Determination of mitotane and metabolite by a simple HPLC-UV method and application in plasma samples

Mitotane (dichlorodifenyldichloroethane, DDD) it is a polychlorinated compound derivative of dichlorodiphenyltrichloroethane (DDT), prescribed in inoperable adrenocortical renal carcinoma and Cushing’s syndrome [1,2]. DDD and its principal metabolite, dichlorodiphenylethene (DDE), can accumulate in fat tissues [3] and plasma concentrations are more related with clinical improvement, than those of dichlorodiphenylacetate (DDA), another metabolite of DDD. HPLC methods constitute a valid alternative to gas chromatography, and plasma concentrations of 14–20 mg/L are considered therapeutic concentrations. The stationary phase was represented by a Higgins Analytical C18 5 μm column (250mmx4.6 mm), at 35 oC. Mobile phase was made by H2O/acetonitrile (10/90, v/v) at 1.0 ml/min. Peaks of interest were monitored at wavelength of 226 nm. Human plasma sample preparation: 200 μl of plasma sample was added with aldrin (as Internal Standard) and 200 μl of acetonitrile for protein precipitation. Samples were vortexed for 30 sec and centrifuged at 12000 rpm x 10 minutes. Clear supernatants (50 μl) were injected in HPLC system. Average recovery was 95%, and the method was linear (r2=0.9988 and 0.9964 for DDD and DDE, respectively) within the range 1-40 mg/L. The LOQ and were 0.2 mg/L and 0.3 mg/L for DDD and 0.066 mg/L and 0.099 mg/L for DDE, respectively. Indeed, the retention time (RT) of DDD and DDE are 7.06 min and 9.42 min and 12.67 min for Internal Standard. Method was validated and applied to 30 plasma samples of 5 different patients affected by adrenocortical renal carcinoma. We analyzed the fluctuations of concentrations over time. In 30 plasma dosages, only 4 samples were within the range of 14-20 mg/L. The mitotane plasma level correlate with plasma DDE levels, according to pearson index. In conclusion, a reliable and rapid HPLC-UV method was developed for the measurement of mitotane and DDE concentrations in plasma samples using aldrin as internal standar