Abstract:

Objective To establish a candidate reference method for the determination of human serum testosterone based on liquid chromatography tandem mass spectrometry（LC-MS/MS）,and to use it into assessing the trueness of clinical method（microparticle chemiluminescence immunoassay）. Methods Testosterone-2,3,4-¹³C₃ was added as internal standard. Serum samples were treated with hexane-ethyl acetate（50∶50,volume fraction）. The upper layer was dried with nitrogen flow. The residual were reconstituted with mobile phase and analyzed by LC-MS/MS. Serum testosterone concentration was calculated by 5-point bracketing method. The isotope dilution liquid chromatography tandem mass spectrometry（ID-LC-MS/MS）（carryover,specificity,linearity,imprecision,accuracy） was validated according to the Clinical and Laboratory Standards Institute（CLSI） C62-A and EP15-A3. The trueness of microparticle chemiluminescence immunoassay was evaluated according to CLSI EP9-A3. Results The ID-LC-MS/MS with 5 min total run time and without carryover was validated over a concentration range of 0.034-22.000 ng/mL,and the specificity was good. The within-run imprecision was ≤2.3%,and the between-run imprecision was ≤2.2%. The bias of analyzing the candidate reference material SRM971（Level male）was 0.2%. The relative biases of analyzing 2017 RELA-A and 2017 RELA-B were -2.9% and -3.3%,respectively. The uncertainty for 2017 RELA-A was 0.11 ng/mL. The linearity between ID-LC-MS/MS and microparticle chemiluminescence immunoassay was good（r=0.963）. However,its average relative bias was large,which total average relative bias was -24.4%,the average relative bias of low concentration group（≤1 ng/mL） was -62.4%,and the average relative bias of high concentration group（>1 ng/mL） was 6.3%. Conclusions The ID-LC-MS/MS established successfully for serum testosterone has good precision and accuracy,with simple preparation and short analysis time and may be used as a candidate reference method.

Key words: Testosterone, Isotope dilution liquid chromatography tandem mass spectrometry, Methodological assessment, Candidate reference method