Establishment And Application Of ID-UPLC-MS/MS Method For Measuring Total Testosterone In Human Serum

Objective To develop a method for the determination of total testosterone in human serum using isotope-diluted ultra-high-performance liquid chromatography-tandem mass spectrometry(ID-UPLC-MS/MS).In order to establish a candidate reference method for serum total testosterone,the sample preparation conditions and chromatographic mass spectrometry detections of this method were optimized.This method was evaluated for its methodological performance,and its ability as a candidate reference method was examined.This UPLC-MS/MS method was used to perform a consistent comparison with several commercially available immunoassay platforms,and then the preparation of mixed serum testosterone working calibrator was explored.The candidate reference methods were used to establish a complete reference system and standardized procedures for serum testosterone.Methods Serum was equilibrated with isotopic internal standard and treated with acidic buffer to release hormones from their binding proteins.The testosterone in the serum samples was extracted by two serial liquid-liquid extraction steps.The first step isolates the lipid fractions from an acidic buffer solution using ethyl acetate and N-hexane.The organic phase is dried down and reconstituted in a basic buffer solution.The second step removes the polar impurities such as phospholipids by N-hexane extraction.Total testosterone in serum is measured via ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)in multiple reaction monitoring(MRM)mode with positive electrospray ionization(ESI).The methodological performance of the method such as precision,accuracy,sensitivity,specificity was comprehensively analyzed and evaluated.The established candidate reference method was used for some application attempts.First,the method was used as a reference method,and three commercially available immunoassay platforms were used as the method to be evaluated.The objective was to evaluate the consistency of the results of the human serum total testosterone.Secondly,the method was used to prepare a mixed serum testosterone reference material,and measurement uncertainty evaluation of the reference material was performed according to relevant regulations.It mainly includes three parts:the certification value uncertainty,the homogeneity uncertainty,and the stability uncertainty.Results The coefficient variation for intra and inter assay of the method were 2.13%(1.40%～2.77%)and 3.44%(3.06%～3.66%),respectively.The recovery ranged from 94.32%to 108.60%for different samples.The limit of detection was 0.50 ng/dL and Limit of quantification was 1.00ng/dL.The Linear range was from 1 to 1000 ng/dL and extended measurement range was from 1 to 10,000 ng/dL.In addition,the extraction efficiency of 3 different levels quality control serum ranged from 85.02%to 93.29%.Moreover,Structural analogs were investigated and didn’t impact on the measurement of testosterone.The QI/CI ratios of 30 serum samples were all within±20%of the average QI/CI ratio of the calibrator,indicating that the method was not affected by interfering substances.The effect of matrix effects on detection was also very small.Compared with the UPLC-MS/MS method,the average bias percentage of the three immunoassay methods was 3.24%for men and 11.63%for women.Using the method to value the mixed serum testosterone reference material.The certified values(reference value±expanded uncertainty,ng/dL)were:50.7±1.7,152.9±2.3,405.7±3.6,792.7±6.9.The expansion factor value k was 2.Conclusions The method has excellent accuracy and precision,high specificity,wide linear range and small matrix effect,especially it performs well in the low value range.It can be used as a routine method of clinical human serum testosterone detection and has the potential to establish a reference method in a traceable reference system.Comparing candidate reference method with immunoassay platforms,some methods show acceptable performance,and some methods need improvement,especially at low testosterone concentration.The mixed serum testosterone reference material reflects the performance advantages of ID-MS,and the obtained reference material also conformed relevant regulations.The ID-MS is an internationally recommended reference method.From the experimental results,a complete set of standardized processes for working calibrator and uncertainty evaluation around candidate reference methods has basically been formed.