An LC–MS/MS method for determination of the bromodomain inhibitor ZEN-3694 and its metabolite ZEN-3791 in human plasma

Abstract

We have developed and validated a novel LC–MS/MS method for the simultaneous quantification of ZEN-3694 and its active metabolite ZEN-3791 in human plasma after protein precipitation. Stable isotope-labeled versions were used as internal standards. Chromatographic separation was achieved on a Kinetex C18 column using 0.1% formic acid in H₂O and 0.1% formic acid in MeOH as mobile phases. Detection was performed via positive electrospray ionization mode with multiple reaction monitoring. The assay exhibited linearity in the concentration range of 5–5000 ng/ml for both analytes. Intra- and inter-assay precision and accuracy were within ±11%. ZEN-3694 and ZEN-3791 recoveries were between 93 and 105%. This LC–MS/MS assay is an essential tool to study ZEN-3694 in an ongoing clinical trial (NCT04840589).

Executive summary

• An LC–MS/MS method was developed and validated to quantitate ZEN-3694 and its major metabolite ZEN-3791, in human plasma.

• A 4.0-min LC–MS/MS method was established in multiple reaction monitoring mode.

• Low sample volumes are needed, as well as simple sample preparation by protein precipitation.

• A novel assay was developed according to US FDA guidelines for bioanalytical method validation, over a linear range of 5–5,000 ng/ml for both ZEN-3694 and ZEN-3791.

• The method is being used to quantitate ZEN-3694 and ZEN-3791 in clinical samples obtained from patients recruited to an ongoing phase I/Ib trial (NCT04840589).

Keywords: :

• assay
• BET bromodomain inhibitor
• chromatography
• clinical
• LC–MS
• mass spectrometry
• ZEN-3694
• ZEN-3791

Financial disclosure

Research reported in this publication includes work by the National Cancer Institute of the National Institutes of Health under grant nos. U24CA247643, UM1CA186690 and P30CA033572. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Acknowledgments

Research reported in this publication includes work by the National Cancer Institute of the National Institutes of Health under grant nos. U24CA247643, UM1CA186690 and P30CA033572. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Additional information

Funding

Research reported in this publication includes work by the National Cancer Institute of the National Institutes of Health under grant nos. U24CA247643, UM1CA186690 and P30CA033572.