Detection of the synthetic peptide ipamorelin in dried blood spots by means of UHPLC-HRMS

Growth hormone-releasing peptides (GHRPs) are synthetic peptides that stimulate the release of growth hormones from the hypothalamus and pituitary gland. Among these, ipamorelin is a highly selective, potent and effective pentapeptide belonging to the class of growth hormone secretagogues (GHS). Because of clinical monitoring and the potential abuse by athletes in amateur and elite sports, detection methods able to identify GHRPs in biological samples are increasingly needed. As an alternative to conventional procedures, testing of Dried Blood Spots (DBS) is gaining increasing attention, since its low volume sampling is easy and scarcely invasive. In this study, an ultra high pressure liquid chromatography–high resolution mass spectrometry method was developed and validated for the screening of ipamorelin in DBS. Ipamorelin was extracted from DBS by liquid extraction and ultrasonic treatment, then detected by the acquisition of high-resolution fragmentation spectra. The method proved satisfactory in terms of sensitivity, specificity, linearity, recovery, precision and ion suppression/enhancement effects. The mass error of the precursor and fragment ions was lower than 5 ppm. Since ipamorelin was successfully detected in DBS with a detection limit of 2.5 ng/mL, the combination of DBS and high resolution mass spectrometry proved to be a suitable and effective method to detect small peptides in blood at low levels. This approach looks promising for targeted and untargeted analysis in clinical monitoring and sport drug testing, especially when unknown synthetic peptides represent the target analytes.