Evaluation of biochemical assays and optimization of LC-MS/MS analysis for the detection of synthetic urine

Ensuring specimen validity is an essential aspect of toxicological laboratories. In recent years, substituting authentic urine specimens for synthetic urine (SU) has become increasingly popular. Such SU products consist of components expected in normal urine and show physiological values for specific gravity and pH. Thus, standard specimen validity testing may fail in revealing adulteration by SU. The present study investigated three methods to distinguish authentic and SU specimens: enzymatic detection of uric acid, the commercially available Axiom Test True SU and liquid chromatography coupled with (tandem) mass spectrometry (LC-MS/MS) analysis of 10 endogenous biomolecules. Additionally, novel direct markers of SU were investigated. Two specimen sets were analyzed by each method. Specimen set A consisted of eight SU products purchased from the Austrian/German market and 43 urine specimens from volunteers of known authenticity, which underwent double-blind analysis. Specimen set B consisted of 137 real urine specimens submitted for drug testing, which were selected due to initial suspicious test results in adulteration testing and reanalyzed by all three methods. Uric acid and LC-MS/MS-based endogenous biomolecule testing showed 100% sensitivity and specificity for set A. The commercial test had 87.5% sensitivity and 97.7% specificity for set A. For set B, uric acid and LC-MS/MS analysis showed almost similar results, even if uric acid was missing one presumptive authentic urine specimen according to LC-MS/MS findings. Nearly half of the SU assignments for the commercial test were presumptive false positives. New SU markers were observed for SU products from the Austrian/German market. One specimen in set B had both an endogenous biomolecule pattern and SU markers suggesting urine dilution with SU. In conclusion, several analytes or methods should be used rather than one, and the most reliable results are achieved if both indirect and direct markers of urine substitution are analyzed.