Establishment Of A Method For The Measurement Of Plasma Renin Activity By Mass Spectrometry

Objective:To study the accurate measurement techniques and methods for plasma renin activity（PRA）,and establish of a candidate reference method for PRA measurements based on Liquid chromatography tandem mass spectrometry（LC-MS/MS）with measurement performance satisfying the requirements of ISO15193 documents.Methods:Calibration of LC-MS/MS and pH Meter,Angiotensin Ⅰ（AngⅠ）standard solution and ¹³C-¹⁵N labeled AngⅠ internal standard solution were used for LC-MS/MS detection to optimize the detection of AngⅠ ion source,ion pair,gas parameters,voltage parameters and other mass spectrometry measurement conditions;single factor analysis was used to optimize the liquid phase measurement conditions such as chromatographic column,mobile phase composition,velocity of flow and so on.Based on the response of AngⅠ chromatogram and mass spectrometry,the gradient of liquid phase elution was optimized.Compare the measurement results of the AngⅠ internal standards of the different¹³C-¹⁵N labeled with different amino acids.Determine the internal standard used in the experiment;take the plasma sample,add the defined ¹³C-¹⁵N AngⅠ internal standard solution and angiotensin-converting enzyme inhibitor,and incubate at 37°C with shaking to make the endogenous renin in the plasma catalyze the angiotensinogen production of AngⅠ.After 3 hours,the low-temperature freezing sample was terminated.The protein was precipitated with an organic solvent,and the supernatant was taken after high-speed centrifugation.Nitrogen was blow dried to concentrate the mobile phase.Based on the isotope dilution principle,LC-MS/MS was used to measure the AngⅠ generated by renin reaction.And establish of standard curve,quantitative analysis of angiogenesis,then calculation of PRA in combination with incubation time.The concentration of angiotensin converting enzyme inhibitor（ACEI）and the kinds of precipitators in plasma sample pretreatment were compared,groped and optimized systematically,and the main performance evaluation indexes of the method were evaluated:linear range,limit of detection,limit of quantitative,precision,method recovery rate etc.Results:（1）Mass spectrometry measurement condition:Electrospray ionization（ESI）positive ion mode analysis was used.The quantitation ion pair of AngⅠ was m/z 433.2→110.2,the qualitative ion pair was m/z 433.2→m/z 70.0,and the internal standard quantification ion pair was m/z 437.3→m/z 110.1,qualitative ion pair m/z 437.3→m/z70.0.（2）Liquid chromatographic conditions:The mobile phase consisted of methanol（containing 0.1%formic acid）-water（containing 0.1%formic acid）,selected Waters Atlantis C18 reversed-phase column（2.1×100mm,3μm）,gradient elution,flow rate0.3ml/min,column temperature 40°C,Injection volume 5μL.（3）Pretreatment condition:ACEI is phenylmethylsulfonyl fluoride,the concentration was 1 mmol/L,incubation time was 3 h,the precipitant was methanol solution（containing 0.1%formic acid）,the ratio of plasma to precipitant was 1:5,and the absolute recovery was 91.59%on average.（4）Method main performance evaluation index:linear range 0.067³3.33 ng·mL^-1·h^-1（R²=0.9998）,limit of detection:0.033 ng·mL^-1·h^-1,limit of quantitative:0.067 ng·mL^-1·h^-1,the intra-assay and inter-assay precisions were 0.40%⁴.86%,0.99%³.94%,respectively,and the average recoveries of low,medium,and high concentrations of plasma samples were 99.01%,101.10%,and 100.83%.Conclusion:PRA candidate reference measurement method has been established based on LC-MS/MS,and the main performance of the method meets ISO15193 document requirements.