Full Length ArticleDetection of the synthetic peptide ipamorelin in dried blood spots by means of UHPLC-HRMS
Highlights
•
Ipamorelin is a synthetic peptide with potential misuse for doping purposes. •
Dried Blood Spots (DBS) are gaining increasing attention in sport drug testing. •
Ipamorelin was detected at ng/mL levels in DBS by UHPLC-QTOF-HRMS instrumentation. •
DBS are a promising matrix for the detection of small peptides in doping control.
Abstract
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.
Introduction
Growth hormone-releasing peptides (GHRPs) are synthetic peptides that stimulate the release of the growth hormone (GH) from the hypothalamus and pituitary gland and modulate food intake, sleep, and cardiac tone [1]. As a consequence, these peptides have received considerable attention in sports drug testing and are included in the World Anti-Doping Agency (WADA) Prohibited List [2]. Among GHRPs, ipamorelin is a potent and selective pentapeptide that belongs to the class of growth hormone secretagogues (GHS). Ipamorelin displays high growth hormone potency and efficacy both in vitro and in vivo. Unlike the others GHRPs, ipamorelin produces an increase of the GH plasma level without increasing the adrenocorticotropin (ACTH) and cortisol levels [3]. For clinical purposes, ipamorelin was administered to increase gastrointestinal mobility in ileum diseases [4]. In a second study, ipamorelin was tested as an alternative to the direct administration of GH in the treatment of children with growth delay [5]. Although ipamorelin and other GHRPs have not received approval for clinical use, several peptides (including ipamorelin) and analogues were identified in black market products [[6], [7], [8], [9]], suggesting their potential misuse for doping purposes.
In sport drug testing, several methods for the detection of GHRP in urine and blood or plasma have been developed [10]. These methods commonly use solid phase extraction (SPE) based on cationic weak exchange stationary phase or protein precipitation, sometimes followed by immunoaffinity purification, followed by liquid chromatography separation coupled to tandem mass spectrometry and/or high resolution mass spectrometry detection [[11], [12], [13], [14], [15], [16], [17], [18], [19], [20]].
Currently, sampling of blood specimens in sports drug testing is considered invasive and require the presence of trained personnel for venous blood sampling. Furthermore, samples shipment and storage at controlled temperature, limited stability and timely analysis, all represent essential prerogatives. Compliance with these requirements implies considerable cost per sample, especially when a limited number of specimens has to be collected. The use of alternative matrices and procedures, including the collection of dried blood spots (DBS), could overcome these limitations, increasing the number of tested blood samples while decreasing the cost of sampling.
DBS is a micro-sampling technique involving the deposit of a minimal volume of capillary blood on a cellulose adsorbent card that can be easily stored in a fresh and dark place for a long time to preserve the analyte stability. The speed and ease of sampling, its low invasiveness and intrusiveness, the low risk of adulteration and the low transportation and storage costs, are among the advantages suggesting the DBS application in several investigation fields, such as new-borns screening [21], therapeutic drug monitoring (TDM) [22,23], clinical chemistry [24], toxicology [[25], [26], [27]], and toxicokinetic [28,29]. In sport drug testing, DBS was taken into consideration as an alternative matrix only recently, and its increasing potential to support result management processes in routine testing has been discussed in the latest literature [10,30]. Some of the existing doping control assays on DBS aim at low molecular mass analytes [31,32] and a limited number of peptide drugs. Recently, Lange et al. developed a LC-HRMS method for the quantitative detection of 46 low molecular mass (<2 kDa) peptide/non-peptide drugs and drug candidates, after a fully automated DBS sample preparation [33]. LC-MS/MS methods for the detection of paginesatide and Synachten® were developed by Moller et al. [34] and Tretzel et al. [35], respectively. A recent screening method for insulin and its analogues was developed by Thomas et al. [36], while a previous study demonstrated the detection of the Insulin-like Growth Factor-1 (IGF-1) in DBS by LC-MS [37]. Recently, Lange and co-workers determined the TGF-β signaling inhibitors sotatercept and luspatercept and the ActRIIA/B-antibody bimagrumab in DBS by LC-HRMS [38].
In the present study, a comprehensive workflow for the specific and sensitive detection of ipamorelin in DBS is presented. Our aim was to develop a simple, suitable and effective procedure based on fast sample extraction followed by a UHPLC-HRMS detection, in order to detect small peptides in blood at low levels.
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Section snippets
Chemicals and reagents
Acetonitrile, citric acid, ipamorelin acetate (98%), MRFA (tetrapeptide Met-Arg-Phe-Ala) and DBS Whatman FTA DMPK-C papers were purchased from Merck Italia (Milan, Italy). Formic acid was supplied by Fisher Scientific Italia (Rodano, Milan, Italy). Ultrapure water for UHPLC use was obtained from a Milli-Q® equipment (Millipore, Bedford, MA, USA). Negative blood was obtained from 4 volunteer subjects. Blood samples were collected in Vacutainer® K2 EDTA tubes and stored at −20 °C until use. Two
UHPLC-HRMS identification
The positive electrospray ionization of ipamorelin results in the predominant doubly protonated ion at m/z 356.7001. Accordingly, all MS experiments were optimized on the precursor ion at 356.7 m/z, corresponding to the [M+2H]2+ ion. Following the collision induced dissociation (CID) experiments of the precursor ion, the fragment ions derived from the y-series at m/z 146.1292 (y1), 490.2820 (y3), 627.3396 (y4) and the b-series at 223.1191 (b2), 420.2041 (b3) and 567.2720 were observed. The
Conclusions
A novel and comprehensive approach for the detection of ipamorelin was successfully developed by combining DBS sampling technique and UHPLC-QTOF-HRMS detection. The rapid sample extraction procedure coupled to highly selective MS/MS acquisition method enabled sensitive detection of ipamorelin spiked at low ng/mL concentration. Furthermore, the use of a HRMS instrument allowed the univocal identification of the analyte of interest and provided at the same time the chance of conducting a reliable
Credit author contribution statement
Conceptualization: E.G., D.D. and A.S.; Methodology: D.D., E.G., E.A. and A.S.; Validation: D.D., E.A. and J.M.; Formal analysis: D.D. and J.M.; Investigation: D.D., E.G. and M.V.; Data curation: D.D., E.A. and E.G.; Writing-original draft preparation: E.G., A.S., P.N., E.A. and M.V.; Writing-review and editing: E.G., A.S., P.N. and M.V.; Supervision, A.S, P.N. and M.V..
All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Also in this study, DBS was spotlighted as a doable micro-sampling strategy in remote world areas, avoiding the cold chain, which at the same time offers the opportunity to carry out a retrospective analysis. GnRH analogues (e.g., goserelin, leuprolide, triptorelin, buserelin, and nafarelin) along with other lower molecular mass analytes (MW < 2 kDa) were mainly detected, after being extracted from an automated DBS sample preparation, below the WADA’s MRPL in the study of Lange et al. [136]. DBS sampling was also involved for the sensitive (LOD = 0.5 ng/L) and reproducible detection of insulin and its synthetic analogs by mass spectrometry [137,138].
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Moreover, authentic DBS collected from a type-I diabetic individual using insulin Aspart were successfully analysed providing proof-of concept data. A simple and effective procedure based on a fast methanolic extraction and a LC-HRMS detection of the growth hormone secretagogue ipamorelin in DBS was recently proposed by Gerace et al. [193]. MS and MS/MS data were collected by a Q-TOF mass spectrometer operating in the positive ESI mode. View all citing articles on ScopusView full text
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