Simplifying and expanding the screening for peptides <2 kDa by direct urine injection,liquid chromatography,and ion mobility mass spectrometry

The analysis of low‐molecular‐mass peptides in doping controls has become a mandatory aspect in sports drug testing and, thus, the number of samples that has to be tested for these analytes has been steadily increasing. Several peptides <2 kDa with performance‐enhancing properties are covered by the list of prohibited substances of the World Anti‐Doping Agency including Desmopressin, LH‐RH, Buserelin, Triptorelin, Leuprolide, GHRP‐1, GHRP‐2, GHRP‐3, GHRP‐4, GHRP‐5,GHRP‐6, Alexamorelin, Ipamorelin, Hexarelin, ARA‐290, AOD‐9604, TB‐500 and Anamorelin. With the presented method employing direct urine injection into a liquid chromatograph followed by ion‐mobility time‐of‐flight mass spectrometry, a facile, specific and sensitive assay for the aforementioned peptidic compounds is provided. The accomplished sensitivity allows for limits of detection between 50 and 500 pg/mL and thus covers the minimum required performance level of 2 ng/mL accordingly. The method is precise (imprecision <20%) and linear in the estimated working range between 0 and 10 ng/mL. The stability of the peptides in urine was tested, and –20°C was found to be the appropriate storage temperature for sports drug testing. Finally, proof‐of‐concept was shown by analysing elimination study urine samples collected from individuals having administered GHRP‐6, GHRP‐2, or LHRH.