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A rapid and sensitive bioanalytical LC–MS/MS method for the quantitation of a novel CDK5 inhibitor 20–223 (CP668863) in plasma: Application to in vitro metabolism and plasma protein-binding studies |
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| Authors: | Veenu Bala Yashpal S Chhonker Richard L Sleightholm Ayrianne J Crawford Michael A Hollingsworth Daryl J Murry |
| Institution: | 1. Clinical Pharmacology Laboratory, Department of Pharmacy Practice and Science, University of Nebraska Medical Center, Omaha, NE, USA
Department of Pharmaceutical Sciences, Mohanlal Sukhadia University, Udaipur, India;2. Clinical Pharmacology Laboratory, Department of Pharmacy Practice and Science, University of Nebraska Medical Center, Omaha, NE, USA;3. Center for Drug Delivery and Nanomedicine, Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA;4. Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA;5. Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA |
| Abstract: | A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (MS/MS) method was developed and validated for the quantitation of the novel CDK5 inhibitor ‘20–223' in mouse plasma. Separation of analytes was achieved by a reverse-phase ACE Excel C18 column (1.7 μm, 100 × 2.1 mm) with gradient elution using 0.1% formic acid (FA) in methanol and 0.1% FA as the mobile phase. Analytes were monitored by MS/MS with an electrospray ionization source in the positive multiple reaction monitoring mode. The MS/MS response was linear over the concentration range 0.2–500 ng/mL for 20–223. The within- and between-batch precision were within the acceptable limits as per Food and Drug Administration guidelines. The validated method was successfully applied to plasma protein binding and in vitro metabolism studies. Compound 20–223 was highly bound to mouse plasma proteins (>98% bound). Utilizing mouse S9 fractions, in vitro intrinsic clearance (CLint) was 24.68 ± 0.99 μL/min/mg protein. A total of 12 phase I and II metabolites were identified with hydroxylation found to be the major metabolic pathway. The validate method required a low sample volume, was linear from 0.2 to 500 ng/mL, and had acceptable accuracy and precision. |
| Keywords: | 20–223 CDK5 inhibitor LC–MS/MS |