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

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.

LC–MS/MS method for the quantitation of a dual PI3K/BRD4 inhibitor SF2523 in mouse plasma: Application to plasma protein binding and metabolism studies

“…The gastrointestinal fluid (GI) stability studies of SF2523 were determined in simulated GIs at a concentrations of 10 μ m as described in our previous report (Aldhafiri et al, 2020; Bala et al, 2020). Simulated GIs were prepared according to United States Pharmacopoeia (USP) specifications.…”

“…The mouse blood–plasma partitioning ratio (B/P) of SF2523 was determined at 0.1 and 1 μg/ml concentrations as described in our previous report (Aldhafiri et al, 2020; Bala et al, 2020). At different time points (0, 30 and 60 min) aliquots (50 μl) were collected for blood analysis, and an additional sample (120 μl) was transferred to a microcentrifuge tube and centrifuged at 4,000 g for 10 min at 4°C to separate 50 μl plasma for analysis.…”

“…A rapid equilibrium dialysis device system (Thermo Scientific, Rockford, IL, USA) was used to evaluate plasma protein binding (PPB) by adding a buffer to the buffer chamber and dosing solution to the sample chamber as described in our previous report (Aldhafiri et al, 2020; Bala et al, 2020). The sample contained mouse plasma spiked with 1 or 10 μ m SF2523 and was added to the sample chamber.…”

“…Metabolic stability was assessed using mouse, rat and human liver microsomes, and mouse liver S9 fractions (XenoTech, LLC, Lenexa, KS, USA) were used for the in vitro assessment of phase I and II metabolism. Incubation with S9 fractions was performed in triplicate, at 2 μ m SF2523 concentration as described in our previous report (Aldhafiri et al, 2020; Bala et al, 2020). The reaction was started by adding 2 μl of SF2523 at a final concentration of 2 μ m .…”

Metabolic stability studies of lead compounds supported by separation techniques and chemometrics analysis

“…Hence, the detection sensitivity decreases when using MS/MS for scanning of unknown metabolites, limiting its usage. However, in case of metabolic stability studies, triple quadrupole instruments provide an excellent alternative to more expensive TOF/Q‐TOF analyzers, enabling both a reliable quantitative analysis (for metabolic stability estimation) and metabolite structure identification [11–19,23,52–60].…”