp,Met,5-HT,DA were completely separated.The correlation coeffections of 9 analytes were more than 0.9991?r²?0.9991?.The LOD was 0.8-9.2 ng·mL^-1 and the LOQ was 2.6-30.5 ng·mL^-1 with RSD%of intra-day precision?10.01%and inter-day precision?12.80%.The relative recoveries ranged from79.82%to 111.11%and absolute recovery ranged from 77.79%to100.25%.Conclusion:The determination method was rapid and simple with high sensitivity with good separation.It could be applied to determine the concentration of neurotransmitters related to diabetic complications.Part II:The establishment of a method for the determination of Hcy and Cys in rat brain tissues and plasmaObjective:To establish an HPLC-ECD method for simultaneously determine the concentration of two substances in rat brain and plasma.Method:Shimadzu high-performance liquid chromatography instrument equipped with an electrochemical detector?ED723?and a DAD detector was adopted in our study.The separation was achieved on C18?4.6 mm×250 mm,5?m?.The mobile phase was consisted of methanol and pH=5.0buffer solution containing 30 mmol·L^-1 sodium acetate and 100?mol·L^-1sodium octane sulfonate with 0.1:99.9,v/v.The whole analysis time was30 min.Detection voltage:900 mv.The column temperature was set at30?.The flow rate was 0.7 mL·min^-1.The injection volume was 20?L.The sample pretreatment method was as follows:the brain tissue was weighed,and ultrapure water was added at a ratio of 1:3?m:v?,and the mixture was manually homogenized with a glass homogenizer.After homogenization,10?L of a 2 mol·L^-1 sodium tetrahydroborate solution was added to 100?L of the homogenate or plasma sample.200?L of 0.4mol·L^-1 perchloric acid was added to the mixed homogenate.After thoroughly mixing,the mixture was centrifuged at 15 000×g,4°C for 15min.The supernatant was directly analyzed.Results:Hcy and Cys were completely separated with good linearity and resolution ranging from0.02 to 4?g·mL^-1?r²?0.991,Rs>2.0?.The LOD was 5-10 ng·mL^-1 and the LOQ was10-20 ng·mL^-1 with intra-day precision of RSD?6.71%and inter-day precision of RSD?9.96%.The relative recoveries were 79.82%-110.18%and the mean extraction recoveries were 84.26%-100.3%.Conclusion:The established method was simple with high sensitivity.It could be applied to separation and determination of neurotransmitters in clinical diabetic complications.Part III:Determination of 11 neurotransmitters in hippocampus,cortex,and plasma of diabetic model rats and drug model ratsObjective:To determine the levels of 11 substances in the hippocampus,cortex,and plasma of rats in the normal group,the diabetic group,and the drugs treated group were to investigate the relationship between the alterations of 11 substances and the complications of diabetes.Methods:Fifty male Sprague-Dawley rats were fed with SPF grade basic diet for one week.Fifty rats were randomly divided into control group,diabetic group,insulin group,vildagliptin group and metformin group.All rats were fed SPF grade basal diet for 8 weeks.Results:Compared with the control group,the concentration of Gly,GABA,Trp,Met,5-HT,and Cys in the cortex of diabetic rats were significantly reduced?p<0.05?.the concentration of Glu,Trp,and Met in the hippocampus of diabetic rats were significantly different?p<0.05?.the concentration of Asp,Glu,Trp,and DA in plasma of diabetic group were significantly differences?p<0.05?.All the three drugs could improve the neurotransmitters concentration changes caused by diabetes.Conclusion:Compared with normal group,the concentration of these neurotransmitters in the central and peripheral system of diabetic rats had altered in differences,suggesting that these neurotransmitters are related to the complications of diabetes.