Dried microsamples: multi-matrix, long-term stability study of doping-relevant peptides
Description du projet
Microsampling provides a wide range of applications that may offer advantages over traditional fluid samples on logistics and bioanalytical workflow. Among microsampling methods, dried matrix spots represent a feasible method for the microsampling of biological matrices to obtain for example dried urine spots (DUS). Moreover, volumetric absorptive microsampling (VAMS) has been recently introduced for the sampling of small, accurate biological fluid volumes. Dried microsamples can usually be stored under ambient conditions, although comprehensive analyte stability assessments are still under research for many compounds. Following the promising results previously obtained from the definition of mid-term stability of some doping-relevant peptides (e.g. GnRH analogues) in urine collected as DUS and VAMS, aim of this research is to carry out a systematic study on the stability in a wider time frame of such compounds in urine sampled as DMS and VAMS and to expand the study to additional peptides and to relevant compounds included into the Athlete Biological Passport (ABP) steroidal module. All variables involved in the sampling process will be assessed: humidity, temperature, light exposure will be evaluated to determine the optimal sampling, storage and transport conditions, and to evaluate results obtained from microsamples. In addition, possible scenarios will be simulated, representing the life cycle of an anti-doping sample: from collection to shipment, storage and handling before being subjected to pretreatment procedures and LC-MS/MS and LC-HRMS analysis. The project goal is to establish feasible and reliable workflows for microsample collection, stably storable and shippable with minimum precautions. These procedures could then be proposed as effective anti-doping strategies to be compared to conventional fluids.
Two different dried microsampling and analysis workflows, based on dried urine spots (DUS)-LC-MS/MS and volumetric absorptive microsampling (VAMS)-LC-MS/MS respectively, were developed and validated for the testing of S2 peptides and of steroids included in the ABP. Validation included sampling time and drying time assays. Moreover, different microsampling volumes (10, 20, 30 μL) were tested to provide better versatility; 30 μL always granted the best sensitivity/selectivity compromise. Eleven-months stability assays showed that both microsampling matrices, stored at 25°C, provided good stability for all analytes at all time points, with end-point stability in the 52-66% range for peptides and in the 79-85% range for steroids. Similarly, both DUS and VAMS at 25°C produced better stability than fluid urine frozen at either -18°C or -80°C, with large differences (28-32.5%) for peptides and marginal ones (2-12%) for peptides. VAMS produced slightly better results than DUS: Stability in the former was 1-8% higher than in in the latter for peptides and 1-3% higher for steroids. Urine microsample storage for 11 months in sub-optimal conditions (up to 35°C, up to 40% HR, 12 h light exposure) caused larger analyte degradation, but again with better performance for VAMS over DUS (-9% vs. -18%, respectively, in comparison to optimal conditions). 11-months stability studies were completed on doping-relevant peptides and ABP-listed steroids, providing positive, interesting results. In particular, innovative microsampling and pretreatment procedures based on dried matrices (DUS and urine VAMS) were optimised for application to urine specimens for anti-doping purposes. Analytical platforms based on LC coupled to different MS detectors were exploited to develop two sets of validated analytical methods, one for the analysis of peptides and another for the analysis of steroids, in dried urine microsamples. Solid, satisfactory performance and good throughput were obtained for each analyte group. After 11 months, peptide stability in dried micro-matrices was above 51% and steroid stability was above 78%. Regarding peptides, stability in the micromatrices at RT was vastly superior to that of frozen or ultra-frozen urine. Results for steroids were less starkly defined, with both microsampling matrices providing marginal advantages in comparison to urine. In all cases, there was no need for any refrigerated or frozen microsample storage. Among the two microsampling strategies, VAMS granted significantly superior performance over DUS for peptides, with much smaller differences for steroids. However, VAMS generally gave better precision, extraction yield and matrix effect in the analytical workflow than DUS. In conclusion, dried urine micromatrices are a very promising tool for enhancing the stability of peptides and steroids in an anti-doping testing perspective. They may also provide reduced storage and shipping requirements and expenses, and simpler pretreatment procedures.