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

• Published in: Journal of Chromatography A Vol. 1508; pp. 73 - 80
• Main Authors: Pitarch-Motellón, J., Roig-Navarro, A.F., Sancho, J.V., Ibáñez, M., Fabregat-Cabello, N., Pozo, O.J., Ventura, Rosa, García Alonso, J.I., Rodríguez-González, Pablo, González Gago, Adriana, Ereño Artabe, Amaia, Van Eenoo, Peter, Deventer, Koen, Dehnes, Yvette, Rzeppa, Sebastian
• Format: Journal Article Web Resource
• Language: English
• Published: Netherlands Elsevier B.V 28.07.2017; Elsevier
• Subjects: Calibration; Carbon Isotopes - chemistry; Chemistry; Chimie; Chromatography, High Pressure Liquid - methods; Chromatography, High Pressure Liquid - standards; Humans; IDMS; Indicator Dilution Techniques; Isotope pattern deconvolution; LC-MS/MS; Physical, chemical, mathematical & earth Sciences; Physique, chimie, mathématiques & sciences de la terre; Reproducibility of Results; Tandem Mass Spectrometry - methods; Tandem Mass Spectrometry - standards; Testosterone; Testosterone - urine; uncertainty; urine; Isotope pattern deconvolution; Testosterone; LC-MS/MS; IDMS; uncertainty; urine
• ISSN: 0021-9673; 1873-3778; 1873-3778
• DOI: 10.1016/j.chroma.2017.05.072

•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 (2ng/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.