Detection of the manipulation of doping control urine samples concerning EPO analyses by means of oral fluid

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.