New Approaches To Identify Urine and Hair Adulteration Attempts in Forensic Toxicology: A Proof-of-Concept Study...

Urine and hair are among the primary biological matrices used for drug and abstinence testing in clinical, forensic, and antidoping settings. A persistent challenge in such analyses is the adulteration of samples: through dilution, substitution, or chemical modification, aimed at concealing the presence of xenobiotics such as drugs of abuse, ethanol, or doping agents. In this study, we investigated whether chemical adulteration, specifically oxidative treatment with hydrogen peroxide (H₂O₂), induces detectable and characteristic changes in the proteomes of urine and hair samples. Using a bottom-up proteomics approach involving LC-HR-MS/MS with data-dependent acquisition, we compared urine samples before and after treatment (10% H₂O₂) and untreated hair samples with those exposed to increasing concentrations of hydrogen peroxide (3%, 6%, 9%, and 12%). Data are available via ProteomeXchange with the identifier PXD064208. We identified distinct peptides, including oxidatively modified forms, that were exclusively present in either untreated or chemically treated groups. In hair samples, the appearance of some of those peptides was dependent on the peroxide concentration. Peptides detectable only after oxidative exposure were of particular interest, as they appeared to be nonphysiological and specific to the adulteration process. These species serve as candidate biomarkers for indirect detection of sample manipulation or for assessing the integrity of compromised samples. The extraction and characterization of these potential marker peptides constitute the primary outcomes of this study. These findings should lay the groundwork for further validation and the development of proteomic methods aimed at enhancing the reliability of drug testing and sample authenticity assessment in forensic and antidoping contexts.