Determination of 19-norandrosterone in human urine by isotope dilution gas chromatography-high resolution mass spectrometry

An accurate and precise method for the determination of total 19-norandrosterone (19-NA), a major metabolite of nandrolone, in human urine was developed based on isotope dilution gas chromatography-high resolution mass spectrometry (ID GC-HRMS). The 19-NA glucuronide, together with deuterated 19-NA (d₄-19-NA) (as the internal standard, IS), was subjected to enzymatic hydrolysis using β-glucuronidase, followed by solid phase extraction (XtractT, mixed-mode column) and liquid–liquid extraction cleanup. The native and deuterated analogues of 19-NA were then derivatised to the corresponding bis(trimethylsilyl) derivatives using N-methyl-N-trimethylsilyltrifluoroacteamide (MSTFA):NH₄I:Dithiothretitol (1,000:2:3 w/w). Identification was achieved under selected ion monitoring of the respective trimethylsilyl derivatives at ion masses m/z 405.26450 and 420.28800 for 19-NA and m/z 409.28920 and 424.31270 for d₄-19-NA within the specific time windows (±1% of the relative retention time to the calibration standard). A linear calibration curve (r ² > 0.9995) was obtained based on seven calibration points (five replicates at each level) in the range 0.05–10 ng/g. The detection limit for 19-NA was found to be 4 pg/g. The method has been applied for the determination of 19-NA by fortifying 19-NA glucuronide at three concentrations (0.2, 2.1 and 7.2 ng/g) in blank urine samples with excellent accuracy and reproducibility. To circumvent the iterative process of exacting matching, a single-point calibration procedure was adopted, where the acceptance criteria for the isotopic ratio in the sample (RF _s) and calibration blends (RF _c) was set close to unity (0.95–1.05). This method was successfully applied in a pilot inter-comparison study, with results in good agreement with the fortified value and other participants’ results (relative standard deviation, RSD < 2.0%) with an expanded relative uncertainty (coverage factor of 2 at 95% confidence level) of 4.7%. It was found in our determination that the main contributors to the uncertainty budget originated from the measurements of the purity of the reference material and the response factor of the calibration standard.