Projets de recherche

Code: 20C17CR

Chapter S4 of WADA’s Prohibited List 2020 (“Hormone and Metabolic Modulators”) lists myostatin propeptide under sub-chapter 4 (“Agents preventing activing receptor IIB activation, Myostatin inhibitors”) as prohibited substance. So far, no approved myostatin propeptide pharmaceuticals are available. On the other hand, myostatin propeptide is sold on the black market (labelled “MyoPro”, “HMP”, “Myostatin-Propeptide (HMP)”, or erroneously “GDF-8” and “Myostatin”). But the administration of black market myostatin propeptide to human test persons will be ethically not justifiable. For that reason we plan a study with rats. The test animals will receive black market myostatin propeptide at a dosage, which can be clearly detected in serum (10 mg/kg BW). After 24, 48 and 168 hours, serum and urine will be collected and tested for myostatin propeptide by electrophoresis and Western blotting. The study will help to clarify (1) how long black market myostatin propeptide is detectable in blood, and (2) if it can also be observed in urine. We have already shown that black market myostatin propeptide can be differentiated from endogenous myostatin propeptide by electrophoresis (SDS-PAGE) and Western blotting.

Main findings

Chapter S4 of WADA’s Prohibited List 2024 (“Hormone and Metabolic Modulators”) lists myostatin propeptide under sub-chapter S4.3. (“Agents preventing activin receptor IIB activation, Myostatin inhibitors such as myostatin-binding proteins (e.g. follistatin, myostatin propeptide)”) as prohibited substance. Currently, myostatin propeptide is only available on the black market. Since administration of black market products to humans is ethically not justifiable, a study with rats was performed.

Aims of the project were:

- an animal study (administration of black market myostatin propeptide (“GDF-8”) and recombinant myostation propeptide standard to rats followed by collection of serum and urine)

- an investigation of the electrophoretic detectability of black market GDF-8 in rat serum and urine after circulation in blood for 24, 48, and 168 hours. Additionally, the detectability of recombinant myostation propeptide standard after 24 hours was also investigated

- an in vitro metabolism study of myostation propeptide using human and rat liver microsomes

Results:

After a single dose administration of black market GDF-8 (10 mg/kg body weight) to rats, the protein was detectable in all serum samples after 24 hours. However, it was no longer traceable in the samples taken after 48 h and 168 h. No GDF-8 was found in the urine samples at all three time-points. Recombinant myostation propeptide was neither detectable in serum nor urine 24 hours post administration.

In order to reveal possible differences in the metabolism of myostation propeptide between humans and rats, it was incubated with human and rat liver microsomes (5, 60, 120, 300 min, 24 hours). The protein proved quite stable - only after 24 hours degradation of the main band was observed.

Conclusions:

Black market myostation propeptide can be detected in rat serum samples by SDS-PAGE and immunoblotting with a monoclonal myostation propeptide-specific antibody. For the extraction from rat serum and urine, polyclonal myostation propeptide-antibodies captured by magnetic beads or coated on ELISA-wells were used. After single dose administration, the protein remained detectable for only 24 hours in rat serum samples. No signals were obtained on Western blots in serum after 48 and 168 hours and at all three time-points in urine samples.

Code: 20C15XD

Clostebol (4-chloro-testosterone; 4-chloro-4-androsten-17-ol-3-one) is an anabolic androgenic steroid (AAS) derivative from testosterone. In humans, among the legitimate therapeutic indications of anabolic steroids, clostebol acetate is approved for topical use in dermatological and ophthalmological preparations. The esterification on the 17 position also permits its oral use, protecting the compound from an extensive firstpass metabolism. Clostebol has also been used in cattle to improve the animal growth. Due to its anabolic properties the International Olympic Committee (IOC) in the past and today the World Antidoping Agency (WADA) have included clostebol and the other AAS in the yearly renewed list of prohibited substances in sports[1]. The human [2,3] and animal[4] metabolism of clostebol allowed to establish adequate methods to 
detect its illicit use. The detection of clostebol intake is traditionally based on the detection of its main metabolite (4-chloro-4-androsten-3a-ol-17-one) excreted into urine glucuronoconjugated (Figure 1.). In order to improve the detection capabilities, antidoping laboratories have developed in the last decade methods based on gas chromatography (GC) coupled to tandem mass spectrometry (MS/MS) lowering the limits of detections or have implemented methods for the direct investigation of phase II metabolites (sulphates) using liquid chromatography coupled to mass spectrometry (LC-MS/MS) [5,6]. The presence of clostebol metabolite in an athlete’s urine sample may be due to its illicit use as anabolic, the ingestion of contaminated meat[7] and finally some case reports describe the accidental contact with cream preparations containing clostebol[8]
. 
In the last years, the improvement of the detection capabilities of the antidoping laboratories has led to a moderate increase of clostebol detection worldwide, and especially in Italy where the use of a cream containing clostebol acetate and neomicine is quite extended. Trofodermin® is a pharmaceutical preparation containing 5% clostebol acetate and 5% neomicine sulphate that can be applied by cream or spray, that can be used for the following treatments: abrasions and erosions of the skin, injuries and wounds, such as varicose ulcers, due to poor blood circulation, bedsores (due to immobility in bed) sores or trauma, fissures (cuts) on the nipple (which can occur during breastfeeding), anal fissures (small cuts around the anus), burn wounds, infected wounds, wounds that delay to form the scar, irritation, redness, and sensitization of the skin that appears after radiotherapy (radiodermatitis), dryness, cracking or peeling of the skin with ulceration. According to the Italian law, a visible symbol on the packaging indicating the presence of a substance included in the WADA list of prohibited substances must be present. Although clostebol is prohibited by all administration routes, the aim of this study is to investigate the presence of clostebol metabolite in urine after an accidental contact with the substance due to the therapeutic application of clostebol acetate in a different individual.

Main findings

The accidental contamination of an individual after getting into close contact with another individual using transdermal clostebol acetate has been demonstrated after different degrees of exposure.This project aimed to search for specific clostebol metabolic markers or concentrations thresholds that may help to distinguish an illicit use of clostebol as AAS after an oral administration from an accidental contamination after a transdermal application, or to distinguish between a transdermal or oral administration, helping to establish adequate criteria to be adopted by the antidoping community. This kind of thresholds have been already applied for other groups of substances

The metabolism of clostebol after oral and transdermal applications has been extensively described using the more common tools used by WADA accredited Laboratories, gas chromatography coupled to mass spectrometry. Ten metabolites were detected after oral administration of which 5 could not be detected after transdermal application under the assay conditions here applied. A suitable criterion to disclose an oral (late excretion) from a transdermal application was achieve. The use of concentrations of any of the metabolite is quite difficult because the variability in the individual absorption, metabolism and/or excretion. Instead, the ratios between specific metabolites M4, M3 and M2 to M1 showed plausible results to discriminated between both administrations.

The proposed metabolic ratios have a fundamental limitation for being applied worldwide by all WADA accredited laboratories. The response ratios (even among isomers) depend on the signals (transitions) selected by every laboratory. To overcome this point the metabolites considered more diagnostic need to be synthetized to allow an estimation of the concentration that would be then independent from the analytical method applied. This would allow in addition to confirm definitively the configuration of the proposed metabolites. Some metabolites are excreted in urine only after oral administrations but their response is much lower and there are more potential structure fitting with the candidates proposed here. The sulfate fraction that was in previous communications considered as a potential solution to the problem presented, were not considered here since this is a fraction not used in ITP conditions. Based on the previous considerations, we propose M2, M3 and M4 as the best targets worth to be synthetized and characterized for this purpose.

Code: 20C13GJ

Objective: To determine urine thresholds consistent with maximum therapeutic dose of: salmeterol.

Study A: Salmeterol
A salmeterol threshold study will be conducted using a similar protocol to that previously used by our team, consisting of two acute urine PK trials 25 over one week; one under normal conditions at baseline, followed by salmeterol dosing for one week and then a second acute PK trial performed under stressful conditions with heat and dehydration22. During the acute phase, we will also assess the effect of salmeterol on muscle strength, peak power and time trial performance utilising a placebo-controlled crossover design. After the second PK trial, subjects are randomly allocated 5 weeks of treatment with placebo or salmeterol (1:1) to assess the effect of salmeterol on muscle mass, fat mass, muscle strength, time trial performance and peak power during cycling measured at six weeks (completion).

Subjects: Thirty six healthy trained men and women (equal male:female) will be recruited for the study, non-smokers, no history of chronic disease, and recreationally active (>5 h/wk). 

Clinical Study Procedure: Pre-intervention will consist of general health screen, lung function test, and VO2 max will be determined by incremental bike ergometer to exhaustion using standard laboratory protocols. A baseline Wingate test protocol will be administered to participants during this period.5 Pharmacokinetic (PK) intervention in all subjects (n=36) will consist of salmeterol delivered by pMDI (200 µg dose) at time 0, followed by urine collection at 0.5, 1, 2, 4, 8 and 24 hours under resting conditions. Salmeterol will be continued to be delivered daily (100 µg twice daily – home administration with supervision monitored by Skype/Facetime) with spot urine collection until the 7th day at which point a 200 µg dose will be administered and a second acute phase PK study will be conducted under identical time (urine collection at 0.5, 1, 2, 4, 8 and 24 hours). The second acute PK study, however, will encompass stressful conditions, namely accumulation of dose (as noted previously using a similar protocol for terbutaline 25) but also with exercise (time trial) under heat (30-35°C) as per our previous work with terbutaline22. Subjects will then be randomised to salmeterol:placebo 1:1 and continue treatment for a further 5 weeks (6 weeks total duration) and undertake a second Wingate test to assess effects of the intervention on exercise performance using our previously applied protocols 5 – further spot urine sampling will not occur during this time. Subjects will be asked to avoid any CYP3A4 inhibitors such as grapefruit juice during the chronic phase. 

Analytical: Urine samples will be analysed by racemic assay (a simple modification based on our previous racemic work 26). We will investigate the alpha-hydroxysalmeterol metabolite and salmeterol in urine, with a total of around 612 samples for the acute and the intermediate chronic spot sampling phase treatment arm.

Code: 20C09GG

Today, the steroid profiling mainly relies on the analysis of glucuronide metabolites. After the selective hydrolysis of the glucuronide moiety, the corresponding free steroid is analyzed by gas chromatography – mass spectrometry (GC-MS) subsequent to silylation of hydroxyl - and keto-groups. The inclusion of sulfate metabolites has previously been difficult due to a non-efficient hydrolysis to the corresponding phase I metabolites. Consequently, an important piece of information about alterations in the steroid metabolism after testosterone doping is not monitored so far. During the last years, the direct analysis of phase II steroid metabolites by liquid chromatography – mass spectrometry (LC-MS) has been described.

 In a previous study, conducted by the current research team, the implementation of sulfate androgen metabolites in the steroid profile was investigated after the intramuscular administration of a single dose of testosterone esters to six healthy volunteers. For this purpose, a LC-MS method was developed and validated which allowed for the quantification of eight conjugated steroids within the same run. As an outcome of this study, a promising potential marker for the intake of exogenous testosterone, the ratio of [testosterone glucuronide/testosterone sulfate]/[epitestosterone glucuronide /epitestosterone sulfate] = (TG/TS/EG/ES). This ratio is further called “combined ratio”. 

The hereby proposed study represents a follow up project aiming at the investigation of the impact of endogenous sulfate steroid metabolites on the steroid profile after additional routes of administration – oral and transdermal, in addition to the already examined one - intramuscular. Also, as an additional element, the current study will be expanded to include male and female subjects. Furthermore, the already existing analytical data for the Phase II metabolites of Testosterone will be enriched. The proposed study is expected to increase knowledge of the usefulness of the combined ratio as a complementary biomarker for testosterone abuse

Code: 20C07MP

Based on earlier results of our group ecdysterone is included in the 2020 Monitoring Program. As ecdysterone containing plants may be part of common human diet, discrimination between common dietary levels, excessive dietary intake of ecdysterone and supplementation for misuse is highly desired.

It is intended to perceive markers for classification of the type of ingestion. This may help to confirm or contradict athletes' claims in future anti-doping investigations.

Within the project high amounts of regular dietary compounds, i.e. spinach or quinoa, will be ingested by volunteers and urinary excretion compared to post administration urines after pure ecdysterone. Concentrations of ecdysterone will be monitored and potential metabolites or additional ecdysteroids from the plants sources will be uncovered. An investigation of the influence of the human microbiome on ecdysteroid metabolism will be included in the project.

Main Findings

In this study, ecdysterone was quantified in spinach and quinoa. Subsequently, quantitative analysis of ecdysterone and its metabolites in urine samples of human volunteers was conducted, following four different administration studies of sautéed spinach, smoothie from sautéed spinach leaves, quinoa and a combination of sautéed spinach and quinoa.

The purpose of the study was to investigate the human excretion of ecdysterone and metabolites following the consumption of ecdysterone-containing food sources, spinach and quinoa.

After all four administrations to humans (n=8, same participants for each) ecdysterone was excreted and quantified in all volunteers. The metabolite 14-deoxy-ecdysterone was also excreted by all volunteers although in certain cases in quantities below the lowest calibration point of the present study. The second metabolite, 14-deoxy-poststerone, was only detected and quantified in the urine of three volunteers (the same for all study arms). The maximum concentration of ecdysterone ranged between 0.08 and 5.5 μg/mL while for 14-deoxy-ecdysterone from 0.02 to 1.45 μg/mL, and for metabolite 14-deoxy-poststerone from 0.03 to 1.9 μg/mL.

Ecdysterone was not always the most abundant analyte found in the urine, since in certain volunteers 14-deoxy-ecdysterone or 14-deoxy-poststerone was excreted in higher amounts. Another aim of the current study was the comparison of ecdysterone and its metabolites excretion after the ingestion of spinach in different preparations and after the ingestion of sole quinoa and combined with spinach. For both sautéed and smoothie administrations the mean (SD) of the total excreted amount (%) of ecdysterone as unchanged plus the metabolites in urine was 1.4 (1.0) %, consequently no significant difference was observed. On the other hand, significant difference was found in the total excreted amount (%) between the consumption of sole quinoa (mean (SD) 2.6 (1.1) %) and combined with spinach (mean (SD) 1.7 (0.9) %).

Code: 20B06DE

A single administration of subcutaneous luspatercept (0.25 mg/kg) is proposed for four healthy participants, two males and two females. While detection of luspatercept has been studied before by spiking standard into samples, the excretion profile of the drug has not yet been determined in an anti-doping setting. These excretion samples are vital for proper validation of the detection method(s) to be applied to athlete samples. Additionally, the detection window of the drug has yet to be determined in serum/plasma and DBS (and, if applicable, urine), which provides important information for results management authorities.

Finally, Phase I clinical data from luspatercept in healthy individuals suggests that there will be an effect on the hematological module of the ABP, if abused by athletes. As such, it is important to provide a well-characterized timeline of these changes.

Main Findings

The main objective of this study was to perform an administration of Reblozyl® (luspatercept-aamt), a newly approved drug to treat anemia, in healthy volunteers and evaluate the detection of luspatercept in serum, dried capillary blood spots (collected with the Tasso M20), and urine for antidoping purpose. Four volunteers, two males and two females, received one subtherapeutic dose of luspatercept (0.25mg/kg) followed 3 weeks after by a second dose. Samples were collected from before administration to 7 weeks after the second dose. Evaluation of Luspatercept detection in the samples was performed after an immunoextraction step with magnetic beads coated with anti-ActRIIB antibodies, followed by electrophoretic separation by SDS-PAGE and a single-blot and immunodetection using a biotinylated anti-ActRIIB. To propose a confirmation analysis, direct detection was also assessed by SDS/SAR-PAGE followed by double-blotting using a second ActRIIB detection antibody or by IEF-PAGE and single-blot. Indirect effects were examined by measuring endogenous EPO concentrations and by evaluating hematological parameters variations using the ABP model.

Despite the supraphysiological dose administered, intense signals were identified for luspatercept in serum from the day after the administration until the end of the study, 7 weeks after the second dose, and detection is likely possible for even longer time. This administration study also confirmed that the drug is excreted unchanged in urine and regularly eliminated, allowing detection in this matrix. 20μL-DBS also showed sufficient sensitivity to detect the drug until the end of the study. The three electrophoretic methods used in Anti-Doping laboratories: SDS-PAGE, SAR-PAGE and IEF-PAGE were all appropriate for both screening and confirmation and could be used for one or the other. They showed very similar sensitivity.

The impact of the luspatercept on indirect markers was evaluated: ABP approach could flag luspatercept administration in some subjects, especially when ABPS and HGB were both atypically increased, but the effects on RET% were limited and the time/amplitude of the effects varied between subject. Endogenous EPO expression was also not strongly affected and was not indicative of the use of an erythropoietic agent.

In conclusion, this study demonstrated that luspatercept can be detected for a long time using electrophoretic methods in all the matrices relevant for doping. Its survey can be easily implemented in antidoping laboratories.

Publication: Marchand A, Miller G, Martin L, Gobbo C, Crouch AK, Eichner D, Ericsson M. Detection of erythropoiesis stimulating agent Luspatercept after administration to healthy volunteers for antidoping purposes. Drug Test Anal. 2022 Jul 5. doi: 10.1002/dta.3341.

Code: 20B05MT

Every year, approximately 2-5% of the urine samples routinely tested for the presence of erythropoiesis-stimulating agents by the Cologne Doping Control Laboratory are "no-shows" with undetectable endogenous (and recombinant) EPO. Possible origins for this phenomenon can be certain urine properties such as extreme specific gravity or low EPO concentration as well as the addition of adulterants such as proteases.

The aim of this research project is to investigate whether EPO detection can also be manipulated by adding oral fluid to doping control urine sample. Saliva contains many different enzymes including carbohydrases and proteases/peptidases, which can potentially interfere with the detection of a highly glycosylated protein such as EPO. In order to elucidate the effects of urinary oral fluid contaminations on EPO analysis, urine samples fortified with different erythropoietins will be mixed with varying amounts of oral fluid collected from healthy volunteers, stored for different periods of time at different temperatures, and subjected to EPO routine analysis.

Moreover, urine samples will be fortified with different amounts of oral fluid and analyzed by means of ultrafiltration, SDS-PAGE separation, tryptic digestion, and LC-HRMS/MS in order to identify saliva-specific proteins suitable as markers to reveal possible oral fluid contaminations in doping control urine samples.

Main Findings

The aim of this study was to investigate this assumption and to develop a detection assay in order to identify present OF in urine doping control samples.

For this purpose, a total of 1080 urine samples were subjected to EPO analysis and evaluated with regard to variations due to the subject, sex, volume of OF, time point of OF-sampling, and storage conditions. The results showed, that OF can indeed lead to masking of ESA abuse. In particular, interindividual differences as well as the sex and the timing of OF-sampling (pre- and post-prandial) were observed to have an impact on the analysis. In addition, the volume of OF in urine is of major relevance, but realistic amounts, which can be achieved e.g. by spitting once or twice, were found to impair the EPO analysis in a significant number of cases.

In order to identify contaminations or urine samples with OF, detection methods targeting human salivary α-amylase (sAA) were developed, as it was found to be a specific and most abundant protein in OF. For this purpose, both a lateral flow strip test (rapid test) and a bottom-up proteomic assay involving tryptic digestion followed by LC-HRMS/MS analysis were evaluated in terms of selectivity, sensitivity, and stability. Carry-over effect as well as linearity were additionally assessed for the bottom-up proteomic approach. Both approaches successfully identified sAA in urine, and the negative controls and OF-enriched samples could be clearly distinguished from each other. However, the naturally excreted level of sAA in urine presented a major challenge. A proof-of-concept study revealed an intersection between individuals with naturally occurring high levels of sAA in urine and those with low levels despite contaminations with OF, e.g. due to degradation processes caused by high concentrations of proteases in OF. First follow-up experiments demonstrated that peptides of the protein “salivary acidic proline-rich phosphoprotein 1/2” could be used as complementary biomarkers, but further research is required to confirm and subsequently optimize this approach in terms of its applicability.

Results of the project were presented at the 41st Cologne Workshop on Doping Analysis 2023, 26.2.-03.03.2023.

Code: 20A16DE

Peptides, IGF-1 and insulin analogues have significant ergogenic properties, however, are very difficult to test for in all samples currently tested for in doping control. To date, only a limited number of specific and targeted samples are tested for these substance. We have developed a high-throughput mass-spectrometry method that will enable anti-doping organisations to screen for large peptides, e.g., GHRHs, insulin analogues and IGF-1 on all samples. This method will enable all WADA-accredited laboratories to effectively screen for these substance.

 1.    To validate a high throughput, sensitive, and inexpensive method to screen several classes of large peptides in urine.
2.    To validate a similar method in serum and plasma.
3.    To test the method for detection of insulin analogues from urine and blood samples from diabetic patients.

Code: 20A13MT

The detection of novel doping-related substances is challenging for doping control laboratories, especially if these compounds are also produced endogenously by the common human metabolism. In these cases not only the detection of these compounds or related metabolites is sufficient but (semi)quantification becomes necessary followed by confirmation of the exogenous source of the urinary metabolites by isotope ratio mass spectrometry. This procedure is well known for testosterone and its metabolites.

The novel doping agent under investigation in this study will be the 11-keto derivative of testosterone and its urinary metabolites. 11-Ketotestosterone (11KT) is described as a potent anabolic doping agent that is of special interest as it additionally showed the ability to lower cortisol levels. It is easily available via internet-based providers and at the moment no analytical strategy exists to detect its misuse. 11KT is closely related to a prohormone called adrenosterone (androste-4-ene-3,11,17-trione) but the proposed method for detection of adrenosterone will not be suitable for 11KT as it is only focusing on the major metabolites also produced in large amounts in the adrenal glands strongly limiting the sensitivity of this approach.

Within this research project, all currently known metabolites of 11KT will be taken into consideration to identify the most promising candidates for doping controls. First, the initial testing procedure will be developed to semi-quantitate the urinary amounts of all compounds of interest. Secondly, a confirmation procedure based on IRMS will be developed and validated. Both methods will be used to investigate an excretion study performed with 11KT in order to identify those metabolites most suitable for sports drug testing. This research will be completed by the investigation of routine doping control samples enabling to identify urinary baseline concentrations and carbon isotope ratios at natural abundance.

Main Findings

In this study, the human metabolism of KT was investigated in order to provide additional means for the detection of KT and its prohormone OHA4. Two volunteers (one female and one male) orally administered 20 mg of KT each, and urine samples were collected for 5 days. Urinary concentrations of KT and its metabolites were investigated, and a reference population encompassing 220 male and female athletes was investigated in order to elucidate preliminary thresholds. As confirmation procedure, an isotope ratio mass spectrometry-based method was developed in order to determine the CIR of KT and relevant metabolites. The developed methods enabled the detection of exogenous KT for more than 20 h after a single oral administration, which is comparable to a single oral testosterone administration.

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.