Advanced Methodologies In The Development And Validation Of Analytical Techniques For Emerging Neurological Therapeutics: Leveraging Liquid Chromatography-Mass Spectrometry (LC-MS) For Comprehensive Structural Elucidation And Pharmacokinetic Profiling

Authors

  • Priti Gupta
  • Vikram
  • Sadique Saqulain
  • Anil Kumar
  • Manoj Kumar Katual
  • Ramesh Kumar
  • Taruna
  • Yash Srivastav
  • Jothieswari Dhamotharan

DOI:

https://doi.org/10.53555/ejac.v20i1.1139

Keywords:

C-MS, neurological therapeutics, pharmacokinetics, structural elucidation, bioanalytical validation, mass spectrometry, drug metabolism, biomarker discovery.

Abstract

Background: The development of effective neurological therapeutics requires precise analytical techniques for drug characterization and pharmacokinetic profiling. Liquid Chromatography-Mass Spectrometry (LC-MS) has emerged as a powerful tool for the structural elucidation and bioanalytical assessment of these drugs. However, challenges such as structural complexity, metabolic variability, and matrix effects necessitate optimized methodologies to ensure accurate drug analysis. Objective: This study aimed to develop and validate LC-MS methodologies for the characterization and pharmacokinetic assessment of four neurological drug candidates: Rivastigmine, Rotigotine, Clobazam, and Natalizumab. The primary objectives were to:
  1. Optimize LC-MS parameters for improved resolution and sensitivity.
  2. Establish structural elucidation workflows using MS/MS spectral analysis.
  3. Assess pharmacokinetic properties in biological matrices.
  4. Validate the analytical method according to regulatory guidelines.
Methods

The study employed an LC-MS system equipped with a C18 and HILIC column, electrospray ionization (ESI), and high-resolution mass analyzers (Q-TOF, Orbitrap). Key experimental workflows included:

  • Chromatographic optimization: Mobile phase selection, gradient optimization, and ionization mode evaluation.
  • Structural elucidation: Fragmentation pattern analysis using MS/MS spectra and database comparisons.
  • Pharmacokinetic profiling: Plasma concentration-time curve analysis to determine Cmax, Tmax, AUC, and half-life.
  • Method validation: Assessment of linearity, accuracy, precision, sensitivity, LOD, LOQ, and robustness following FDA/EMA guidelines.
Results
  1. LC-MS Optimization: The best chromatographic performance was achieved using a C18 column for Rivastigmine and Clobazam, and a HILIC column for Rotigotine and Natalizumab. An acetonitrile-water gradient with 0.1% formic acid provided optimal separation.
  2. Structural Characterization: MS/MS fragmentation revealed distinct molecular patterns, confirming drug identity with database validation scores >95%.
  3. Pharmacokinetics: The drugs exhibited varied absorption and elimination properties:
    • Rivastigmine: Short half-life (t1/2 = 2.8 h), requiring frequent dosing.
    • Rotigotine: Longer elimination time (t1/2 = 3.5 h), supporting sustained delivery.
    • Clobazam: High plasma levels (Cmax = 350 ng/mL) and extended half-life = 7.0 h, ideal for once-daily dosing.
    • Natalizumab: Prolonged circulation (t1/2 = 14.2 h), supporting monthly dosing regimens.
  4. Validation Outcomes: The developed method met FDA/EMA acceptance criteria with R² > 0.99, precision CV < 6%, LOD = 0.5 ng/mL, and LOQ = 2.0 ng/mL.
Conclusion: The optimized LC-MS method demonstrated high sensitivity, specificity, and reproducibility for the analysis of neurological therapeutics. These findings have significant implications for drug development, regulatory approval, and personalized medicine. Future advancements should incorporate ion mobility spectrometry (IMS-MS), AI-driven spectral analysis, and clinical pharmacokinetic applications to further enhance LC-MS methodologies in neuropharmacology.  

Author Biographies

  • Priti Gupta

    PhD Scholar and Assistant Professor, Department of Pharmaceutical Sciences and Pharmaceutical Chemistry, Dr. K.N Modi University  and Sai Tirupati University, Rajasthan, India

  • Vikram

    Assistant Professor, University College of Pharmacy, Guru Kashi University Talwandi Sabo, Punjab, India

  • Sadique Saqulain

    Phd Scholar, Department of Pharmaceutical Sciences, Madhyanchal Professional University (MPU),Bhopal, Madhya Pradesh, India

  • Anil Kumar

    Head & Assistant Professor,  Department of Chemistry (PG), Sahibganj College Sahibganj,  Jharkhand, India

  • Manoj Kumar Katual

    Associate Professor & Dean, Faculty of Pharmacy, Guru Kashi University, Bhatinda, Punjab, India

  • Ramesh Kumar

    Former Assistant Professor, Lord Shiva College of Pharmacy, Sirsa, Haryana, India

  • Taruna

    Assistant Professor,  Department of Chemistry, HPTU, Himachal Pradesh, India

  • Yash Srivastav

    Assistant Professor, Department of Pharmacy, Shri Venkateshwara University, Gajraula, Uttar Pradesh, India

  • Jothieswari Dhamotharan

    Professor and Principal Sri Venkateswara College of Pharmacy,  RVS Nagar,  Chittoor,  Andhra Pradesh, India

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Published

30-01-2025

Issue

Vol. 20 No. 1 (2025)

Section

Articles