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

Anal Bioanal Chem. 2020 Jun;412(15):3765-3777. doi: 10.1007/s00216-020-02634-4. Epub 2020 Apr 16.


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." Graphical abstract.

Keywords: Doping; Dried blood spots (DBS); Growth hormone–releasing peptides (GHRP); Hematocrit (Hct); Sport; TAP blood collection device.

Publication types

  • Validation Study

MeSH terms

  • Chromatography, Liquid / instrumentation
  • Chromatography, Liquid / methods
  • Doping in Sports
  • Dried Blood Spot Testing / instrumentation*
  • Dried Blood Spot Testing / methods
  • Equipment Design
  • Hematocrit
  • Humans
  • Limit of Detection
  • Oligopeptides / blood*
  • Peptides / blood
  • Substance Abuse Detection / instrumentation*
  • Substance Abuse Detection / methods
  • Tandem Mass Spectrometry / instrumentation
  • Tandem Mass Spectrometry / methods


  • Oligopeptides
  • Peptides
  • growth hormone releasing hexapeptide
  • growth hormone-releasing peptide-2