Supramolecular solvents for simplifying sample preparation in the detection of small peptides in DBS

Code: DBS23SP02SR

Doping testing of small peptides (SPs) is mostly performed in urine using mixed-mode weak cation sorbents prior to LC-MS/MS. Complementary information can be obtained in a cost-effective way using dried blood spot (DBS) samples. However, many issues still need to be addressed before considering it reliable for routine use. A major issue is the demand for simpler and faster sample processing, which is mainly based on DBS treatment with boiling water or methanol followed by sample cleanup and eluate evaporation. On the other hand, there is the need for higher SP recoveries (e.g. most of them are below 30%), and lower LODs (e.g. although no MRPLs have been set for SPs in DBS, achieved LODs are above the requested MRPLs established for SPs in urine, i.e. 1-2 ng/mL). Other issues include the demand for more thorough studies about hematocrit effects, SP shipping and storage stability, and the timing and procedure for addition of internal standards. This project aims to develop an efficient and high throughput analytical platform for reliable testing and confirmation of SPs in DBS samples. The approach will be based on the combination of the capability of supramolecular solvents (SUPRASs) to integrate efficient compound extraction and sample clean-up with the power of liquid chromatography-mass spectrometry (LC-QTOF and LC-QQQ) for SP detection.

The analytical platform will be applied to the detection of 58 peptidic drugs and metabolites, most of them routinely analysed by the WADA accredited laboratory of the Health Institute Carlos III (ISCIII, Madrid) within the ITP for urinary SPs. They will include growth hormone releasing peptides and secretagogues, growth hormone fragments, antidiuretic hormones, gonadorelin releasing peptides and growth factor modulators) Some of the selected SPs such as GHRP-1, GHRP-2, GHRP-6, hexarelin, alexamorelin, triptorelin AOD9604, desmopressin, TB-500 are unstable in urine. The capability of SUPRASs for simultaneous SP extraction from DBS and sample cleanup will be investigated using SUPRASs made up of very different nanostructures (e.g. hexagonal inverted or ribbon-shaped aggregates, sponges, cubosomes, etc.) that offer mixed-interaction mechanisms, including dispersion, ionic, hydrogen bonding, dipole-dipole, CH-π and/or polar hydrophobicity. Five marketed collection devices based on cellulose and hydrophilic porous materials will be comparatively evaluated in terms of SP recoveries and matrix effects. Addition of internal standards on DBS and SUPRAS (before extraction) will be compared. Robustness will be evaluated in terms of hematocrit effect. The optimized SUPRAS-LC-MS platforms for SP detection will be validated according to WADA guidelines and an analytical workflow that includes the proposed MRPLs will be delivered. The proposed method will be validated by the ISCIII lab using LC-QTRAP-MS/MS. As a proof of concept, the SUPRAS-based method will be applied to the analysis of DBS samples obtained from the subcutaneous administration of a single dose of GHRP-2 and Ipamorelin to two intervention groups, each consisting of 10 elite athletes.