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

Abstract

17Three quantification methodologies, namely calibration with internal standard (Cal-IS, non-weighted), 18 weighted calibration with internal standard (wCal-IS) and isotope pattern deconvolution (IPD) have been 19 used for the determination of testosterone in urine by LC-MS/MS. Uncertainty has been calculated and 20 compared for the three methodologies through intra-and inter-laboratory reproducibility assays. IPD 21showed the best performance for the intra-laboratory reproducibility, with RSD and combined uncertainty 22 values below 4% and 9% respectively. wCal-IS showed similar performance, while Cal-IS where not constant 23 and clearly worse at the lowest concentration assayed (2 ng/mL) reaching RSD values up to 16%. The inter-24 laboratory assay indicated similar results although wCal-IS RSD (20%) was higher than IPD (10%) and Cal-IS 25 get worse with RSD higher than 40% for the lowest concentration level. Uncertainty budgets calculated for 26 the three procedures revealed that intercept and slope were the most important factors contributing to 27 uncertainty for Cal-IS. The main factors for wCal-IS and IPD were the volumes of sample and/or standard 28 measured. 29 30 INTRODUCTION 31

From fundamentals in calibration to modern methodologies: A tutorial for small molecules quantification in liquid chromatography–mass spectrometry bioanalysis

“…The IPD approach showed equal accuracy compared to a fortified calibration (SAM), achieving recoveries within 90-110% and coefficients of variation below 5% for all samples tested [149]. More recently, the same group evaluated the uncertainty of this approach in an interlaboratory study, including four different World Anti-Doping Agency accredited laboratories [152]. The IPD was performed for the quantification of testosterone in urine samples and the quantification performance compared to an EC surrogate analyte approach.…”

Application of the adaptive Monte Carlo method for uncertainty evaluation in the determination of total testosterone in human serum by triple isotope dilution mass spectrometry

“…Isotope dilution mass spectrometry (IDMS) is widely recognized as one of the most accurate methods for high-order reference measurement procedure (RMP) and has been witnessed many elaborations and developments over the years [21][22][23][24][25]. However, only a few publications dealt with IDMS measurement uncertainty [26][27][28][29][30]. In most of these publications, the measurement uncertainties were determined according to the relative standard uncertainties of several main factors.…”