UC Davis

The abuse of erythropoiesis stimulating agents (ESAs), such as recombinant human erythropoietin (rhEPO), is of great concern to the safety, integrity, and fair competition of horseracing. The current methods of definitive rhEPO detection utilize Liquid Chromatography-Mass Spectrometry (LC-MS) to unambiguously identify the presence of human-specific peptides in equine samples. However, confirmation and quantitation become challenging with rhEPO concentrations less than 0.1 ng/mL. This project concentrated on the derivatization of tryptic peptides of rhEPO 46VNFYAWK52 (T6) and 144VYSNFLR150 (T17) to increase ionization efficiency and lower current limits of detection. A total of eleven derivatization reagents were tested: 5-dimethylamino naphthalene-1-sulfonyl chloride (Dansyl Chloride), nα-(2,4-dinitro-5fluorophenyl)-l-valinamide (DNFP-L-V), (carboxymethyl)trimethylammonium chloride hydrazide (Girard’s Reagent T), 2,4,6-triphenylpyrylium tetrafluoroborate (TPP), 2,4,6-trimethylpyrylium tetrafluoroborate (TMP), (n-succinimidyloxycarbonyl-methy)tris(2,4,6-trimethoyxlphenyl)phosphonium bromide (TMPP-Ac-OSu), cyanine3-nhs ester (CY3-NHS), dipyrrolidino(n-succinimidyloxy)carbenium hexafluorophosphate (HSPyU), n,n,n′,n′- tetramethyl-o-(n-succinimidyl)uronium tetrafluoroborate (TSTU), tandem mass tag pro zero label reagent (TMTpro Zero), and 6-aminoquinolyl-n-hydroxysuccinimidyl carbamate (AQC). Following the selection of AQC as the optimal derivatization reagent, the reaction protocol was optimized through a basic buffer selection and sample clean-up process. To assess method performance, equine serum samples without any detectable rhEPO were spiked with an rhEPO standard solution at various concentrations. The samples were purified and rhEPO isolated using immunopurification, followed by tryptic digestion, solid phase extraction cleanup using Hydrophilic-Lipophilic Balance cartridges, derivatization with AQC in a sodium tetraborate buffer, and analysis using LC-HRMS. Accuracy and precision of the method could not be calculated because integrated ion area counts for all multiply charged species and associated qualifying ions expected from derivatized peptides were not present.