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A Review: HPLC Method Development and Validation

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International Journal of Analytical and Bioanalytical Chemistry

Universal Research Publications. All rights reserved

ISSN-2231-5012
Review Article
A Review: HPLC Method Development and Validation
Santosh Kumar Bhardwaj a,b *, K. Dwivedia and D. D. Agarwala
a
School of Studies in Chemistry, Jiwaji University, Gwalior, India.
b
Shimadzu Analytical India Pvt Ltd, Delhi, India.
Email: [email protected]
Received 04 November 2015; accepted 20 November 2015
Abstract
HPLC is the dominant separation technique in modern pharmaceutical and biomedical analysis because it results in highly
efficient separations and in most cases provides high detection sensitivity. Most of the drugs in multi component dosage
forms can be analyzed by HPLC method because of the several advantages like rapidity, specificity, accuracy, precision
and ease of automation in this method. HPLC methods development and validation play important roles in new discovery,
development, manufacture of pharmaceutical drugs and various other studies related to humans and animals. An analytical
procedure is developed to test a defined characteristic of the drug substance or drug product against established acceptance
criteria for that characteristic. This review gives information regarding various stages involved in development and
validation of HPLC method. Validation of HPLC method as per ICH Guidelines covers all the performance characteristics
of validation, like Accuracy, precision, specificity, linearity, range and limit of detection, limit of quantification, robustness
and system suitability testing.
© 2015 Universal Research Publications. All rights reserved
Keywords: HPLC, Method development, Validation.

1.0 Introduction: pressure through the column. The principle of separation

High Performance Liquid Chromatography (HPLC) was followed is the adsorption of solute on stationary phase
derived from the classical column chromatography and, is based on its affinity towards stationary phase. (Figure-1)
one of the most important tools of analytical chemistry The technique of HPLC has following features.6
today.1In the modern pharmaceutical industry, high-  High resolution
performance liquid chromatography (HPLC) is the major  Small diameter, Stainless steel, Glass column
and integral analytical tool applied in all stages of drug  Rapid analysis
discovery, development, and production.2 HPLC is the  Relatively higher mobile phase pressure
method of choice for checking peak purity of new chemical  Controlled flow rate of mobile phase
entities, monitoring reaction changes is in synthetic
procedures or scale up, evaluating new formulations and
carrying out quality control / assurance of the final drug
products.3 The Goal of HPLC method is to try & separate,
quantify the main drug, any reaction impurities, all
available synthetic intermediates and any degradants. 4High
Performance Liquid Chromatography is now one of the
most powerful tools in analytical chemistry. It has the
ability to separate, identify, and quantify the compounds
that are present in any sample that can be dissolved in a
liquid. HPLC is the most accurate analytical methods Fig-1: Flow Diagram of HPLC
widely used for the quantitative as well as qualitative
analysis of drug product and used for determining drug 2.0 HPLC Method Development:
product stability. 5 HPLC principle is the solution of sample Methods are developed for new products when no official
is injected into a column of porous material (stationary methods are available. Alternate method for existing (Non-
phase) and liquid phase (mobile phase) is pumped at higher Pharmacopoeial) products are to reduce the cost and time

International Journal of Analytical and Bioanalytical Chemistry 2015; 5(4): 76-81

76
for better precision and ruggedness. When alternate method 2.2 Selection of chromatographic conditions
proposed is intended to replace the existing procedure During initial method development, a set of initial
comparative laboratory data including merit/demerits are conditions (detector, column, mobile phase) is selected to
made available. The goal of the HPLC-method is to try & obtain the first “scouting” chromatograms of the sample. In
separate, quantify the main active drug, any reaction most cases, these are based on reversed-phase separations
impurities, all available synthetic inter-mediates and any on a C18 column with UV detection. A decision on
degradants.7 developing either an isocratic or a gradient method should
Steps involved in Method development are. 6,7 be made at this point.
 Understanding the Physicochemical properties of 2.2.1 Selection of Column:
drug molecule. A column is of course, the starting and central piece of
 Selection of chromatographic conditions. a chromatograph. A appropriately selected column
 Developing the approach of analysis. can produce a good chromatographic separation which
 Sample preparation provides an accurate and reliable analysis. An improperly
 Method optimization used column can often generate confusion, inadequate, and
 Method validation (figure-2) poor separations which can lead to results that are invalid
or complex to interpret.9The heart of a HPLC system is the
column. Changing a column will have the greatest effect on
the resolution of analytes during method development.
Choosing the best column for application requires
consideration of stationary phase chemistry, retention
capacity, particle size, and column dimensions. The three
main components of an HPLC column are the hardware,
the matrix, and the stationary phase. There are several types
of matrices for support of the stationary phase, including
silica, polymers, alumina, and zirconium. Silica is the most
common matrix for HPLC columns. Silica matrices are
robust, easily derivatized, manufactured to consistent
sphere size, and does not tend to compress under pressure.
Silica is chemically stable to most organic solvents and to
low pH systems. One short coming of a silica solid support
is that it will dissolve above pH 7. In recent years, silica
supported columns have been developed for use at high pH.
The nature, shape and particle size of the silica support
effects separation. Smaller particle results in a greater
number of theoretical plates, or increased.The nature of the
stationary phase will determine whether a column can be
used for normal phase or reverse phase chromatography.
Normal phase chromatography utilizes a polar stationary
phase and a non-polar mobile phase. Generally, more polar
compounds elute later than non-polar compounds.
Commonly used reverse phase columns and their uses are
Figure -2: Steps involved in HPLC Method development listed below. Propyl (C3), Butyl (C4), and Pentyl (C5)
phases are useful for ion-pairing chromatography (C4) and
2.1 Understanding the physicochemical properties of peptides with hydrophobic residues, and other large
drug molecules: molecules. C3–C5 columns generally retain non-polar
Physicochemical properties of a drug molecule play an solutes more poorly when compared to C8 or C18 phases.
important role in method development. For Method Examples include Zorbax SB-C3, YMC-Pack C4, and Luna
development one has to study the physical properties like C5. These columns are generally less stable to hydrolysis
solubility, polarity, pKa and pH of the drug molecule. than columns with longer alkyl chains. Octyl (C8, MOS)
Polarity is a physical property of a compound. It helps phases have wide applicability. This phase is less retentive
an analyst, to decide the solvent and composition than the C18 phases, but is still quite useful for
of the mobile phase. 6 The solubility of molecules can be pharmaceuticals, nucleosides, and steroids.10Selection of
explained on the basis of the polarity of molecules. Polar, the stationary phase/column is the first and the most
e.g. water, and nonpolar, e.g. benzene, solvents do not mix. important step in method development. The development of
In general, like dissolves like i.e., materials with similar a rugged and reproducible method is impossible without the
polarity are soluble in each other. Selection of diluents is availability of a stable, high performance column. To avoid
based on the solubility of analyte. The acidity or basicity of problems from irreproducible sample retention during
a substance is defined most typically by the pH value. method development, it is important that columns be stable
Selecting a proper pH for ionizable analytes often leads to and reproducible. The separation selectivity for certain
symmetrical and sharp peaks in HPLC.7 components vary between the columns of different

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77
 Fugire-3 : Method validation parameters

Table 1: Important Information concerning sample composition and properties

Number of Compounds present
Chemical Structures (functionality) of compounds
Molecular weight of compounds
PKa values of compounds
UV Spectra of compounds
Concentration range of compound in sample of interest
Sample Solubility

manufacturer as well as between column production ionized or neutral. Alteration of the mobile-phase pH is one
batches from the same manufacturer. Column dimensions, of the greatest tools in the “chromatographer’s toolbox”
silica substrate properties and bonded stationary phase allowing simultaneous change in retention and selectivity
characteristics are the main ones. The use of silica-based between critical pair of components.12
packing is favored in most of the present HPLC columns  Effect of organic modifier: -Selection of the organic
due to several physical characteristics.6 modifier type is relatively simple in reverse phase
2.2.2 Selection of Chromatographic mode: HPLC, The usual choice is between acetonitrile and
chromatographic modes based on the analyte’s molecular methanol (rarely THF). Gradient elution is usually
weight and polarity. All case studies will focus on reversed- employed with complex multicomponent samples
phase chromatography (RPC), the most common mode for since it may not be possible to get all components
small organic molecules. Ionizable compounds (acids and eluted between k (retention factor) 1 and 10 using a
bases) are often separated by RPC with buffered mobile single solvent strength under isocratic conditions. 12
phases (to keep the analytes in a non-ionized state) or with 2.2.4 Selection of detector and wavelength:
ion-pairing reagents.8 After the chromatographic separation, the analyte of
2.2.3 Optimization of Mobile phase: interest is detected by using suitable detectors. Some
Buffer Selection: Different buffers such as potassium commercial detectors used in LC are: ultraviolet (UV)
phosphate, sodium phosphate and acetate were evaluated detectors, fluorescence detectors, electrochemical detectors,
for system suitability parameters and overall refractive index (RI) detectors and mass spectrometry (MS)
chromatographic performance. detectors. The choice of detector depends on the sample
Effect of pH.:- If analytes are ionisable, the proper mobile- and the purpose of the analysis. In case of multicomponent
phase pH must be chosen based on the analyte pKa so the analysis the absorption spectra may have been shifted to
target analyte is in one predominate ionization state, longer or shorter wavelengths compared to the parent
International Journal of Analytical and Bioanalytical Chemistry 2015; 5(4): 76-81
78
compound. Therefore the UV spectra of target analyte and temperature, sample amounts, injection volume, and
impurities must be taken and overlaid with each other, and diluents solvent type. This is used to find the desired
the spectra should be normalized due to different amounts balance between resolution and analysis time after
present in the mixture. A wavelength must be chosen such satisfactory selectivity has been achieved. The parameters
that adequate response is for most of the analytes can be involved include column dimensions, column-packing
obtained.12,13 particle size and flow rate. These parameters may be
2.3. Developing the approach for analysis: changed without affecting capacity factor or selectivity.10
While developing the analytical method on RP-HPLC the 2.6 Method Validation :
first step which is followed, the selections of various Validation of an analytical method is the process by which
chromatographic parameters like selection of mobile phase, it is established by laboratory studies, that the performance
selection of column, selection of flow rate of mobile phase, characteristics of the method meet the requirements for the
selection of pH of mobile phase. All of these parameters intended analytical application. Validation is required for
are selected on the basis of trials and followed by any new or amended method to ensure that it is capable of
considering the system suitability parameters. Typical giving reproducible and reliable results, when used by
parameters of system suitability are e.g. retention time different operators employing the same equipment in the
should be more than 5 min, the theoretical plates should be same or different laboratories. The type of validation
more than 2000, the tailing factor should be less than 2, program required depends entirely on the particular method
resolution between 2 peaks should be more than 5, % and its proposed applications.13Results from method
R.S.D. of the area of analyte peaks in standard validation can be used to judge the quality, reliability and
chromatograms should not be more than 2.0 %.like other. consistency of analytical results; it is an integral part of any
Detection wavelength is usually isobestic point in the case good analytical practice. Use of equipment that is within
of simultaneous estimation of 2 components.6 specification, working correctly and adequately calibrated
2.4 Sample preparation: is fundamental to the method validation process. Analytical
Sample preparation is a critical step of method methods need to be validated or revalidated.16
development that the analyst must investigate. For • Before their introduction into routine use;
example, the analyst should investigate if centrifugation • Whenever the conditions change for which the method
(determining the optimal rpm and time) shaking and/or has been validated
filtration of the sample is needed, especially if there are • Whenever the method is changed
insoluble components in the sample. The objective is to Typical parameters recommended by FDA, USP, and ICH
demonstrate that the sample filtration does not affect the are as follow.16,18
analytical result due to adsorption and/or extraction of 1. Specificity
leachable. The effectiveness of the syringe filters is largely 2. Linearity & Range
determined by their ability to remove 3. Precision
contaminants/insoluble components without leaching  Method precision (Repeatability)
undesirable artifacts (i.e., extractable) into the filtrate. The  Intermediate precision (Reproducibility)
sample preparation procedure should be adequately 4. Accuracy (Recovery)
described in the respective analytical method that is applied 5. Solution stability
to a real in-process sample or a dosage form for subsequent 6. Limit of Detection (LOD)
HPLC analysis. The analytical procedure must specify the 7. Limit of Quantification (LOQ)
manufacturer, type of filter, and pore size of the filter 8. Robustness
media. 12The purpose of sample preparation is to create a 9. System suitability
processed sample that leads to better analytical results Specificity : selectivity of an analytical method as its
compared with the initial sample. The prepared sample ability to measure accurately an analyte in the presence of
should be an aliquot relatively free of interferences interference, such as synthetic precursors, excipients,
that is compatible with the HPLC method and that will not enantiomers, and known (or likely) degradation products
damage the column.13,15 that may be expected to be present in the sample matrix. 17
2.5 Method optimization : Linearity and range: The linearity of an analytical
Most of the optimization of HPLC method development procedure is its ability (within a given range) to obtain test
has been focused on the optimization of HPLC results, which are directly proportional to the concentration
conditions.14 The mobile phase and stationary phase of analyte in the sample. A linear relationship should be
compositions need to be taken into account. Optimization evaluated across the range of the analytical procedure. It is
of mobile phase parameters is always considered first as demonstrated directly on the drug substance by dilution of
this is much easier and convenient than stationary phase a standard stock solution of the drug product components,
optimization. To minimize the number of trial using the proposed procedure. Linearity is usually
chromatograms involved, only the parameters that are expressed as the confidence limit around the slope of the
likely to have a significant effect on selectivity in the regression line.16-18 For the establishment of linearity,
optimization must be examined. Primary control variables minimum of five concentrations are recommended by ICH
in the optimization of liquid chromatography (LC) methods guideline.19 The range of an analytical method is the
are the different components of the mobile phase interval between the upper and lower levels that have been
determining acidity, solvent, gradient, flow rate, demonstrated to be determined with precision, accuracy

International Journal of Analytical and Bioanalytical Chemistry 2015; 5(4): 76-81

79
and linearity using the method.17 Robustness: is defined as the measure of the ability of an
Precision: The precision of an analytical procedure analytical method to remain unaffected by small but
expresses the closeness of agreement (degree of scatter) deliberate variations in method parameters (e.g. pH, mobile
between a series of measurements obtained from multiple phase composition, temperature and instrumental settings)
sampling of the same homogeneous sample under the and provides an indication of its reliability during normal
prescribed conditions. Precision may be considered at three usage. Determination of robustness is a systematic process
levels: repeatability, intermediate precision and of varying a parameter and measuring the effect on the
reproducibility.19The precision of an analytical procedure is method by monitoring system suitability and/or the analysis
usually expressed as the standard deviation or relative of samples.18,19
standard deviation of series of measurements. Precision System Suitability:System suitability tests are an integral
may be either the degree of reproducibility or of the part of liquid chromatographic methods. They are used to
repeatability of the analytical procedure under normal verify that the detection sensitivity, resolution and
conditions. Intermediate precision (also known as reproducibility of the chromatographic system are adequate
ruggedness) expresses within laboratories variations, as on for the analysis to be done. The tests are based on the
different days, or with different analysts or equipment concept that the equipment, electronics, analytical
within same laboratory. Precision of an analytical operations and samples to be analyzed constitute an integral
procedure is determined by assaying a sufficient number of system that can be evaluated as such. Factors, such as the
aliquots of a homogeneous sample to be able to calculate peak resolution, number of theoretical plates, peak tailing
statistically valid estimates of standard deviation or relative and capacity have been measured to determine the
standard deviation.20 suitability of the used method.16-20
Accuracy (Recovery):The accuracy of an analytical 3. Conclusion
procedure expresses the closeness of agreement between In recent years development of the analytical methods for
the value which is accepted either as a conventional true identification, purity evaluation and quantification of drugs
value or an accepted reference value and the value found. lt has received a great deal of attention in the field of
is determined by applying the method to samples to which pharmaceutical analysis.This review describes HPLC
known amounts of analyte have been added. These should method development and validation in general way. A
be analysed against standard and blank solutions to ensure general and very simple approach for the HPLC method
that no interference exists. The accuracy is then calculated development for the separation of compounds was
from the test results as a percentage of the analyte discussed. Knowledge of the physiochemical properties of
recovered by the assay. It may often be expressed as the the primary compound is of utmost importance prior to the
recovery by the assay of known, added amounts of any HPLC method development. The selection of buffer
analyte.18,19 and mobile phase composition (organic and pH) plays a
Solution stability: During validation the stability of dramatic role on the separation selectivity. Final
standards and samples is established under normal optimization can be performed by changing the gradient
conditions, normal storage conditions, and sometimes in slope, temperature and flow rate as well as the type and
the instrument to determine if special storage conditions are concentration of mobile-phase modifiers. Optimized
necessary, for instance, refrigeration or protection from method is validated with various parameters (e.g.
light.18 specificity, precision, accuracy, detection limit, linearity,
Limit of Detection (LOD) : Limit of detection (LOD) of etc.) as per ICH guidelines.
an individual procedure is the lowest amount of analyte in a Abbreviations
sample that can be detected but not necessarily quantitated HPLC High Performance Liquid Chromatography
as an exact value. In analytical procedures that exhibit ICH International conference on Harmonization
baseline noise, the LOD can be based on a signal-to-noise Id Internal Diameter
(S/N) ratio (3:1), which is usually expressed as the LC Liquid Chromatography
concentration of analyte in the sample. The signal-to-noise LOD Limit of Detection
ratio is determined by: s = H/h Where H = height of the LOQ Limit of Quantitation
peak corresponding to the component. h = absolute value m Meter
of the largest noise fluctuation from the baseline of the mm Mili meter
chromatogram of a blank solution.18-20 MS Mass Spectrometry
Limit of Quantification (LOQ) : The limit of Quantitation ODS Octyl decyl silane
(LOQ) or Quantitation limit of an individual analytical RI Refractive index
procedure is the lowest amount of analyte in a sample that THF Tetrahydrofuran
can be quantitatively determined with suitable precision USP United states Pharmacopeia
and accuracy. For analytical procedures such as HPLC that μm Micron
exhibit baseline noise, the LOQ is generally estimated from References:
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Source of support: Nil; Conflict of interest: None declared

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