BACKGROUND: Doping is the use of prohibited substances by athletes to improve their performance. World Anti-Doping Agency (WADA)-accredited laboratories require various metabolite reference standards of the prohibited chemical substances or drugs for routine quality control. Therefore, it was proposed to develop efficient synthetic methodologies for highly pure reference materials of Phase II metabolites of octopamine, norfenefrine and etilefrine, which are prohibited in sports by WADA under the S6 stimulant category. The reference materials were characterized using various analytical techniques. New highperformance liquid chromatography with diode-array detection (HPLC-DAD) methods were developed for purity assessment.RESULTS: The synthesized Phase II metabolite reference standards, i.e. octopamine sulfate, norfenefrine sulfate and etilefrine sulfate, were confirmed by 1 H NMR, 13 C NMR, liquid chromatography-high-resolution mass spectrometry (LC-HRMS), attenuated total reflectance Fourier transform infrared and thermogravimetric analysis. In the LC-HRMS study, the mass error value of synthesized compounds was less than 10 ppm (error) which confirms the identity of the reference materials. New HPLC-DAD method were developed to ensure the purity of the reference materials. We used the HILIC column as metabolite reference standards are highly polar. The mobile phase was composed of water and acetonitrile in fixed composition. The HPLC-DAD purity of the developed reference materials was observed as 100%.CONCLUSION: We have developed reproducible synthetic routes for octopamine sulfate, norfenefrine sulfate and etilefrine sulfate, which are prohibited in sports by WADA. The synthesized metabolites were characterized using different advanced analytical techniques. These reference standards will be helpful to all WADA-accredited laboratories in routine anti-doping testing.
