Optimizing Antibiotic Analysis: A Comprehensive Guide to HPLC Process of Assessment and Quantification

Year : | Volume : 01 | Issue : | Page : –

    Manjeet Baliyan


This paper provides a comprehensive overview of the evolution and applications of
chromatography, with a focus on High-Pressure Liquid Chromatography (HPLC) in
pharmaceutical analysis. Chromatography, initially discovered by Russian botanist Mikhail
Tswett in the 19th century, has seen significant advancements since its inception, leading to
various chromatography types and applications.

The narrative traces the historical development, highlighting Tswett’s pioneering work in plant
color categorization and the emergence of true chromatography. The 1940s witnessed the
evolution of chromatography types, while the 1960s marked a pivotal moment with Csaba
Harwath’s creation of the first liquid chromatographic equipment. In the late 1960s, Kirkland’s
incorporation of a flow cell for a UV detector played a crucial role in advancing High-
Performance Liquid Chromatography (HPLC).

The focus then shifts to HPLC, a powerful separation method extensively used in pharmaceutical
analysis. The paper details the principles of chromatography, advantages of HPLC, and the
various types of separation it offers, including Normal Phase and Reverse Phase

Chromatography. The instrumentation of HPLC is explored, and the crucial role of buffers in
HPLC separations is emphasized.

The selection of solvents, columns, and mobile phases in HPLC is discussed in detail, addressing
the importance of solubility, absorbance, and water in mobile phases. The paper also delves into
degassing techniques and highlights the significance of system suitability parameters for method

The latter part of the paper provides an in-depth exploration of analytical method development
using HPLC, covering key considerations such as chromatography mode, detector selection, and
optimization of mobile phases. The validation parameters for HPLC methods, including system
suitability, efficiency, retention time, and resolution factor, are thoroughly explained in
accordance with ICH guidelines.

Keywords: Chromatography, High-Pressure Liquid Chromatography (HPLC), Pharmaceutical Analysis, Analytical Method Development, Gas Chromatography (GC)

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1. Ettre LS. 1941–1951: The golden decade of chromatography. Analyst. 1991 Jan
2. Ettre LS. The birth of partition chromatography. LC-GC North America. 2001 May
3. Coskun O. Separation techniques: chromatography. Northern clinics of Istanbul.
4. Ravisankar P, Navya CN, Pravallika D, Sri DN. A review on step-by-step analytical
method validation. IOSR J Pharm. 2015 Oct;5(10):7-19.
5. Kirthi A, Shanmugam R, Prathyusha MS, Basha DJ. A review on bioanalytical method
development and validation by RP-HPLC. Journal of global trends in pharmaceutical
sciences. 2014;5(4):2265-71.
6. Harvey DT. The Essence of Chromatography (Poole, Colin F.).
7. Žuvela P, Skoczylas M, Jay Liu J, Ba̧czek T, Kaliszan R, Wong MW, Buszewski B.
Column characterization and selection systems in reversed-phase high-performance
liquid chromatography. Chemical reviews. 2019 Jan 3;119(6):3674-729.
8. Dolan J. A guide to HPLC and LC-MS buffer selection. ACE HPLC columns. 2009:1-20.
9. Kalakuntla RR, Kumar KS. Bioanalytical method validation: A quality assurance auditor
view point. Journal of Pharmaceutical Sciences and Research. 2009 Sep 1;1(3):1.
10. Riek U, Ramirez S, Wallimann T, Schlattner U. A versatile multidimensional protein
purification system with full internet remote control based on a standard HPLC system.
Biotechniques. 2009 May;46(6):ix-xii.
11. Majors RE. The cleaning and regeneration of reversed-phase HPLC columns. LC GC
NORTH AMERICA. 2003 Jul 1;21(1):19-27.
12. Dewi LP, Wahyono D, Puspitasari I, Humardewayanti R, Lukitaningsih E. Bioanalytical
and validation high-performance liquid chromatography method for simultaneous
quantification cefotaxime and ciprofloxacin in human plasma. Journal of Applied
Pharmaceutical Science. 2024 Jan 4;14(1):221-9.
13. Rajput G, Patel P, Gupta GD, Kurmi BD. A mini-review on simultaneous quantification
of active pharmaceutical ingredients by UV and quality by design assisted HPLC method.

M. Development and validation of a bioanalytical method for quantification of LNA-i-
miR-221, a 13-mer oligonucleotide, in rat plasma using LC–MS/MS. Journal of
Pharmaceutical and Biomedical Analysis. 2018 Feb 20;150:300-7.
27. Muralidharan S, Chiang JH, Chin JB, Ghan SC. Bioanalytical method development and
validation of griseofulvin nanoparticles using RP-HPLC. Journal of Young pharmacists.
28. Ackermann BL, Berna MJ, Murphy AT. Advances in high throughput quantitative drug
discovery bioanalysis. Integrated strategies for drug discovery using mass spectrometry.
2005 Jun 24:315-58.
29. Tiwari G, Tiwari R. Bioanalytical method validation: An updated review. Pharmaceutical
methods. 2010 Oct 1;1(1):25-38.
30. Chaudhari YA, Patil VR, Gujar RR, Patil KR, Nangare S. A concise review on analytical
profile of chlorthalidone. Research Journal of Pharmaceutical Dosage Forms and
Technology. 2022 Mar 4;14(1):63-71.

Subscription Review Article
Received January 12, 2024
Accepted January 19, 2024

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