Enhanced urinary stability and detection window of peptide hormones and growth factors by dried urine microsampling

Code: 17A20LM 

Urine samples collected worldwide for anti-doping testing are not always shipped in refrigerated conditions from collection sites to WADA accredited laboratories, in particular when several transport days are required. Commensal urethral microbiota, urinary pathogens and environmental bacteria may contaminate urine and the enzymatic activity by microbial contamination can cause severe modifications to the excreted compounds, in particular to doping-relevant peptides such as hormones and growth factors. Since delays are inevitable and sample refrigeration is not always possible and effective, sound methods for preserving and stabilizing urine samples are desirable.
This Project involves the use of Volumetric Absorptive Microsampling (VAMSTM) and Dried Urine Spot (DUS) strategies for the collection of dried microsamples processed by means of streamlined workflows to be developed ad hoc, for high-throughput LC-MS/MS analysis of several peptide hormones and growth factors (i.e. GHRP-1, GHRP-2, GHRP-6, hexarelin, alexamorelin, triptorelin, AOD9064, CJC-1293, desmopressin, TB-500, hCG and ACTH). DUS and VAMS approaches represent promising alternatives to the procedures currently performed by anti-doping laboratories, allowing analyte stabilization by water loss and the consequent broadening of their detection window. This innovative sampling produces logistics savings due to the small transported volume (by air shipments and through customs) and to the possibility to keep the specimens at room temperature, with significant implications on overall analysis cost. 
The ultimate goal of this project is to establish and validate feasible but reliable protocols for the collection of urine microvolumes, stably storable and shippable with no particular precautions. As a proof of concept, instrumental analytical methods will be developed, validated and applied for the stability assessment of several peptide hormones and grow factors in dried urine microsamples stored at different conditions. In parallel, stability will be assessed in classic fluid urine stored at temperature-controlled conditions and thorough comparisons with dried microsamples will be carried out.

Main Findings: 

•    Innovative microsampling and pretreatment procedures based on dried matrices (DUS and urine VAMS) were optimised for the application to urine specimens for anti-doping purposes. Important experimental parameters were studied and specifically optimised. 
•    Original MS, HRMS and FT-IR methods were used for peptide chemical identity confirmation; then LC-MS/MS and LC-HRMS methods were developed for the simultaneous analysis of the peptides of interest in dried urine microsamples. After suitable study of the experimental conditions, the final methods provided good, solid performance within relatively short run times.
•    The LC-MS/MS and LC-HRMS methods were validated according to current guidelines, with good results for all assays and all analytes, and with similar or better performances than those of comparable published methods.
•    The microsampling, pretreatment and analysis workflow was successfully applied to the study of peptide stability in dried matrices.
•    Mid-term stability assays were carried out, both on dried and fluid urine-based matrices, with outstanding results. In fact, DUS and urine VAMS were remarkably stable, even though they were kept at RT, with all studied peptides recovered in the 80-95% range at the end of the study period (3 months). The stability of the chosen peptides, which are known to be prone to degradation under common storage conditions, was greatly enhanced in comparison to fluid urine stored at freezing (-20°C) and ultra-freezing (-80°C) temperatures. Analyte loss in fluid urine at -80°C was regularly 5-10% larger at study end than the loss in dried matrices, with widely worse losses (up to 35% more) for fluid urine kept at -20°C.
The use of innovative microsampling media offers significantly interesting perspectives towards the development of engineered, highly reliable devices.