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Published on: December 18, 2020
Development and validation of an LC-MS/MS assay for the quantification of cintirorgon (LYC-55716) in mouse plasma and
Paniz Heydari1, Wenlong Li1, Alfred H Schinkel2
1Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacoepidemiology & Clinical Pharmacology, Universiteitsweg 99, 3584 CG Utrecht, the Netherlands; The Netherlands Cancer Institute, Department of Pharmacy & Pharmacology, Louwesweg 6, 1066 EC Amsterdam, the Netherlands.
Abstract:
Cintirorgon (LYC-55716) is a promising first-in-class antitumor agent as a RORγ agonist in the treatment against various types of cancer. To support preclinical mouse studies, a bioanalytical method was developed and successfully applied for quantification of cintirorgon in mouse plasma and tissue homogenates using LC-MS/MS. The method was fully validated in mouse plasma and partial validation was performed in eight different homogenates originating from brain, kidney, liver, lung, small intestine, small intestine content, spleen, and testis. Sample preparation was performed using 96-well plates for fast and efficient analysis. Protein precipitation was done by addition of 20 µL acetonitrile containing monensin as internal standard to 10 µL sample. Chromatographic separation was achieved on a Polaris 3 C18-A column using gradient elution with 0.2% (v/v) formic acid and 0.2% (v/v) ammonium hydroxide in water (A) and methanol (B) as eluents. The total run time was 3 min. Detection was carried out with a triple quadrupole mass spectrometer with electrospray ionization operated in the positive ion-mode. Quantification could be accomplished within a linear validated concentration range of 5-4,000 ng/mL (10-4,000 ng/mL in brain homogenates) with an intra- and inter-day precision between 4.6-14.7% and 5.1-15.6% (including the LLOQ), respectively, and accuracies between 89.1%-111.2%. The method was successfully applied to a preclinical study with cintirorgon in mice.
Insights
A new bioanalytical method quantifies cintirorgon, a RORγ agonist antitumor drug, in mouse plasma and tissues. This validated LC-MS/MS method supports preclinical cancer research.
Area of Science:
- Pharmacology
- Analytical Chemistry
- Oncology
Background:
- Cintirorgon (LYC-55716) is a novel first-in-class antitumor agent targeting RORγ.
- RORγ agonists show potential in treating various cancers.
Purpose of the Study:
- To develop and validate a bioanalytical method for quantifying cintirorgon in mouse biological matrices.
- To support preclinical studies of cintirorgon in mice.
Main Methods:
- Quantification of cintirorgon using Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS).
- Sample preparation involved protein precipitation using acetonitrile and monensin as internal standard on 96-well plates.
- Chromatographic separation on a Polaris 3 C18-A column with gradient elution.
- Detection via triple quadrupole mass spectrometry in positive ion mode.
Main Results:
- The method was fully validated in mouse plasma and partially in eight tissue homogenates.
- Validated linear concentration range: 5–4,000 ng/mL (plasma), 10–4,000 ng/mL (brain).
- Intra- and inter-day precision ranged from 4.6–14.7% and 5.1–15.6%, respectively.
- Accuracies were between 89.1%–111.2%.
Conclusions:
- A robust and validated LC-MS/MS bioanalytical method for cintirorgon was successfully developed.
- The method is suitable for quantifying cintirorgon in mouse plasma and various tissue homogenates for preclinical studies.
- This supports the advancement of cintirorgon as a potential cancer therapeutic.
