TY - JOUR
T1 - A rapid and sensitive bioanalytical LC–MS/MS method for the quantitation of a novel CDK5 inhibitor 20–223 (CP668863) in plasma
T2 - Application to in vitro metabolism and plasma protein-binding studies
AU - Bala, Veenu
AU - Chhonker, Yashpal S.
AU - Sleightholm, Richard L.
AU - Crawford, Ayrianne J.
AU - Hollingsworth, Michael A.
AU - Murry, Daryl J.
N1 - Funding Information:
The work was supported by the State of Nebraska through the Pediatric Cancer Research Group, the University of Nebraska Medical Center, the Fred & Pamela Buffett Cancer Center Support Grant from the National Cancer Institute under award number P30 CA036727, National Cancer Institute SPORE grant P50-CA127297, and National Cancer Institute Fellowship F31 CA224942-01A1. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The compound 20-223 was synthesized by Dr. Yogesh A. Sonawane and Dr. Amarnath Natarajan, University of Nebraska Medical Center. We also acknowledge the helpful discussions with Robert J. Classon at Shimadzu Scientific Instruments, Columbia, MD.
Funding Information:
The work was supported by the State of Nebraska through the Pediatric Cancer Research Group, the University of Nebraska Medical Center, the Fred & Pamela Buffett Cancer Center Support Grant from the National Cancer Institute under award number P30 CA036727, National Cancer Institute SPORE grant P50‐CA127297, and National Cancer Institute Fellowship F31 CA224942‐01A1. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The compound 20‐223 was synthesized by Dr. Yogesh A. Sonawane and Dr. Amarnath Natarajan, University of Nebraska Medical Center. We also acknowledge the helpful discussions with Robert J. Classon at Shimadzu Scientific Instruments, Columbia, MD.
Publisher Copyright:
© 2020 John Wiley & Sons, Ltd.
PY - 2020/8/1
Y1 - 2020/8/1
N2 - 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.
AB - 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.
KW - 20–223
KW - CDK5 inhibitor
KW - LC–MS/MS
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U2 - 10.1002/bmc.4859
DO - 10.1002/bmc.4859
M3 - Article
C2 - 32307720
AN - SCOPUS:85084795393
VL - 34
JO - Biomedical Chromatography
JF - Biomedical Chromatography
SN - 0269-3879
IS - 8
M1 - e4859
ER -