The Establishment Of A Candidate Reference Measurement Procedure For Serum/Plasma Aldosterone And Its Application

Objectives:To establish an accurate and reliable reference measurement procedure of plasma/serum aldosterone based on isotope dilution liquid chromatography-tandem mass spectrometry(ID-LC/MS/MS)technology.Methods:ID-LC/MS/MS system consists of ACQUITY UPLC I-CLASS chromatography system and Xevo TA-S mass spectrometry system(Waters,America).Aldosterone pure powder,purchased from Sigma Aldrich,is dissolved in 40%methanol in water solution to make aldosterone standard working solution for quantification.Two points quantification method is adopted and conditions of sample preparation and parameters on ID-LC/MS/MS system are optimized.According to relevant EP documents,Specifications of this method,such as limit of quantity(LOQ)value,imprecision,relevant recovery ratio,matrix effects and trueness,are evaluated.Biological variation specifications derived from Westgard database are used for criteria.Results:Aldosterone D-8 is selected as the internal standard,and solid phase extraction with principle of negative ion exchange by Oasis Max ?Elution plate is used for sample preparation,and super-pure water and methanol are used for flow solution on chromatography system,and MRM model with ESI-ion source is adopted on mass spectrometry system with 359.16/189.14 as the quantification ion tunnel and 359.16/331.23 as the qualification ion tunnel.Single measurement interval is 6 minutes and no interferences are found.LOQ value is lower than 20 pg/mL.The repeatability(N=5)and within-laoratory imprecision(N=25)of low,medium and high concentration samples are 2.06%?2.44%and 2.54%?3.66%,respectively,which meet 1/2 of the optimum imprecision specification requirement(?3.675%).The average relevant recovery ratios(N=6)of all low,medium and high concentration aldosterone standard solutions added range from 98.76%to 99.11%,which meet 1/2 of the optimum bias specification requirement(?3.1%)derived from the biological variation database.Conclusions:We have established an ID-LC/MS/MS for aldosterone measurement with high sensitivity and specificity,which is suitable and reliable for being regarded as a candidate reference measurement procedure for aldosterone standardization.Objectives:To prepare aldosterone candidate secondary reference materials(SRMs)of three concentration levels for providing traceability of aldosterone measurement for manufacturers and clinical laboratories,improving measurement quality of aldosterone and enhancing its progress of standardization.Methods:Fresh left human sera were collected in the Endocrine laboratory for the preparation of SRMs and the target values of SRMs are assigned by the aldosterone candidate reference measurement procedure(CRMP)according to ISO 35 guidelines.The stability,homogeneity,frozen-thawing effects and commutability of SRMs are evaluated as well according to ISO 35.Results:The target values(N=4)of three concentration aldosterone SRMs are 54.61 ±1.99(pg/g)?139.26±3.47(pg/g)?226.58±7.39(pg/g),respectively.Good homogeneity and stability at 80? for a half year is validated and no obvious frozen-thawing effects are found.Attributed to insufficient specificity of aldosterone clinical measurement procedures and the lack of enough sample volumes,only Diasorin,Antu and one ID-LC/MS/MS methods are included for commutability analysis and aldosterone SRMs are confirmed to possess commutability between Diasorin and ID-LC/MS/MS method.Obvious difference among different measurement procedures in measuring aldosterone was found in proficiency testing of aldosterone in 2019 with SRMs of three concentration levels as samples.After calibration by using SRMs of high and low levels,the comparability of measurement results of SRMs at medium level by participant laboratories improved quite a lot.Conclusions:Aldosterone SRMs of three concentration levels based on human pooled sera are prepared,may adopted for manufactures or laboratories to set traceability chain of aldosterone measurement,therefore,be hopefully enhancing the standardization of aldosterone measurement.Objectives:To evaluate the correlation and comparability of aldosterone measurement results of two CLIAs and one ID-LC/MS/MS method,and to verify the commutability of aldosterone candidate secondary reference materials,quality controls of manufacturers and external quality assessment samples.Current situation of aldosterone measurement standardization is analyzed by intergrating the data of aldosterone external quality assessment programs(EQAPs)in China with method comparability.Methods:According to EP14-A3 and EP9-A3 guidelines,45 individual left fresh plasma specimens with initial aldosterone measurement results ranging from 30?1000pg/mL were collected,split into aliquots and stored at-80?.Precision specifications were verified before sample testing according to EP15-A3.Deming regression analysis,R2 and Concordance Correlation Coefficient(CCC)were adopted as indicators to evaluate the correlation and comparability of methods.Bland-Ataman plots were used to analysis the absolute and relevant bias between methods.Commutability was evaluated according to EP14-A3 with 95%prediction intervals as criteria.Results:Precision specifications of all three methods meet the requirements for methods comparison.Good correlation(R2=0.985)and comparability(CCC=0.967)was demonstrated between Diasorin and LC-MS/MS,while unsatisfied correlation and comparability were found between Antu and LC-MS/MS or Diasorin.The average relevant bias of all pairs made by three methods exceed the lowest bias requirement(18.6%)derived from the biological variation database.Only the correlation of Diasorin and LC-MS/MS meets the requirement for commutability assessment,and aldosterone candidate secondary reference materials show commutability on Diasorin and LC-MS/MS methods.Aldosterone EQAPs data show remarkable differences exist between-group and within-group measurement results when groups are divided by different reagents manufactured by different manufacturers.Conclusions:Differences are found on aldosterone measurement results by different measurement methods,and the insufficient specificity and diversified traceability of methods are two main causes and still have a long way to go.The implementation of aldosterone reference measurement procedure,commutable aldosterone reference materials and national programs to enhance harmonization/standardization of aldosterone measurement,such as external quality assessment programs,is one of keys to enhancing the standardization and mutal-recognition of aldosterone measurement results and standardized screening and diagnosis criteria of primary aldosteronism.