International Journal of Trend in Scientific Research and Development (IJTSRD)

Volume 7 Issue 4, July-August 2023 Available Online: www.ijtsrd.com e-ISSN: 2456 – 6470

A Review: HPLC Method Development and Validation

Ajay Sanjay Salvi, Mohini S. Khamkar, Dr. Lahu D. Hingane
Department of Quality Assurance, Aditya Pharmacy College, Beed, Maharashtra, India

ABSTRACT How to cite this paper: Ajay Sanjay

Due to its very effective separations and often high detection Salvi | Mohini S. Khamkar | Dr. Lahu D.
sensitivity, HPLC is the most widely used separation method in Hingane "A Review: HPLC Method
contemporary pharmaceutical and biomedical analysis. The majority Development and Validation" Published
of medications in multiple component dosage forms can be examined in International
Journal of Trend in
using the HPLC method due to its many benefits, including speed,
Scientific Research
specificity, accuracy, precision, and ease of automation. The and Development
development and validation of HPLC procedures are crucial to novel (ijtsrd), ISSN:
discoveries, the creation of pharmaceutical medications, and 2456-6470,
numerous other investigations involving both humans and animals. Volume-7 | Issue-4, IJTSRD58595
To compare a defined characteristic of the drug substance or drug August 2023,
product to predetermined acceptance criteria for that characteristic, pp.34-39, URL:
an analytical technique is designed. This review provides details on www.ijtsrd.com/papers/ijtsrd58595.pdf
the numerous steps that go into developing and validating an HPLC
technique. According to ICH Guidelines, validating an HPLC Copyright © 2023 by author (s) and
International Journal of Trend in
technique include testing for system appropriateness as well as
Scientific Research and Development
accuracy, precision, specificity, linearity, range and limit of Journal. This is an
detection, limit of quantification, robustness, and other performance Open Access article
characteristics. distributed under the
terms of the Creative Commons
KEYWORDS: HPLC, Method development, Validation Attribution License (CC BY 4.0)
(http://creativecommons.org/licenses/by/4.0)
INTRODUCTION
High Performance Liquid Chromatography (HPLC) stationary phase, or sample solution, is injected into a
was derived from the classical column porous column, and the mobile phase, or liquid phase,
chromatography and, is one of the most important is pumped through the column at a higher pressure.
tools of analytical chemistry today. In the modern The adsorption of solute on stationary phase based on
pharmaceutical industry, high performance liquid its affinity towards stationary phase is the separation
chromatography (HPLC) is the major and integral principle that is used. The HPLC method has the
analytical tool applied in all stages of drug discovery, following characteristics.
development, and production. The preferred approach High definition
for testing the peak purity of new chemical entities, Quick analysis,
keeping track of reaction changes during scale-up or Stainless steel, glass column, and small diameter
synthesis processes, assessing new formulations, and Regulated mobile phase flow rate
performing quality control and assurance on finished Somewhat higher mobile phase pressure
pharmaceutical products is HPLC. The purpose of the HPLC Method Development:
HPLC approach is to attempt to quantify and separate When there are no official methods for a novel
the primary drug, any contaminants from reactions, product, methods are devised. Reduce the cost and
all readily available synthetic intermediates, and any time for current (non-pharmacopoeial) items by using
degradants. One of the most effective tools in alternative methods. for increased robustness and
analytical chemistry nowadays is high performance precision. Comparative laboratory data with merits or
liquid chromatography. Every material that can demerits are made available when an alternative
dissolve in a liquid can have its constituents approach is suggested to replace the current
separated, identified, and quantified using this procedure. The fundamental objective of the HPLC
technique. HPLC is one of the most precise analytical method is to attempt and quantify the separation and
techniques that is frequently used to analyse quantification of the main active medication, any
pharmacological products both quantitatively and reactive impurities, all readily available synthetic
qualitatively as well as to assess their stability. The intermediaries, and any degradants.

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 International Journal of Trend in Scientific Research and Development @ www.ijtsrd.com eISSN: 2456-6470
Steps involved in Method development are. polymers, alumina, and zirconium, are used to
Understanding the Physicochemical properties of support the stationary phase. The most typical matrix
drug molecule. for HPLC columns is silica. Silica matrices have a low
Selection of chromatographic conditions. tendency to compress under pressure, are resilient,
Developing the approach of analysis. simple to derivatize, and are produced with uniform
Sample preparation spherical sizes.
Method optimization Most organic solvents and low pH solutions have
Method validation
little effect on the chemical stability of silica. A silica
Understanding drug molecule physicochemical solid support's drawback is that it will dissolve above
characteristics: pH 7. For application at high pH, silica-supported
A drug's physicochemical characteristics are columns have recently been created.
important for essential part in the development of
Separation is influenced by the silica support's
methods. One must study the physical characteristics composition, shape, and particle size. Increased or
of the drug molecule, such as its solubility, polarity,
more theoretical plates are produced by smaller
pKa, and pH, in order to build a method. A particles. Whether type of chromatography—normal
compound's physical characteristic of polarity. An
phase or reverse phase—a column is best suited for
analyst can use it to choose the mobile phase's solvent depends on the characteristics of the stationary phase.
and chemical component.6 6 The polarity of the
molecules can be used to explain their solubility. Normal phase chromatography utilizes a polar
Solvents that are nonpolar, like benzene, and polar, stationary phase and a non-polar mobile phase.
like water, do not combine. Like generally dissolves Generally, more polar compounds elute later than
like, which means that substances with comparable non-polar compounds. Commonly used reverse phase
polarities can be dissolved in one another. The choice columns and their uses are listed below. Propyl (C3),
of diluents depends on how soluble the analyte is. The Butyl (C4), and Pentyl (C5) Phases are helpful for
pH value is often used to determine whether a large molecules, hydrophobic peptides, and ion-
substance is acidic or basic. In HPLC, choosing the pairing chromatography (C4). Comparing C3-C5
right pH for ionizable analytes frequently produces phases to C8 or C18 phases, non-polar solutes are
symmetrical and sharp peaks. often retained less well by C3-C5 columns. Zorbax
SB-C3, YMC-Pack C4, and Luna C5 are a few
Choosing the chromatographic parameters: examples. Compared to columns with longer alkyl
To get the first "scouting" chromatograms of the
chains, these columns are typically less resistant to
sample, a set of basic settings (detector, column,
hydrolysis. The applications of octyl (C8, MOS)
mobile phase) are chosen during initial technique
phases are numerous. While less retention than the
development. On reversed-phase separations on a
C18 stages, this phase is nonetheless very helpful for
C18 column with UV detection, they are typically
drugs, nucleosides, and steroids. 10 In developing a
based. At this point, a choice should be taken
method, choosing the stationary phase or column is
regarding whether to develop an isocratic or a
the first and most crucial step. Without the
gradient methodology.
availability of a stable, high performance column, it is
Column Selection: impossible to build a robust and reproducible
A chromatograph's beginning point and centrepiece is procedure. The column production batches from the
a column, of course. An accurate and trustworthy same manufacturer as well as columns from other
analysis can be produced by a good chromatographic manufacturers varied in the separation selectivity for
separation from a well-chosen column. A poorly used specific components. The key ones include column
column can frequently produce unclear, insufficient, dimensions, silica substrate parameters, and bonded
and poor separations, which can produce results that stationary phase qualities. Due to a number of
are unreliable or difficult to understand. physical properties, silica-based packing is preferred
in most current HPLC columns.
The column is the brains of an HPLC setup. During
technique development, changing a column will have Chromatographic mode selection:
the biggest impact on the resolution of analytes. The Polarity and molecular weight-based chromatographic
stationary phase chemistry, retention capability, modes. Reversed-phase chromatography (RPC), the
particle size, and column dimensions must all be most typical method for tiny organic compounds, will
taken into account when selecting the optimum be the main topic of all case studies. Ion-pairing
column for an application. Hardware, a matrix, and a reagents or buffered mobile phases, which retain the
stationary phase are the three major parts of an HPLC analytes in a non-ionized state, are frequently used in
column. Several types of matrices, including as silica,

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 International Journal of Trend in Scientific Research and Development @ www.ijtsrd.com eISSN: 2456-6470
RPC to separate ionizable chemicals (acids and than 2000, the tailing factor should be less than 2,
bases). resolution between 2 peaks should be greater than 5,
and the R.S.D. of the area of analyte peaks in
Mobile phase optimization:
standard chromatograms should not be greater than
Buffer Choice: The system appropriateness
2.0%, among other parameters that indicate a system
parameters and overall chromatographic performance
is suitable.
of various buffers, including potassium phosphate,
sodium phosphate, and acetate, were assessed. When two components are estimated simultaneously,
the detection wavelength is often at its isobestic point.
Impact of pH:
If analytes can be ionised, the correct mobile phase Sample preparation:
pH must be selected based on the analyte's pKa so The analyst must look at the sample preparation phase
that the target analyte is in one predominant of method development. For instance, if the sample
ionisation state, either ionised or neutral. One of the contains insoluble components, the analyst should
best techniques in the "chromatographer's toolbox" determine whether centrifugation (choosing the best
for changing both retention and selectivity between rpm and time), shaking, and/or filtration of the sample
important pairs of components simultaneously is are necessary. The purpose is to show that the sample
adjusting the pH of the mobile-phase. filtration has no impact on the analytical outcome
caused by leachable adsorption and/or extraction.
Impact of organic modifier:
Syringe filters' efficiency is largely dependent on its
Choosing an organic modifier type in reverse phase
capacity to filter out impurities and insoluble
HPLC is quite straightforward. Acetonitrile and
substances without introducing unwanted artefacts
methanol are often the options (rarely THF). Gradient
(i.e.,extractables) into the filtrate. Whether using an
elution is typically used with complicated
actual in-process sample or a dosage form for a future
multicomponent samples since it may not be viable to
HPLC analysis, the sample preparation process
elute all components using a single solvent strength
should be adequately specified in the applicable
under isocratic conditions between k (retention factor)
analytical technique. The manufacturer, type, and
1 and 10. 12
pore size of the filter media must be mentioned in the
Choosing a wavelength and detector: analytical technique. The goal of sample preparation
Following chromatographic separation, the desired is to transform a raw sample into a processed sample
analyte is identified using the appropriate detectors. that yields superior analytical results to the raw
Commercial detectors that are utilised in LC include sample. The prepared sample should be an aliquot
mass spectrometry (MS) detectors, UV detectors, that is compatible with the HPLC process and won't
fluorescence detectors, electrochemical detectors, and harm the column, and it should be reasonably clear of
detectors that measure refractive index (RI). interferences.
The sample and the goal of the analysis influence the Method optimization:
detector selection. In the case of multicomponent The improvement of HPLC conditions has received
analysis, the absorption spectra may have been altered the majority of attention throughout HPLC method
from the parent chemical to longer or shorter development optimization. It is necessary to consider
wavelengths. Due to the various levels of the compositions of the fixed and mobile phases.
contaminants in the combination, the UV spectra of Mobile phase parameter optimization is always
the target analyte and the impurities must be collected prioritised since it is more practical and
and overlaid, and the spectra must be normalised. It is straightforward than stationary phase optimization.
necessary to select a wavelength that will allow for a Only the parameters that are likely to have a
sufficient response for the majority of the analytes. significant impact on selectivity in the optimization
Creating the analytical strategy: must be looked at in order to reduce the amount of
The selection of several chromatographic parameters, trial chromatograms required. The various elements
such as the mobile phase, column, flow rate, and pH of the mobile phase serve as primary control variables
of the mobile phase, is the initial step in the in the optimization of liquid chromatography (LC)
development of an analytical technique for RP- techniques.
HPLC. Calculating the gradient, flow rate, temperature,
These parameters are all chosen through testing, and sample amounts, injection volume, and solvent type
the system suitability parameters are taken into of the diluents. Following satisfactory selectivity, this
account afterwards. Retention time should be greater is utilised to identify the ideal balance between
than 5 minutes, theoretical plates should be greater resolution and analysis time. The variables include
flow rate, column packing particle size, and column

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 International Journal of Trend in Scientific Research and Development @ www.ijtsrd.com eISSN: 2456-6470
dimensions. Changes to these parameters won't have of the regression line is typically used to express
an impact on selectivity or capacity factor. linearity. The ICH recommendation recommends
using a minimum of five concentrations to establish
Method Validation:
An analytical method is validated when it has been linearity. The range between the upper and lower
proven through laboratory tests that its performance levels that can be determined using an analytical
characteristics are appropriate for the intended method with verified precision, accuracy, and
analytical application. Any new or modified method linearity is known as the range of the method.
needs to be validated to make sure it can produce Precision: The degree of scatter between a set of
repeatable and reliable results when applied by measurements obtained from multiple sampling of the
various operators using the same equipment in the same homogeneous sample under the specified
same or other laboratories. The specific approach and conditions is expressed as the closeness of agreement
the applications it is intended for determine exactly (precision) of an analytical procedure. There are three
what kind of validation program is necessary. A types of accuracy: repeatability, intermediate
crucial component of any sound analytical procedure, precision, and reproducibility. The standard deviation
method validation data can be used to assess the or relative standard deviation of a sequence of data is
calibre, dependability, and consistency of analytical typically used to express the precision of an analytical
findings. The method validation process is technique. The degree of reproducibility or
fundamentally dependent on the use of equipment repeatability of the analytical technique under ideal
that is within specification, operating correctly, and circumstances might be referred to as precision.
having a sufficient calibration. It is necessary to Ruggedness, or intermediate precision, expresses
validate or revalidate analytical techniques. variability within laboratories, such as on different
Before they are used frequently days or with different analysts or equipment within
Whenever the criteria for which the method has the same laboratory. By testing an adequate number
been approved change of aliquots of a homogeneous sample, one can
Whenever the technique is modified evaluate the precision of an analytical technique by
calculating statistically accurate estimates of the
The following are typical parameters that the
standard deviation or relative standard deviation.
FDA, USP, and ICH recommend.
1. Specificity Accuracy (Recovery): The accuracy of an analytical
2. Range and Linearity technique expresses how closely the value found and
3. Precision the value accepted as either a conventional true value
or an approved reference value agree. Applying the
Technique specificity (Repeatability)
technique to samples that have known dosages of
Intermediate accuracy (Reproducibility)
4. Accuracy (Recovery) analyte added yields the result. To make sure there is
5. Stability of the solution no interference, these should be compared to both
6. Limit of Detection (LOD) standard and blank solutions.
7. Limit of Quantification (LOQ) The accuracy is then computed as a percentage of the
8. Robustness analyte recovered by the assay using the test findings.
9. System Suitability The recovery by the test of known, added amounts of
Specificity: Selectivity of an analytical method is the analyte is a common way to represent it.
capacity to detect an analyte accurately in the Stability of solutions: During validation, the stability
presence of interfering substances, such as synthetic of standards and samples is established under normal
precursors, excipients, enantiomers, and known or circumstances, normal storage circumstances, and
likely degradation products that may be anticipated to occasionally in the instrument to ascertain whether
be present in the sample matrix. special storage circumstances, such as refrigeration or
Range and linearity: The capacity of an analytical protection from light, are required.
technique to produce test results that are inversely Limit of Detection (LOD): The lowest amount of
proportional to the concentration of analyte in the analyte in a sample that can be detected but not
sample is known as linearity. It is important to assess necessarily quantitated as an accurate value is known
a linear relationship over the entire analytical as the limit of detection (LOD) of a specific method.
procedure. By diluting a standard stock solution of The LOD can be predicated on a signal-to-noise (S/N)
the drug product's constituent parts according to the ratio (3:1), which is typically reported as the
suggested process, it is directly proven on the drug concentration of analyte in the sample, in analytical
substance. The confidence limit surrounding the slope techniques that exhibit baseline noise. The signal-to-

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 International Journal of Trend in Scientific Research and Development @ www.ijtsrd.com eISSN: 2456-6470
noise ratio is calculated using the formula: s = H/h, temperature, flow rate, type, and concentration of
where H is the height of the component-specific peak. mobile-phase modifiers can all be altered for the final
The highest noise deviation from the chromatogram optimization. According to ICH criteria, the
of a blank solution's baseline, expressed in absolute optimised method is verified using a variety of factors
terms, is given by the formula h. (such as specificity, precision, accuracy, detection
limit, linearity, etc.).
Limit of Quantification (LOQ): The smallest
amount of analyte in a sample that can be Abbreviations
quantitatively identified with adequate precision and HPLC High Performance Liquid Chromatography
accuracy is known as the limit of quantification ICH International conference on Harmonization
(LOQ), also known as the quantitation limit of a Id Internal Diameter
specific analytical process. The LOQ is typically
estimated from a determination of S/N ratio (10:1) for LC Liquid Chromatography
analytical processes like HPLC that exhibit baseline LOD Limit of Detection
noise, and is typically confirmed by injecting LOQ Limit of Quantitation
standards that yield this S/N ratio and have an m Meter
acceptable percent relative standard deviation as well.
mm Mili meter
Robustness: Robustness is a measure of an analytical MS Mass Spectrometry
method's capacity to stay unaffected by minute but
ODS Octyl decyl silane
intentional changes in method parameters (such as
pH, mobile phase composition, temperature, and RI Refractive index
instrument settings), and it shows how reliable the THF Tetrahydrofuran
method will be under typical conditions. Determining USP United states Pharmacopeia
robustness is a systematic process that involves µm Micron
changing a parameter and evaluating the impact on
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