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 (r > 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 (C) and (T) values for the biomarkers in the systemic circulation were WLD A (5.59 ± 0.34 ng/mL, T 1.00 ± 0.00 h), WNONE (6.28 ± 0.41 ng/mL, T 0.95 ± 0.11 h), WLD B (6.45 ± 2.87 ng/mL, T 0.95 ± 0.11 h), WF A (6.50 ± 0.27 ng/mL, T 1.00 ± 0.00 h), and 12 DEOXY (5.68 ± 0.39 ng/mL, T 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.