Determination of prohibited, small peptides in urine for sports drug testing by means of nano-liquid chromatography/benchtop quadrupole orbitrap tandem-mass spectrometry

In the present study, a screening assay was developed comprising 11 prohibited peptides (<1.5 kDa) that are sufficiently purified from urine using weak cation exchange with subsequent determination of all substances by means of nanoUHPLC separation coupled to high resolution tandem mass spectrometry. These peptides included Gonadorelin (LH-RH), Desmopressin and 9 growth hormone releasing peptides (GHRP-1, -2, -4, -5, -6, Hexarelin, Alexamorelin, Ipamorelin and a GHRP-2 metabolite); however, the procedure is expandable to further target analytes or metabolites. The method was validated with a main focus on qualitative result interpretation considering the parameters specificity, linearity (0–500 pg/mL), recovery (45–95%), precision (<20% at 100 pg/mL), limits of detection (2–10 pg/mL), robustnesss and ion suppression. The proof-of-principle was shown by analysing excretion study urine samples for LHRH, Desmopressin and GHRP-2.

► We developed a combined screening procedure for prohibited peptides.
► It is more sensitive and effective than single procedures.
► It is expandable to further peptides/metabolites.
► It provides highest specificity due to HRMS.