Pharmaceutical Chemistry Journal

Vol. 38, No. 4, 2004

STRUCTURE OF CHEMICAL COMPOUNDS,

METHODS OF ANALYSIS AND PROCESS CONTROL

VALIDATION OF HPLC TECHNIQUES

FOR PHARMACEUTICAL ANALYSIS

N. A. �pshtein1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 38, No. 4, pp. 40 � 56,
April, 2004.

Original article submitted June 18, 2002.

Validation (evaluation of suitability) of an analytical

technique is a procedure aimed at obtaining experimentally
justified evidence of the ability of this technique to give results
characterized by the required accuracy and precision
[1 � 7].2 All analytical techniques used for the development
of pharmaceuticals and for the determination of their quality
characteristics have to be validated. In the case of using
methods stipulated and described in the State Pharmacopoeia,
it is not necessary to evaluate their suitability, provided
that the analyses are conducted with strict observation
of the text of each particular article. In most other cases, especially
in cases of modification of the drug composition, the
scheme of synthesis, or the analytical procedure, it is necessary
to re-evaluate the suitability of the analytical techniques.

This paper is aimed at (i) considering the peculiarities of

validation of HPLC techniques for pharmaceutical analysis,

(ii) critically assessing the main approaches to evaluation of

the validation characteristics, and (iii) providing practical
recommendations and criteria for finding correct solutions.
The USSR State Pharmacopoeia (valid in the Russian
Federation) introduced the section �Statistical Analysis of
Biological Test Results� in 1968 (Xth Ed.) and the section �
Statistical Processing of Chemical Experimental Data� in
1988 (XIth Ed.). These sections are devoted to problems involved
in the metrological attestation of analytical techniques.

In 1987, the United States Food and Drug Administration

(FDA) issued practical guides on the main principles of vali

1
�Akrikhin� Chemico-Pharmaceutical Joint-Stock Company, Staraya
Kupavna, Moscow Region, Russia.

2
Here and below the terms �accuracy� and �precision� (repeatability,
reproducibility) are treated in accordance with the State Standard GOST R
ISO 5725�1�2002. Previously, accuracy had the meaning of correctness,
but now correctness is described in terms of �trueness.� In justifying the
suitability of techniques related to the qualitative determination of substances,
statistical processing of the experimental results is obligatory [8].

dation [5] and on the presentation of samples and analytical

data pertaining to the validation of methods [6]. In 1993, the
International Conference on Harmonization (ICH) developed
generalized recommendations on the validation of analytical
procedures; these documents were published in 1994 and
treated in more detail in 1995 [1 � 3]. In 1994, the US FDA
Center for Drug Evaluation and Research (CDER) issued a
guide on the validation of chromatographic methods [4].
These documents and some review papers and monographs
[9 � 13] provided a basis for extensive implementation of the
procedure of validation of analytical methods. The Internet
offers the Laboratory Guide to Method Validation and Related
Topics at http://www.eurachem.ul.pt /. In recent years,
new guides have become available from CDER [15], Waters
Company [16], and Labcompliance [17 � 19]. Special sections
are devoted to these problems in national and international
pharmacopoeias and guides on the validation of analytical
procedures [7, 20 � 22]. Also available are computer
program packages, such as ELISA Method Validation Templates
(Waters) and LaChrom 2000 Validation Manager
(Merck) representing electronic tables with incorporated
functions of statistical processing of the results of measurements
and issuing validation certificates, and monographs on
the related subjects [23 � 34].

Tables 1 and 2 summarize the most recent recommendations

concerning selection of the validation characteristics
depending on the type of analytical procedures. A comparative
analysis of these data shows that, according to the
United States Pharmacopoeia (USP-26), methods of dissolution
testing have to be validated only with respect to precision
(repeatability, reproducibility), while the other characteristics
�may require validation, depending on the specific
test nature.� In contrast, according to the FDA CDER guidelines
[15], procedures used for quantitative analysis and dissolution
testing need the same validation characteristics.

212

0091-150X/04/3804-0212 � 2004 Plenum Publishing Corporation

Validation of HPLC Techniques for Pharmaceutical Analysis

Moreover, according to CDER [15], all methods of quantitative

analysis have to be characterized with respect to robustness
(see below). Robustness is not included in USP-26 [7]
because this characteristic has to be studied at the stage of
development of an analytical procedure, rather than in the
course of validation.

Since any analytical situation poses a multifactor problem,

validation has to include at least testing of the analytical
system as a whole under the conditions stipulated by the description.
3 The second task is evaluation of the stability (robustness)
of the analytical system with respect to small variations
of the main factors (for example, the ratio of the mobile
phase components, pH, temperature, etc.). This problem is
less important than the first one because it is usually solved
at the stage of development of a given analytical procedure.
For this reason, problems pertaining to robustness are only
briefly mentioned in this review.

Let us consider the main stages of validation of the

HPLC techniques used in pharmacy.

1. TESTING THE ANALYTICAL PROCEDURE AS A

UNIFIED SYSTEM UNDER THE CONDITIONS
STIPULATED IN THE DESCRIPTION
Figure 1 shows the general scheme of evaluation of the
suitability of an analytical procedure, which takes into account
specific features of HPLC. As can be seen, testing an
analytical procedure as a whole in the general case allows the
following validation characteristics to be determined [1 � 7]:

(i) specificity; (ii) precision; (iii) linearity; (iv) accuracy; (v)

suitability range; (vi) limit of detection; (vii) limit of
quantitation; and (viii) stability of solutions.4 Depending on
a particular type of the analytical procedure (HPLC technique)
only a part rather than all of the above characteristics
may be required (see Tables 1 and 2).
1.1.
Confirming
the
Specificity
of
a
Given
Analytical
Procedure
(Separating
Power
of
a
Chromatographic
System)
By the specificity of a system is meant its ability to detect
a given substance unambiguously (reliably) in the presence
of other components (including impurities) that may be
present in the samples [1 � 4]. The proof of specificity depends
on the task of a given procedure and on the availability
of reference samples of the main impurities.

1.1.1. The specificity of procedures aimed at determination

of the content of a parent substance, the parameters
of solubility, and the homogeneity of dosage. In order
to confirm the specificity of these procedures, it is usually required
that peaks of the substances to be determined are sufficiently
well resolved between themselves and from peaks
of the main impurities, the system components (e.g., of the
sample solvent), and the placebo. For this purpose, the sepa3

According to this concept, instrumentation, electronics, analytical procedures,

and analyzed samples constitute a unified analytical system, which
can be considered as a whole [7].

Sometimes, the stability of phases and solutions is considered within the

framework of the problem of robustness.

Specificity (Section 1.1)

2.
Precision (Section 1.2)
2.1
Repeatability (Section 1.2.1)
2.2
Intermediate precision
(Section 1.2.2)
Recommended requirements
to the repeatability of sample
injections (Section 3.3)

2.3Reproducibility. Checked
in special cases
(Section 1.2.3)
3.Linearity (Section 1.3)
4.Accuracy (Section 1.4)
5.Suitability range (Section 1.5)
6.Limit of detection (Section 1.6)
7.Limit of quantitation
(Section 1.7)
8.Stability of solutions
(Section 1.8.)
It is expedient to determine
these validation characteristics
in the course of proof of the
analytical system accuracy
(using statistical parameters
determined for the calibration
graph) (Sections 1.3; 1.4;
1.6.2; and 1.7.2)

9.
Robustness (stability) of HPLC
procedures with respect to small
Criteria of suitability of a given

variations in the main system

chromatographic system
factors (ratio of the mobile

(Section 3)

phase components, pH,

temperature, etc.). Robustness
is usually studied in the stage
of development of the given
HPLC tecnique (Section 2)

Fig. 1. The general scheme of validation of HPLC-based analytical

procedures.

ration of peaks is confirmed by a set of chromatograms, at

least of (a) the test solution, (b) the reference parent substance
solution, (c) the solvent (blank), (d) the placebo (for
filled drugs), and (e) the solution used for the evaluation of
suitability of the chromatographic system. The degree of
peak separation is usually described in terms of the separation
coefficient R
. Recommended values of this coefficient

are given below in the Section 3.3.

It is recommended to confirm the specificity by investigation

of the �purity� of peaks of the parent substance to be
determined [4, 27]. This test is usually performed using a diode
matrix detector and a special program evaluating spectral
homogeneity of the measured peak (e.g., Peak Purity
Millennium, Waters). The principle of evaluation of the peak
purity is as follows. The sample chromatograms are mea

sured at various detector wavelengths .

(numbered n
). Each
point of the peak is characterized by a spectrum, which is
mathematically described by a vector in the n-dimensional
space of the values of absorption (in absorption units, AU) at
the preset n
wavelengths (the length of this vector is proportional
to the substance concentration in the solution studied).
The difference between the spectra is evaluated by the angle
between the corresponding vectors (called the spectral con
TABLE 1. Validation Characteristics according to th United States
Pharmacopoeia (2003)

Category

Characteristic
II
I Quantita-Limiting III IV
tive tests tests
Accuracy Yes Yes MB MB No
Precision Yes Yes No Yes No
Specificity Yes Yes Yes MB Yes
Limit of detection No No Yes MB No
Limit of quantitation No Yes No MB No
Linearity Yes Yes No MB No
Suitability range Yes Yes MB MB No

Notes: Yes = usually studied; No = usually not studied; MB = may

be required (depending on a specific test nature). Category I includes
analytical methods intended for determination of the content
of the main component in parent substances or ready-to-use medicinal
forms; category II includes methods of determination of impurities
and decomposition products; category III includes methods of
determination of the parameters of dissolution, drug release, etc.;
category IV includes identification tests; the terms �accuracy� and
�precision� (repeatability, reproducibility) are treated in accordance
with the State Standard GOST R ISO 5725�1-2002.

trast angle #). If .

= 0, the spectra are considered similar (homogeneous).
This implies that the value of absorption in one
spectrum measured at a given wavelength .
can be obtained
from the value in another spectrum measured at the same
wavelength by multiplying by a certain constant factor. In order
to evaluate the spectral homogeneity of a chromatographic
peak, the spectral contrast angles .
are calculated for
all points of this peak relative to the angle at the peak maximum
and then the maximum value .
p (purity angle) is determined.

Two spectra determined for the same substance can differ,

for example, because of the influence of the baseline
noise. In order to take this into account, a threshold spectral
angle #
th is determined (i) by determining the maximum
spectral angle between pints of the baseline with maximum
noise levels or (ii) by taking six chromatograms of a standard
sample solution, determining the maximum purity angle .
p
for each chromatogram, and considering the maximum of
these values as the threshold spectral angle #
th. This threshold
angle characterizes the level below which the difference
between two spectra can be considered insignificant. The obtained
.
values are compared to #. For .
< #, the peak is

p
thpth

considered spectrally homogeneous; otherwise the peak is

influenced by the presence (i.e., additional absorption) of another
substance. In practice, it is possible to use a simplified
procedure, whereby the chromatograms of the same peak are
measured at two or three wavelengths and the
chromatograms are checked for the proportionality (see
above) at all points. The latter method is less reliable, be-

N. A. �pshtein
TABLE 2. Validation Characteristics Recommended for Various
Tests by the United States FDA (CDER and CBER) [15]

Type of analytical procedure

Tests for impurities

Qualitative
determina-

Chatacteristic

Identifica

tion and dis

tion

quantitative limiting

solution
tests

Accuracy No Yes No Yes

Repeatability No Yes No Yes
Intermediate precision No Yes1 No Yes1
Specificity Yes2 Yes Yes Yes4
Limit of detection No No3 Yes No
Limit of quantitation No Yes No No
Linearity No Yes No Yes
Suitability range No Yes No Yes
Robustness No Yes No3 Yes

Notes: Yes = usually studied; No = usually not studied; 1 in cases

where the reproducibility is studied, there is no need to specially determine
the intermediate precision; 2 insufficient specificity of a
given analytical procedure can be compensated by introducing additional
tests; 3 may be required in some cases (e.g., when the limit of
detection is close to the rated limiting content of an impurity); 4 insufficient
specificity of a given analytical procedure can be compensated
by determining the content of impurities.

cause the spectrum can be influenced by variations in the

mobile phase composition (e.g., under gradient HPLC conditions),
or by deviations from the Lambert � Beer law at high
levels of absorption. Therefore, negative results of evaluation
of the spectral homogeneity of peaks in the gradient HPLC
should be critically assessed and checked for optical densities
not exceeding 1 AU.

It should be noted that specificity is rarely checked using

chromatomass spectroscopy (HPLC � MS) � because of the
high cost of this procedure � and the chemical and other
analyses of the eluate fractions corresponding to the parent
substance, because of tedious procedures.

1.1.2. The specificity of procedures aimed at determination

of impurities. In evaluating the specificity of such
procedures, it is necessary to differentiate between two cases.
Case
1. The main impurities are known and available.
In order to confirm the specificity of a procedure used for determining
the impurities in a parent substance, it is necessary
to show that (i) this procedure allows the peaks of the main
products of decomposition of the parent substance to be detected
and that (ii) the peaks of impurities are sufficiently
well separated between themselves and from peaks of the
parent substance and the system components (solvents). Developers
of the technology of a parent substance have to
prove additionally that (iii) the proposed procedure allows
determining the main impurities related to the technological
process and that (iv) all such impurities present at an amount
of .
0.1% are identified [7].
Validation of HPLC Techniques for Pharmaceutical Analysis

In order to confirm the specificity of a procedure used for

determining impurities in parent substances, it is necessary to
demonstrate that (i) this procedure allows the peaks of the
main rated impurities to be detected and that (ii) the peaks of
these impurities are sufficiently well separated between
themselves and from peaks of the parent substance and the
system components (solvents).

The specificity is confirmed by a set of chromatograms,

including at least those of (a) a model solution of the parent
substance and the main impurities (prepared by adding these
known impurities or their solutions to the parent compound
or its mixture with the placebo), (b) the solvent, (c) the placebo
(for filled drugs), (d) the test solution, and (e) the solution
used for the evaluation of suitability of the chromatographic
system.

In addition, it is recommended to confirm the specificity

of the given analytical procedure by data on the �purity� of
the main peaks in the chromatograms of test solutions.

Case
2. The main impurities are unknown (unidentified)
or absent. In this case, it is expedient to use special experiments
involving modification of the parent compound
(sometimes called �stress testing� [2, 15, 27]), whereby a solution
of the given drug (or of the parent compound) is subjected
to factors leading to its partial destruction with the formation
of related identifiable compounds. The degree of decomposition
can be readily determined from the decrease in
the area of the main peak in the chromatogram of the final
solution relative to that in the initial solution. The specificity
of the given analytical procedure is judged by separation of
the peaks of impurities between themselves and from the
peak of the main component and by the peak purity of the
parent compound.

In particular, the specificity of HPLC procedures can be

proved using the following methods of chemical modification.

(i) Hydrolysis with 0.1 N solutions of HCl or NaOH at

room temperature or at elevated temperatures. Example: a
granulate was treated with 0.1 N HCl solution for 5 h at
60�C, after which the sample was extracted according to the
proposed procedure and analyzed by HPLC.
(ii) Oxidation with a 3% hydrogen peroxide solution,
0.05 M iodine solution, etc. Example: captopril was partly
oxidized with 0.05 M iodine solution (in order to obtain
captopril disulfide � the main technological impurity [22]),
extracted according to the proposed procedure, and analyzed
by HPLC.
(iii) Thermal decomposition by heating to 60 � 100�C.
Example: a granulate was kept for 7 days at 80�C. The resulting
solid products of decomposition can be stored and used
as control substances in the tests for suitability of a chromatographic
system.
(iv) Photochemical decomposition under illumination
(e.g., UV irradiation).
(v) Chemical addition reactions, for example, drug
bromination at multiple bonds.
A mixture of the initial substance and the products of its
chemical modification can be used for preparing solutions
used for the evaluation of suitability of the chromatographic
system.

The duration of action used for the chemical modification

of drugs is selected taking into account the following
factors.

(i) The peaks of the products of drug modification are to

be clearly distinguishable in the chromatogram. Therefore,
the treatment duration must be sufficient to provide for a not
less than 10% decrease in the main peak height (area), which
is proportional to the drug content.
(ii) The peaks of the products of drug modification have
to be sufficiently well separated from the main peak, but the
main peak height (area) must be comparable with that in the
initial test solution. According to [27], it is recommended
that the main peak intensity would decrease by no more than
30%. However, this requirement is not as critical, since the
stronger decomposition of the parent compound can be compensated
by adding it in the necessary amount.
(iii) It is desired that the percentage �content� of the
products of drug modification determined by the method of
internal normalization would be close to the level of maximum
permissible content of a single impurity.
Further steps in the proof of specificity of the analytical
procedure in the case under consideration are the same as in
case 1. The validation characteristics additionally include
chromatograms obtained in the course of experiments on the
chemical modification of the parent compound.

Data presentation. The proof of the specificity of an analytical

procedure is presented in the form of a set of
chromatograms (see above) with discussion of the obtained
results. These data are supplemented by the results of calculation
of (i) the separation coefficient R for two peaks of the

most closely spaced components, (ii) the column efficiency

N, and (iii) the asymmetry parameters (tailing factors) of the
main analytical peaks.

It is necessary to make the following remark. Solving the

problem of detection and separation of impurities by HPLC
is still an art, with the results depending on the skill of developers.
It is very important to select the optimum chromatographic
columns and mobile phases. Moreover, it is known
that, depending on the column loading, it is possible (a) to
observe no additional peaks of impurities, (b) to find a certain
number of such peaks, a part of which cannot be quantitatively
characterized, or (c) to reveal a very large number of
additional measurable peaks upon overloading the column
with respect to the main drug component. Therefore, in developing
and validating the methods of impurity determination
(especially in the case of parent compounds), it is expedient
to check for the possible presence of additional peaks
(i.e., impurities) by chromatography of drug solutions with
elevated concentrations providing overloading of the column
with respect to the main component. However, this approach
is usually inapplicable in the case of ready-to-use drugs containing
large amounts of auxiliary substances.
1.2. Confirming the Precision of a Given Analytical Procedure
The precision (repeatability, reproducibility) of an analytical
procedure characterizes the random scatter (variation)
of the results relative to the mean value. It should be emphasized
that, in order to obtain reliable results, the sample must
have a homogeneous composition. The results have to be statistically
treated. Variants leading to rough errors (e.g., using
variation range R or the �three sigma� rule [8]) should be rejected.
In evaluating the suitability of HPLC procedures (actually,
in determining the metrological characteristics), it is
necessary to use measuring vessels of class A or special calibrated
measuring vessels and take all other possible measures
to increase the reproducibility and accuracy of HPLC
measurements [27].

The precision (repeatability, reproducibility) of an analytical

procedure is evaluated in terms of the standard deviation
(SD) of the relative standard deviation (percentage
RSD) determined in a series of measurements and calculated
by the formulas

SD .

#( Xi #X )2 (m #1) ,

i1

100SD

RSD(%) .
.
(1)

According to the ICH recommendations [4] on the validation

of chromatographic procedures, the characteristics of
precision are considered at three levels: repeatability, intermediate
precision, and reproducibility (Table 3).

In [4, 27] and in many other papers, the set of validation

characteristics of HPLC procedures includes the injection repeatability.
However, the author believes that this is incorrect:
this parameter characterizes the quality of injector (e.g.,
syringe) rather than the suitability of a proposed procedure.

TABLE 3. Main Precision Characteristics for the Validation of

HPLC Procedures According to ICH [4]

Precision characteristic Conditions of determination

Repeatability Injection Determined for the same sample

(Rt ) repeatability
preparation by the same analyst
using the same instrument
(chromatograph) during a short
period of time

Intra-assay
precision
Intermediate � Determined for the same sample

precision (Ip )
preparation by different analysts
using various instruments
(chromatographs) during a prolonged
period of time (not less
than two days)

Reproducibility � The same, in various laboratories

(Rp )

N. A. �pshtein
This characteristic is not included in the list of CDER [15]
and USP-26 [7]. In HPLC validation, data on the injection
repeatability should be provided as additional material required
in cases of search for the �bottleneck� of a proposed
method (dispersion analysis).

1.2.1. Injection repeatability and intra-assay precision.

In the general case, repeatability (R p) characterizes the
reproducibility of a given analytical procedure for the same
sample preparation, as performed by the same analyst using
the same instrument (chromatograph) during a relatively
short period of time. For evaluating the repeatability in a
given laboratory, the same analyst prepares samples of a
model mixture or the same batch of a parent compound or a
drug:
(a) not less that nine samples of solutions covering the
rated range of concentrations. For example: a homogenized
powder of triturated tablets from the same batch is used to
prepare drug solutions with concentrations equal to 50, 100,
and 150% of the reference sample solution concentration according
to the proposed procedure, or
(b) not less than six samples of solutions in the region of
concentrations close to the nominal value. For example: a
homogenized powder of a parent compound or triturated tablets
from the same batch is used to prepare six solutions with
nominal concentration according to the proposed procedure.
Note that each sample solution is (i) prepared independently
of the other solutions and (ii) chromatographed at least
three times.

Each solution is characterized by the drug content Xi

(i =1, �, N ), the average value X .
Xi

N , the standard
deviation SD, the relative standard deviation RSD of particular
measurements, and the confidence interval (for P = 95%)
of the average value.
It is required to show that the average results are statistically
equivalent (e.g., in terms of the Student t-criterion) or,
which is more convenient for the practical analysis, that
RSD .
1.0% for determination of parent compounds,
RSD .
2.0% for drugs, or RSD .
10.0% for impurities
[13, 27].5

1.2.2. Intermediate precision. This value characterizes

the reproducibility of results obtained in the same laboratory
by different analysts using various instruments
(chromatographs) during a prolonged period of time (not less
than two days) for the same homogeneous sample or a model
drug mixture according to the proposed analytical procedure.
Typically, not less than six solutions are prepared with
concentrations close to the nominal value (see the preceding
section). Each sample solution is (i) prepared independently
of the other solutions and (ii) chromatographed at least three
times.

5
For small values of the standard deviation (SD << 1), the t-criterion may
give statistically significant differences even for close (almost identical)
values of the compared average concentrations. This is related to the fact
that this criterion is proportional to the ratio of systematic and random errors.
Validation of HPLC Techniques for Pharmaceutical Analysis

These solutions are characterized by the average drug

content according to the results obtained by each of the analysts,
Xi and Xj (i, j =1, �, N ). These data are statistically
processed and characterized by generalized average values
X 1 .
XN and X 2 .
XN and the corresponding

ij

standard deviations (SDi, SDj ) and relative standard deviations

(RSDi, RSDj ) of particular measurements.

First, it is required to show that the proposed procedure

of determination of the drug content and the impurity
coincentraiton provides for the statistically equivalent standard
deviations SDi and SDof the results obtained by differ-

ent analysts (in terms of the Fisher F-criterion). Then, it is

necessary to demonstrate that the average results of these
(for certainty, two) analysts are statistically reliably
(P = 95%) identical in terms of the t-criterion calculated as

t .
X X#| |1 2 .
X X#| |1 2 ,
#1
2
2
2SD SD #1
2
2
2SD SD
m1 m2 m

where X 1, X 2 are the average results of analyses performed

by analysts 1 and 2 and SD1,SD2 are the standard deviations
in the particular series of m1 and m2 parallel determinations
(usually m1= m2= m ). This t value is compared to tabulated
values of the Student criterion t (P = 95%, f = m1+ m2 � 2),
where P = 95% is the confidence probability and
f = m1+ m2 � 2 is the number of degrees of freedom. If the
calculated parameter t is lower than the tabulated value, the
difference of average values can be considered as statistically
insignificant with a 95% confidence probability. Otherwise,
the average results differ to a greater extent than that admitted
by random errors in both series [35].

In pactice, validation of the procedures of determination

of the content of impurities is sometimes performed using a
less strict method [13], by showing that the scatter (RSD) of
the results of one analyst (characterized by a greater standard
deviation (SDor SD) relative to the average result of an
ij

other analyst (with a lower SDor SD) does not exceed a

ij

certain preset level, for example, so that RSD .

10% for impurities
with a rated content up to 1%, RSD .
25% for an impurity
content within 0.1 � 1%, and RSD .
50% for an impurity
level below 0.1%.

It should be noted that, according to USP-26 [7], it is in

most cases sufficient to determine only the repeatability for
proper validation of an analytical procedure, while the intermediate
precision and reproducibility characteristics should
be determined for procedures included in the pharmacopoeial
articles.

1.2.3. Reproducibility. This characteristic is determined

by comparing the results obtained upon analysis of the same
samples in different laboratories using a proposed analytical
procedure. The necessary statistical methods are described in
monograph [35, Chapter 8.4] and in the State Standard
GOST R ISO 5725-2002. It should be noted that for reliable
evaluation of the statistical significance of the difference between
the results obtained in such investigation, it is necessary
that the round robin tests involve not less than five
laborqtories [35].

In practice, however, the reproducibility is usually evaluated

using two or three laboratories and characterized by less
strict estimates. For example, validation of a procedure proposed
for the quantitative determination of a parent compound
is performed by demonstrating the statistical equivalence
of the standard deviations SDand SDof the results

ij

obtained in different laboratories (in terms of the Fisher

F-criterion). Then, it is demonstrated that the scatter (RSD)
of the results of analyses in one laboratory (characterized by
the maximum standard deviation (SDor SD) relative to the

ij

average results of analyses in other laboratories (with lower

SDor SDvalues) does not exceed a certain preset level [13].

ij

The full-scale reproducibility of analytical procedures is

rarely validated because (i) it is necessary to involve certified
laboratories capable of reproducing the proposed procedure
with high precision and (ii) this requires high organizational
facilities and expenditures.

1.3. Confirming Linearity of the Response to Drug Concentration

Linearity characterizes the ability of a proposed analytical
procedure to give (within the suitability range) a response
signal with the magnitude Y (e.g., peak height or area) directly
proportional to the amount C (concentration) of a drug
to be determined: Y = a + bC. According to ICH recommendations
[3], the linearity in practice is first visually estimated
from the linear appearance of the plot of Y versus C.Ifthe
plot appears linear, this relation is studied by methods of regression
analysis in terms of the linear equation Y = a + bC.

For the analytical procedures for determining the content

of a parent compound, CDER recommends establishing the
criterion of linearity at a level of the correlation coefficient r
not lower than 0.999 [4]. However, even such a high level of
correlation may be accompanied by significant deviations
from linearity in the regions of high and low drug concentrations
[13]. For this reason, ICH [3] recommends that the linearity
be validated by a plot of the difference Y�C showing
deviations (residuals) of the calculated values yi = a + bC
from the measured Yvalues as the function of the concentra

tion C. The �outbursts� of the points (xi, y) relative to the

ii

regression model can be determined by calculating the parameter

t using the formula [36]

| yi #Yi |

,
1(Y .
y)2

t .

SD 1##

N (N #
1) SD

Here, yi and Yi are the calculated and experimentally measured

values of the response, respectively; y = #yi /N; N is
the total number of experimental points (xi , y), and

i
#( y #Y )2

#( yi .
y)2

ii

SD0 .

, SD .

N #2

N #1

The calculated t value is compared to tabulated values of the

Student criterion t (P = 95%, f = N -2). If the calculated parameter
is greater than the tabulated value, the given point
can be considered as deviating from the adopted regression
model with a 95% confidence probability.

In practice, validation of the procedures of determination

of the content of impurities with respect to linearity is sometimes
performed proceeding from a correlation coefficient of
r #0.98 [13]. According to our estimates, use of the calibration
graphs with such correlation coefficients may lead to
RSD values on the order of 20% and above. Therefore, it
would be more correct to establish the criterion of linearity at
least at the level of r #0.990.

The linearity should be validated based on the analysis of

at least five solutions with various concentrations covering
the entire suitability range of a proposed analytical procedure
[35]. According to ICH recommendations [3], the linearity
can be demonstrated directly by using the reference parent
substance (dilutions of a standard solution) and#or model artificial
mixtures including components of the drug studied.
The most adequate approach consists in taking thoroughly
weighed aliquots of the drug components and preparing solutions
according to the proposed procedure, since all operations
of the analyst should correspond strictly to those stipulated
in the description. In practice, however, an �intermediate�
approach recommended by ICH [3] is frequently
employed. According to this, solutions are partly prepared
using weighed aliquots of the drug components and the other
are obtained by diluting these stock solutions. It should be
emphasized that the linearity of a proposed analytical procedure
should be confirmed in the course of validation of the
accuracy, which reduces expenditures and saves time. The
usual procedures are as follows.
(a) For the analysis of parent compounds, it is common
practice to prepare a reference solution of the compound
with a concentration at or above the upper limit of the expected
concentration interval (suitability range of the proposed
procedure). Then, a series of dilutions is prepared so
as to cover the entire range. Each solution is studied in a series
of two or three injections.
(b) For the analysis of ready-to-use drugs, the linearity is
frequently checked in the same way as for the parent compound
(i.e., using solutions of the parent compound as described
in (a)). However, it is incorrect to ignore the possibility
that auxiliary components (placebo) may influence the results.
Therefore, it is more correct to validate the linearity
using model mixtures of the parent compound and placebo.
(c) For the determination of impurities using the method
of internal normalization of the peak areas or heights, it is
possible to evaluate the linearity by preparing dilutions of the
reference sample solution (with a concentration equivalent to
the rated value) in the model impurity solution so as to obtain
N. A. �pshtein
drug concentrations in the range from 0.05% (dilution by a
factor of 2000!) to 2.5%. Instead of making some dilutions, it
is possible to use a proportional decrease in the volume of
applied sample (which saves the mobile phase).

Data presentation. The validation characteristics include

(i) a regression equation of the Si = a + bC type (for example,
S = 15536 + 15833969C [mg#ml]), (ii) the correlation
coefficient (e.g., r = 0.9998), and (iii) a plot visually
confirming the linearity of relationship between S and C.

1.4. Confirming the Accuracy of a Given Analytical Procedure

The accuracy characterizes the proximity of the experimental
results, obtained using a proposed analytical procedure,
to the �true� value in the entire suitability range of this
procedure. The accuracy represents a combination of the random
and systematic error.6 In order to provide for accurate
HPLC determinations, it is recommended to use standard solutions
with concentrations close to within 10% of the test
solution concentration.

The accuracy of analytical procedures should be determined

using homogeneous samples with exactly known concentrations
of the compounds to be determined. For validation
purposes a series of such solutions is prepared using the
reference parent compound. According to ICH recommendations
and USP-26 [1, 7, 15], the accuracy can be expressed
both in the classical form, as the difference X � .
between
the average experimental value (X ) and the true value (#)
with the corresponding confidence interval #X,7

( X ##) ##X ,
(2)

and in an alternative (and more illustrative) form, in terms of

the percentage recovery of the known amount of the compound
to be determined,

(found content )

R .
100%. (3)

(introduced content )

The author believes that the content recovery testing

should be preferred for evaluating the accuracy. This approach
provides a more illustrative characteristic of the reliability
of results obtained using a proposed analytical procedure
and reveals the need for additional checks in the case of
a significant systematic error (see below). On the other hand,
the recovery defined by formula (3) incompletely characterizes
the accuracy, since this quantity is also random and requires
knowledge of the corresponding confidence interval.
Thus, it is recommended to determine both the recovery R
(%) and the confidence interval at a preset probability
(P = 95%), representing the accuracy in a form analogous to
expression (2):

R ##R.
(4)

Obviously, the proposed methods should not involve significant

systematic errors. In the absence of systematic er

6
Accordiong to the State Standard GOST R ISO 5725-1�2002 the systematic
error usually characterizes �trueness.�

The value of #X characterizes random errors.

Validation of HPLC Techniques for Pharmaceutical Analysis

rors, the error is determined by the precision. Based on the

permissible RSD values (see above), experimental experience,
and analysis of the published data [13, 27], it is possible
to draw the conclusion that there is no need to verify a
proposed analytical procedure in the absence of a significant
systematic error, provided that it is established that the recovery
with allowance of the confidence interval does not fall
outside the following limits:

99.0�101.0%, for the quantitative analysis of parent substances

with a high rated content of the active component
(98% and above);

98.0�102%, for the quantitative analysis of parent substances

with a lower rated content of the active component
(98% and below) and ready-to-use drugs;

90.0�110%, for the quantitative determination of impurities

with a rated maximum content of up to 1%;

75�125%, for the quantitative determination of impurities

with a rated maximum content from -0.1 to 1%;

50.0�150%, for the quantitative determination of impurities

with a rated maximum content below 0.1%.

1.4.1. The procedure of accuracy evaluation. ICH recommends

making three determinations (i.e., analyze three
model mixtures) for three different concentrations. However,
this approach does not allow the accuracy to be determined
together with linearity and other validation characteristics.
The author believes that the accuracy should be evaluated using
not less than nine determinations at various concentrations
covering the entire range of suitability of the proposed
procedure. This provides for the possibility of determining
this characteristic simultaneously with the calibration graph
parameters and their statistical characteristics (for evaluating
the linearity according to Section 1.3), the limit of
quantitation (Section 1.7.2), and the limit of detection (Section
1.6.2). It should be emphasized that these determinations
should include all stages of the proposed analytical procedure.
For parent substances, the accuracy of analysis is usually
determined by comparison to a reference sample. According
to this, the reference sample (or a high-purity substance) is
analyzed using the standard procedure and the results are
compared to data in the certificate of the reference sample or
to the results of analysis of the high-purity parent substance
performed by an alternative method (e.g., titration) with
known accuracy and precision. It is recommended that, for
parent substances with a high rated content of the active
component, the average recovery should be not less than
99 � 101% at each level [13].

For ready-to-use drugs, the accuracy is evaluated

through the analysis of mixtures containing known amounts
of the parent compounds and placebo; for the quantitative
determination of impurities, this characteristic is determined
by the analysis of such mixtures containing known amounts
of these impurities. These analyses are performed using two
principal methods.

The method of recovery of a parent compound introduced

into the placebo (matrix). This method, used for the

analysis of drugs comprising mixtures of parent and auxiliary

compounds, is based on determining the recovery of a
known amount of the parent substance introduced into the
placebo. The placebo is prepared separately and then introduced
in a nominal (or proportional) amount into measuring
flasks. Then, thoroughly weighed amounts of the parent
compound or its concentrated solutions are added so that
(upon filling the flasks to the marks) the sample concentrations
would cover the entire expected suitability range of the
proposed analytical procedure. For example, a parent compound
can be introduced into a placebo solution at a level of
80, 100, and 120% of the nominal concentration indicated on
the label (or the reference solution concentration).

The method of standard additives. This method is generally

analogous to that described above but is applied only
when it is impossible to prepare a solution of placebo free
from the parent compound or when this compound is present
in the placebo in an unknown amount (e.g., in biological
samples). In these cases, the reference sample of the parent
compound is added at an amount of 50, 80, 100, 120, and
150% of its expected content in the analyzed solution. Using
the proposed procedure, the amount of the parent compound
is determined (found content) and compared to the known
additive (introduced content). Alternatively, it is possible to
compare the results of analyses performed using the proposed
method and the data obtained for the same samples by
validated alternative methods.

For the validation of analytical procedures intended for

the analysis of ready-to-use drugs, it is expedient to use the
same reference substance for preparing both model mixtures
and standard solutions. This eliminates errors related to the
possible uncertainty of the composition indicated on the label
of the reference sample and allows using commercial
samples instead of special reference compounds. At a low
concentration of the parent compound in the mixture (when
it is impossible to add a thoroughly weighed amount of this
compound), one may add a known amount of concentrated
solution and then fill the measuring flask with a solvent stipulated
by the proposed analytical procedure.

For the quantitative determination of impurities, the accuracy

of evaluation has certain peculiarities. In this case, the
method of recovery of a parent compound introduced into
the placebo and the method of standard additives have limited
applicability because these validation procedures require
large amounts of identified impurities. In this case, the accuracy
is most frequently checked using the method described
below.

The method of internal normalization with or without

response factors. According to this method, identified impurities
are characterized by the response factors with respect
to the parent compounds determined by the analytical procedure
under consideration. These coefficients depend on the
mobile phase composition and the analytical wavelength. For
reproduction (or modification) of the analytical procedure,
the detector wavelength is not changed, while the mobile
phase composition can be corrected for the difference in the
220

parameters of chromatographic columns. In the case of a

considerable change in this composition (see Section 4), it is
necessary to re-determine at least the values of the response
factors. For determining unidentified and accidental impurities,
these factors are conventionally taken equal to unity (assuming
that the sensitivity for these impurities is the same as
that for the parent compound). If the reference samples of
impurities and decomposition products are unavailable, the
validation has to be performed using alternative methods.

1.4.2. Testing for systematic error. The analytical procedure

can be checked for the absence of systematic errors
by one of the three methods considered below.
Method based on the Student t-criterion without regression
analysis [8]. Each sample (whose total number is
N .
5) with known values .
(introduced content) of the component
to be determined is analyzed in m =3�6 parallel determinations.
The total data array is characterized by the dispersion
(SD) and the Student criterion8

#.
xm

||

t .

SD

This t value is compared to tabulated values of the Student

criterion t (P, f = m � 1). If the calculated parameter is
greater than the tabulated value for P = 95% and f = m �1.
t > t (P, f ), the results obtained using the proposed method
can be considered as involving a systematic error #. This error
is calculated by the formula

| x ##|

#.
100%.
(5)

The standard deviation SD0 in a particular analysis is calculated

using the set of all m parallel determinations performed
for N (or g in the notation of [8]) samples. This allows
the SD0 value to be reduced and the sensitivity of determination
of the systematic error to be increased with a
simultaneous decrease in the confidence interval for the results
of analysis, #X = t SD0.

m, where t is the Student criterion

for f = m � 1 degrees of freedom.
The algorithm of these calculations is as follows. Each
kth sample is characterized by the deviation of the experimental
value from average, di .
Xi .
X, and the dispersion

.
m .

SD2
k .
.
d 2 .
(m #1). Then, the difference between the

.
i1 .
maximum and minimum values of the dispersion SDk
2 is
checked to be insignificant in terms of the Fisher F-criterion.
If this difference is actually insignificant, the SD02 value is
calculated as the sum of square deviations for all samples di

vided by the number of samples,

8
This t value is essentially the ratio of the systematic error |x ##| and the
random error SD.

m.
0

N. A. �pshtein
N
SD2.
k
SD0
2 .
1k
N
.

Finally, the Student t-criterion is calculated as

t .
(|#.
x|.
m) SD0 (6)

and compared to tabulated values of the Student criterion

t (P, f ) for f = N(m � 1) degrees of freedom.

It should be emphasized that, for small (practically insignificant)

systematic error and small random error, the calculated
t-criterion can be greater than the tabulated value. However,
a small systematic error can be ignored in practice
when the analytical problem does not require high accuracy
of determination. This approach is also valid for other methods
of evaluation of the accuracy of a proposed analytical
procedure.

Method of regression analysis with the Student t-criterion

[35, 38]. This method seems to be the most effective,
since it provides for the possibility of using the calibration
graph for the accuracy evaluation and determination of some
other validation characteristics (see the generalized scheme
in Fig. 1).

For simultaneous determination of the constant and variable

systematic errors, not less than N = 5 samples with
known values of the parent compound are studied and the relationship
between the �introduced content� (m) and the

�found content� (mf ) is processed by least squares in terms

of the equation mf = a + bm. Using these data, the parame

ters t =|a|#SDand t=|1 �b.#SDare calculated and com

a 0 b 0

pared to the critical (tabulated) values of the Student criterion

t (P, f ) for the confidence probability P = 95% and f = N �2
degrees of freedom. Using the results of regression analysis,
it is possible to judge with 95% probability about the absence
of a constant systematic error, provided that t .
t (P,

f = N � 2), and the absence of a linear variable systematic error,

provided that tb
.
t (P, f = N � 2).

It is expedient to perform validation of the accuracy of an

analytical procedure together with checking for the linearity
of the system response (area or height of the peak) as a function
of the concentration of the parent compound to be determined
(in fact, linearity of the calibration graph in terms of
the criteria described in Section 1.3) and with finding the
limit of detection (Section 1.6.2) and the limit of quantitation
(Section 1.7.2).

Method of regression analysis with the Fisher F-criterion.

This method is based on the assumption that a linear
variable systematic error can be ignored. This assumption is
justified because HPLC in pharmacy is used in a relatively
narrow range of sample solution concentrations.

The measurements are performed for not less than N .

5
samples (model mixtures) with known values of the parent
compound, after which the relationship between the �introduced
content� (mt ) and �found content� (mf ) is processed
Validation of HPLC Techniques for Pharmaceutical Analysis

by least squares in terms of the relation mf = a + bm. The ab-

sence of a constant systematic error is confirmed by the insignificance

of the coefficient a [35] at a commonly accepted
confidence probability level of P = 95%. For this purpose,
the Fisher criterion calculated is as

FPf 1 N �, f 2 N �2

(,1 ) .
2 2 (7),

N 1 SD N 2)

SD (�)� (�

01 02

02 N 2)

SD2(�

where SDand SDare the standard deviations obtained

01 02

for the above relations without the free term (mf = bm) and

with the free term (mf = a + bm). If the calculated F value is

smaller than the tabulated (critical) values F (P = 95%, f1, f),

the free term a is in fact insignificant and the error is absent

to within a 95% confidence probability.

1.4.3. Data presentation and evaluation of the systematic

error. The final judgment about the accuracy of a proposed
analytical procedure can be made upon validation of
its specificity, precision (repeatability, reproducibility), and
linearity.9 It is necessary to indicate a particular method of
normalization of the content of impurities (weight fractions,
percentage of the area under the peak of the main component,
etc.). The validation results are presented by data on the
recovery of the amount of introduced parent compound with
a confidence interval, R ##R, or the difference between the
average experimental value X and the true value .
with the
corresponding confidence interval: ( X ##) ##X.
The proposed procedure involves no significant systematic
error, provided that the recovery with allowance of the
confidence interval does not fall outside the following limits:

99.0�101.0%, for the quantitative analysis of parent substances

with a high rated content of the active component
(98% and above);

98.0�102%, for the quantitative analysis of parent substances

with a lower rated content of the active component
(98% and below) and ready-to-use drugs;

90.0�110%, for the quantitative determination of impurities

with a rated maximum content of up to 1%;

75�125%, for the quantitative determination of impurities

with a rated maximum content from � 0.1 to 1%;

50.0�150%, for the quantitative determination of impurities

with a rated maximum content below 0.1%.

The numerical result of a particular analysis, X (or R),

must contain the last significant digit in the same position as
that in the numerical value of the error of determination, #X
(or #R) [37]. The number of significant digits in the latter
value is determined as follows. If the first significant digit in
the error is #3, then #X is expressed by a value with one significant
digit; should the first significant digit in the error

9 It is expedient to confirm the linearity together with determining the accuracy.

be <3, then #X is expressed by a value with two significant

digits.

1.5. Validation of the Suitability Range of a Given Analytical

Procedure
The range of suitability of a given analytical procedure is
the interval between minimum and maximum concentrations
(amounts) of a compound to be determined in which (i) the
linearity is observed, (ii) the characteristics of repeatability
fall within permissible (preset) limits, and (iii) the accuracy
is maintained at a sufficiently high level [1 � 3]. This interval
must contain all values of the concentrations (amounts),
which can be encountered in the course of routine analyses.
The range of suitability of a given analytical procedure is expressed
in the same units as the results of analyses.

It should be noted that, in practice, it is not necessary to

determine the maximum possible range of suitability for an
HPLC procedure. If it were necessary, this range could be determined
using threshold RSD values obtained in the course
of validation of the linearity and precision. For example,
RSD must not exceed 3% for HPLC procedures aimed at determination
of the parent compounds and 10% for procedures
of impurity determination [13].

In practice, it is sufficient to show that a given range of

suitability covers �with margin� the rated limiting concentrations
of the substances to be determined, as indicated in the
corresponding pharmacopoeial articles. For this reason, it is
recommended that the range of suitability of a given analytical
procedure be not less than the following intervals [3, 7].

(i) For the quantitative determination of the main component

concentration in parent compounds and ready-to-use
drugs: from 80 to 120% of the nominal content (i.e., the concentrations
of test solutions should range within 80 � 120%
relative to the concentration of a reference sample solution
used accordsing to the proposed procedure).
(ii) For evaluation of the homogeneity of dosage: from
70 to 130% of the nominal content, provided that a wider interval
is not required (in special cases such as aerosols).
(iii) For dissolution tests: #20% (absolute percentage) of
the rated value of drug release. For example, for monitoring
the behavior of a drug with delayed release of a parent compound,
for which the release is rated as 20% within the first
hour and up to 90% within a 24-h period of time, the suitability
range must extend from 0 to 110% of the nominal drug
content.
(iv) For the quantitative determination of impurities by
the method of external standard: from 50 to 120% of the
nominal content. It should be noted that, in view of the possibility
of RSD values amounting up to 50% (Section 1.2.2), it
would be more correct to establish the upper limit at 150% of
the nominal content. For impurities exhibiting a very high biological
activity, toxicity, or unpredictable behavior, the limits
of detection and quantitation must correspond to the level
at which these impurities have to be controlled.10
(v) In cases where the quantitative determination of a
parent compound and the detection of impurities are per
formed jointly and only the reference sample solution of the
main component at a nominal concentration is used, the suitability
range must extend from the rated LPI value (more
precisely, from half of this value, see the preceding point) up
to 120% of the nominal concentration of the parent compound.
If the LPI value is unknown (e.g., during the development
of an analytical procedure) the initial lower limit of the
suitability range according to the ICH recommendations
should be established at 0.1% of the nominal concentration
of the parent compound for a daily drug dose below 1 g;
should this dose exceed 1 g, the lower limit of the suitability
range should be reduced to 0.05% of the nominal drug concentration.

1.6. Determining the Limit of Detection of a Given Analytical

Procedure
The limit of detection (LOD) is determined as the minimum
concentration of analyzed substance in the sample,
which (i.e., the corresponding response) can be detected under
preset conditions [3, 4, 7]. For HPLC procedures used for
the determination of parent compounds and ready-to-use
drugs, the LOD is required for the detection of limiting impurity
concentrations. Obviously, the LOD may depend on the
HPLC detectors and pumps. For this reason, this characteristic
has to be rated for various instruments, including those
available for potential users. Irrespective of the method used
for evaluation, it is necessary to have a chromatogram showing
that a response peak exceeding the baseline noise is actually
observed at a concentration corresponding to the LOD of
the substance to be detected.

According to USP-26 [7], it is usually not necessary to

determine the actual LOD of the analyzed substances (except
for analytical procedures intended for monitoring the cleanness
of technological equipment). In most other cases, it is
sufficient to show that the impurity of interest is reliably detected
at a preset level (see Section 1.6.5).

1.6.1. Determining the LOD from the Standard Deviation

of the Response and the Slope of the Calibration
Curve. According to this method [3], the LOD value is calculated
by the formula
LOD=3/3(SD#b ), (8)

assuming that the response � concentration relation is linear

in the range from the maximum possible concentration of the
analyzed compounds down to zero. It is recommended to determine
the slope b of the calibration curve using reference
sample solutions with concentrations in the vicinity of the
LOD (see Section 1.7).

The value of the standard deviation SD can be determined

using one of the two methods:

10 For the analysis of impurities, it was recommended [27, p. 695] to perform

tests in the range from the limit of quantitation with respect to the
main component (typically, below 0.1%) up to a 5% concentration in solution.

N. A. �pshtein
(a) Using the calibration curve S = a + bC constructed
using the results of analyses for a series of the reference sample
solutions with decreasing concentrations in the region of
the LOD. Using these data, a regression equation S = a + bC
of the area under peak S versus concentration C is calculated
and the standard deviation SD of the free term is deter-
a

mined. Alternatively, the standard deviation is determined

for the intersections of several regression lines of the calibration
curve with the ordinate axis, constructed using several
series of reference sample solutions with concentrations in
the region of the LOD.

(b) Using the standard deviation of the area under the

peak in the chromatograms of an impurity with concentrations
within 0.01 � 0.05% of the nominal drug concentration
determined using the given analytical procedure, typically
for ten sequential injections.
1.6.2. Determining the LOD Using the Free Term and
the Coordinates of the Midpoint of the Calibration
Curve. This is the most convenient way to determine the
LOD. According to this method, the calibration curve is described
by the regression equation Y = a + bC, where Y is the
response (peak area of height) and C is the concentration. For
this relation, the LOD is calculated by the formula
2C
tP f (, )
SD0

LOD .

, (9)

##tP f ) .
0

Ya (, SD

where C and Y are the coordinates of the midpoint; a is the

free term; SD0 is the standard deviation of the experimental
values from the calculated ones; nis the number of parallel

determinations for each experimental point (typically,

nh =2�3); t (P, f = N � 2) is the Student criterion (F = 99%
[35]) for f = N � 2 degrees of freedom; and N is the number
of experimental points on the calibration graph.

It should be noted that a different formula for the LOD

was presented in [35], according to which the background
(i.e., the free term) did not influence this value. The error has
been eliminated in the new edition of this monograph.

1.6.3. Determining the LOD for the Signal-to-Noise

Ratio S#N
#3. The method of evaluation of the LOD using
the S#N
ratio (for S#N
#3) [3, 4] should not be used for the
validation of HPLC procedures because the baseline noise
strongly depends on the experimental conditions. On a scale
comparable with the noise intensity, the baseline appears as a
broken line of complicated shape and admits only a very subjective
estimate of the maximum noise N
.
1.6.4. Evaluating the LOD from the Minimum Concentration
for Which the RSD is Below a Preset Value.
According to calculations [39], the relative error of the LOD
amounts to at least 20%. For this reason, the LOD can be theoretically
evaluated as the minimum solution concentration
for which the RSD for the peak area (height) of the analyzed
compound for five sequential determinations does not exceed
20%. However, this approach is not convenient in practice
because it requires numerous analyses. For example, it
Validation of HPLC Techniques for Pharmaceutical Analysis

was pointed out [40] that evaluation of the LOD by this

method in accordance with FDA recommendations (defining
the LOD as the minimum concentration for which the RSD
does not exceed 15%) would require 288 determinations.

In order to take into account the influence of the drug

matrix on the LOD, it was suggested [30] to take into account
the so-called �specific LOD.� These values are determined
using mixtures of a given parent substance with a placebo
instead of a solution, which takes into account the effect
of asymmetry of the peaks of other components on the LOD.
According to ICH [3], the LOD has to be refined using various
columns and instruments because this characteristic depends
on the noise level (and, hence, on the working life of
the column, on the properties of the detector, etc.) and on the
peak shape.

1.6.5. Alternative Proof of the Reliable Detection of

Impurities in Parent Substances and Ready-to-Use
Drugs. For a test solution concentration of about 1 mg#ml,
the response signal at a level of not less than 1 V can be
readily obtained. For an impurity concentration of 0.01%,
this corresponds to a response on the order of 100 #V, which
is one order of magnitude higher than the noise level of modern
detectors. For validating the reliable detection of impurities,
it is sufficient to present the chromatogram of a reference
(or test) solution diluted D =2L times, where L is an integer
indicating the ratio of the main component
concentration to the minimum rated concentration of the impurity.
This chromatogram must visually confirm the possibility
of reliably detecting impurities at the level of about
half of their rated content. For example, if the content of an
individual impurity must not exceed 0.5%, the above ratio is
D = 2(100#0.5) = 400. For the normalized total impurity
content, D = 2000, which corresponds to an individual impurity
content of 100#D = 0.05% (the British Pharmacopoeia
does not take into account impurities with concentrations below
0.05%, except for highly toxic substances). In many
cases, this approach eliminates the need to establish the LOD
during the determination of impurities in parent compounds
and ready-to-use drugs.
Data presentation. According to ICH [3], it is necessary
to validate the LOD value and indicate the method of determination.
If the LOD was calculated from experimental data,
it is necessary to present the results of analysis of the required
number of samples with the content of a detected
component close to the LOD. If the LOD was estimated by
visual assessment of chromatograms, it is necessary to present
chromatograms confirming reliable detection of the
peaks of parent compounds or impurities.

The number of significant digits in the LOD [39] is determined

as follows. If the first significant digit in the LOD
value is > 3, then this limit is expressed by a value with one
significant digit and rounded to the closest integer; should
the first significant digit in the LOD be #3, then the LOD
can be is expressed by values with two significant digits and
the second digit should be rounded to 0 or 5.

1.7. Validation of the Limit of Quantitation of a Given

Analytical Procedure
The limit of quantitation (LOQ ) is the minimum concentration
of analyzed substance that can be determined at an acceptable
precision (repeatability, reproducibility) and accuracy
under rated conditions of analysis by a given method
[1, 15]. The ICH [3] and USP-26 [7] recommend several
methods of determining LOQ for the impurity analysis.

1.7.1. Evaluating the LOQ for the signal-to-noise ratio

S#N
=10. This is the most widely used method of determining
the LOQ. However, since this procedure makes use
of the peak heights (rather than areas), it is more suited for
the HPLC techniques using this measure of the response.
This approach does not take into account the requirements on
reproducibility of the HPLC procedure.
According to this method, an initial reference solution of
the analyzed compound is determined for which the signal-
to-noise ratio is at least 30 [27]. Then, this solution is sequentially
diluted until this ratio decreases to S#N
#10. The
corresponding concentration is considered as the LOQ. For
this evaluation of the LOQ, it is recommended to determine
the maximum baseline noise near a peak of the analyzed
compound, in the interval of retention times within #10W

0.5,
where W0.5 is the full width at half maximum (FWHM) of
this peak [22].
For HPLC procedures used in pharmacy, it would be interesting
to study the influence of the maximum noise N
on
the relative standard deviation of results for a nearly Gaussian
peak shape. According to [27],

50

RSD[%] .
, (10)

SN .

where S is the detector signal intensity. A simple calculation

shows that, at S#N
= 10, the noise influence alone can make
RSD as large as #5% (!).

1.7.2. Evaluating the LOQ using the calibration

curve. The ICH [3] recommends determining the LOQ by
the formula
LOQ = 10(SD#b ), (11)
where b is the slope of the calibration curve and SD is the
standard deviation of the response signal. The peculiarities of
this approach were considered in Section 1.6.1. A significant
disadvantage of this approach (as well as of some others)
recommended by ICH is the inability to take into account the
requirement of acceptable reproducibility (see above).

The author believes that a more correct estimate of the

LOQ from the calibration graph, with allowance for the
reproducibility requirements, can be obtained in the following
way [41]. First, the values of response Y (area under the
peak of the analyzed component) within the limits of the calibration
curve Y = a + bC are used to calculate (i) the theoretical
values of C =(Y � a)#b and (ii) the standard deviations
224

(SD) and relative standard deviations (RSD = SD#C .

100)

cc

of the results of analysis. Then, the plot of RSD versus C is

used to determine the concentration Cp corresponding to a
permissible RSD value preset for the analytical procedure
under consideration. This permissible concentration Cp is
taken to be LOQ with allowance for the RSD (i.e., for the
reproducibility) requirements.

The values of standard deviations of the results of analyses

are calculated by the formula [35]

SD

11 .
SDb .
2
.
Ys #Y .
2

SD .

####.
.
(12)

Nm .
b #.
SD0 .

for f = N � 2 degrees of freedom, where SD0 is the standard

deviation of the linear relation (calibration curve), N is the
number of experimental points on the calibration curve, Y is

the response (area under the peak), m is the number of parallel

(independent) determinations, b is the slope of the calibration
curve, SDb is the corresponding standard deviation, and
Y = Yk#N is the average response of all experimental points
used for constructing the calibration graph.
1.7.3. Other methods. Other methods of determining the
LOQ are not theoretically justified and did not find wide use.
In particular, it was suggested to determine the LOQ as the
minimum concentration of the analyzed compound for which
six sequential injections give RSD .
3.0% [27, p.695] or
RSD .
2.0% [29]. With this approach, the LOQ is essentially
defined as the concentration at which the RSD becomes
greater than a certain preset value.
1.7.4. Allowance of the matrix effect on the LOQ.It
was suggested to determine the so-called specific LOQ p30].
This characteristic is determined under the same conditions
as described above for the LOD, but in a real test solution,
and therefore takes into account the influence of asymmetry
of the other peaks on the LOQ of the analyzed compound.
Data presentation. The LOQ value is presented with indication
of the method used for its determination. If this
characteristic is evaluated using the signal-to-noise ratio, it is
necessary to present chromatograms showing the reliability
of detection of the peak due to the analyzed component or
impurity. If the LOQ was determined by calculation or extrapolation,
it is necessary to present the results of analysis of
a sufficiently large number of samples with the content of the
analyzed compound close to the LOQ level.

1.8. Stability of Solutions.

1.8.1. Confirming the stability of solutions using the
regression relation for the area under the peak versus the
time. As a rule, the analytical solutions are used for a relatively
short period of time (th ) for which the content of the
analyzed substances (proportional to the areas under the corresponding
peaks) remains practically unchanged. In order to
confirm this statistically, it is sufficient to determine the coefficient
b in the regression relation St = S0+ bt (or
S� S0= bt ) between the area Sunder the peak at the time t
tt

N. A. �pshtein
(within the interval from 0 to th ) and the area S0 at the initial
moment, calculate the parameter tb =|b |/SDb, and compare
this value with the critical (tabulated) Student criterion
t (P = 99%, f = N � 1) for the confidence probability
P = 99% and f = N � 1 degrees of freedom, where N is the
number of experimental points on the above interval. If tb is
smaller than the tabulated t (P = 99%, f = N � 1) value, the
coefficient b is statistically insignificant and, hence, there is
no significant difference between the areas under the peak
(and, hence, the solution concentration) at the initial time and
at any other moment up to the last experimental point.

1.8.2. Methods of evaluation. The stability of test and

reference solutions ST (%) can be evaluated using the following
methods.
(i) For continuous testing within a relatively short period
of time � by the ratio of the area Sunder the peak of the ant
alyzed compound (or impurity) at the moment t to the initial
area S:

ST = 100S#S0. (13)

(ii) Irrespective of the duration of experiment � by the

ratio of the amount m(concentration) of the analyzed comt

pound (or impurity) at the moment t to the initial value m0:

ST = 100m#m0. (14)

The test and reference solutions are prepared according

to the given procedure and analyzed with certain time intervals.
The maximum possible duration of measurements depends
on the practical requirements on the possible storage
time of the given solution (which may not necessarily be
equal to the maximum rated storage time). The solutions can
be considered stable until the difference |100 � ST | does not
exceed the relative error of determination of the main component
(or impurity) according to the given analytical procedure.
11

Data presentation. The stability of solutions is confirmed

by data on the storage times of solutions and mobile
phases used in the analyses.

2. EVALUATION OF THE STABILITY OF ANALYTICAL

SYSTEMS WITH RESPECT TO SMALL VARIATIONS
OF PARAMETERS (ROBUSTNESS)
The robustness of an analytical procedure is the characteristic
of its stability with respect to small variations of the
system parameters possible under real conditions. This stability
is usually evaluated in terms of the RSD of the results
of analyses compared to the analogous data obtained using
strictly observed conditions according to the validated analytical
procedure. A more correct procedure based on evaluation
of the statistical significance of the difference between
these results in terms of the Student t-criterion is rarely applied
to the HPLC techniques.

11 The absolute value of |100 � ST | is taken because ST can be greater than

100% due to random errors.
Validation of HPLC Techniques for Pharmaceutical Analysis

Robustness is usually studied at the stage of development

of the analytical procedure. Typical parameters capable of influencing
the results of analyses and, hence, studied in the
course of validation, are as follows:11

(i) the content of the organic solvent in the eluent

( .
2%);
(ii) the amount of additives (salts, ion-pair reagents, etc.)
in the eluent ( .
10%);
(iii) pH of the buffer solution ( .
0.5);
(iv) HPLC column temperature ( .
5 �C);
(v) time of extraction of the analyzed compound from a
drug eluent ( .
20%);
(vi) extractant composition ( .
5%);
(vii) eluent concentration gradient ( .
2%);
(viii) mobile phase flow rate;
(ix) column type and#or manufactirer.
The parameters (called critical) influencing the results of
analyses should be indicated in the validation report. The description
of the given analytical procedure must provide the
corresponding warning remarks. In order to decrease the
number of tests necessary for the validation of robustness of
a given analytical procedure, it is expedient to use the methods
of experiment planning, which are capable of quantitatively
following the effect of several parameters (factors)
varied simultaneously [32 � 34, 42].

There is no need for special additional investigations of

robustness if no critical parameters were revealed in the
course of development and#or implementation of the proposed
analytical procedure.

Data presentation. The validation report should include

the chromatograms and tables showing the effects of each
critical parameter on the results of analyses (in comparison
to the normal values) and the absence of such effects for the
other most important parameters (see above). It is desired
that the proposed analytical procedure be robust with respect
to all the important parameters, which makes this procedure
suitable for routine laboratory use.

3. CRITERIA OF SUITABILITY
OF A CHROMATOGRAPHIC SYSTEM
USP-26 [7] stipulates the System Suitability Test (SST)
intended to establish that the separating power and
reproducibility of a given chromatographic system provide
for the adequate solution of the task of analysis. It is assumed
that the equipment, electronics, analytical procedures, and
samples comprise a unified system investigated as a whole.
Upon chromatography of a special SST solution, the results
are checked for obeying the SST requirements (see below).
The SST solution must contain the analyzed compound and
all other additives necessary for evaluating the suitability of
the given system for implementing the required analyses.

12 Values in parentheses indicate recommended limits of variation relative

to the values stipulated by the given procedure.

The additives may include other analyzed components, identified

impurities, possible decomposition products, and
substances playing the role of internal standards.

Effective means for finding SST solutions is provided by

experiments on the chemical modification of a particular
drug under consideration (see Section 1.1.2). The solutions
and substances obtained in such experiments contain the
products of destruction of the main drug components and#or
compounds structurally close to the analyzed parent substances.
Such solutions are expediently used in the tests for
suitability of a given chromatographic system and frequently
allow one to reject expensive standard samples of impurities.

The author believes that the suitability of a given chromatographic

system is adequately described by the following
main criteria.

(i) Criteria of the separating power of the chromatographic

system.
(ii) Criteria of reliable determination of the beginning
and end of a peak (and#or of the peak height) of the analyzed
compound on the chromatogram.
(iii) Criteria of reproducibility of the results of measurements
(repeatability of injections) (iv);
Additional criteria should be introduced only provided
that critical parameters (see above) were established in the
course of robustness evaluation.

The SST criteria should not be overloaded by numerous

extra parameters, since this may lead to a situation where
only HPLC columns and equipment used for the validation
purposes will be formally suitable for all analytical procedures.

3.1. Separating Power of a Chromatographic System

The criteria of separating power include the separation
coefficient R , the peak-to-valley ratio h#v (see below), and

the relative retention times of components.

3.1.1. Relative retention times. For analytical procedures

intended for the quantitative analysis of drugs, homogeneous
dosing tests, and determination of the drug�s dissolution
characteristics, it is usually sufficient to characterize
the separating power by the relative retention times of several
substances, including the main drug component, a structurally
close compound, and#or an internal standard. The relative
retention time of the main component is usually taken
equal to unity.
The author believes that the retention times should not be
used as characteristics of suitability of a chromatographic
system because these values depend on the eluate flow rate,
temperature, and dead volume of the system. On the other
hand, the retention times do not influence the accuracy and
precision of an analytical procedure (provided that other suitability
requirements are satisfied). It is more correct to indicate
the recommended values (or intervals) of retention times
of the main components in the description of the analytical
procedure and characterize the other substances by their relative
retention times.
N. A. �pshtein
3.1.2. Peak separation coefficient. For analytical procedures
intended for the determination of impurities, the system
suitability requirements should indicate the minimum
possible values of the peak separation coefficient R at least
s

for the main (rated) impurities. In the case of HPLC procedures

intended for the quantitative analysis of drugs, homogeneous
dosing tests, and determination of the drug dissolution
characteristics, it may be necessary to introduce the peak
separation coefficient into the system suitability requirements
if (i) a high content of some impurity is admitted, (ii)
there is a possibility of partial overlap between the peaks of
the main components, or (iii) the peaks of the placebo components
occur in the vicinity of the main analytical peaks.

The minimum possible values of the separation coefficient

R
are determined proceeding from the following

considerations.

If the peaks are not significantly different in heights and

possess nearly Gaussian shapes, their complete separation at
the baseline level requires that R .
1.5. This condition is rec-

ommended by the British Pharmacopoeia and by the EEC

Pharmacopoeia.

If the peaks have significantly different heights (e.g.,

used in the determination of impurities) and#or exhibit tailing,
CDER recommends that R .
2.0 [4]. It should be noted

that this situation is most frequently encountered in practice

during the analysis of drugs.

For separating peaks with large tailing factors ( > 2.5, see
below), it is recommended to restrict the separation coefficient
to R .
2.5. This situation is quite rare, being only typi

cal of drugs containing several heteroatoms capable of forming

strong hydrogen bonds with silanol groups of the sorbent.
Higher �critical� R values are usually unnecessary, be-
s

cause the condition R > 2.5 ensures good separation virtu-

ally at the baseline level. On the other hand, it should be

noted that, due to the availability of highly effective columns
and computer-optimized systems, values of R < 1.2 cannot

be justified, except for (i) the isomer peaks, (ii) the peaks of
some components in complex multicomponent mixtures
(such as extracts of biological objects, antibiotics, alkaloids,
and multivitamin preparations), and (iii) the peaks of minor
components (impurities, aromatic and color additives, etc.).

It should be noted that the British Pharmacopoeia (BP

2001, Appendix III, p. A143) indicates that, in tests for the
content of related compounds (when the peaks of main components
and impurities are incompletely separated), the system
suitability requirements may describe the peak separation
in terms of the peak-to-valley ratio p#v = hp #hv, where hp
is the peak height relative to the extrapolated baseline and h

is the height of the lowest point in the valley separating the

peaks (relative to the same extrapolated baseline). This parameter
is rarely used during the analysis of impurities in
drugs: sometimes it is convenient for the description of impurity
peaks occurring immediately in front of the main analytical
peak. For suitability of the chromatographic system, it
is usually required that hp > hv. For example, in the analysis

of fluoxetin hydrochloride, it is required that

h #h .
1.1#0.1=9.

pv

3.2. Criteria of Reliable Determination of the Beginning

and End (and#or the Height) of a Chromatographic Peak
The peak asymmetry is described in terms of the tailing
fgactor T0.05 [7]. This parameter, which characterizes the
asymmetry at a level of 0.05% of the peak height, is especially
important for the procedures of quantitative analyses.
According to BP 2001 [22], the recommended values fall
within T0.05 = 0.8 � 1.5. Under this condition, the peaks are
sufficiently symmetric and exhibit no significant tailing,
which favors reliable determination of the beginning and end
of the peak (and, hence, of the peak area) and reduces the
probability of overlap. An analysis of the corresponding articles
of the BP 2001 and USP-26 showed that the permissible
T0.05 interval extends from 0.75 to 2.5. Asymmetric peaks
with T0.05 outside the 0.75 � 2.5 range should be avoided: for
peaks with T0.05 on the order of 3 and above or 0.7 and below,
the boundaries of peaks practically cannot be determined.

An additional criterion of peak separation is provided

by the efficiency of a chromatographic column (N ) with
respect to the main analytical peak. This parameter characterizes
the peak width at half height (FWHM) and is calculated
by the conventional formula [7]. The ICH recommends
using columns with efficiencies N .
2000 theoretical plates,
which can be considered as the lower limit for the procedures
of impurity determination in parent compounds. According
to practical experience it is usually sufficient to require that
N .
1000 theoretical plates.

In some cases in the HPLC determination of the main

drug components, USP-26 admits N .
400 theoretical plates.
However, even this is not the minimum possible level: rapid
analyses are sometimes performed using short chromatographic
columns with 3.5-#m (and smaller) sorbent particles,
which provide for a relatively low error of determination
(< 1.5%) even at N .
250 � 300 theoretical plates. The above
criteria of suitability of the chromatographic system with respect
to the N value are not absolute: this parameter must
only provide for the required accuracy and precision of the
proposed analytical procedure.

3.3. Criteria of Reproducibility of the Results of HPLC

Measurements
The criterion of reproducibility of the results of HPLC
measurements with respect to the repeatability of injections
is usually formulated in terms of the relative standard deviation
RSD of the peak areas or heights. In the quantitative
analysis of drugs, the permissible value is usually restricted
at RSD .
2.0% for the main components and RSD .
5.0%
for impurities. Higher RSD values should be avoided or their
validity should be additionally justified.

The quantitative analysis of parent compounds requires

increased accuracy because the rated content of the main active
component is typically allowed to differ from 100% only
Validation of HPLC Techniques for Pharmaceutical Analysis

within 1 � 3%. For this reason, it is usually required that the

sequentially measured chromatograms (three to six) have
RSD of the peak areas or heights not exceeding 1%. However,
even this value does not always provide an acceptable
confidence interval. For this reason, PR 2001 [22] suggested
a formula for determining the maximum possible RSD values
depending on the upper rated limit (B ) of the drug content
and the number of repeated injections m. According to
this, the possible RSD (Table 4) is calculated for a series of
injections of a reference solution as

BK m

RSD.
, (15)

max

t
%, m #1

90

Here, K = 0.349 is a constant calculated as

(.K .
06 2)( .
t 090 6 ), where 0.6.
2 is the �required�

.%, 5

RSD for six injections at B = 1.0; m is the number of repeated

injections of the reference solution (3 .
m .
6);
t 90%(m � 1) is the Student coefficient for a 90% confidence
probability (two-sided) and f = m � 1 degrees of freedom. A
disadvantage of this approach is introduction of the constant
K involving an empirical �required RSD = 0.6.
2.

For example, if the rated content of the main component

is 98.0 � 101.0%, the RSD for a suitable chromatographic
system can be determined for B = 1.0% (Table 4). According
to this table, the RSD of the peak area (height) must not exceed
0.21% for three parallel determinations and 0.42% for
six determinations.

4. REVALIDATION OF HPLC PROCEDURES

Repeated validation may be required in the following
cases [7]: (i) modification of the ready-to-use drug; (ii) modification
of the parent drug synthesis technology; (iii) modification
of the analytical procedure.
According to the USP, users of the analytical procedures
described in this pharmacopoeia and the National Formulary
need not perform revalidation and only have to check for the
suitability of method under real analytical conditions [7].
This implies that, if a laboratory makes a reference to some
method stipulated in the USP, it is obliged to use this procedure
exactly as described (nonmodified) in order to avoid the
need for complete revalidation of a �modified� method. Obviously,
all procedures of sample preparation for the tests
have to be thoroughly performed without any deviations
from the validated procedure.

A special feature of HPLC (and other chromatographic

techniques) is the dependence of the parameters of the chromatographic
system on the sorbent type (C8, C18, etc) and
on the grade and even manufacturer of a particular sorbent.
For this reason, it is usual practice in HPLC (and other chromatographic
techniques) to perform an objectively unavoidable
modification of the chromatographic conditions as compared
to those stipulated by the validated procedure, with observation
of the criteria of suitability of the chromatographic

TABLE 4. Maximum Permissible Values of the Relative Standard

Deviation RSD Depending on the Upper Limiting Content (B)ofthe
Analyzed Compound and the Number of Sequential Injections (m)

m =3 m =4 m =5 m =6

B,%

Maximum permissible RSD, %

1.0 0.21 0.30 0.37 0.42

1.5 0.31 0.44 0.55 0.64
2.0 0.41 0.59 0.73 0.85
2.5 0.52 0.74 0.92 1.06
3.0 0.62 0.89 1.10 1.27
4.0* 0.83 1.19 1.47 1.70
5.0* 1.04 1.49 1.83 2.13
*

Values calculated in this study.

system. Until now, there is no commonly accepted opinion

which kinds of modification of the chromatographic system
can be considered insignificant and requiring additional
revalidation of the entire procedure. Nevertheless, some acceptable
limits of variation of the main parameters have been
published [22, 31], which can be considered as a basis for assessing
the need for revalidation even of an �insignificantly
modified� analytical procedure.

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