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Abstract

HPLC has become the workhorse of analytical separations due to its versatility, sensitivity, and precision. However, optimizing and validating an HPLC method for specific analytes requires an intricate adjustment of parameters. This review provides a comprehensive overview of the key steps involved in developing and validating a robust HPLC method. Initially, we delve into the critical factors influencing method development, including analyte properties, sample preparation strategies, column selection, mobile phase optimization, and detector choice. We detail the importance of resolution, peak shape, and retention time control in achieving optimal separation. Next, we dissect the validation process, highlighting essential parameters like linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, specificity, robustness, and system suitability. We discuss established protocols and regulatory guidelines for each parameter, emphasizing the principles behind their evaluation. Furthermore, we explore advanced method development approaches, such as hyphenation with mass spectrometry (MS) for enhanced analyte identification and quantitative analysis. We also briefly touch upon emerging trends in HPLC, including microfluidic chips and green chromatography practices. This review serves as a valuable resource for both novice and experienced analysts, offering a roadmap for navigating the intricacies of HPLC method development and validation, ultimately paving the way for reliable and reproducible analytical results.

Keywords

HPLC, method development, validation, chromatography, separation, analysis, accuracy, precision, specificity, robustness, LOD, LOQ.

Reference

  1. Sharma BK. Instrumental Methods of Chemical Analysis, 24th Edition, Pg: 68-110.
  2. Gurdeep R Chatwal, Sham Anand. Instrumental Methods of Chemical Analysis, pg:185-190.
  3. Khan MC, Reddy NK, Ravindra G, Reddy KVSRK, Dubey PK. Development and validation of a stability indicating HPLC method for simultaneous determination of four novel fluoroquinolone dimers as potential antibacterial agents. J Pharmaceut Biomed Anal, 59, 2012, 162–166.
  4. Blanchet B, Sabourea C, Benichou AS, Billemont B, Taieb SR, Alain D. Development and validation of an HPLC-UV- visible method for sunitinibquantifcation in human plasma. ClinChimActa, 404, 2009, 134–139.
  5. FDA Guidance for Industry. Analytical Procedures and Method Validation, Chemistry, Manufacturing, and Controls Documentation, Center for Drug Evaluation and Research (CDER) and Center for Biologics Evaluation and Research (CBER), 2000.
  6. Korany MA, Mahgoub H, Ossama TF, Hadir MM. Application of artificial neural networks for response surface modeling in HPLC method development. J Adv Res, 3, 2012, 53–63.
  7. Ferrarini A, Huidobro AL, Pellati F, Barbas C. Development and validation of a HPLC method for the determination of sertraline and three non-chiral related impurities. J Pharmaceut Biomed Anal, 53, 2010, 122–129.
  8. Collier JW, Shah RB, Bryant AR, Habib MJ, Khan MA, Faustino PJ. Development and application of a validated HPLC method for the analysis of dissolution samples of levothyroxine sodium drug products. J Pharmaceut Biomed Anal, 54, 2011, 433–438.
  9. Singh S, Bakshi M. Guidance on conduct of stress tests to determine inherent stability of drugs. Phrama Tech, 24, 2000, 1- 14.
  10. Swartz ME, Jone MD, Fowler P, Andrew MA. Automated HPLC method development and transfer. LcGc N. Am, 75, 2002, 49-50.
  11. Synder LR, Kirkland JJ, Glajach JLX. In Practical HPLC Methods Development. John Wiley, New York, 295, 1997, 643- 712.
  12. Swartz M, Murphy MB. New Fronties in Chromatography. Am Lab, 37, 2005, 22-27.
  13. Debebe Z, Nekhai S, Ashenaf M, David BL, Kalinowski DS, RG Victor, Byrnes WM, Richardson DR, Karla PK. Development of a sensitive HPLC method to measure invitro permeability of E- and Z-isomeric forms of thiosemicarbazones in Caco-2 monolayers. J Chromatogram B, 906, 2012, 25–32.
  14. www.agilent.com/chem/store (Accessed on 11/01/2024)
  15. Dolan JW. Peak tailing and resolution. LcGc N. Am, 20, 2002, 430-436.
  16. Qiang Fu, Shou M, Chien D, Markovich R, Rustum AM. Development and validation of a stability-indicating RP-HPLC method for assay of betamethasone and estimation of its related compounds. J Pharmaceut Biomed Anal, 51, 2010, 617– 625.
  17. Nguyen AT, Aerts T, Dam DW, Deyn PPD. Biogenic amines and their metabolites in mouse brain tissue: Development, optimization and validation of an analytical HPLC method. J Chromatogra B, 878, 2010, 3003–3014.
  18. International Conference on Harmonization of technical Requirements for Registration of Pharmaceuticals for Human use, ICH
  19. Harmonized Tripartite guideline-Validation of Analytical procedures: Text and methodology Q2 (R1), Current step 4 version., London 2005.
  20. Singh R. HPLC method development and validation-an overview. Journal of Pharmaceutical Education & Research. 2013 Jun 1;4(1).
  21. Sonia K, Lakshmi KS. HPTLC method development and validation: An overview. Journal of Pharmaceutical Sciences and Research. 2017 May 1;9(5):652.
  22. Ahuja S, Rasmussen H, editors. HPLC method development for pharmaceuticals. Elsevier; 2011 Sep 21

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Sayali V. Ganjiwale
Corresponding author

Department of Pharmaceutical Quality Assurance, P. Wadhwani College of Pharmacy, Yavatmal, Maharashtra, India

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A. P. Dewani
Co-author

Department of Pharmaceutical Quality Assurance, P. Wadhwani College of Pharmacy, Yavatmal, Maharashtra, India

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A. V. Chandewar
Co-author

Department of Pharmaceutical Quality Assurance, P. Wadhwani College of Pharmacy, Yavatmal, Maharashtra, India

Sayali V. Ganjiwale*, A. P. Dewani, A. V. Chandewar, A Comprehensive Overview of HPLC Method Development and Validation, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 1, 802-811. https://doi.org/10.5281/zenodo.10581387

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