(T1030-06-35) A Validated LC-MS/MS Assay for the Simultaneous Determination of Nebivolol, Dasatinib and Devimistat and Its Application to Pharmacokinetic Study in Rats

Purpose: Cancer is very common and a leading cause of death worldwide. In 2020, GLOBOCSN data reported 19.3 million new cancer cases and 10 million deaths, accounting for nearly one in six deaths [1]. Among these cases, female breast cancer stood out as the most diagnosed cancer, with 2.3 million new cases (11.7%) and 685,000 death globally in 2020 [2]. Triple-negative breast cancer (TNBC) comprises about 10-15% of all breast cancers, which carries the worst prognosis and survival rates among all breast cancers. Median survival varies but remain approximately 18 months or less [3]. Consequently, there is a pressing need to discover safe and effective therapies in the high priority in TNBC research. Monotherapy for the aggressive high-grade TNBC often yields limited efficacy, prompting the exploration of combination targeting multiple pathways as an emerging strategy. Furthermore, combination therapy not only enhance effectiveness compared to monotherapy but also may allow for the use of lower doses of individual agents [4]. Therefore, a simple, rapid, and sensitive method for quantifying drugs in samples is particularly crucial. In the present study, a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of nebivolol (NBV), dasatinib (DAS) and devimistat (CPI), which may be used for TNBC therapy.

Methods: A validated LC-MS/MS method was developed for simultaneous quantification of NBV, DAS and CPI. Deuterated compounds served as internal standards (IS), while acetonitrile via a liquid-liquid extraction method. Separation occurred on a C18 column (Waters Acquity® UPLC BEH, 1.7 μm, 2.1×50 mm) with an isocratic mobile phase comprising water with 0.1% formic acid (aqueous phase) and acetonitrile (organic phase) at a ratio of 3:7, v/v. Electrospray positive ionization (ESI) positive mode was utilized to determine the three drugs with transitions 406/151 for NBV, 488/401 for DAS, and 389/91 for CPI. The method was validated in plasma in the concentration range of 0.5-100 ng/mL, adhering to the bioanalytical method validation guidance from US Food and Drug Administration (US FDA). Linearity, selectivity, sensitivity, accuracy, precision (inter-day and intra-day), extraction method, matrix effect and recovery were established. To access method suitability, Sprague Dawley rats (n=3) were administered a single dose of NBV (1.9 mg/kg), DAS (3.6 mg/kg), and CPI (4.8 mg/kg) via intravenous injection from the tail vein. Blood samples (150 μL/timepoint) were collected via tail snipping at specified intervals (10’, 30’, 1h, 2h, 3h, 4h, 6h, 8h, and 24h), followed by plasma extraction and storage at -80 ℃ until analysis. A volume of ten microliter of plasma sample were required for the LC-MS/MS assay. All procedures adhered to the approved animal protocol by the University of Houston Institutional Animal Care and Use Committee.

Results: The retention times for NBV, DAS and CPI were 1.12, 1.08, and 1.85 min, respectively, with a total run time per sample of 3 min. Calibration curves of NBV, DAS and CPI exhibited linearity in the range of 0.5-100 ng/mL with r2 >0.99, specifically 0.9991±0.0005 for NBV, 0.9977±0.002 for DAS, and 0.9961±0.0026 for CPI (n=3). In the matrix of plasma, no interfering peaks were detected at the retention times of analytes and their respective IS. The lower limit of quantification (LLOQ) was 0.5 ng/mL for all analytes. Intra-day and inter-day precision ranged from 1.3% to 12.8% for NBV, 1.3% to 8.0% for DAS, and 6.6% to 14.5% for CPI across quality control (QC) samples at LLOQ and low, medium, and high concentrations (n=6, each). Accuracy values (bias %) for all analytes were between 92.4-110.7%(NBV), 89.9-102.3%(DAS), and 96.1-106.5% (CPI) at four different concentration levels (n=6). The matrix effect and recovery ranged from 88.2 to 105.3% and 81.4 to 114.1%, respectively (n=6), all falling within acceptable limits. No carryover effect was observed in all analytes. The developed method was successfully applied to determine PK parameters on rats (n=3). revealing Cmax values of 89.4±18.9 ng/mL for NBV, 210.0±42.3 ng/mL for DAS, and 1573.3±300.2 ng/mL for CPI. The method proved suitable for analyzing serial plasma samples at various timepoints.

Conclusion: A rapid, specific, and sensitive LC-MS/MS method was developed and validated to determine NBV, DAS and CPI simultaneously in rat plasma. All parameters were in the acceptable range. The advantages of this method are: 1) a small plasma sample volume requirement (10 μL), 2) straightforward and rapid sample preparation, 3) efficient with a short runtime of 3 minutes per sample, 4) high sensitivity with a lower limit of quantification (LLOQ) of 0.5 ng/mL, and 5) excellent specificity, with no interference from other peaks. The method was successfully applied to ascertain drug concentrations in pharmacokinetic studies conducted on rats.

References: 1. Hyuna Sung, J.F., Rebecca L Siegel, Mathieu Laversanne, Isabelle Soerjomataram, Ahmedin Jemal, Freddie Bray, CA Cancer J Clin. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin., 2021. 71(3): p. 209-249.
2. Breast Cancer. 2023 [cited 2023 July 21]; Available from: https://www.who.int/news-room/fact-sheets/detail/breast-cancer.
3. Schmid P, A.S., Rugo HS, et al., Atezolizumab and Nab-Paclitaxel in advanced triple-negative breast cancer. N Engl J Med, 2018. 379: p. 2108-2121.
4. Mir, M.A., et al., Targeting different pathways using novel combination therapy in triple negative breast cancer. Current Cancer Drug Targets, 2020. 20(8): p. 586-602.

Acknowledgements: We extend our sincere gratitude to Dr. Rongjin Sun from the University of Houston for his invaluable assistance in the PK study. The funding source was supported by Congressionally Directed Medical Research Programs (CDMRP) (HT94252410011).


Table 1. Intra-day and inter-day accuracy and precision of 3 analytes in rat plasma matrix


Table 2. Matrix effect and extraction recovery of 3 analytes in rat plasma


Figure 1 PK profiles of nebivolol, dasatinib and devimistat in rat plasma
Route: intravenous injection via tail vein.
Dose: nebivolol (NBV) 1.9 mg/kg, dasatinib (DAS) 3.6 mg/kg; devimistat (CPI) 4.8 mg/kg