Accurate analysis of testosterone in human serum using a heart-cutting 2D-UPLC–MS/MS procedure

Abstract Pathology laboratories are encouraged to participate in External Quality Assurance (EQA) schemes to estimate the degree of possible bias in their test results. In such schemes, typically the median of results reported by laboratories is used as the “target value” for assessment of accuracy. However, the ideal situation is where reference values with low measurement uncertainties are provided by a reference laboratory using reference methodologies. The National Measurement Institute of Australia (NMIA) recently provided reference values for an EQA for testosterone in human serum coordinated by the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP). Reference values were determined using a novel isotope-dilution two-dimensional ultra-performance liquid chromatography tandem mass spectrometry (2D-UPLC–MS/MS) procedure. Samples were subjected to solvent extraction and solid-phase extraction clean-up prior to instrument analysis. A second analytical method employing different sample clean-up and analysis procedures was also developed for confirmatory analyses of samples. In this method, serum extracts were cleaned-up using high performance liquid chromatography (HPLC) and analysed by gas chromatography-high resolution mass spectrometry (GC-HRMS). The availability of two orthogonal methods for sample analysis facilitated the rigorous investigation of bias in the 2D-UPLC–MS/MS method. Reference values were assigned to 6 EQA samples containing testosterone at 0.2 ng/g–5.4 ng/g. The uncertainties in the reference values were low and ranged from 3.3%–9.0% at a level of confidence of 95%. The reference values can be used by laboratories in the EQA to obtain information on bias in their methods and allow an accuracy-based grading of their performance.