Research And Application Of Renin Analysis Methodology Based On Liquid Chromatography-tandem Mass Spectrometry(LC-MS/MS)

Objective:To establish a simple,rapid,specific,sensitive,reproducible and accurate method for the detection of renin by combining the pretreatment methods of internal standard correction,enzyme inhibitors,organic reagents and high performance liquid chromatography-tandem mass spectrometry（LC-MS/MS）,and apply it to the study of related diseases in healthy people and hypertensive patients.The consistency of the results will be compared with the traditional method.Methods:Firstly,the mass spectrometry needle pump was applied to inject the sample directly,and the standard sample was applied to optimize the conditions and parameters of mass spectrometry,and then the parameters of liquid phase conditions were optimized to obtain the LC-MS/MS measurement conditions required in this experiment.Plasma was incubated under suitable conditions to make renin activity,promote the reaction,prevent the degradation of AngⅠ（angiotensinⅠ）produced during the reaction.In the end,the reaction was terminated within a certain time.The content of AngⅠwas measured and the plasma renin activity was calculated combined with the incubation time.The pretreatment steps of serum samples were optimized step by step,which includes the linear range of the instrument,the interference of bovine serum albumin,the selection of deproteinized precipitant and volume,the concentration and volume of enzyme inhibitor and the selection of incubation time.A LC-MS/MS method for the determination of Ang I in plasma was established by using positive ion electrospray ionization model,standard curve determined by standard and accurate quantification combined with isotope-labeled internal standard method.Finally,the linear range,detection limit,quantitative detection limit,inter-day and intra-day precision,inter-day and intra-day recovery and sample stability of the established method were investigated.Results:Ang I and internal standard were detected by mass spectrometry needle pump direct injection and LC-MS/MS,and the optimized MRM parameters were obtained,including ion pair information of parent ion and daughter ion,DP,EP,CEP,CE and CXP values.In the optimization of liquid phase conditions using standards,the liquid phase optimization results include:C18 reversed phase chromatographic column（5μm,4.6×150 mm）,organic phase A is methanol containing 0.1%formic acid,water phase B is pure water containing 0.1%formic acid.The pretreatment conditions for the formation of Ang I reaction by renin were optimized.Bovine serum albumin was selected as blank plasma,acetonitrile was selected as deproteinizing reagent,methanol-water（1:1,V/V,0.05%FA）was selected as the re-solubilizing solvent,PMSF（phenylmethylsulfonyl fluoride）was selected as the inhibitor to terminate the reaction,and the incubation time of the reaction was 3 hours.Under various optimized conditions of this experiment,plasma renin activity（PRA）was calculated by using LC-MS/MS technique combined with the incubation time.The detection time of the entire LC-MS/MS method is 15 min.The linear range of the instrument and the method is 0.5-100 ng·m L^-1,the detection limit is 0.2 ng·m L^-1,and the quantitative detection limit is 0.5 ng·m L^-1.The intra-batch precision in low,medium and high concentrations was 7.29%to 10.72%,3.76%to 8.94%and 3.93%to 5.99%,respectively.The inter-batch precision of each concentration for three successive days was 6.71%,1.36%and 1.23%,respectively.The recovery in low,medium and high concentrations was 70.79%to 101.28%,75.68%to 110.45%and 65.27%to 81.37%,respectively.The relative standard deviations of the recovery rates of each concentration were 13.91%,13.77%and 8.84%,respectively.Finally,the method established in this study was compared with the results of the same clinical samples,showing commendably consistent trend.Conclusions:In this study,a method for calculating PRA by measuring the content of Ang I produced by reaction and incubation time was established,which combined protein precipitation,optimization of pretreatment,enzyme inhibition and LC-MS/MS techniques.The results reveal that the pretreatment step in this study is simple and convenient,the incubation condition is suitable,and the detection sensitivity of the mass spectrometer is significantly improved after incubation.Moreover,the method has good repeatability,good inter-batch precision and intra-batch precision,and good recovery rates of low,medium and high concentration.In this experiment,the internal standard correction method was used for accurate quantification,and this method was applied to the analysis and detection of plasma renin activity in healthy people and patients with hypertension,and the results were consistent with those of the traditional method.