Dispersive liquid-liquid microextraction followed by high-performance liquid chromatography as an efficient and sensitive technique for determination of nandrolone and testosterone in human urine

Dispersive liquid-liquid microextraction (DLLME) and high performance liquid chromatography – UV detection was presented for extraction and determination of nandrolone and testosterone in human urines. Chloroform at microliter volume level and acetonitrile were used as extraction and dispersive solvents, respectively. The main advantages of method are high speed, high enrichment factor, high recovery, good repeatability and extraction solvent volume at µL level. The influence of several variables (e.g. type and volume of disperser and extraction solvents, ionic strength, etc.) on the performance of the sample preparation step was carefully evaluated. Under the optimum conditions, the calibration graphs were linear in the range of 5–500 μg L−1 with detection limit of 2.5 μg L−1 for both of them. The relative standard deviation (R.S.D.s) for five replicate measurements of nandrolone and testosterone were 9.4% and 8.8%, respectively. The relative recoveries of nandrolone and testosterone in urine sample at spiking level of 25.0 μg L−1 are ranged between 86.4% and 98%. DLLME combined with HPLC-UV is a fast, simple and efficient method for the determination of nandrolone and testosterone in human urines.