J Pharm Educ Res Vol. 4, Issue No.

1, June 2013

HPLC method development and validation- an overview

Ranjit Singh
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014
E-mail: [email protected]

Received: May 14, 2013; Accepted: May 22, 2013

ABSTRACT
HPLC method development and validation play important role in the discovery, development and manufacture of
pharmaceutical products. This article mainly focuses on the optimization of HPLC conditions and other important
perspectives during method development and validation. Various critical steps related to analytical method
development and validation is discussed. A sequence of events required for method development and analytical
validation are described. The steps involved in developing a stability-indicating HPLC method influences the
analysis of degradation products/impurities in stability study and its validation demonstrate the suitability for its
intended purpose.

Key words: Method validation, Method development, International conference on Harmonization (ICH), High Pressure
Liquid Chromatography (HPLC),

INTRODUCTION
Analytic method development and validation are key
elements of any pharmaceutical development program.
HPLC analysis method is developed to identify, quantity
or purifying compounds of interest. This technical brief
will focus on development and validation activities as
applied to drug products. Various steps for HPLC method
development are given in Fig.1.
Effective method development ensures that laboratory
resources are optimized, while methods meet the
objectives required at each stage of drug development.
Method validation, required by regulatory agencies at
certain stages of the drug approval process, is defined as
the “process of demonstrating that analytical procedures
are suitable for their intended use” 1,2.
To know information concerning your compound or
analyte is worth. Understand its physical and chemical
Fig.1. Steps involved in HPLC method development
characteristics allow you to select the most appropriate
HPCL method development from your vast literature.
it’s organic soluble or water soluble, as this helps to select
Information concerning sample, for example, molecular
the best mobile phase and column to be used in your
mass, structure and functionality, pKa values and UV
HPLC Method development.
spectra, solubility of compound(s) should be compiled.
The requirement of removal of insoluble impurities Various detectors include: UV/Visible, photodiode
by filtration, centrifugation, dilution or concentration array detector, fluorescence detector, conductivity detector,
to control the concentration, extraction (liquid or solid refractive index detector, electrochemical detector, Mass
phase), derivatization for detection etc. should be checked. spectrometer detector, evaporative light scattering detector.
For pure compound, determine sample solubility whether UV-Vis detectors are typical in many laboratories as they
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can detect a wide array of compounds organic, pH, flow rate, temperature, wavelength, and
column age) and standardization (integration, wavelength,
Analytic methods are intended to establish the
standard concentration, and response factor correction).
identity, purity, physical characteristics and potency of
During the preliminary method development stage, all
the drugs that we use. Methods are developed to support
individual components should be investigated before
drug testing against specifications during manufacturing
the final method optimization. This gives the scientist
and quality release operations, as well as during long-
a chance to critically evaluate the method performance
term stability studies. Methods may also support safety
in each component and streamline the final method
and characterization studies or evaluations of drug
optimization 8. The percentage of time spent on each
performance. Once a stability-indicating method is in
stage is proposed to ensure the scientist will allocate
place, the formulated drug product can then be subjected
sufficient time to different steps. In this approach, the
to heat and light in order to evaluate potential degradation
three critical components for a HPLC method (sample
of the API in the presence of formulation excipients 3,4.
preparation, HPLC analysis and standardization) will first
The validation of an analytic method demonstrates the be investigated individually 9-11.
scientific soundness of the measurement or characterization.
The degraded drug samples obtained are subjected
It is required to varying extents throughout the regulatory
to preliminary chromatographic separation to study the
submission process. The validation practice demonstrates
number and types of degradation products formed under
that an analytic method measures the correct substance,
various conditions 12. Scouting experiments are run
in the correct amount, and in the appropriate range for the
and then conditions are chosen for further optimization
intended samples. It allows the analyst to understand the 13
. Resolving power, specificity, and speed are key
behavior of the method and to establish the performance
chromatographic method attributes to keep in mind during
limits of the method 5,6. The goal is to identify the critical
method development 14. Selectivity can be manipulated by
parameters and to establish acceptance criteria for method
combination of different factors like solvent composition,
system suitability 7. The HPLC system with its different
type of stationary phase, mobile phase, buffers and pH.
components is shown in Figure 2.
Changing solvents and stationary phases are the most
comfortable approaches to achieve the separation. The
proper range of pH is an important tool for separation of
ionizable compounds. Acidic compounds are retained
at low pH while basic compounds are more retained at
higher pH. The neutral compounds remain unaffected. The
pH range 4-8 is not generally employed because slight
change in pH in this range would result in a dramatic shift
in retention (up-slope or down-slope of curve) (Figure 3).
However, by operating at pH extremes (2-4 or 8-10), not
only is there a 10-30 fold difference in retention that can

Fig 2: HPLC system with its different components

METHOD DEVELOPMENT
The three critical components for a HPLC method are:
sample preparation (% organic, pH, shaking/sonication,
Figure 3: Reversed-phase retention behavior as pH is varied
sample size, sample age), HPLC analysis conditions (%
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be exploited in method development but also the method mechanically stable, and have a porous structure similar
can be made more robust which is a desirable outcome to that of silica. However, zirconia’s main advantage over
with validation in min 15,16. silica is that it is very stable in a wide range of eluent pH;
indeed the ZirChrom®-EZ and ZirChrom®-MS phases
Method development within the different pH range
are stable over the pH range of 1-10.
from 1 to 12 for better chromatographic resolution between
two or more peak of an analyte depends upon three main Separation of many samples can be enhanced by
factors that are -Column efficiency, Selectivity, Retention selecting the right column temperature. Higher column
time. The ionizable analytes are either bases or acids and temperature reduces system backpressure by decreasing
it effects above three factors dramatically with change in mobile phase viscosity, which in turn allows use of longer
pH. Retention time can be improved by changing the pH columns with higher separation efficiency. However, an
that will lead to easy separation of ionizable analytes from overall loss of resolution between mixture components in
non-ionized form. By changing the mobile phase pH can many samples occurs by increasing column temperature
also improve column efficiency because it altered both the .The optimum temperature is dependent upon nature of
ionization of the analyte and the residual silanols and it the mixture components. The overall separation can be
also minimizes secondary interactions between analytes improved by simultaneous changes in column temperature
and the silica surface that will lead to poor peak shape. To and mobile phase composition 19-21. Recently, normal phase
achieve optimum resolution, it require change in the pH HPLC is back popular with the birth of HILIC technology
of mobile phase. Method development can proceeds by that proved to improve reproducibility in separating
investigating parameters of chromatographic separations polar and hydrophilic compounds such as peptides,
first at low pH and then at higher pH until optimum results carbohydrate, vitamins, polar drugs and metabolites.
are achieved 17. In order to develop a HPLC method effectively, most
of the effort should be spent in method development
Mobile phase composition (or solvent strength) plays
and optimization as that will improve the final method
an important role in RP-HPLC separation. Acetonitrile
performance 22,23. Method validation is important to
(ACN), methanol (MeOH) and tetrahydrofuran (THF)
complete method development.
are commonly used solvents in RP-HPLC having low
UV cut-off of 190, 205 and 212nm respectively. These METHOD VALIDATION
solvents are miscible with water. Mixture of acetonitrile
and water is the best initial choice for the mobile phase Validation is defined by the International Organization
during method development. An HPLC column packed for Standardization (ISO) as “verification, where the
with stationary phase of C18-bonded silica (C18 Column) specified requirements are adequate for an intended use”,
and C8-bonded silica (C8 Column) is used in RP-HPLC where the term verification is defined as “provision of
separation of a wide range of organic compounds. The objective evidence that a given item fulfills specified
separation selectivity for certain components vary between requirements” 24.
the columns of different manufacturer as well as between The applicability and scope of an analytical method
column production batches from the same manufacturer. should be defined before starting the validation process.
Column dimensions, silica substrate properties and bonded It includes defining the analytes, concentration range,
stationary phase characteristics are the main ones. The use description of equipment and procedures, validation level
of silica-based packing is favoured in most of the present and criteria required. The validated range is defined by
HPLC columns due to several physical characteristics. IUPAC as “the interval of analyte concentration within
Silica substrates are available in spherical or irregular which the method can be regarded as validated” 25,26. This
shapes and can be prepared with different surface areas, range does not have to be the highest and lowest possible
pore sizes and particle sizes, which make them suitable levels of the analyte that can be determined by the method.
for most HPLC applications. Totally porous silica particles Instead, it is defined on the basis of the intended purpose
with 5 µm diameter provide the desired characteristics for of the method 27,28. The method can be validated for use
most HPLC separations 14,16,18. Zirconia-based columns as a screening (qualitative), semi-quantitative (e.g. 5-10
are revolutionary HPLC phases. Zirconia particles are
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ppm) or quantitative method. It can also be validated the two graphical evaluations using HPLC is shown in
for use on single equipment, different equipments in the Figure 4 where Rc = Line of constant response 36,37.
laboratory, different laboratories or even for international
use at different climatic and environmental conditions.
The criteria of each type of validation will of course
be different with the validation level required 24,29. The
various validation parameters include linearity, accuracy,
precision, ruggedness, robustness, LOD, LOQ and
selectivity or specificity.
Linearity
The linearity of an analytical procedure is its ability
(within a given range) to obtain test results that are directly
proportional to the concentration of analyte in the sample
30,31
. It is essential to determine the useful range at which
the instrumental response is proportional to the analyte
concentration. Generally, a value of co-relation coefficient
(r2) > 0.998 is considered as evidence of an acceptable
fit of the data to the regression line 32. Significance of
deviation of intercept of calibration line from the origin
Figure 4: Graphical presentations of linearity plot of caffeine
value of zero can be evaluated statistically by determining sample using HPLC
confidence limits for the intercept, generally at the 95 % Accuracy
level 33,34.
Accuracy is defined by ISO as “closeness of
Linearity is determined by a series of three to six agreement between a measured quantity value and a
injections of five or more standards. Peak areas (or true quantity value of a measurand”. It is a qualitative
heights) of the calibration standards are usually plotted characteristic that cannot be expressed as a numerical
in the Y-axis against the nominal standard concentration, value. It has an inverse relation to both random and
and the linearity of the plotted curve is evaluated through systematic errors, where higher accuracy means lower
the value of the co-relation coefficient (r2). Because errors 33,38. Accuracy is evaluated by analyzing test drug at
deviations from linearity are sometimes difficult to detect, different concentration levels. Typically, known amounts
two additional graphical procedures can be used. The of related substances and the drug substance in placebo
first one is to plot deviations from regression line versus are spiked to prepare an accuracy sample of known
concentration or versus logarithm of concentration. For concentration of related substance. Samples are prepared
linear ranges, the deviations should be equally distributed in triplicate. ICH recommends accuracy evaluation using
between positive and negative values. Another approach a minimum of nine determinations over a minimum of
is to divide signal data by their respective concentrations three concentration levels covering the range specified.
yielding the relative responses 35. It is determined by comparing the found concentration
A graph is plotted with the relative responses on with the added concentration. The methods of determining
Y-axis and the corresponding concentrations on X-axis on accuracy include analysis of analysis of an analyte of
a log scale. The obtained line should be horizontal over known purity (i.e., reference material), comparisons of
the full linear range. At higher concentrations, there will results of the proposed analytical procedure with those
typically be a negative deviation from linearity. Parallel of a second well-characterized procedure and standard
horizontal lines are drawn in the graph corresponding to, addition method. The accuracy may also be inferred once
for example, 95 % and 105 % of the horizontal line. The precision, linearity and specificity have been established
method is linear up to the point where the plotted relative
39,40
.
response line intersects the 95 % line. A comparison of
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Precision Selectivity and Specificity

It expresses closeness of agreement (degree of scatter) Selectivity and specificity are sometimes used
between a series of measurements obtained from multiple interchangeably to describe the same concept in method
sampling of the same homogeneous sample under the validation. Selectivity of an analytical method is defined
prescribed conditions. Precision may be considered at by the ISO as “property of a measuring system, used with
three levels: repeatability, intermediate precision and a specified measurement procedure, whereby it provides
reproducibility 29,41. Repeatability is also referred to as measured quantity values for one or more measurands
intra-assay precision. It is a measure of precision of such that the values of each measurand are independent of
analysis in one laboratory by one operator using one other measurands or other quantities in the phenomenon,
piece of equipment over a relatively short time-span. body, or substance being investigated” 24. Specificity is the
It is degree of agreement of results when experimental ability to assess unequivocally the analyte in the presence
conditions are maintained as constant as possible, and of components that may be expected to be present. The
expressed as RSD of replicate. ICH recommends a specificity of a test method is determined by comparing test
minimum of nine determinations covering the specified results from an analysis of samples containing impurities,
range for the procedure (e.g., three concentrations/three degradation products, or placebo ingredients with those
replicates as in the accuracy experiment), or a minimum obtained from an analysis of samples without impurities,
of six determinations at 100% of the test concentration degradation products, or placebo ingredients. Specificity
for evaluation of repeatability which should be reported can best be demonstrated by resolution between the analyte
as standard deviation, relative standard deviation peak and the other closely eluting peak(s) 29,44.
(coefficient of variation) or confidence interval. ICH Detection limit (LOD) and Quantitation limit (LOQ)
defines intermediate precision as long-term variability of
the measurement process and is determined by comparing LOD of an analytical procedure is the lowest
the results of a method run within a single laboratory concentration of an analyte in a sample which can be
over a number of weeks. It is also called as interday detected but not necessarily quantitated as an exact value
precision 40. It reflects discrepancies in results obtained where as LOQ is the lowest amount of analyte in a sample
by different operators, from different instruments, with which can be quantitatively determined with suitable
standards and reagents from different suppliers, with precision and accuracy. ICH guidelines describe three
columns from different batches or a combination of these methods for determining LOD and LOQ that include:
but in the same laboratory 29. The objective of intermediate Visual evaluation
precision validation is to verify that the method will It may be used for both non instrumental and
provide same results in the same laboratory once the instrumental methods. The LOD and LOQ is determined
development phase is over. Reproducibility expresses by analysis of samples with known concentrations of
precision of analysis of the same sample by different analyte and by establishing the minimum level at which
analysts in different laboratories using operational and the analyte can be reliably detected or quantified with
environmental conditions that may differ but are still acceptable accuracy and precision respectively.
within the specified parameters of the method 24,42. The
objective is to verify that the method will provide the
same results despite differences in room temperature
and humidity, variedly experienced operators, different
characteristics of equipments (e.g., delay volume of an
HPLC system), variations in material and instrument
conditions (e.g. in HPLC, mobile phase composition, pH,
flow rate of mobile phase), equipments and consumables
of different ages, columns from different suppliers or
different batches and solvents, reagents and other material Figure 5: Limit of detection and limit of quantitation via signal to
with different quality 29,43. noise ratio (S/N)

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S/N Ratio approach impurities known to be unusually potent or to produce

This method can only be applied to analytical toxic or unexpected pharmacological effects 40.
procedures which exhibit baseline noise. It is determined
by comparing measured signals from samples with known
low concentrations of analyte with those of blank samples
and establishing minimum concentration at which the
analyte can be reliably detected. A S/N ratio of 3:1 is
considered acceptable for estimating LOD (with Relative
Standard Deviation (RSD) ≤ 10%) whereas for whereas
for LOQ S/N ratio of 10:1 is considered appropriate (with
Relative Standard Deviation (RSD) ≤ 3%) described in
Figure 5.
Standard deviation of the response and slope
Fig. 6: Range
The LOD and LOQ may be expressed as:
Robustness
LOD = 3.3 x σ/S and LOD = 10 x σ/S
The robustness of an analytical procedure is a measure
where σ = the standard deviation of the response, S = the
of its capacity to remain unaffected by small, but deliberate
slope of the calibration curve of analyte. The slope S may
variations in method parameters and provides an indication
be estimated from the calibration curve of the analyte.
of its reliability during normal usage. For determination
The value of σ may be taken from as standard deviation
of method robustness, a number of chromatographic
of analytical background responses of an appropriate
parameters, for example, flow rate, column temperature,
number of blank samples.
injection volume, detection wavelength or mobile phase
Alternatively, it can be taken as residual standard composition are varied within a realistic range and
deviation of a regression line or standard deviation of quantitative influence of the variables is determined. If
y-intercepts if regression lines obtained after samples influence of the parameter is within a previously specified
containing an analyte in the range of LOD and LOQ 40. tolerance, the parameter is said to be within the method’s
Range robustness range. Obtaining data on these effects will
allow to judge whether a method needs to be revalidated
The range of an analytical procedure is the interval when one or more of parameters are changed, for example
between the upper and lower concentration of analyte in to compensate for column performance over time 46,47.
the sample (including these concentrations) for which Variation in method conditions for robustness should be
it has been demonstrated that the analytical procedure small and reflect typical day-to-day variation. Critical
has a suitable level of precision, accuracy and linearity parameters are identified during the method development
(Figure 6) 45. It is established by confirming that the process. Only these critical method parameters should
analytical procedure provides an acceptable degree of be investigated for robustness. Common critical method
linearity, accuracy and precision when applied to samples parameters can be divided into two categories. The HPLC
containing amounts of analyte within or at the extremes of conditions include HPLC column (lot, age, and brand),
the specified range of the analytical procedure. The range Mobile-phase composition (pH ± 0.05 unit, organic content
of an analytical method varies with its intended purpose. ± 2%) and HPLC instrument (dwell volume, detection
It is generally 80-120% of the test concentration for assay wavelength ± 2 nm, column temperature ± 5°C, flow
of a drug substance or a finished (drug) product, 70-130% rate). The sample preparation variations include sample
of the test concentration for content uniformity, ±20% solvent (pH ± 0.05 unit, organic content ± 2%), sample
over the specified range for dissolution testing, reporting preparation procedure (shaking time, different membrane
level of an impurity to 120% of the specification for filters) and HPLC solution stability. The variations in
determination of an impurity and it should commensurate chromatographic parameters for robustness study are
with LOD or LOQ (the control level of impurities), for given in the Table 29, 40.
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J Pharm Educ Res Vol. 4, Issue No. 1, June 2013

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