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

Abstract

The added value of dried blood spot (DBS) samples complementing the information obtained from commonly routine doping control matrices is continuously increasing in sports drug testing. In this project, a robotic-assisted non-destructive hematocrit measurement from dried blood spots by near-infrared spectroscopy followed by a fully automated sample preparation including strong cation exchange solid-phase extraction and evaporation enabled the detection of 46 lower molecular mass (< 2 kDa) peptide and non-peptide drugs and drug candidates by means of LC-HRMS. The target analytes included, amongst others, agonists of the gonadotropin-releasing hormone receptor, the ghrelin receptor, the human growth hormone receptor, and the antidiuretic hormone receptor. Furthermore, several glycine derivatives of growth hormone-releasing peptides (GHRPs), arguably designed to undermine current anti-doping testing approaches, were implemented to the presented detection method. The initial testing assay was validated according to the World Anti-Doping Agency guidelines with estimated LODs between 0.5 and 20 ng/mL. As a proof of concept, authentic post-administration specimens containing GHRP-2 and GHRP-6 were successfully analyzed. Furthermore, DBS obtained from a sampling device operating with microneedles for blood collection from the upper arm were analyzed and the matrix was cross-validated for selected parameters. The introduction of the hematocrit measurement method can be of great value for doping analysis as it allows for quantitative DBS applications by managing the well-recognized "hematocrit effect."

Evaluation of dried blood spots as an alternative sample matrix for equine antidoping analysis

“…As with current approaches for routine post‐race and out‐of‐competition samples, complete coverage for all classes of molecules will likely require several different extraction and instrumental analysis approaches to reach the desired levels of coverage. Several of these classes of agents have been evaluated in DBS for human antidoping efforts though further evaluation of the use of DBS in the horse should be undertaken if coverage for these drug classes is desired 7,8,17–19 …”

“…DBS have been used for over 50 years and offer many advantages in the analysis of various compounds with minimal sample volume 1,2 . The use of DBS in antidoping analysis has recently become more feasible with the advances in analytical sensitivity and specificity afforded by modern liquid chromatography–mass spectrometry (LC‐MS) instrumentation 1,3–8 …”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…Substances stimulating GH secretion such as GH releasing peptides (GHRPs) or GH secretagogues (GHS) are prohibited in sports and, accordingly, analyzed also in routine doping controls. Extending the use of DBS in this regard, Lange et al established a fully automated analytical approach allowing for the determination of the hematocrit of blood spotted on DBS cards followed by extraction, SPE‐based purification, evaporation, and reconstitution prior to LC‐HRMS/MS analysis of 22 ghrelin receptor agonists plus representative metabolites and 12 gonadorelin receptor agonists 123 . Nondestructive hematocrit analyses were accomplished by means of near infrared spectroscopy, and spots were extracted onto a strong cation‐exchange SPE cartridge and eluted and concentrated to 100 μl for subsequent analysis.…”

Dried blood spots for anti‐doping: Why just going volumetric may not be sufficient

“…A simple option to estimate the HCT is to correlate the scanned surface area of the spot in combination to the known applied volume of whole blood 52 . A more advanced application of non‐destructive HCT measurement during an anti‐doping study has been presented by Lange et al using NIR 53 . However, they concluded that differences in temperature or humidity might influence the molecular vibrations of the DBS matrix, thus negatively influencing the outcome of the used DBS NIR spectrometry measurement.…”

“…52 A more advanced application of non-destructive HCT measurement during an anti-doping study has been presented by Lange et al using NIR. 53 However, they concluded that differences in temperature or humidity might influence the molecular vibrations of the DBS matrix, thus negatively influencing the outcome of the used DBS NIR spectrometry measurement. The use of reflectance-based DBS HCT measurement does not have this limitation.…”