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 C¬¬¬¬¬18 cartridge. A multi-step gradient elution of water (A) and acetonitrile (B) with flow rate 1-1.5ml/min with curve index 5of 20% to 80% of B at various intervals and variable detection at UV max of 203nm and 242nm. Validation of the method was performed in terms of recovery with 87.2-106%, intra- and inter-assay variability of 0.93-21.28% and 0.40-9.18% respectively and linearity between 10-1200ng/ml. E2 shows the least LOD (25.16ng/ml) and LOQ (229.8ng/ml) followed by cortisol (45.64ng/ml and 138.32ng/ml) T (72.946ng/ml and 221.04ng/ml) and DHP (75.83ng/ml and 138.32ng/ml). The developed chromatograph was applied to analyse plasma hormonal level in amur carp.