Sample validity lessons learned from challenging cases and proposed biomarkers for synthetic urine detection

Toxicology laboratories rely on a combination of on-site and laboratory based validity testing to detect dilution, adulteration, and substitution in urine samples collected for drugs of abuse testing. At Melbourne Pathology, Sample Validity Testing (SVT) includes on-site checks such as temperature, creatinine, color, and supervised collection, as well as routine laboratory testing of all specimens for creatinine, pH, and oxidants. Additional validity testing is performed when a specimen is deemed suspicious, including osmolality and the biomarkers urea, uric acid, amylase, and γ-glutamyltransferase (GGT). We describe three cases in which SVT was critical in identifying inauthentic specimens, and one where SVT resulted in the inaccurate identification of an authentic case as inauthentic due to a medical condition. We also present the SVT results from seven synthetic urine (SU) samples detected in routine casework. Urea and uric acid proved somewhat successful at identifying SU, present in three of the SU samples. GGT and amylase proved the most effective at identifying SU, with none of the SU samples containing the enzymes. These biomarker assays were rapid, cost‑effective, and readily implemented using existing biochemistry workflows. Our findings highlight that no single validity measure is sufficient in isolation; instead, results should be interpreted collectively and on a case‑by‑case basis. As SU formulations continue to evolve, laboratories may benefit from adopting additional, non‑routine validity tests tailored to their operational capacity.