LC-ESI-MS/MS method validation for simultaneous quantification of FDA-approved anticancer agents futibatinib and binimetinib in rat plasma: Insights from preclinical pharmacokinetics.

This study investigates the combination of FGFR inhibitor futibatinib (FTB) and MEK inhibitor binimetinib (BTB) for KRASmt NSCLC therapy. An analytical method was developed and validated for measuring FTB and BTB concentrations in rat plasma, adhering to USFDA guidelines. Using liquid-liquid extraction on 45-µL plasma samples, a 6.5-min run time was achieved. The linear calibration curve ranged from 2 to 100 ng/mL. Intra-day and inter-day accuracy ranged between 92.06% and 100.08%. Four blank injections post high-concentration samples resolved significant carryover. Extraction recoveries averaged 92.06% to 102.37% across concentrations. No significant endogenous interference was detected in blank plasma. The LLOQ for both drugs was 2.0 ng/mL. Selectivity, matrix effects, stability, and dilution integrity met the acceptance criteria. The method assessed FTB and BTB interaction potential in combination therapy at 5 mg/kg. The findings provide essential pharmacokinetics insights for future clinical trials.

Simultaneous Estimation of Quercetin and trans-Resveratrol in Cissus quadrangularis Extract in Rat Serum Using Validated LC-MS/MS Method: Application to Pharmacokinetic and Stability Studies.

Cissus quadrangularis is a nutrient-rich plant with a history of use in traditional medicine. It boasts a diverse range of polyphenols, including quercetin, resveratrol, ß-sitosterol, myricetin, and other compounds. We developed and validated a sensitive LC-MS/MS method to quantify quercetin and t-res biomarkers in rat serum and applied this method to pharmacokinetic and stability studies. The mass spectrometer was set to negative ionization mode for the quantification of quercetin and t-res. Phenomenex Luna (C18(2), 100 A, 75 × 4.6 mm, 3 µ) column was utilized to separate the analytes using an isocratic mobile phase consisting of methanol and 0.1% formic acid in water (82:18). Validation of the method was performed using various parameters, including linearity, specificity, accuracy, stability, intra-day, inter-day precision, and the matrix effect. There was no observed significant endogenous interference from the blank serum. The analysis was completed within 5.0 min for each run, and the lower limit of quantification was 5 ng/mL. The calibration curves showed a linear range with a high correlation coefficient (r2 > 0.99). The precision for intra- and inter-day assays showed relative standard deviations from 3.32% to 8.86% and 4.35% to 9.61%, respectively. The analytes in rat serum were stable during bench-top, freeze-thaw, and autosampler (-4 °C) stability studies. After oral administration, the analytes showed rapid absorption but underwent metabolism in rat liver microsomes despite being stable in simulated gastric and intestinal fluids. Intragastric administration resulted in higher absorption of quercetin and t-res, with greater Cmax, shorter half-life, and improved elimination. No prior research has been conducted on the oral pharmacokinetics and stability of anti-diabetic compounds in the Ethanolic extract of Cissus quadrangularis EECQ, making this the first report. Our findings can provide the knowledge of EECQ's bioanalysis and pharmacokinetic properties which is useful for future clinical trials.

Simultaneous estimation of five biomarkers of neuroprotective herb Ashwagandha NMITLI-118R AF1 in rat plasma and brain using LC-ESI-MS/MS: Application to its pharmacokinetic and stability studies.

Withania Somnifera (WS) is a popular nutritional supplement in the USA, Europe, and Asia, known for its pharmacological effects on neurological disorders. However, the bioanalytical method development, validation, and pharmacokinetics of WS NMITLI-118R AF1 biomarkers Withanolide A (WLD A), Withanone (WNONE), Withanolide B (WLD B), Withaferin A (WF A), and 12 Deoxywithastramonolide (12 DEOXY) in rats have not been comprehensively explored. This study aimed to develop and validate a sensitive and selective LC-ESI-MS/MS method for these biomarkers in male Sprague Dawley rats plasma and brain matrix. Rats were divided into eight groups, each containing five rats. A plant extract of NMITLI-118R AF1 at 50 mg/kg was orally administered to the rats for in-vivo pharmacokinetic investigation. All the analytes had a linear calibration curve (r2 > 0.999), and intra-day and inter-day precision (%) were found in the range of 2.46 - 13.71% and accuracy were within the acceptable range (±15%). The biomarkers of NMITLI-118R AF1 were found stable in in-vitro plasma and simulated gastro-intestinal fluids. The observed (Cmax) and (Tmax) values for the biomarkers in the systemic circulation were WLD A (5.59 ± 0.34 ng/mL, Tmax 1.00 ± 0.00 h), WNONE (6.28 ± 0.41 ng/mL, Tmax 0.95 ± 0.11 h), WLD B (6.45 ± 2.87 ng/mL, Tmax 0.95 ± 0.11 h), WF A (6.50 ± 0.27 ng/mL, Tmax 1.00 ± 0.00 h), and 12 DEOXY (5.68 ± 0.39 ng/mL, Tmax 1.00 ± 0.00 h). In contrast to the old method, our approach exhibits a lower limit of quantification (LLOQ), shorter run time (less than10 min), and enables the detection of WF A and WNONE in fresh rat plasma by other quantitative analysis of mass spectrometry (m/z) [M]+. Shows high sample volumes for both, larger plasma volumes, costlier sample collection techniques dried blood spot (DBS), more expensive solid phase extraction techniques (SPE) and longer analysis time 14 min. Moreover, our method requires a smaller sample volume 10 µL, offers faster analysis time 4 min, and achieves a higher sensitivity 1 ng/mL. This is the first report of a comprehensive study on in-vitro and in-vivo pharmacokinetics of NMITLI-118R AF1 biomarkers, which may aid in further pre-clinical and clinical trial investigations.