Validation of a robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis
Michael T Davis1, Nicole M Anders1,2, A Dimitrios Colevas3
1The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, Maryland, USA.
Abstract:
EBNA1 is an Epstein Barr virus (EBV) protein expressed in all EBV-associated cancers. EBNA1 plays a critical role in the replication and maintenance of EBV episomes in latently infected cells. VK-2019 was developed as a highly specific inhibitor of EBNA1 DNA binding activity and is currently in phase 1 development as a treatment for EBV-associated carcinomas. A sensitive and reliable method was developed to quantify VK-2019 in human plasma using liquid chromatography with tandem mass spectrometry to perform detailed pharmacokinetic studies. VK-2019 was extracted from plasma using protein precipitation with acetonitrile. Separation of VK-2019, two purported metabolites, and the internal standard, VK-2019-d6, was achieved with a Zorbax XDB C18 column using a gradient flow over 6 min. VK-2019 was detected using a SCIEX 4500 triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The assay range was 0.5-500 ng/mL and proved to be accurate and precise. Dilutions of 1:10 were accurately quantified. VK-2019 was stable in plasma at -70°C for approximately 18 months. The method was applied to assess the total plasma concentrations of VK-2019 in a patient who received a single and multiple oral daily doses of 120 mg.
Insights
A new method accurately quantifies VK-2019, an Epstein-Barr virus (EBV) inhibitor targeting EBNA1, in human plasma. This assay supports pharmacokinetic studies for VK-2019, a potential treatment for EBV-associated cancers.
Area of Science:
- Pharmacology
- Analytical Chemistry
- Virology
Background:
- Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is crucial for EBV episome maintenance in infected cells.
- EBNA1 is expressed in all EBV-associated cancers, making it a therapeutic target.
- VK-2019 is an inhibitor of EBNA1 DNA binding activity in early clinical development for EBV-associated carcinomas.
Purpose of the Study:
- To develop and validate a sensitive and reliable bioanalytical method for quantifying VK-2019 in human plasma.
- To support pharmacokinetic (PK) studies of VK-2019 during its Phase 1 clinical development.
Main Methods:
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for quantification.
- Acetonitrile-mediated protein precipitation was used for plasma sample extraction.
- A C18 column with a 6-minute gradient elution and positive electrospray ionization detection on a triple quadrupole mass spectrometer were utilized.
Main Results:
- The validated assay demonstrated accuracy and precision across the 0.5-500 ng/mL range.
- The method accurately quantified 1:10 plasma dilutions.
- VK-2019 exhibited stability in plasma stored at -70°C for at least 18 months.
Conclusions:
- A robust LC-MS/MS method was successfully developed and validated for quantifying VK-2019 in human plasma.
- This assay is suitable for supporting pharmacokinetic assessments in clinical trials.
- The method's reliability and the drug's stability are critical for evaluating VK-2019's therapeutic potential in EBV-driven malignancies.
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