Determination of Mitotane and principal metabolite by a simple HPLC-UV method and its validation in human plasma sample

Introduction Mitotane (DDD) is prescribed to inoperable adrenocortical renal carcinoma and Cushing’s syndrome. DDD and its principal metabolite, dichlorodiphenylethene (DDE), can accumulate in fat tissues and their plasma concentrations are associated with clinical improvement more than those of dichlorodiphenylacetate (DDA). The therapeutic range of plasma concentrations is 14–20 mg/L. Therefore, therapeutic drug monitoring (TDM) is required to exploit the maximum therapeutic benefit with acceptable toxicity. Methods Chromatographic conditions: stationary phase was represented by a Higgins Analytical C18 5 μm column (250mmx4.6 mm), maintained at 35 oC. Mobile phase was made by H2O/acetonitrile (10/90, v/v), and pumped at 1.0 ml/min. Peaks of interest were monitored at wavelength of 226 nm. Human plasma samples (200 μl) were added with aldrin (as Internal Standard) and 200 μl of acetonitrile for protein precipitation. Clear supernatants (50 μl) were injected within the HPLC apparatus. Results The average recovery of analytes 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 values of limit of quantitation and detection 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. It is worth noting that sample preparation and run time are short enough to allow the analysis of at least 3 samples per hour (20 min total run). Indeed, the retention time (RT) of DDD and DDE are 7.06 min and 9.42 min, respectively, while the RT of the internal standard is 12.67 min. Finally, the method validation completely fulfilled FDA guidelines. Conclusions A reliable and rapid HPLC-UV method was developed for the measurement of mitotane and DDE concentrations in plasma samples using aldrin as internal standard for better accuracy and precision over the range of drug concentrations expected after the administration of mitotane at standard doses.