Advancing peptide analysis in dried blood spots: application potential and stability study of doping-relevant peptides

Code: 20A12LM

Current illicit doping practices include the use of peptide hormones and their peptide release factors, with different kinds of activity (gonadotrophic, corticotrophic, growth factor, etc.), with the common purpose of increasing sport performance. These substances are included in the WADA Prohibited List, section S2. One of the main analytical hurdles for the detection of peptides is their low stability in blood (and other biological fluids). This makes sample storage and shipping critical steps, possibly causing the decrease of peptide levels to amounts that are no longer detectable due to degradation.

Dried microsampling provides logistic and analytical advantages over fluid samples: water loss can effectively stop most degradation and biotransformation reactions, leading to higher peptide stability and to more favorable, cheaper storage and transportation conditions. Among microsampling methods, dried blood spots (DBS) is the most well-known and widely applied, but more innovative alternatives are also available (“smart DBS”), such as special cards and devices that obviate some drawbacks of “classical DBS”, like dependency of sampling volume on haematocrit and lack of sampling accuracy. Other dried microsampling approaches, such as volumetric absorption microsampling (VAMS), exist as well. Following the promising results obtained from stability studies on doping-relevant peptides in urine microsamples (Project funded within the 2017 Scientific Research Grants, successfully developed and concluded by this research team), aim of the present project is to carry out a systematic study on the stability of prohibited peptides in dried blood spots. The most important variables involved in the sampling process will be studied, such as humidity, temperature and light exposure, to determine optimal sampling, storage and shipping conditions, and to evaluate the results obtained from microsamples.

The project goal is to establish feasible and reliable workflows for dried blood microsample collection, which could be proposed as effective strategies for anti-doping testing.

Main Findings

Background – With the introduction of dried blood spots (DBS) into official anti-doping testing workflows, scientific studies on their performance have been increasing. DBS-based analytical platforms can be advantageous over conventional methods of blood drawing and handling. Increased analyte stability due to lack of water (and consequent increased reliability of delayed analyses) is one of the most attractive features of dried microsampling, however it has to be verified for each analyte or at least for each chemical class of analytes. Project aims – Prompted by the results obtained from previous WADA-funded projects regarding doping-relevant peptide stability in urine-derived dried micromatrices, we have undertaken the task of assessing the mid-term (3-month) stability of 19 peptides in DBS. Both classical and “smart” DBS platforms were tested; the latter are based on microfluidics to obtain fixed-volume DBS from blood drops. Peptide stability in DBS stored under subpar and worst-case conditions was also evaluated. Results were compared with stability data obtained from plasma samples stored at -20 or -80°C (Figure 1). Results – Validated, original LC-MS/MS methods were developed for the simultaneous determination of the 19 analytes. Using them, peptide stability was reliably evaluated. Stability in DBS stored at room temperature was always good (78-84% recovery) and significantly (5-35%) higher than stability in plasma stored at freezing temperatures. It was also observed that subpar and worst-case handling and storage conditions can have a noticeable impact on analyte stability in DBS (up to 15% lower after 90 days), without compromising overall reliability and storage, handling, and cost advantages. Conclusions – The one-year project was successfully completed according to the planned timeline. Innovative microsampling, preparation and analysis platforms were developed, based on classical and microfluidic DBS exploitation. The results of mid-term stability assays confirmed that the dried microsampling approach is a viable alternative to classical venipuncture and plasma analysis, providing better stability and sampling feasibility coupled to considerably lower shipping and storage costs. Future plans – A systematic study on the long-term (1+ years) stability of peptides in DBS would be the natural prosecution of the present project. Other forms of microsampling could also be tested, from volumetric adsorptive (VAMS) devices to microfluidic or membrane-based devices for the automatic creation of dried plasma spots (DPS) from blood drops to water-soluble supports, and others. Finally, more peptides could be added to the current analytical panel, to increase the validity and significance of the produced results for anti-doping testing advancement.