Gestrinone

Dried urine spots have recently been proposed as an alternative matrix in the anti-doping field. Drying urine may open the opportunity to limit microbial and thermal degradation of the prohibited substances during transportation to the anti-doping laboratories without the need for refrigeration or freezing. In this study, a multi-targeted initial testing procedure was developed for the determination of 237 prohibited drugs/metabolites from 11 different classes in dried urine spots. The comparability between two different microsampling techniques (i.e., Whatman^® FTA DMPK-C cards and Mitra^® tips) was evaluated. The developed method was then used to evaluate the stability of the target compounds in urine for 7 days under different environmental conditions to simulate the transportation of the urine samples from the collection sites to anti-doping laboratories. Sample preparation consists of (i) extraction of the analytes from the collection device using a mixture of acetonitrile/methanol (1/1) for 30 min at 40 °C, (ii) enzymatic hydrolysis, and (iii) sample concentration by solid-phase extraction. Analysis was performed using liquid chromatography coupled to high-resolution mass spectrometry. The entire workflow was validated in terms of specificity (analytes were distinguishable from the matrix interferences), sensitivity (only with the Mitra^® tips the limits of detection comply with the World Anti-Doping Agency's requirements for the majority of the target compounds), carry-over (no signals in the negative urine injected after the positive urine), matrix effect (16-28% for Mitra^® tips and 22-35% for DMPK-C cards), and extraction yield (Mitra^® tips: 51-88%; DMPK-C cards: 40-76%). As proof of concept, authentic urine samples were analyzed: results obtained in dried urine were compared with those of fluid urine, providing good agreement. Stability studies showed that the target compounds were stable for the whole duration of the study (7 days) at -20 and 4 °C in both fluid and dried urine. At 50 °C or at 20-25 °C, several thiazide-based compounds were completely degraded to their degradation product in the first 24 h or after 3-4 days in fluid urine, whereas in dried urine the compounds were detectable for the entire duration of the study.

Numerous bioactive compounds containing carbonyl groups, including aldehydes and ketones, are widely acknowledged as potential biomarkers for several diseases and are implicated in the development of metabolic disorders. However, the detection of carbonyl metabolites is hindered by challenges, such as poor ionization efficiency, low biological concentration, instability, and complexity of the sample matrix. To overcome these limitations, we developed a Girard derivatization-based enrichment (GDBE) strategy for capturing and comprehensively profiling carbonyl metabolites in biological samples. A functionalized resin, named carbonyl capture and reporter-ion installation (CCRI) resins, was synthesized to selectively capture carbonyl metabolites via a Girard reaction. After unwanted metabolites were removed, the hydrazone derivatives were cleaved from the solid-phase resins and subjected to LC-MS analysis. The proposed GDBE strategy exhibits exceptional selectivity for capturing and enriching carbonyl metabolites. Moreover, this method surpasses current detection limits by enhancing the MS sensitivity and facilitating structural characterization of hydrazone derivatives by a specific MS/MS fragmentation signature. Using the GDBE method, 957 potential carbonyl metabolites were successfully identified in liver tissue from alcohol-fed mice. Among them, 76 carbonyl metabolites were annotated, indicating the potential of this strategy for the efficient nontargeted profiling of the carbonyl submetabolome in complex biological samples.