Isolation, enrichment, and analysis of erythropoietins in anti-doping analysis by receptor-coated magnetic beads and liquid chromatography-mass spectrometry.

Human erythropoietin (hEPO) is an erythropoiesis stimulating hormone frequently employed in antianemia therapy. Its capability to increase the amount of red blood cells however makes hEPO and its derivatives also attractive to dishonest athletes aiming at an artificial and illicit enhancement of their endurance performance. A major objective of the international antidoping fight is the elimination of drug misuse and prevention of severe adverse effects caused by nontherapeutic administrations of highly potent drugs. The emergence of novel and innovative erythropoietin-mimetic agents (EMAs) has been continuously growing in the last years, and the option of using dedicated monoclonal antibodies (mAb) for analytical and sample preparation approaches is gradually reaching limits. In the present study the common ability and property of all EMAs, to bind on the human erythropoietin receptor (hEPOR), is therefore exploited. An alternative methodology to isolate and analyze EMAs, in particular endogenous EPO and the recombinant forms EPOzeta, darbepoetin alfa, and C.E.R.A., from human urine is described, employing conventional ultrafiltration for preconcentration of the target analytes followed by EMA-specific isolation via hEPOR-bound magnetic beads. Analytical data were generated by means of gel-based electrophoretic analysis and nanoliquid chromatography/high resolution/high accuracy tandem mass spectrometry. Limits of detection enabled by the established sample preparation protocols were approximately 20 pg/mL for EPOzeta, 30 pg/mL for darbepoetin alfa, and 80 pg/mL for C.E.R.A.

Introduction of a PEGylated EPO conjugate as internal standard for EPO analysis in doping controls.

Immunopurification of doping control samples is a mandatory necessity in erythropoietin (EPO) analysis during a confirmation procedure; moreover, it has become common practice to also immunopurify samples for the initial testing procedure. Typically used materials (e.g., Stemcell purification plate and MAIIA purification kit) rely on anti-EPO antibodies for purification. Also, the detection of EPO after electrophoretic separation and western blotting is based on a monoclonal anti-EPO antibody, clone AE7A5, directed against a 26 amino acid sequence of the N-terminal region of human EPO. While the electrophoretic separation and blot transfer efficiency can be monitored with reference standards and quality control samples, it is presently not possible to monitor the functionality of the entire sample preparation procedure. The reliance on antibodies for both purification and detection has complicated the implementation of an internal standard (ISTD). In this study, customized EPO-polyethylene glycol (PEG) conjugates were synthesized as potential ISTDs and assessed as to their compatibility with existing sample preparation procedures for urine and blood sample analysis using the most common immunopurification techniques. Moreover, probing for the impact of the ISTD on sodium N-lauroylsarcosinate ("sarcosyl") polyacrylamide gel electrophoresis (SAR-PAGE)-based EPO analysis concerning potential interference with target analytes was conducted. The presented data demonstrate that a 12-kDa PEG residue attached to human EPO represents a particularly useful construct to serve as ISTD for erythropoietin-receptor agonist (ERA) analysis. The conjugate is applicable to both urine and blood testing using the commonly employed purification techniques, supporting and improving result interpretations especially concerning specimens where the natural abundance of human EPO is low.