Identification of Synthetic Urine by Analysis of Stable Carbon and Nitrogen Isotope Ratios and Comparison to Established GC‐MS/MS and LC‐MS/MS Analysis

ABSTRACT Manipulation of urine samples is a recurring issue in doping control and forensic analyses, for instance through the use of synthetic urine products designed to mimic human urine. This study evaluated analytical approaches for identifying synthetic urine and mixtures of synthetic and authentic urine: gas chromatography tandem mass spectrometry (GC‐MS/MS) for analysis of endogenous urinary steroids, elemental analyzer isotope ratio mass spectrometry (EA‐IRMS) of carbon and nitrogen, and urea‐nitrogen compared to an established liquid chromatography mass spectrometry (LC‐MS/MS) method incorporating synthetic urine markers. All methods correctly identified synthetic urine in a double‐blind sample set. As an initial testing procedure in doping control, GC‐MS/MS identified synthetic urine samples through absence of endogenous steroids. However, EA‐IRMS was superior to MS/MS methods in identifying mixtures of synthetic and authentic urine. Synthetic urine isotope ratios of total urinary carbon and nitrogen and/or urea nitrogen (δ 13 C ≤ −29.7‰, δ 15 N ≤ +0.4‰) were clearly differentiated from authentic urine isotope ratios (δ 13 C ≥ −26.1‰, δ 15 N ≥ +1.6‰), reflecting the synthetic origin of constituents. Mixtures of synthetic and authentic urine up to 50:50 displayed isotope signatures inconsistent with human urine, enabling reliable detection of adulteration. The LC‐MS/MS approach detected mixed samples with high proportions of synthetic urine by combining biomolecule profiling with synthetic urine‐specific markers. Overall, the findings demonstrate that EA‐IRMS is a complementary tool for identifying synthetic or manipulated urine samples, especially when traditional biomarkers or steroid profiles are inconclusive. The method enhances the reliability of doping control and forensic urine authenticity testing.