Recent improvements in sports drug testing concerning the initial testing for peptidic drugs (< 2 kDa) – sample preparation, mass spectrometric detection, and data review

Abstract

In this work, a novel initial testing assay based on liquid chromatography-mass spectrometry is presented, enabling the detection of peptidic drugs and drug candidates (< 2 kDa) prohibited in sports. The assay covers representatives and metabolites of gonadotropin releasing hormone and its analogs (GnRHs), growth hormone secretagogues (GHS), growth hormone releasing peptides (GHRPs), and the Vasopressin-analog Desmopressin. The general objective of this work was to reduce sample preparation efforts to a minimum while preserving highest possible sensitivity and specificity of the assay, demonstrating limits of detection between 50 and 200 pg/mL.Here, a "dilute-and-inject" strategy provides the simplest conceivable sample preparation procedure. Furthermore, the combination of well-established strategies for the determination of peptides, such as two-dimensional liquid chromatography, dimethyl sulfoxide (DMSO)-assisted electrospray ionization, high resolution mass spectrometric detection and a tailored reporter template, which facilitates data review enormously, provides a high-throughput initial testing assay for lower molecular mass peptidic and peptide-related analytes.

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC-HRMS

“…Due to the discovery of glycine-modified analogues, the list of analytes was extended preventively with a series of nine glycine-modified peptides, mainly GHRPs. In addition, GHRP-1 that could not be detected in urine before [10], and felypressin, an ADH receptor agonist, were implemented for the first time in an anti-doping detection procedure. The vast majority of the drug candidates are still under development, in clinical trials or were discontinued.…”

“…As shown in Table 1, a total of 46 target analytes were included within this study, comprising "classical" peptide drugs such as ipamorelin (pentapeptide) or goserelin (decapeptide) and non-peptide drugs (mimetics) such as anamorelin as well as several potentially performance-enhancing Gly-derivatives of the GHRPs. Moreover, GHRP-1, which was stable in urine only as its metabolite [10], was successfully analyzed in DBS. Felypressin, a new vasoconstrictor related to vasopressin, was determined for the first time.…”

“…Subsequently, detection methods were developed and the analytical spectrum was continuously expanded. While initially mainly SPE extracts from urine samples were used for the detection of lower molecular mass peptides by anti-doping laboratories [1,[3][4][5][6][7][8][9], more recent approaches include a total of 21-36 target peptides or their metabolites which can be directly detected from urine by LC-HRMS ("dilute-and-inject") [10][11][12]. The growing list of analytes comprises agonists of the ghrelin receptor (e.g.…”

Annual banned‐substance review – Analytical approaches in human sports drug testing

“…Whether this modification is eliminated by in vivo metabolic reactions yielding established target analytes of GHRPs and GHRH remains to be shown; if the modification is sufficiently resistant to proteolytic activities in vivo, test methods will potentially necessitate adaptation. A flexible strategy employing a dilute‐and‐inject approach combined with online‐trapping and LC‐HRMS(/MS) was presented by Görgens et al, covering a total of 35 peptidic drugs or respective urinary metabolites including (amongst others) 11 growth hormone releasing factors and 12 luteinizing hormone (LH) releasing hormones . Online trapping was accomplished by means of isocratic loading of 40 μL of urine onto a phenyl‐hexyl trapping column (3 × 10 mm, 2.6 μm particle size), and the analytes were subsequently backflushed for separation onto a C‐18 analytical column (2 × 50 mm, 2.7 μm particle size).…”

The 36th Manfred Donike workshop on doping analysis

“…Finally, tips and tricks of improved peptide hormone analysis in routine doping control settings targeting especially growth hormone releasing peptides (GHRPs) were presented by Görgens et al, followed by an outlook on a potential future use of alternative matrices. Exploiting the continuously growing instrumental sensitivity and improving sample preparation strategies, Thomas et al convincingly illustrated the utility of dried blood spots for the analysis of synthetic insulins in doping controls …”