Development and validation of a liquid chromatography coupled to a diode array detector (LC-DAD) method for measuring...

Area of Science:

• Pharmacology and Toxicology
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Mitotane (o,p '-DDD) is the primary therapeutic agent for adrenocortical carcinoma (ACC).
• Accurate plasma concentration monitoring of mitotane is essential for effective treatment and dose adjustment in ACC patients.

Purpose of the Study:

• To develop and validate a simple, reliable, and straightforward method for determining mitotane (o,p '-DDD) concentrations in human plasma.
• To establish a robust assay for therapeutic drug monitoring (TDM) and dose management of mitotane in patients with adrenocortical cancer.

Main Methods:

• Plasma samples were spiked with varying concentrations of mitotane (DDD) and an internal standard (p,p '-DDD).
• Protein precipitation with acetonitrile was followed by HPLC-DAD analysis using a reversed-phase C18 column.
• Method validation included assessment of sensitivity, selectivity, precision, linearity, accuracy, recovery, and matrix effects.

Main Results:

• The developed HPLC-DAD method demonstrated excellent linearity (R2 > 0.9987) over the 1.00–50.00 µg/mL range with high extraction efficiency (97.80 %–105.50 %).
• The assay showed high sensitivity (analytical sensitivity 0.98 µg/mL, LOQ 1.00 µg/L), good precision (CV < 9.98 %), and no carry-over or significant matrix effects.
• Results correlated well with a previously used GC-MS method (r = 0.88), indicating high accuracy and reliability.

Conclusions:

• The developed and validated method for mitotane (o,p '-DDD) determination in plasma is simple, robust, efficient, and sensitive.
• This assay is suitable for therapeutic drug monitoring and precise dose management, aiding in achieving optimal therapeutic index for mitotane in adrenocortical cancer patients.