Pharmaceutical Chemistry Journal
4, 2004
STRUCTURE OF CHEMICAL COMPOUNDS,
METHODS OF ANALYSIS AND PROCESS CONTROL
VALIDATION OF HPLC TECHNIQUES
FOR PHARMACEUTICAL ANALYSIS
N. A. Épshtein1
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 re-
sults 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 Pharmaco-
poeia, it is not necessary to evaluate their suitability, pro-
vided that the analyses are conducted with strict observation
of the text of each particular article. In most other cases, es-
pecially in cases of modification of the drug composition, the
scheme of synthesis, or the analytical procedure, it is neces-
sary 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 in-
volved in the metrological attestation of analytical tech-
niques.
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 sub-
stances, statistical processing of the experimental results is obligatory [8].
212
Vol. 38, No.
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 Re-
lated 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 sec-
tions are devoted to these problems in national and interna-
tional pharmacopoeias and guides on the validation of ana-
lytical procedures [7, 20 – 22]. Also available are computer
program packages, such as ELISA Method Validation Tem-
plates (Waters) and LaChrom 2000 Validation Manager
(Merck) representing electronic tables with incorporated
functions of statistical processing of the results of measure-
ments and issuing validation certificates, and monographs on
the related subjects [23 – 34].
Tables 1 and 2 summarize the most recent recommenda-
tions concerning selection of the validation characteristics
depending on the type of analytical procedures. A compara-
tive analysis of these data shows that, according to the
United States Pharmacopoeia (USP-26), methods of dissolu-
tion testing have to be validated only with respect to preci-
sion (repeatability, reproducibility), while the other charac-
teristics “may require validation, depending on the specific
test nature.” In contrast, according to the FDA CDER guide-
lines [15], procedures used for quantitative analysis and dis-
solution testing need the same validation characteristics.
0091-150X/04/3804-0212 © 2004 Plenum Publishing Corporation
Validation of HPLC Techniques for Pharmaceutical Analysis 213

Moreover, according to CDER [15], all methods of quantita- Specificity (Section 1.1)
tive analysis have to be characterized with respect to robust-
Recommended requirements
ness (see below). Robustness is not included in USP-26 [7] 2. Precision (Section 1.2) to the repeatability of sample
because this characteristic has to be studied at the stage of 2.1 Repeatability (Section 1.2.1) injections (Section 3.3)
2.2 Intermediate precision
development of an analytical procedure, rather than in the (Section 1.2.2)
course of validation. 2.3 Reproducibility. Checked
in special cases
Since any analytical situation poses a multifactor prob- (Section 1.2.3)
lem, validation has to include at least testing of the analytical 3. Linearity (Section 1.3)
system as a whole under the conditions stipulated by the de-
It is expedient to determine
scription.3 The second task is evaluation of the stability (ro- 4. Accuracy (Section 1.4) these validation characteristics
bustness) of the analytical system with respect to small varia- in the course of proof of the
analytical system accuracy
tions of the main factors (for example, the ratio of the mobile 5. Suitability range (Section 1.5) (using statistical parameters
phase components, pH, temperature, etc.). This problem is determined for the calibration
graph) (Sections 1.3; 1.4;
less important than the first one because it is usually solved 1.6.2; and 1.7.2)
6. Limit of detection (Section 1.6)
at the stage of development of a given analytical procedure.
For this reason, problems pertaining to robustness are only
briefly mentioned in this review. 7. Limit of quantitation
(Section 1.7)
Let us consider the main stages of validation of the
HPLC techniques used in pharmacy. 8. Stability of solutions
(Section 1.8.)
1. TESTING THE ANALYTICAL PROCEDURE AS A
UNIFIED SYSTEM UNDER THE CONDITIONS 9. Robustness (stability) of HPLC
STIPULATED IN THE DESCRIPTION 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)
Figure 1 shows the general scheme of evaluation of the phase components, pH,
suitability of an analytical procedure, which takes into ac- temperature, etc.). Robustness
is usually studied in the stage
count specific features of HPLC. As can be seen, testing an of development of the given
analytical procedure as a whole in the general case allows the HPLC tecnique (Section 2)

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 tech-
nique) 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 de-
tect a given substance unambiguously (reliably) in the pres-
ence of other components (including impurities) that may be
present in the samples [1 – 4]. The proof of specificity de-
pends 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 determi-
nation of the content of a parent substance, the parame-
ters of solubility, and the homogeneity of dosage. In order
to confirm the specificity of these procedures, it is usually re-
quired that peaks of the substances to be determined are suf-
ficiently 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 sepa-
3
According to this concept, instrumentation, electronics, analytical proce-
dures, and analyzed samples constitute a unified analytical system, which
can be considered as a whole [7].
4
Sometimes, the stability of phases and solutions is considered within the
framework of the problem of robustness.
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 sub-
stance 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 separa-
tion coefficient Rs. Recommended values of this coefficient
are given below in the Section 3.3.
It is recommended to confirm the specificity by investi-
gation of the “purity” of peaks of the parent substance to be
determined [4, 27]. This test is usually performed using a di-
ode matrix detector and a special program evaluating spec-
tral 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 l (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 propor-
tional 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-
214 N. A. Épshtein

TABLE 1. Validation Characteristics according to th United States TABLE 2. Validation Characteristics Recommended for Various
Pharmacopoeia (2003) Tests by the United States FDA (CDER and CBER) [15]
Category Type of analytical procedure
II Tests for impurities Qualitative
Characteristic
I III IV Chatacteristic determina-
Quantita- Limiting Identifica-
tion and dis-
tive tests tests tion quantitative limiting solution
Accuracy Yes Yes MB MB No tests
Precision Yes Yes No Yes No Accuracy No Yes No Yes
Specificity Yes Yes Yes MB Yes Repeatability No Yes No Yes
Limit of detection No No Yes MB No Intermediate precision No Yes1 No Yes1
Limit of quantitation No Yes No MB No Specificity Yes2 Yes Yes Yes4
Linearity Yes Yes No MB No Limit of detection No No3 Yes No
Suitability range Yes Yes MB MB No Limit of quantitation No Yes No No
Notes: Yes = usually studied; No = usually not studied; MB = may Linearity No Yes No Yes
be required (depending on a specific test nature). Category I in- Suitability range No Yes No Yes
cludes analytical methods intended for determination of the content Robustness No Yes No3 Yes
of the main component in parent substances or ready-to-use medici-
nal forms; category II includes methods of determination of impuri- Notes: Yes = usually studied; No = usually not studied; 1 in cases
ties and decomposition products; category III includes methods of where the reproducibility is studied, there is no need to specially de-
determination of the parameters of dissolution, drug release, etc.; termine the intermediate precision; 2 insufficient specificity of a
category IV includes identification tests; the terms “accuracy” and given analytical procedure can be compensated by introducing addi-
“precision” (repeatability, reproducibility) are treated in accordance tional tests; 3 may be required in some cases (e.g., when the limit of
with the State Standard GOST R ISO 5725–1-2002. detection is close to the rated limiting content of an impurity); 4 in-
sufficient specificity of a given analytical procedure can be compen-
sated by determining the content of impurities.

trast angle q). If q = 0, the spectra are considered similar (ho-

mogeneous). This implies that the value of absorption in one
spectrum measured at a given wavelength l can be obtained
from the value in another spectrum measured at the same
wavelength by multiplying by a certain constant factor. In or-
der to evaluate the spectral homogeneity of a chromato-
graphic peak, the spectral contrast angles q are calculated for
all points of this peak relative to the angle at the peak maxi-
mum and then the maximum value qp (purity angle) is deter-
mined.
Two spectra determined for the same substance can dif-
fer, for example, because of the influence of the baseline
noise. In order to take this into account, a threshold spectral
angle qth 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 qp
for each chromatogram, and considering the maximum of
these values as the threshold spectral angle qth. This thresh-
old angle characterizes the level below which the difference
between two spectra can be considered insignificant. The ob-
tained qp values are compared to qth. For qp < qth, the peak is
considered spectrally homogeneous; otherwise the peak is
influenced by the presence (i.e., additional absorption) of an-
other 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-
cause the spectrum can be influenced by variations in the
mobile phase composition (e.g., under gradient HPLC condi-
tions), 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 densi-
ties 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 determi-
nation 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 de-
termining 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 de-
tected 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). De-
velopers 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 ³ 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 pla-
cebo (for filled drugs), (d) the test solution, and (e) the solu-
tion used for the evaluation of suitability of the chromato-
graphic 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 (unidenti-
fied) or absent. In this case, it is expedient to use special ex-
periments involving modification of the parent compound
(sometimes called “stress testing” [2, 15, 27]), whereby a so-
lution of the given drug (or of the parent compound) is sub-
jected to factors leading to its partial destruction with the for-
mation of related identifiable compounds. The degree of de-
composition 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 modifica-
tion.
(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 result-
ing solid products of decomposition can be stored and used
as control substances in the tests for suitability of a chro-
matographic system.
(iv) Photochemical decomposition under illumination
(e.g., UV irradiation).
(v) Chemical addition reactions, for example, drug
bromination at multiple bonds.
215
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 modifica-
tion 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 com-
pensated 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 maxi-
mum 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 an-
alytical 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 calcu-
lation of (i) the separation coefficient Rs 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 devel-
opers. It is very important to select the optimum chromato-
graphic 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 cer-
tain number of such peaks, a part of which cannot be quanti-
tatively 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 de-
veloping and validating the methods of impurity determina-
tion (especially in the case of parent compounds), it is expe-
dient 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 con-
taining large amounts of auxiliary substances.
216 N. A. Épshtein

1.2. Confirming the Precision of a Given Analytical Pro- This characteristic is not included in the list of CDER [15]
cedure and USP-26 [7]. In HPLC validation, data on the injection
The precision (repeatability, reproducibility) of an ana- repeatability should be provided as additional material re-
lytical procedure characterizes the random scatter (variation) quired in cases of search for the “bottleneck” of a proposed
of the results relative to the mean value. It should be empha- method (dispersion analysis).
sized that, in order to obtain reliable results, the sample must 1.2.1. Injection repeatability and intra-assay preci-
have a homogeneous composition. The results have to be sta- sion. In the general case, repeatability (Rp) characterizes the
tistically treated. Variants leading to rough errors (e.g., using reproducibility of a given analytical procedure for the same
variation range R or the “three sigma” rule [8]) should be re- sample preparation, as performed by the same analyst using
jected. In evaluating the suitability of HPLC procedures (ac- the same instrument (chromatograph) during a relatively
tually, in determining the metrological characteristics), it is short period of time. For evaluating the repeatability in a
necessary to use measuring vessels of class A or special cali- given laboratory, the same analyst prepares samples of a
brated measuring vessels and take all other possible mea- model mixture or the same batch of a parent compound or a
sures to increase the reproducibility and accuracy of HPLC drug:
measurements [27]. (a) not less that nine samples of solutions covering the
The precision (repeatability, reproducibility) of an ana- rated range of concentrations. For example: a homogenized
lytical procedure is evaluated in terms of the standard devia- powder of triturated tablets from the same batch is used to
tion (SD) of the relative standard deviation (percentage prepare drug solutions with concentrations equal to 50, 100,
RSD) determined in a series of measurements and calculated and 150% of the reference sample solution concentration ac-
by the formulas cording to the proposed procedure, or
(b) not less than six samples of solutions in the region of
m concentrations close to the nominal value. For example: a
SD = å( X i - X )2 ( m - 1) , homogenized powder of a parent compound or triturated tab-
i =1
lets from the same batch is used to prepare six solutions with
100SD nominal concentration according to the proposed procedure.
RSD(%) = . (1) Note that each sample solution is (i) prepared independ-
X
ently of the other solutions and (ii) chromatographed at least
According to the ICH recommendations [4] on the vali- three times.
dation of chromatographic procedures, the characteristics of Each solution is characterized by the drug content X i
precision are considered at three levels: repeatability, inter- (i = 1, …, N ), the average value X = S X i N , the standard
mediate precision, and reproducibility (Table 3). deviation SD, the relative standard deviation RSD of particu-
In [4, 27] and in many other papers, the set of validation lar measurements, and the confidence interval (for P = 95%)
characteristics of HPLC procedures includes the injection re- of the average value.
peatability. However, the author believes that this is incor- It is required to show that the average results are statisti-
rect: this parameter characterizes the quality of injector (e.g., cally equivalent (e.g., in terms of the Student t-criterion) or,
syringe) rather than the suitability of a proposed procedure. 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
TABLE 3. Main Precision Characteristics for the Validation of 1.2.2. Intermediate precision. This value characterizes
HPLC Procedures According to ICH [4] the reproducibility of results obtained in the same laboratory
Precision characteristic Conditions of determination by different analysts using various instruments
Repeatability Injection Determined for the same sample (chromatographs) during a prolonged period of time (not less
(Rt ) repeatability preparation by the same analyst than two days) for the same homogeneous sample or a model
using the same instrument drug mixture according to the proposed analytical procedure.
(chromatograph) during a short Typically, not less than six solutions are prepared with
period of time
concentrations close to the nominal value (see the preceding
Intra-assay
precision
section). Each sample solution is (i) prepared independently
Intermediate – Determined for the same sample
of the other solutions and (ii) chromatographed at least three
precision (Ip ) preparation by different analysts times.
using various instruments
(chromatographs) during a pro- 5
longed period of time (not less For small values of the standard deviation (SD << 1), the t-criterion may
than two days) give statistically significant differences even for close (almost identical)
values of the compared average concentrations. This is related to the fact
Reproducibility – The same, in various laboratories that this criterion is proportional to the ratio of systematic and random er-
(Rp ) rors.
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 ana-
lysts, X i and X j (i, j = 1, …, N ). These data are statistically
processed and characterized by generalized average values
X 1 = S X i N and X 2 = S X j N and the corresponding
standard deviations (SDi, SDj ) and relative standard devia-
tions (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 stan-
dard deviations SDi and SDj of 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
| X 1 - X 2| | X 1 - X 2|
t= = ,
SD12 SD 22 SD12 + SD 22
+ laboratories capable of reproducing the proposed procedure
m1 m2 m with high precision and (ii) this requires high organizational
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 admit-
ted 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 (SDi or SDj ) relative to the average result of an-
other analyst (with a lower SDi or SDj) does not exceed a
certain preset level, for example, so that RSD £ 10% for im-
purities with a rated content up to 1%, RSD £ 25% for an im-
purity content within 0.1 – 1%, and RSD £ 50% for an impu-
rity 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 inter-
mediate 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 be-
217
tween the results obtained in such investigation, it is neces-
sary that the round robin tests involve not less than five
laborqtories [35].
In practice, however, the reproducibility is usually evalu-
ated using two or three laboratories and characterized by less
strict estimates. For example, validation of a procedure pro-
posed for the quantitative determination of a parent com-
pound is performed by demonstrating the statistical equiva-
lence of the standard deviations SDi and SDj of the results
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 (SDi or SDj) relative to the
average results of analyses in other laboratories (with lower
SDi or SDj values) does not exceed a certain preset level [13].
The full-scale reproducibility of analytical procedures is
rarely validated because (i) it is necessary to involve certified
facilities and expenditures.
1.3. Confirming Linearity of the Response to Drug Con-
centration
Linearity characterizes the ability of a proposed analyti-
cal procedure to give (within the suitability range) a response
signal with the magnitude Y (e.g., peak height or area) di-
rectly proportional to the amount C (concentration) of a drug
to be determined: Y = a + bC. According to ICH recommen-
dations [3], the linearity in practice is first visually estimated
from the linear appearance of the plot of Y versus C. If the
plot appears linear, this relation is studied by methods of re-
gression 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 concentra-
tions [13]. For this reason, ICH [3] recommends that the lin-
earity be validated by a plot of the difference Y–C showing
deviations (residuals) of the calculated values yi = a + bC
from the measured Yi values as the function of the concentra-
tion Ci. The “outbursts” of the points (xi, yi ) relative to the
regression model can be determined by calculating the pa-
rameter t using the formula [36]
| y i -Y i |
t= ,
1 (Y i - y ) 2
SD 0 1+ +
N ( N - 1) × SD 2y
Here, yi and Yi are the calculated and experimentally mea-
sured values of the response, respectively; y = Syi /N; N is
the total number of experimental points (xi , yi ), and
218
S( y i - Y i ) 2 S( y i - y ) 2
SD 0 = , SD y = . factor of 2000!) to 2.5%. Instead of making some dilutions, it
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 pa-
rameter 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 some-
times performed proceeding from a correlation coefficient of
r ³ 0.98 [13]. According to our estimates, use of the calibra-
tion 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 ³ 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 ar-
tificial mixtures including components of the drug studied.
The most adequate approach consists in taking thoroughly
weighed aliquots of the drug components and preparing solu-
tions according to the proposed procedure, since all opera-
tions of the analyst should correspond strictly to those stipu-
lated in the description. In practice, however, an “intermedi-
ate” 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 proce-
dure 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 ex-
pected concentration interval (suitability range of the pro-
posed procedure). Then, a series of dilutions is prepared so
as to cover the entire range. Each solution is studied in a se-
ries 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 com-
pound (i.e., using solutions of the parent compound as de-
scribed in (a)). However, it is incorrect to ignore the possibil-
ity that auxiliary components (placebo) may influence the re-
sults. 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. Épshtein
drug concentrations in the range from 0.05% (dilution by a
is possible to use a proportional decrease in the volume of
applied sample (which saves the mobile phase).
Data presentation. The validation characteristics in-
clude (i) a regression equation of the Si = a + bC type (for ex-
ample, S = 15536 + 15833969C [mg/ml]), (ii) the correla-
tion 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 Pro-
cedure
The accuracy characterizes the proximity of the experi-
mental results, obtained using a proposed analytical proce-
dure, to the “true” value in the entire suitability range of this
procedure. The accuracy represents a combination of the ran-
dom and systematic error.6 In order to provide for accurate
HPLC determinations, it is recommended to use standard so-
lutions with concentrations close to within 10% of the test
solution concentration.
The accuracy of analytical procedures should be deter-
mined using homogeneous samples with exactly known con-
centrations of the compounds to be determined. For valida-
tion purposes a series of such solutions is prepared using the
reference parent compound. According to ICH recommenda-
tions and USP-26 [1, 7, 15], the accuracy can be expressed
both in the classical form, as the difference X – m between
the average experimental value (X ) and the true value (m)
with the corresponding confidence interval DX ,7
( X - m ) ± DX , (2)
and in an alternative (and more illustrative) form, in terms of
the percentage recovery of the known amount of the com-
pound 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 ap-
proach provides a more illustrative characteristic of the reli-
ability of results obtained using a proposed analytical proce-
dure 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 character-
izes the accuracy, since this quantity is also random and re-
quires 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 ± DR . (4)
Obviously, the proposed methods should not involve sig-
nificant systematic errors. In the absence of systematic er-
6
Accordiong to the State Standard GOST R ISO 5725-1–2002 the system-
atic error usually characterizes “trueness.”
7
The value of DX 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 experi-
ence, and analysis of the published data [13, 27], it is possi-
ble 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 recov-
ery with allowance of the confidence interval does not fall
outside the following limits:
99.0–101.0%, for the quantitative analysis of parent sub-
stances with a high rated content of the active component
(98% and above);
98.0–102%, for the quantitative analysis of parent sub-
stances 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 impuri-
ties with a rated maximum content of up to 1%;
75–125%, for the quantitative determination of impuri-
ties with a rated maximum content from -0.1 to 1%;
50.0–150%, for the quantitative determination of impuri-
ties with a rated maximum content below 0.1%.
1.4.1. The procedure of accuracy evaluation. ICH rec-
ommends 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 us-
ing not less than nine determinations at various concentra-
tions 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 (Sec-
tion 1.6.2). It should be emphasized that these determinations
should include all stages of the proposed analytical proce-
dure.
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 intro-
duced into the placebo (matrix). This method, used for the
219
analysis of drugs comprising mixtures of parent and auxil-
iary 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 intro-
duced 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 concentra-
tions would cover the entire expected suitability range of the
proposed analytical procedure. For example, a parent com-
pound 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 gen-
erally 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 pro-
posed 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 la-
bel 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 stip-
ulated by the proposed analytical procedure.
For the quantitative determination of impurities, the ac-
curacy 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 lim-
ited applicability because these validation procedures require
large amounts of identified impurities. In this case, the accu-
racy is most frequently checked using the method described
below.
The method of internal normalization with or without
response factors. According to this method, identified impu-
rities are characterized by the response factors with respect
to the parent compounds determined by the analytical proce-
dure 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 impuri-
ties, these factors are conventionally taken equal to unity (as-
suming 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 pro-
cedure 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 re-
gression analysis [8]. Each sample (whose total number is
N ³ 5) with known values m (introduced content) of the com-
ponent to be determined is analyzed in m = 3 – 6 parallel de-
terminations. The total data array is characterized by the dis-
persion (SD0) and the Student criterion8
| m - x| m
t=
SD 0
This t value is compared to tabulated values of the Stu-
dent 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 d. This er-
ror is calculated by the formula
| x - m|
d= 100%. (5)
m ters ta = |a|/SD0 and tb = |1 – b|/SD0 are calculated and com-
The standard deviation SD0 in a particular analysis is cal-
culated using the set of all m parallel determinations per-
formed for N (or g in the notation of [8]) samples. This al-
lows the SD0 value to be reduced and the sensitivity of deter-
mination of the systematic error to be increased with a
simultaneous decrease in the confidence interval for the re-
sults of analysis, DX = t SD0/ m, where t is the Student cri-
terion 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 experimen-
tal value from average, d i = X i - X , and the dispersion
æm ö the criteria described in Section 1.3) and with finding the
SD 2k = ç å d i2 ÷ ( m - 1). Then, the difference between the
è i =1 ø
maximum and minimum values of the dispersion SD 2k is
checked to be insignificant in terms of the Fisher F-criterion.
If this difference is actually insignificant, the SD 20 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 - m | and the
random error SD0/ m.
N. A. Épshtein
N
å SD k2
k =1
SD 20 = .
N
Finally, the Student t-criterion is calculated as
t = (| m - x |× m ) SD 0 (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 insig-
nificant) systematic error and small random error, the calcu-
lated t-criterion can be greater than the tabulated value. How-
ever, 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 meth-
ods of evaluation of the accuracy of a proposed analytical
procedure.
Method of regression analysis with the Student t-cri-
terion [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 vari-
able systematic errors, not less than N = 5 samples with
known values of the parent compound are studied and the re-
lationship between the “introduced content” (mt ) and the
“found content” (mf ) is processed by least squares in terms
of the equation mf = a + bmi. Using these data, the parame-
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 ta £ t (P,
f = N – 2), and the absence of a linear variable systematic er-
ror, 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 func-
tion of the concentration of the parent compound to be deter-
mined (in fact, linearity of the calibration graph in terms of
limit of detection (Section 1.6.2) and the limit of quantitation
(Section 1.7.2).
Method of regression analysis with the Fisher F-crite-
rion. 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 ³ 5
samples (model mixtures) with known values of the parent
compound, after which the relationship between the “intro-
duced 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 + bmt. The ab-
sence of a constant systematic error is confirmed by the in-
significance of the coefficient a [35] at a commonly accepted
confidence probability level of P = 95%. For this purpose,
the Fisher criterion calculated is as
F ( P, f 1 = N – 1, f 2 = N – 2 ) =
SD 201 ( N – 1) – SD 202 ( N – 2 )
, linearity is observed, (ii) the characteristics of repeatability
SD 202 ( N – 2)
where SD01 and SD02 are the standard deviations obtained
for the above relations without the free term (mf = bmt ) and
with the free term (mf = a + bmt ). If the calculated F value is
smaller than the tabulated (critical) values F (P = 95%, f1, f2),
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 system-
atic error. The final judgment about the accuracy of a pro-
posed 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 compo-
nent, etc.). The validation results are presented by data on the
recovery of the amount of introduced parent compound with
a confidence interval, R ± DR , or the difference between the
average experimental value X and the true value m with the
corresponding confidence interval: ( X - m ) ± DX .
The proposed procedure involves no significant system-
atic 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 sub-
stances with a high rated content of the active component
(98% and above);
98.0–102%, for the quantitative analysis of parent sub-
stances 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 impuri-
ties with a rated maximum content of up to 1%;
75–125%, for the quantitative determination of impuri-
ties with a rated maximum content from – 0.1 to 1%;
50.0–150%, for the quantitative determination of impuri-
ties 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, DX
(or DR) [37]. The number of significant digits in the latter
value is determined as follows. If the first significant digit in
the error is ³ 3, then DX is expressed by a value with one sig-
nificant digit; should the first significant digit in the error
9
It is expedient to confirm the linearity together with determining the accu-
racy.
221
be < 3, then DX is expressed by a value with two significant
digits.
1.5. Validation of the Suitability Range of a Given Ana-
lytical 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
(7),
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 ex-
pressed 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 de-
termined 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 de-
termination of the parent compounds and 10% for proce-
dures of impurity determination [13].
In practice, it is sufficient to show that a given range of
suitability covers “with margin” the rated limiting concentra-
tions 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 analyti-
cal procedure be not less than the following intervals [3, 7].
(i) For the quantitative determination of the main compo-
nent concentration in parent compounds and ready-to-use
drugs: from 80 to 120% of the nominal content (i.e., the con-
centrations 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 in-
terval 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 com-
pound, for which the release is rated as 20% within the first
hour and up to 90% within a 24-h period of time, the suitabil-
ity 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 possi-
bility 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 bi-
ological activity, toxicity, or unpredictable behavior, the lim-
its 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-
222
formed jointly and only the reference sample solution of the
main component at a nominal concentration is used, the suit-
ability 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 com-
pound. If the LPI value is unknown (e.g., during the develop-
ment 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 con-
centration.
1.6. Determining the Limit of Detection of a Given Ana-
lytical Procedure
The limit of detection (LOD) is determined as the mini-
mum concentration of analyzed substance in the sample,
which (i.e., the corresponding response) can be detected un-
der 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 impu-
rity concentrations. Obviously, the LOD may depend on the
HPLC detectors and pumps. For this reason, this characteris-
tic 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 show-
ing that a response peak exceeding the baseline noise is actu-
ally 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 clean-
ness of technological equipment). In most other cases, it is
sufficient to show that the impurity of interest is reliably de-
tected at a preset level (see Section 1.6.5).
1.6.1. Determining the LOD from the Standard Devi-
ation of the Response and the Slope of the Calibration
Curve. According to this method [3], the LOD value is cal-
culated by the formula
LOD = 3.3(SDa/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 de-
termine 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 deter-
mined using one of the two methods:
10
For the analysis of impurities, it was recommended [27, p. 695] to per-
form 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 solu-
tion.
N. A. Épshtein
(a) Using the calibration curve S = a + bC constructed
using the results of analyses for a series of the reference sam-
ple 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 SDa of the free term is deter-
mined. Alternatively, the standard deviation is determined
for the intersections of several regression lines of the calibra-
tion 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 concentra-
tions 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 de-
scribed 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 × t ( P, f ) × SD 0 nj
LOD = , (9)
Y - a + t ( P, f ) × SD 0 nj
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; nj is 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 sub-
jective estimate of the maximum noise N¢.
1.6.4. Evaluating the LOD from the Minimum Con-
centration 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 the-
oretically 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 ex-
ceed 20%. However, this approach is not convenient in prac-
tice 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 ac-
count the so-called “specific LOD.” These values are deter-
mined using mixtures of a given parent substance with a pla-
cebo 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 vari-
ous columns and instruments because this characteristic de-
pends 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 mV, which
is one order of magnitude higher than the noise level of mod-
ern detectors. For validating the reliable detection of impuri-
ties, it is sufficient to present the chromatogram of a refer-
ence (or test) solution diluted D = 2L times, where L is an in-
teger indicating the ratio of the main component
concentration to the minimum rated concentration of the im-
purity. This chromatogram must visually confirm the possi-
bility 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 impu-
rity content of 100/D = 0.05% (the British Pharmacopoeia
does not take into account impurities with concentrations be-
low 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 deter-
mination. If the LOD was calculated from experimental data,
it is necessary to present the results of analysis of the re-
quired 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 pres-
ent chromatograms confirming reliable detection of the
peaks of parent compounds or impurities.
The number of significant digits in the LOD [39] is de-
termined 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.
223
1.7. Validation of the Limit of Quantitation of a Given
Analytical Procedure
The limit of quantitation (LOQ ) is the minimum concen-
tration of analyzed substance that can be determined at an ac-
ceptable precision (repeatability, reproducibility) and accu-
racy 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 ra-
tio S/N¢ = 10. This is the most widely used method of deter-
mining 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 sig-
nal-to-noise ratio is at least 30 [27]. Then, this solution is se-
quentially 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 ± 10W0.5,
where W0.5 is the full width at half maximum (FWHM) of
this peak [22].
For HPLC procedures used in pharmacy, it would be in-
teresting to study the influence of the maximum noise N¢ on
the relative standard deviation of results for a nearly Gaussi-
an peak shape. According to [27],
50
RSD[%] = , (10)
S N¢
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 follow-
ing way [41]. First, the values of response Y (area under the
peak of the analyzed component) within the limits of the cal-
ibration curve Y = a + bC are used to calculate (i) the theoret-
ical values of C = (Y – a)/b and (ii) the standard deviations
224
(SDc) and relative standard deviations (RSD = SDc/C ´ 100)
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 analy-
ses are calculated by the formula [35]
2 2
SD 0 1 1 æ SD b ö æ Y s -Y ö
SD c = + +ç ÷÷ ç ÷
b N m çè b ø
ç SD
è 0
÷
ø reference solutions ST (%) can be evaluated using the fol-
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, Ys is
the response (area under the peak), m is the number of paral-
lel (independent) determinations, b is the slope of the calibra-
tion 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 in-
dication 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 ex-
trapolation, 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 rela-
tively short period of time (th ) for which the content of the
analyzed substances (proportional to the areas under the cor-
responding peaks) remains practically unchanged. In order to
confirm this statistically, it is sufficient to determine the coef-
ficient b in the regression relation St = S0 + bt (or
St – S0 = bt ) between the area St under the peak at the time t
N. A. Épshtein
(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.
(12) 1.8.2. Methods of evaluation. The stability of test and
lowing methods.
(i) For continuous testing within a relatively short period
of time — by the ratio of the area St under the peak of the an-
alyzed compound (or impurity) at the moment t to the initial
area S0:
ST = 100St /S0. (13)
(ii) Irrespective of the duration of experiment — by the
ratio of the amount mt (concentration) of the analyzed com-
pound (or impurity) at the moment t to the initial value m0:
ST = 100mt /m0. (14)
The test and reference solutions are prepared according
to the given procedure and analyzed with certain time inter-
vals. The maximum possible duration of measurements de-
pends 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 com-
ponent (or impurity) according to the given analytical proce-
dure.11
Data presentation. The stability of solutions is con-
firmed by data on the storage times of solutions and mobile
phases used in the analyses.
2. EVALUATION OF THE STABILITY OF ANALYTI-
CAL SYSTEMS WITH RESPECT TO SMALL VARIA-
TIONS OF PARAMETERS (ROBUSTNESS)
The robustness of an analytical procedure is the charac-
teristic of its stability with respect to small variations of the
system parameters possible under real conditions. This sta-
bility 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 ana-
lytical procedure. A more correct procedure based on evalua-
tion of the statistical significance of the difference between
these results in terms of the Student t-criterion is rarely ap-
plied 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 in-
fluencing 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 de-
scription 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 meth-
ods of experiment planning, which are capable of quantita-
tively 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 pro-
posed 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.
225
The additives may include other analyzed components, iden-
tified 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 sub-
stances. 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 chro-
matographic system is adequately described by the following
main criteria.
(i) Criteria of the separating power of the chromato-
graphic 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 measure-
ments (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 proce-
dures.
3.1. Separating Power of a Chromatographic System
The criteria of separating power include the separation
coefficient Rs , the peak-to-valley ratio h/v (see below), and
the relative retention times of components.
3.1.1. Relative retention times. For analytical proce-
dures intended for the quantitative analysis of drugs, homo-
geneous dosing tests, and determination of the drug’s disso-
lution characteristics, it is usually sufficient to characterize
the separating power by the relative retention times of sev-
eral substances, including the main drug component, a struc-
turally close compound, and/or an internal standard. The rel-
ative 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 suit-
ability requirements are satisfied). It is more correct to indi-
cate 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 rela-
tive retention times.
226
3.1.2. Peak separation coefficient. For analytical proce-
dures intended for the determination of impurities, the sys-
tem suitability requirements should indicate the minimum
possible values of the peak separation coefficient Rs at least
for the main (rated) impurities. In the case of HPLC proce-
dures intended for the quantitative analysis of drugs, homo-
geneous dosing tests, and determination of the drug dissolu-
tion characteristics, it may be necessary to introduce the peak
separation coefficient into the system suitability require-
ments 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 com-
ponents occur in the vicinity of the main analytical peaks.
The minimum possible values of the separation coeffi-
cient Rs 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 Rs ³ 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 tail-
ing, CDER recommends that Rs ³ 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 coeffi-
cient to Rs ³ 2.5. This situation is quite rare, being only typi-
cal of drugs containing several heteroatoms capable of form-
ing strong hydrogen bonds with silanol groups of the sorbent.
Higher “critical” Rs values are usually unnecessary, be-
cause the condition Rs > 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 Rs < 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 com-
ponents and impurities are incompletely separated), the sys-
tem suitability requirements may describe the peak separa-
tion in terms of the peak-to-valley ratio p/v = hp/hv, where hp
is the peak height relative to the extrapolated baseline and hv
is the height of the lowest point in the valley separating the
peaks (relative to the same extrapolated baseline). This pa-
rameter is rarely used during the analysis of impurities in
drugs: sometimes it is convenient for the description of im-
purity peaks occurring immediately in front of the main ana-
lytical peak. For suitability of the chromatographic system, it
is usually required that hp > hv. For example, in the analysis
N. A. Épshtein
of fluoxetin hydrochloride, it is required that
hp/hv ³ 1.1/0.1 = 9.
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 espe-
cially 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 arti-
cles 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 be-
low, the boundaries of peaks practically cannot be deter-
mined.
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 charac-
terizes the peak width at half height (FWHM) and is calcu-
lated by the conventional formula [7]. The ICH recommends
using columns with efficiencies N ³ 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 ³ 1000 theoretical plates.
In some cases in the HPLC determination of the main
drug components, USP-26 admits N ³ 400 theoretical plates.
However, even this is not the minimum possible level: rapid
analyses are sometimes performed using short chromato-
graphic columns with 3.5-mm (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 re-
spect 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 devia-
tion 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 ac-
tive component is typically allowed to differ from 100% only
Validation of HPLC Techniques for Pharmaceutical Analysis 227

within 1 – 3%. For this reason, it is usually required that the TABLE 4. Maximum Permissible Values of the Relative Standard
sequentially measured chromatograms (three to six) have Deviation RSD Depending on the Upper Limiting Content (B) of the
RSD of the peak areas or heights not exceeding 1%. How- Analyzed Compound and the Number of Sequential Injections (m)
ever, even this value does not always provide an acceptable m=3 m=4 m=5 m=6
B, %
confidence interval. For this reason, PR 2001 [22] suggested Maximum permissible RSD, %
a formula for determining the maximum possible RSD val- 1.0 0.21 0.30 0.37 0.42
ues depending on the upper rated limit (B ) of the drug con- 1.5 0.31 0.44 0.55 0.64
tent and the number of repeated injections m. According to 2.0 0.41 0.59 0.73 0.85
this, the possible RSD (Table 4) is calculated for a series of 2.5 0.52 0.74 0.92 1.06
injections of a reference solution as 3.0 0.62 0.89 1.10 1.27
4.0* 0.83 1.19 1.47 1.70
BK m
RSD max = , (15) 5.0* 1.04 1.49 1.83 2.13
t 90% , m - 1
*
Values calculated in this study.
Here, K = 0.349 is a constant calculated as
K = ( 0.6 2 ) × ( t 0.90%,5 6 ), where 0.6/ 2 is the “required”
RSD for six injections at B = 1.0; m is the number of re- system. Until now, there is no commonly accepted opinion
peated injections of the reference solution (3 £ m £ 6); which kinds of modification of the chromatographic system
t 90%(m – 1) is the Student coefficient for a 90% confidence can be considered insignificant and requiring additional
probability (two-sided) and f = m – 1 degrees of freedom. A revalidation of the entire procedure. Nevertheless, some ac-
disadvantage of this approach is introduction of the constant ceptable limits of variation of the main parameters have been
K involving an empirical “required RSD = 0.6/ 2. published [22, 31], which can be considered as a basis for as-
For example, if the rated content of the main component sessing the need for revalidation even of an “insignificantly
is 98.0 – 101.0%, the RSD for a suitable chromatographic modified” analytical procedure.
system can be determined for B = 1.0% (Table 4). According
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