Recent studies report that nandrolone, a widely used anabolic steroid by athletes to enhance their performance, is produced endogenously in the humans. However, the detection of nandrolone has mainly been indicated by the measurement of its urinary metabolites (19-norandrosterone and 19-noretiocholanone). The aim of this study was to develop a sensitive and specific ELISA for nandrolone to investigate whether the parent steroid can be detected in the urine of healthy volunteers. Nandrolone antibodies were produced by immunising sheep with nandrolone-3-CMO-KLH immunogen and used with HRP-donkey anti-sheep IgG conjugate as tracer to develop the ELISA method. Cross-reactivity values of anti-nandrolone antibody with major interfering steroids including nandrolone metabolites were minimal except for testosterone (1.8%) and dihydrotestosterone (3.98%). A sensitive standard curve for nandrolone ELISA has been constructed with good reproducibility and a minimum detection limit of 12.75 pmole/L (3.5 pg/mL). The assay was evaluated for specificity, sensitivity, parallelism, accuracy and imprecision and all found to be satisfactory. The validity of the urinary nandrolone assay was confirmed by the excellent correlation between the ELISA results and those obtained by LC/MS/MS (ELISANand=1.06 LC/MS/MS Nand+0.032, R2=0.98, p<0.001). Urinary nandrolone excretion in healthy volunteers who were known not to have taken any anabolic steroids was assessed in exercising and non-exercising individuals. In non-exercising females, endogenous urinary nandrolone levels were found to range from 0.014–2.122 ng/mL (0.069–8.98 nmol/ day), and 0.017–1.291 nmol/day for exercising females. In non-exercising males, the levels ranged from 0.018–0.486 ng/mL (0.078–2.341 nmol/day), and 0.041–2.44 nmol/day for exercising males. In conclusion, a simple, rapid and sensitive ELISA method has been developed to estimate urinary excretion of nandrolone, and used for the detection of this steroid in urine. It seems likely that nandrolone as such, though at parts per billion, is excreted in the urine of healthy subjects not knowingly ingesting steroids
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