Evaluation of uncertainty sources in the determination of testosterone in urine by calibration-based and isotope dilution quantification using ultra high performance liquid chromatography tandem mass spectrometry

- Uncertainty assessment through intra- and inter-laboratory reproducibility.
- Internal standard calibration and isotope pattern deconvolution (IPD) were compared.
- IPD showed the lowest combined uncertainty (9%) and inter-laboratory RSD (10%).
- Uncertainty budget were calculate to find the main uncertainty sources.
- IPD is a promising way to improve longitudinal fluctuations in steroid profiling.

Three quantification methodologies, namely calibration with internal standard (Cal-IS, non-weighted), weighted calibration with internal standard (wCal-IS) and isotope pattern deconvolution (IPD) have been used for the determination of testosterone in urine by LC-MS/MS. Uncertainty has been calculated and compared for the three methodologies through intra- and inter-laboratory reproducibility assays. IPD showed the best performance for the intra-laboratory reproducibility, with RSD and combined uncertainty values below 4% and 9% respectively. wCal-IS showed similar performance, while Cal-IS where not constant and clearly worse at the lowest concentration assayed (2 ng/mL) reaching RSD values up to 16%. The inter-laboratory assay indicated similar results although wCal-IS RSD (20%) was higher than IPD (10%) and Cal-IS get worse with RSD higher than 40% for the lowest concentration level. Uncertainty budgets calculated for the three procedures revealed that intercept and slope were the most important factors contributing to uncertainty for Cal-IS. The main factors for wCal-IS and IPD were the volumes of sample and/or standard measured.