Quantitative Analysis Of Endogenous Carbohydrates In Human Urine And Serum Using Hydrophilic Interaction Liquid Chromatography Coupled With Tandem Mass Spectrometry

The rapid growth of aging population has brought many social problems.Chronic diseases,such as high blood pressure,heart disease,diabetes and cancer,endangered the people's health.The best way to prevent these diseases is early diagnosis and early treatment,which improves the therapuitic efficiency and living quality of the patients.As a new branch of science,metabonomics has advantages in the early diagnosis,prevention and treatment of disease.Metabolic abnormalities lead to abnormal proteins.And long-term metabolic disturbance can greatly increase the gene mutation,which leads to gene mutations in the end and causes many irreversible major diseases.A simple and fast method for quantification of endogenous carbohydrates in human urine and serum was developed using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.The quantification method can be used in the expanded metabonomics study.Firstly,in our study a simple and fast method for quantification of 15 endogenous carbohydrates in human urine was developed using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.Chromatographic conditions,mass spectrometric conditions and sample preparation method were all optimized.The methanol/acetonitrile?50:50,v/v?mixture was used to effectively remove the protein and impurities in urine,with the ratio of 5 times volume of organic solvents to 1 volume of urine sample.The chromatographic separation for endogenous carbohydrates in urine was accomplished by using an ACQUITY UPLC BEH Amide column?2.1×100 mm,1.7 ?m?.Mobile phase A was composed of 10 m M ammonium acetate in H2O/ACN?95:5,v/v?,and mobilephase B consisted of 10 m M ammonium acetate in ACN/H2O?95:5,v/v?.The compounds of interest were detected by MS/MS innegative ion multiple reaction monitoring?MRM?mode.Method validation including linearity,precision,matrix effects,recovery and stability was performed.The calibration curves showed a good linearity?correlation coefficient of r > 0.99?with the LOQ in the range of 0.075-30 nmol·L^-1 for most of compounds.The intra-and inter-dayprecision?CV?ranged from 2.53%-7.64% and 3.28%-7.34%,respectively.The recovery was in the range of 85.5%-115%.The matrix effect of most chemicals was in the range of 80%-120%.The stability data in the range of 93.1%-106%,indicatingthat post-preparative samples stored at 4 ? for 72 h was stable under routine laboratory analysis.These results met the requirements of Guidance on Bioanalysis: method validation and analysis of study samples.Finally,concentrations of twelve carbohydrates in human urine?mean±SE?were deternined: melibiose 2668.1±134.4 nmol·L^-1;sucrose 5070.6±277.6 nmol·L^-1;maltose 15214.5±442.5 nmol·L^-1;maltotriose 849.7±42.6 nmol·L^-1;glucose 173965.0±4918.9 nmol·L^-1;fructose 22172.5±1463.6 nmol·L^-1;raffinose 292.1±17.6 nmol·L^-1;inosine 2083.5±93.4 nmol·L^-1;adenosine 2645.5±78.2 nmol·L^-1;mannitol 83925.0±3938.6 nmol·L^-1;erythritol 375805.0±12094.6 nmol·L^-1;arabitol 192921.1±5689.2 nmol·L^-1.Concentration of three carbohydrates in human urine,namely maltotetraose;xylose and ribose were below the limit of quantitation.Then,targeted analysis of 16 endogenous carbohydrates for expanded metabonomics approach was applied to 24 sera from healthy women,using HILIC-MS/MS.According to the previous report's optimized chromatography conditions,mass spectrometry conditions and sample preparation method,we established this method.And method validationwas partly performed,The calibration curves showed a good linearity?correlation coefficient of r > 0.999?with the lowest LOD of 0.3 nmol·L^-1and lowest LOQ of 1 nmol·L^-1;The precision?CV?ranged from 1.30%-4.66%,which was far less than 15%;The recovery was in the range of 86.8%-103%,which indicated that the results also met the requirements of quantification in biological samples.At last,concentrations of sixteen carbohydrates in human serum?mean±SE?were deternined: Raffinose 162.5±2.7 nmol·L^-1;Fucose 3271.3±30.9 nmol·L^-1;Ribose 8700.1±180.5 nmol·L^-1;Xylose 3818.3±50.0 nmol·L^-1;Maltose 1499.3±27.9 nmol·L^-1;Maltotriose 1446.1±36.0 nmol·L^-1;Maltotetraose 446.0±1.2 nmol·L^-1;Glucose 2751250.0±14419.3 nmol·L^-1;Fructose 7500.0±234.7 nmol·L^-1;Inosine 2381.2±73.2 nmol·L^-1;Adenosine 9.9±0.1 nmol·L^-1;Sucrose 170.6±8.7 nmol·L^-1;Mannitol 2415.0±72.1 nmol·L^-1;Melibiose 149.0±0 nmol·L^-1;Erythrito l4229.6±37.1 nmol·L^-1;Arabitol 1213.3±6.8 nmol·L^-1,but glucose,fructose and inosine were analyzed with samples diluted 20 times.In the end,the results show that a simple,accurate,sensitive and specific LC–MS/MS method for determination of endogenous carbohydrates in human urine and serum was developed and validated.The method was applied to the analysis of normal human urine,and could be used in the validation of biomark and metabolic mechanism research in the future.