Simultaneous determination of fish steroidal hormones using RP-HPLC with UV detection by multi-step gradient elution technique

A reversed phase high performance liquid chromatography (HPLC) method with UV detection by multi-step gradient elution method has been developed for simultaneous determination of testosterone, 17β-estradiol, 17α, 20β-dihydroxy-4- pregnen-3-one and cortisol in fish. The method described includes limited sample preparation with single step SPE using C18 cartridge.