Development and Validation of an LC-MS/MS Method for the Simultaneous Quantification of Abiraterone, Enzalutamide, and Their Major Metabolites in Human Plasma

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Abiraterone acetate and enzalutamide are 2 novel drugs for the treatment of metastatic castration-resistant prostate cancer. The metabolism of these drugs is extensive. Major metabolites are N-desmethyl enzalutamide, enzalutamide carboxylic acid, abiraterone N-oxide sulfate, and abiraterone sulfate; of which N-desmethyl enzalutamide is reported to possess antiandrogen capacities. A liquid chromatography-tandem mass spectrometry method for simultaneous quantification of abiraterone, enzalutamide, and the main metabolites has been developed and validated to support therapeutic drug monitoring.


Human plasma samples of patients treated with abiraterone or enzalutamide were harvested at the clinic and stored at −20°C. Proteins were precipitated by acetonitrile, and the final extract was injected on a Kinetex C18 column and separated with gradient elution. Analytes were detected by liquid chromatography-mass spectrometry (Triple Quad 6500).


The method was validated over various linear ranges: 1–100 ng/mL for abiraterone, 5–500 ng/mL for enzalutamide and enzalutamide carboxylic acid, 10–1000 ng/mL for N-desmethyl enzalutamide, 30–3000 ng/mL for abiraterone N-oxide sulfate, and 100–10,000 ng/mL for abiraterone sulfate. Intra-assay and interassay variabilities were within ±15% of the nominal concentrations for quality control samples at medium and high concentrations and within ±20% at the lower limit of quantification, respectively.


The described method for simultaneous determination of abiraterone and enzalutamide was validated successfully and provides a useful tool for therapeutic drug monitoring in patients treated with these agents.

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