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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. 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 dation [5] and on the presentation of samples and analytical
technique is a procedure aimed at obtaining experimentally data pertaining to the validation of methods [6]. In 1993, the
justified evidence of the ability of this technique to give re- International Conference on Harmonization (ICH) developed
sults characterized by the required accuracy and precision generalized recommendations on the validation of analytical
[1 – 7].2 All analytical techniques used for the development procedures; these documents were published in 1994 and
of pharmaceuticals and for the determination of their quality treated in more detail in 1995 [1 – 3]. In 1994, the US FDA
characteristics have to be validated. In the case of using Center for Drug Evaluation and Research (CDER) issued a
methods stipulated and described in the State Pharmaco- guide on the validation of chromatographic methods [4].
poeia, it is not necessary to evaluate their suitability, pro- These documents and some review papers and monographs
vided that the analyses are conducted with strict observation [9 – 13] provided a basis for extensive implementation of the
of the text of each particular article. In most other cases, es- procedure of validation of analytical methods. The Internet
pecially in cases of modification of the drug composition, the offers the Laboratory Guide to Method Validation and Re-
scheme of synthesis, or the analytical procedure, it is neces- lated Topics at http://www.eurachem.ul.pt /. In recent years,
sary to re-evaluate the suitability of the analytical techniques. new guides have become available from CDER [15], Waters
This paper is aimed at (i) considering the peculiarities of Company [16], and Labcompliance [17 – 19]. Special sec-
validation of HPLC techniques for pharmaceutical analysis, tions are devoted to these problems in national and interna-
(ii) critically assessing the main approaches to evaluation of tional pharmacopoeias and guides on the validation of ana-
the validation characteristics, and (iii) providing practical
lytical procedures [7, 20 – 22]. Also available are computer
recommendations and criteria for finding correct solutions.
program packages, such as ELISA Method Validation Tem-
The USSR State Pharmacopoeia (valid in the Russian
plates (Waters) and LaChrom 2000 Validation Manager
Federation) introduced the section “Statistical Analysis of
(Merck) representing electronic tables with incorporated
Biological Test Results” in 1968 (Xth Ed.) and the section “
functions of statistical processing of the results of measure-
Statistical Processing of Chemical Experimental Data” in
ments and issuing validation certificates, and monographs on
1988 (XIth Ed.). These sections are devoted to problems in-
the related subjects [23 – 34].
volved in the metrological attestation of analytical tech-
Tables 1 and 2 summarize the most recent recommenda-
niques.
In 1987, the United States Food and Drug Administration tions concerning selection of the validation characteristics
(FDA) issued practical guides on the main principles of vali- depending on the type of analytical procedures. A compara-
tive analysis of these data shows that, according to the
1
“Akrikhin” Chemico-Pharmaceutical Joint-Stock Company, Staraya
United States Pharmacopoeia (USP-26), methods of dissolu-
Kupavna, Moscow Region, Russia. tion testing have to be validated only with respect to preci-
2
Here and below the terms “accuracy” and “precision” (repeatability, sion (repeatability, reproducibility), while the other charac-
reproducibility) are treated in accordance with the State Standard GOST R
teristics “may require validation, depending on the specific
ISO 5725–1–2002. Previously, accuracy had the meaning of correctness,
but now correctness is described in terms of “trueness.” In justifying the test nature.” In contrast, according to the FDA CDER guide-
suitability of techniques related to the qualitative determination of sub- lines [15], procedures used for quantitative analysis and dis-
stances, statistical processing of the experimental results is obligatory [8]. solution testing need the same validation characteristics.

212
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]: Fig. 1. The general scheme of validation of HPLC-based analytical
(i) specificity; (ii) precision; (iii) linearity; (iv) accuracy; (v) procedures.
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 ration of peaks is confirmed by a set of chromatograms, at
may be required (see Tables 1 and 2). least of (a) the test solution, (b) the reference parent sub-
1.1. Confirming the Specificity of a Given Analytical stance solution, (c) the solvent (blank), (d) the placebo (for
Procedure (Separating Power of a Chromatographic System) filled drugs), and (e) the solution used for the evaluation of
By the specificity of a system is meant its ability to de- suitability of the chromatographic system. The degree of
tect a given substance unambiguously (reliably) in the pres- peak separation is usually described in terms of the separa-
ence of other components (including impurities) that may be tion coefficient Rs. Recommended values of this coefficient
present in the samples [1 – 4]. The proof of specificity de- are given below in the Section 3.3.
pends on the task of a given procedure and on the availability It is recommended to confirm the specificity by investi-
of reference samples of the main impurities. gation of the “purity” of peaks of the parent substance to be
1.1.1. The specificity of procedures aimed at determi- determined [4, 27]. This test is usually performed using a di-
nation of the content of a parent substance, the parame- ode matrix detector and a special program evaluating spec-
ters of solubility, and the homogeneity of dosage. In order tral homogeneity of the measured peak (e.g., Peak Purity
to confirm the specificity of these procedures, it is usually re- Millennium, Waters). The principle of evaluation of the peak
quired that peaks of the substances to be determined are suf- purity is as follows. The sample chromatograms are mea-
ficiently well resolved between themselves and from peaks sured at various detector wavelengths l (numbered n ). Each
of the main impurities, the system components (e.g., of the point of the peak is characterized by a spectrum, which is
sample solvent), and the placebo. For this purpose, the sepa- mathematically described by a vector in the n-dimensional
space of the values of absorption (in absorption units, AU) at
3
According to this concept, instrumentation, electronics, analytical proce- the preset n wavelengths (the length of this vector is propor-
dures, and analyzed samples constitute a unified analytical system, which
can be considered as a whole [7].
tional to the substance concentration in the solution studied).
4
Sometimes, the stability of phases and solutions is considered within the The difference between the spectra is evaluated by the angle
framework of the problem of robustness. 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
cause the spectrum can be influenced by variations in the
spectrum measured at a given wavelength l can be obtained
mobile phase composition (e.g., under gradient HPLC condi-
from the value in another spectrum measured at the same
tions), or by deviations from the Lambert – Beer law at high
wavelength by multiplying by a certain constant factor. In or-
levels of absorption. Therefore, negative results of evaluation
der to evaluate the spectral homogeneity of a chromato-
of the spectral homogeneity of peaks in the gradient HPLC
graphic peak, the spectral contrast angles q are calculated for
should be critically assessed and checked for optical densi-
all points of this peak relative to the angle at the peak maxi-
ties not exceeding 1 AU.
mum and then the maximum value qp (purity angle) is deter- It should be noted that specificity is rarely checked using
mined. chromatomass spectroscopy (HPLC – MS) — because of the
Two spectra determined for the same substance can dif- high cost of this procedure — and the chemical and other
fer, for example, because of the influence of the baseline analyses of the eluate fractions corresponding to the parent
noise. In order to take this into account, a threshold spectral substance, because of tedious procedures.
angle qth is determined (i) by determining the maximum 1.1.2. The specificity of procedures aimed at determi-
spectral angle between pints of the baseline with maximum nation of impurities. In evaluating the specificity of such
noise levels or (ii) by taking six chromatograms of a standard procedures, it is necessary to differentiate between two cases.
sample solution, determining the maximum purity angle qp Case 1. The main impurities are known and available.
for each chromatogram, and considering the maximum of In order to confirm the specificity of a procedure used for de-
these values as the threshold spectral angle qth. This thresh- termining the impurities in a parent substance, it is necessary
old angle characterizes the level below which the difference to show that (i) this procedure allows the peaks of the main
between two spectra can be considered insignificant. The ob- products of decomposition of the parent substance to be de-
tained qp values are compared to qth. For qp < qth, the peak is tected and that (ii) the peaks of impurities are sufficiently
considered spectrally homogeneous; otherwise the peak is well separated between themselves and from peaks of the
influenced by the presence (i.e., additional absorption) of an- parent substance and the system components (solvents). De-
other substance. In practice, it is possible to use a simplified velopers of the technology of a parent substance have to
procedure, whereby the chromatograms of the same peak are prove additionally that (iii) the proposed procedure allows
measured at two or three wavelengths and the determining the main impurities related to the technological
chromatograms are checked for the proportionality (see process and that (iv) all such impurities present at an amount
above) at all points. The latter method is less reliable, be- of ³ 0.1% are identified [7].
Validation of HPLC Techniques for Pharmaceutical Analysis 215

In order to confirm the specificity of a procedure used for A mixture of the initial substance and the products of its
determining impurities in parent substances, it is necessary to chemical modification can be used for preparing solutions
demonstrate that (i) this procedure allows the peaks of the used for the evaluation of suitability of the chromatographic
main rated impurities to be detected and that (ii) the peaks of system.
these impurities are sufficiently well separated between The duration of action used for the chemical modifica-
themselves and from peaks of the parent substance and the tion of drugs is selected taking into account the following
system components (solvents). factors.
The specificity is confirmed by a set of chromatograms, (i) The peaks of the products of drug modification are to
including at least those of (a) a model solution of the parent be clearly distinguishable in the chromatogram. Therefore,
substance and the main impurities (prepared by adding these the treatment duration must be sufficient to provide for a not
known impurities or their solutions to the parent compound less than 10% decrease in the main peak height (area), which
or its mixture with the placebo), (b) the solvent, (c) the pla- is proportional to the drug content.
cebo (for filled drugs), (d) the test solution, and (e) the solu- (ii) The peaks of the products of drug modification have
tion used for the evaluation of suitability of the chromato- to be sufficiently well separated from the main peak, but the
graphic system. main peak height (area) must be comparable with that in the
In addition, it is recommended to confirm the specificity initial test solution. According to [27], it is recommended
of the given analytical procedure by data on the “purity” of that the main peak intensity would decrease by no more than
the main peaks in the chromatograms of test solutions. 30%. However, this requirement is not as critical, since the
Case 2. The main impurities are unknown (unidenti- stronger decomposition of the parent compound can be com-
fied) or absent. In this case, it is expedient to use special ex- pensated by adding it in the necessary amount.
periments involving modification of the parent compound (iii) It is desired that the percentage “content” of the
(sometimes called “stress testing” [2, 15, 27]), whereby a so- products of drug modification determined by the method of
internal normalization would be close to the level of maxi-
lution of the given drug (or of the parent compound) is sub-
mum permissible content of a single impurity.
jected to factors leading to its partial destruction with the for-
Further steps in the proof of specificity of the analytical
mation of related identifiable compounds. The degree of de-
procedure in the case under consideration are the same as in
composition can be readily determined from the decrease in
case 1. The validation characteristics additionally include
the area of the main peak in the chromatogram of the final
chromatograms obtained in the course of experiments on the
solution relative to that in the initial solution. The specificity
chemical modification of the parent compound.
of the given analytical procedure is judged by separation of
Data presentation. The proof of the specificity of an an-
the peaks of impurities between themselves and from the alytical procedure is presented in the form of a set of
peak of the main component and by the peak purity of the chromatograms (see above) with discussion of the obtained
parent compound. results. These data are supplemented by the results of calcu-
In particular, the specificity of HPLC procedures can be lation of (i) the separation coefficient Rs for two peaks of the
proved using the following methods of chemical modifica- most closely spaced components, (ii) the column efficiency
tion. N, and (iii) the asymmetry parameters (tailing factors) of the
(i) Hydrolysis with 0.1 N solutions of HCl or NaOH at main analytical peaks.
room temperature or at elevated temperatures. Example: a It is necessary to make the following remark. Solving the
granulate was treated with 0.1 N HCl solution for 5 h at problem of detection and separation of impurities by HPLC
60°C, after which the sample was extracted according to the is still an art, with the results depending on the skill of devel-
proposed procedure and analyzed by HPLC. opers. It is very important to select the optimum chromato-
(ii) Oxidation with a 3% hydrogen peroxide solution, graphic columns and mobile phases. Moreover, it is known
0.05 M iodine solution, etc. Example: captopril was partly that, depending on the column loading, it is possible (a) to
oxidized with 0.05 M iodine solution (in order to obtain observe no additional peaks of impurities, (b) to find a cer-
captopril disulfide — the main technological impurity [22]), tain number of such peaks, a part of which cannot be quanti-
extracted according to the proposed procedure, and analyzed tatively characterized, or (c) to reveal a very large number of
by HPLC. additional measurable peaks upon overloading the column
(iii) Thermal decomposition by heating to 60 – 100°C. with respect to the main drug component. Therefore, in de-
Example: a granulate was kept for 7 days at 80°C. The result- veloping and validating the methods of impurity determina-
ing solid products of decomposition can be stored and used tion (especially in the case of parent compounds), it is expe-
as control substances in the tests for suitability of a chro- dient to check for the possible presence of additional peaks
matographic system. (i.e., impurities) by chromatography of drug solutions with
(iv) Photochemical decomposition under illumination elevated concentrations providing overloading of the column
(e.g., UV irradiation). with respect to the main component. However, this approach
(v) Chemical addition reactions, for example, drug is usually inapplicable in the case of ready-to-use drugs con-
bromination at multiple bonds. 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 217

These solutions are characterized by the average drug tween the results obtained in such investigation, it is neces-
content according to the results obtained by each of the ana- sary that the round robin tests involve not less than five
lysts, X i and X j (i, j = 1, …, N ). These data are statistically laborqtories [35].
processed and characterized by generalized average values In practice, however, the reproducibility is usually evalu-
X 1 = S X i N and X 2 = S X j N and the corresponding ated using two or three laboratories and characterized by less
standard deviations (SDi, SDj ) and relative standard devia- strict estimates. For example, validation of a procedure pro-
tions (RSDi, RSDj ) of particular measurements. posed for the quantitative determination of a parent com-
First, it is required to show that the proposed procedure pound is performed by demonstrating the statistical equiva-
of determination of the drug content and the impurity lence of the standard deviations SDi and SDj of the results
coincentraiton provides for the statistically equivalent stan-
obtained in different laboratories (in terms of the Fisher
dard deviations SDi and SDj of the results obtained by differ-
F-criterion). Then, it is demonstrated that the scatter (RSD)
ent analysts (in terms of the Fisher F-criterion). Then, it is
of the results of analyses in one laboratory (characterized by
necessary to demonstrate that the average results of these
the maximum standard deviation (SDi or SDj) relative to the
(for certainty, two) analysts are statistically reliably
(P = 95%) identical in terms of the t-criterion calculated as average results of analyses in other laboratories (with lower
SDi or SDj values) does not exceed a certain preset level [13].
| X 1 - X 2| | X 1 - X 2| The full-scale reproducibility of analytical procedures is
t= = , rarely validated because (i) it is necessary to involve certified
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
facilities and expenditures.
where X 1 , X 2 are the average results of analyses performed 1.3. Confirming Linearity of the Response to Drug Con-
by analysts 1 and 2 and SD1, SD2 are the standard deviations centration
in the particular series of m1 and m2 parallel determinations Linearity characterizes the ability of a proposed analyti-
(usually m1 = m2 = m ). This t value is compared to tabulated
cal procedure to give (within the suitability range) a response
values of the Student criterion t (P = 95%, f = m1 + m2 – 2),
signal with the magnitude Y (e.g., peak height or area) di-
where P = 95% is the confidence probability and
rectly proportional to the amount C (concentration) of a drug
f = m1 + m2 – 2 is the number of degrees of freedom. If the
calculated parameter t is lower than the tabulated value, the to be determined: Y = a + bC. According to ICH recommen-
difference of average values can be considered as statistically dations [3], the linearity in practice is first visually estimated
insignificant with a 95% confidence probability. Otherwise, from the linear appearance of the plot of Y versus C. If the
the average results differ to a greater extent than that admit- plot appears linear, this relation is studied by methods of re-
ted by random errors in both series [35]. gression analysis in terms of the linear equation Y = a + bC.
In pactice, validation of the procedures of determination For the analytical procedures for determining the content
of the content of impurities is sometimes performed using a of a parent compound, CDER recommends establishing the
less strict method [13], by showing that the scatter (RSD) of criterion of linearity at a level of the correlation coefficient r
the results of one analyst (characterized by a greater standard not lower than 0.999 [4]. However, even such a high level of
deviation (SDi or SDj ) relative to the average result of an- correlation may be accompanied by significant deviations
other analyst (with a lower SDi or SDj) does not exceed a from linearity in the regions of high and low drug concentra-
certain preset level, for example, so that RSD £ 10% for im- tions [13]. For this reason, ICH [3] recommends that the lin-
purities with a rated content up to 1%, RSD £ 25% for an im- earity be validated by a plot of the difference Y–C showing
purity content within 0.1 – 1%, and RSD £ 50% for an impu- deviations (residuals) of the calculated values yi = a + bC
rity level below 0.1%. from the measured Yi values as the function of the concentra-
It should be noted that, according to USP-26 [7], it is in tion Ci. The “outbursts” of the points (xi, yi ) relative to the
most cases sufficient to determine only the repeatability for regression model can be determined by calculating the pa-
proper validation of an analytical procedure, while the inter- rameter t using the formula [36]
mediate precision and reproducibility characteristics should
be determined for procedures included in the pharmacopoeial | y i -Y i |
articles. t= ,
1.2.3. Reproducibility. This characteristic is determined 1 (Y i - y ) 2
SD 0 1+ +
by comparing the results obtained upon analysis of the same N ( N - 1) × SD 2y
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 Here, yi and Yi are the calculated and experimentally mea-
GOST R ISO 5725-2002. It should be noted that for reliable sured values of the response, respectively; y = Syi /N; N is
evaluation of the statistical significance of the difference be- the total number of experimental points (xi , yi ), and
218 N. A. Épshtein

S( y i - Y i ) 2 S( y i - y ) 2 drug concentrations in the range from 0.05% (dilution by a


SD 0 = , SD y = . factor of 2000!) to 2.5%. Instead of making some dilutions, it
N -2 N -1
is possible to use a proportional decrease in the volume of
applied sample (which saves the mobile phase).
The calculated t value is compared to tabulated values of the Data presentation. The validation characteristics in-
Student criterion t (P = 95%, f = N - 2). If the calculated pa- clude (i) a regression equation of the Si = a + bC type (for ex-
rameter is greater than the tabulated value, the given point ample, S = 15536 + 15833969C [mg/ml]), (ii) the correla-
can be considered as deviating from the adopted regression tion coefficient (e.g., r = 0.9998), and (iii) a plot visually
model with a 95% confidence probability. confirming the linearity of relationship between S and C.
In practice, validation of the procedures of determination 1.4. Confirming the Accuracy of a Given Analytical Pro-
of the content of impurities with respect to linearity is some- cedure
times performed proceeding from a correlation coefficient of The accuracy characterizes the proximity of the experi-
r ³ 0.98 [13]. According to our estimates, use of the calibra- mental results, obtained using a proposed analytical proce-
tion graphs with such correlation coefficients may lead to dure, to the “true” value in the entire suitability range of this
RSD values on the order of 20% and above. Therefore, it procedure. The accuracy represents a combination of the ran-
would be more correct to establish the criterion of linearity at dom and systematic error.6 In order to provide for accurate
least at the level of r ³ 0.990. HPLC determinations, it is recommended to use standard so-
The linearity should be validated based on the analysis of lutions with concentrations close to within 10% of the test
at least five solutions with various concentrations covering solution concentration.
the entire suitability range of a proposed analytical procedure The accuracy of analytical procedures should be deter-
[35]. According to ICH recommendations [3], the linearity mined using homogeneous samples with exactly known con-
can be demonstrated directly by using the reference parent centrations of the compounds to be determined. For valida-
substance (dilutions of a standard solution) and/or model ar- tion purposes a series of such solutions is prepared using the
tificial mixtures including components of the drug studied. reference parent compound. According to ICH recommenda-
The most adequate approach consists in taking thoroughly tions and USP-26 [1, 7, 15], the accuracy can be expressed
weighed aliquots of the drug components and preparing solu- both in the classical form, as the difference X – m between
tions according to the proposed procedure, since all opera- the average experimental value (X ) and the true value (m)
tions of the analyst should correspond strictly to those stipu- with the corresponding confidence interval DX ,7
lated in the description. In practice, however, an “intermedi- ( X - m ) ± DX , (2)
ate” approach recommended by ICH [3] is frequently
employed. According to this, solutions are partly prepared and in an alternative (and more illustrative) form, in terms of
using weighed aliquots of the drug components and the other the percentage recovery of the known amount of the com-
are obtained by diluting these stock solutions. It should be pound to be determined,
emphasized that the linearity of a proposed analytical proce- ( found content )
R= ´ 100%. (3)
dure should be confirmed in the course of validation of the ( introduced content )
accuracy, which reduces expenditures and saves time. The
usual procedures are as follows. The author believes that the content recovery testing
(a) For the analysis of parent compounds, it is common should be preferred for evaluating the accuracy. This ap-
practice to prepare a reference solution of the compound proach provides a more illustrative characteristic of the reli-
with a concentration at or above the upper limit of the ex- ability of results obtained using a proposed analytical proce-
pected concentration interval (suitability range of the pro- dure and reveals the need for additional checks in the case of
posed procedure). Then, a series of dilutions is prepared so a significant systematic error (see below). On the other hand,
as to cover the entire range. Each solution is studied in a se- the recovery defined by formula (3) incompletely character-
ries of two or three injections. izes the accuracy, since this quantity is also random and re-
(b) For the analysis of ready-to-use drugs, the linearity is quires knowledge of the corresponding confidence interval.
frequently checked in the same way as for the parent com- Thus, it is recommended to determine both the recovery R
pound (i.e., using solutions of the parent compound as de- (%) and the confidence interval at a preset probability
scribed in (a)). However, it is incorrect to ignore the possibil- (P = 95%), representing the accuracy in a form analogous to
ity that auxiliary components (placebo) may influence the re- expression (2):
sults. Therefore, it is more correct to validate the linearity R ± DR . (4)
using model mixtures of the parent compound and placebo. Obviously, the proposed methods should not involve sig-
(c) For the determination of impurities using the method nificant systematic errors. In the absence of systematic er-
of internal normalization of the peak areas or heights, it is
possible to evaluate the linearity by preparing dilutions of the 6
Accordiong to the State Standard GOST R ISO 5725-1–2002 the system-
reference sample solution (with a concentration equivalent to atic error usually characterizes “trueness.”
7
the rated value) in the model impurity solution so as to obtain The value of DX characterizes random errors.
Validation of HPLC Techniques for Pharmaceutical Analysis 219

rors, the error is determined by the precision. Based on the analysis of drugs comprising mixtures of parent and auxil-
permissible RSD values (see above), experimental experi- iary compounds, is based on determining the recovery of a
ence, and analysis of the published data [13, 27], it is possi- known amount of the parent substance introduced into the
ble to draw the conclusion that there is no need to verify a placebo. The placebo is prepared separately and then intro-
proposed analytical procedure in the absence of a significant duced in a nominal (or proportional) amount into measuring
systematic error, provided that it is established that the recov- flasks. Then, thoroughly weighed amounts of the parent
ery with allowance of the confidence interval does not fall compound or its concentrated solutions are added so that
outside the following limits: (upon filling the flasks to the marks) the sample concentra-
99.0–101.0%, for the quantitative analysis of parent sub- tions would cover the entire expected suitability range of the
stances with a high rated content of the active component proposed analytical procedure. For example, a parent com-
(98% and above); pound can be introduced into a placebo solution at a level of
98.0–102%, for the quantitative analysis of parent sub- 80, 100, and 120% of the nominal concentration indicated on
stances with a lower rated content of the active component the label (or the reference solution concentration).
(98% and below) and ready-to-use drugs; The method of standard additives. This method is gen-
90.0–110%, for the quantitative determination of impuri- erally analogous to that described above but is applied only
ties with a rated maximum content of up to 1%; when it is impossible to prepare a solution of placebo free
75–125%, for the quantitative determination of impuri- from the parent compound or when this compound is present
ties with a rated maximum content from -0.1 to 1%; in the placebo in an unknown amount (e.g., in biological
50.0–150%, for the quantitative determination of impuri- samples). In these cases, the reference sample of the parent
ties with a rated maximum content below 0.1%. compound is added at an amount of 50, 80, 100, 120, and
1.4.1. The procedure of accuracy evaluation. ICH rec- 150% of its expected content in the analyzed solution. Using
ommends making three determinations (i.e., analyze three the proposed procedure, the amount of the parent compound
model mixtures) for three different concentrations. However, is determined (found content) and compared to the known
this approach does not allow the accuracy to be determined additive (introduced content). Alternatively, it is possible to
together with linearity and other validation characteristics. compare the results of analyses performed using the pro-
The author believes that the accuracy should be evaluated us- posed method and the data obtained for the same samples by
ing not less than nine determinations at various concentra- validated alternative methods.
tions covering the entire range of suitability of the proposed For the validation of analytical procedures intended for
procedure. This provides for the possibility of determining the analysis of ready-to-use drugs, it is expedient to use the
this characteristic simultaneously with the calibration graph same reference substance for preparing both model mixtures
parameters and their statistical characteristics (for evaluating and standard solutions. This eliminates errors related to the
the linearity according to Section 1.3), the limit of possible uncertainty of the composition indicated on the la-
quantitation (Section 1.7.2), and the limit of detection (Sec- bel of the reference sample and allows using commercial
tion 1.6.2). It should be emphasized that these determinations samples instead of special reference compounds. At a low
should include all stages of the proposed analytical proce- concentration of the parent compound in the mixture (when
dure. it is impossible to add a thoroughly weighed amount of this
For parent substances, the accuracy of analysis is usually compound), one may add a known amount of concentrated
determined by comparison to a reference sample. According solution and then fill the measuring flask with a solvent stip-
to this, the reference sample (or a high-purity substance) is ulated by the proposed analytical procedure.
analyzed using the standard procedure and the results are For the quantitative determination of impurities, the ac-
compared to data in the certificate of the reference sample or curacy of evaluation has certain peculiarities. In this case, the
to the results of analysis of the high-purity parent substance method of recovery of a parent compound introduced into
performed by an alternative method (e.g., titration) with the placebo and the method of standard additives have lim-
known accuracy and precision. It is recommended that, for ited applicability because these validation procedures require
parent substances with a high rated content of the active large amounts of identified impurities. In this case, the accu-
component, the average recovery should be not less than racy is most frequently checked using the method described
99 – 101% at each level [13]. below.
For ready-to-use drugs, the accuracy is evaluated The method of internal normalization with or without
through the analysis of mixtures containing known amounts response factors. According to this method, identified impu-
of the parent compounds and placebo; for the quantitative rities are characterized by the response factors with respect
determination of impurities, this characteristic is determined to the parent compounds determined by the analytical proce-
by the analysis of such mixtures containing known amounts dure under consideration. These coefficients depend on the
of these impurities. These analyses are performed using two mobile phase composition and the analytical wavelength. For
principal methods. reproduction (or modification) of the analytical procedure,
The method of recovery of a parent compound intro- the detector wavelength is not changed, while the mobile
duced into the placebo (matrix). This method, used for the phase composition can be corrected for the difference in the
220 N. A. Épshtein

N
parameters of chromatographic columns. In the case of a
considerable change in this composition (see Section 4), it is
å SD k2
k =1
SD 20 = .
necessary to re-determine at least the values of the response N
factors. For determining unidentified and accidental impuri-
ties, these factors are conventionally taken equal to unity (as- Finally, the Student t-criterion is calculated as
suming that the sensitivity for these impurities is the same as
that for the parent compound). If the reference samples of
t = (| m - x |× m ) SD 0 (6)
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- and compared to tabulated values of the Student criterion
cedure can be checked for the absence of systematic errors t (P, f ) for f = N(m – 1) degrees of freedom.
by one of the three methods considered below. It should be emphasized that, for small (practically insig-
Method based on the Student t-criterion without re- nificant) systematic error and small random error, the calcu-
gression analysis [8]. Each sample (whose total number is lated t-criterion can be greater than the tabulated value. How-
N ³ 5) with known values m (introduced content) of the com- ever, a small systematic error can be ignored in practice
ponent to be determined is analyzed in m = 3 – 6 parallel de- when the analytical problem does not require high accuracy
terminations. The total data array is characterized by the dis- of determination. This approach is also valid for other meth-
persion (SD0) and the Student criterion8 ods of evaluation of the accuracy of a proposed analytical
procedure.
| m - x| m Method of regression analysis with the Student t-cri-
t= terion [35, 38]. This method seems to be the most effective,
SD 0
since it provides for the possibility of using the calibration
graph for the accuracy evaluation and determination of some
This t value is compared to tabulated values of the Stu- other validation characteristics (see the generalized scheme
dent criterion t (P, f = m – 1). If the calculated parameter is in Fig. 1).
greater than the tabulated value for P = 95% and f = m – 1. For simultaneous determination of the constant and vari-
t > t (P, f ), the results obtained using the proposed method able systematic errors, not less than N = 5 samples with
can be considered as involving a systematic error d. This er- known values of the parent compound are studied and the re-
ror is calculated by the formula lationship between the “introduced content” (mt ) and the
“found content” (mf ) is processed by least squares in terms
| x - m|
d= 100%. (5) of the equation mf = a + bmi. Using these data, the parame-
m ters ta = |a|/SD0 and tb = |1 – b|/SD0 are calculated and com-
pared to the critical (tabulated) values of the Student criterion
The standard deviation SD0 in a particular analysis is cal- t (P, f ) for the confidence probability P = 95% and f = N – 2
culated using the set of all m parallel determinations per- degrees of freedom. Using the results of regression analysis,
formed for N (or g in the notation of [8]) samples. This al- it is possible to judge with 95% probability about the absence
lows the SD0 value to be reduced and the sensitivity of deter- of a constant systematic error, provided that ta £ t (P,
mination of the systematic error to be increased with a f = N – 2), and the absence of a linear variable systematic er-
simultaneous decrease in the confidence interval for the re- ror, provided that tb £ t (P, f = N – 2).
sults of analysis, DX = t SD0/ m, where t is the Student cri- It is expedient to perform validation of the accuracy of an
terion for f = m – 1 degrees of freedom. analytical procedure together with checking for the linearity
The algorithm of these calculations is as follows. Each of the system response (area or height of the peak) as a func-
kth sample is characterized by the deviation of the experimen- tion of the concentration of the parent compound to be deter-
tal value from average, d i = X i - X , and the dispersion mined (in fact, linearity of the calibration graph in terms of
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 limit of detection (Section 1.6.2) and the limit of quantitation
maximum and minimum values of the dispersion SD 2k is (Section 1.7.2).
Method of regression analysis with the Fisher F-crite-
checked to be insignificant in terms of the Fisher F-criterion.
rion. This method is based on the assumption that a linear
If this difference is actually insignificant, the SD 20 value is
variable systematic error can be ignored. This assumption is
calculated as the sum of square deviations for all samples di- justified because HPLC in pharmacy is used in a relatively
vided by the number of samples, 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
8
This t value is essentially the ratio of the systematic error | x - m | and the compound, after which the relationship between the “intro-
random error SD0/ m.
duced content” (mt ) and “found content” (mf ) is processed
Validation of HPLC Techniques for Pharmaceutical Analysis 221

by least squares in terms of the relation mf = a + bmt. The ab- be < 3, then DX is expressed by a value with two significant
sence of a constant systematic error is confirmed by the in- digits.
significance of the coefficient a [35] at a commonly accepted 1.5. Validation of the Suitability Range of a Given Ana-
confidence probability level of P = 95%. For this purpose, lytical Procedure
the Fisher criterion calculated is as The range of suitability of a given analytical procedure is
F ( P, f 1 = N – 1, f 2 = N – 2 ) = the interval between minimum and maximum concentrations
(amounts) of a compound to be determined in which (i) the
SD 201 ( N – 1) – SD 202 ( N – 2 ) (7),
, linearity is observed, (ii) the characteristics of repeatability
SD 202 ( N – 2) 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),
where SD01 and SD02 are the standard deviations obtained which can be encountered in the course of routine analyses.
for the above relations without the free term (mf = bmt ) and The range of suitability of a given analytical procedure is ex-
with the free term (mf = a + bmt ). If the calculated F value is pressed in the same units as the results of analyses.
smaller than the tabulated (critical) values F (P = 95%, f1, f2), It should be noted that, in practice, it is not necessary to
the free term a is in fact insignificant and the error is absent determine the maximum possible range of suitability for an
to within a 95% confidence probability. HPLC procedure. If it were necessary, this range could be de-
1.4.3. Data presentation and evaluation of the system- termined using threshold RSD values obtained in the course
atic error. The final judgment about the accuracy of a pro- of validation of the linearity and precision. For example,
posed analytical procedure can be made upon validation of RSD must not exceed 3% for HPLC procedures aimed at de-
its specificity, precision (repeatability, reproducibility), and termination of the parent compounds and 10% for proce-
linearity.9 It is necessary to indicate a particular method of dures of impurity determination [13].
normalization of the content of impurities (weight fractions, In practice, it is sufficient to show that a given range of
percentage of the area under the peak of the main compo- suitability covers “with margin” the rated limiting concentra-
nent, etc.). The validation results are presented by data on the tions of the substances to be determined, as indicated in the
recovery of the amount of introduced parent compound with corresponding pharmacopoeial articles. For this reason, it is
a confidence interval, R ± DR , or the difference between the recommended that the range of suitability of a given analyti-
average experimental value X and the true value m with the cal procedure be not less than the following intervals [3, 7].
corresponding confidence interval: ( X - m ) ± DX . (i) For the quantitative determination of the main compo-
The proposed procedure involves no significant system- nent concentration in parent compounds and ready-to-use
atic error, provided that the recovery with allowance of the drugs: from 80 to 120% of the nominal content (i.e., the con-
confidence interval does not fall outside the following limits: centrations of test solutions should range within 80 – 120%
99.0–101.0%, for the quantitative analysis of parent sub- relative to the concentration of a reference sample solution
stances with a high rated content of the active component used accordsing to the proposed procedure).
(98% and above); (ii) For evaluation of the homogeneity of dosage: from
98.0–102%, for the quantitative analysis of parent sub- 70 to 130% of the nominal content, provided that a wider in-
stances with a lower rated content of the active component terval is not required (in special cases such as aerosols).
(98% and below) and ready-to-use drugs; (iii) For dissolution tests: ± 20% (absolute percentage) of
90.0–110%, for the quantitative determination of impuri- the rated value of drug release. For example, for monitoring
ties with a rated maximum content of up to 1%; the behavior of a drug with delayed release of a parent com-
75–125%, for the quantitative determination of impuri- pound, for which the release is rated as 20% within the first
ties with a rated maximum content from – 0.1 to 1%; hour and up to 90% within a 24-h period of time, the suitabil-
50.0–150%, for the quantitative determination of impuri- ity range must extend from 0 to 110% of the nominal drug
ties with a rated maximum content below 0.1%. content.
The numerical result of a particular analysis, X (or R), (iv) For the quantitative determination of impurities by
must contain the last significant digit in the same position as the method of external standard: from 50 to 120% of the
that in the numerical value of the error of determination, DX nominal content. It should be noted that, in view of the possi-
(or DR) [37]. The number of significant digits in the latter bility of RSD values amounting up to 50% (Section 1.2.2), it
value is determined as follows. If the first significant digit in would be more correct to establish the upper limit at 150% of
the error is ³ 3, then DX is expressed by a value with one sig- the nominal content. For impurities exhibiting a very high bi-
nificant digit; should the first significant digit in the error 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
9
(v) In cases where the quantitative determination of a
It is expedient to confirm the linearity together with determining the accu- parent compound and the detection of impurities are per-
racy.
222 N. A. Épshtein

formed jointly and only the reference sample solution of the (a) Using the calibration curve S = a + bC constructed
main component at a nominal concentration is used, the suit- using the results of analyses for a series of the reference sam-
ability range must extend from the rated LPI value (more ple solutions with decreasing concentrations in the region of
precisely, from half of this value, see the preceding point) up the LOD. Using these data, a regression equation S = a + bC
to 120% of the nominal concentration of the parent com- of the area under peak S versus concentration C is calculated
pound. If the LPI value is unknown (e.g., during the develop- and the standard deviation SDa of the free term is deter-
ment of an analytical procedure) the initial lower limit of the mined. Alternatively, the standard deviation is determined
suitability range according to the ICH recommendations for the intersections of several regression lines of the calibra-
should be established at 0.1% of the nominal concentration tion curve with the ordinate axis, constructed using several
of the parent compound for a daily drug dose below 1 g; series of reference sample solutions with concentrations in
should this dose exceed 1 g, the lower limit of the suitability the region of the LOD.
range should be reduced to 0.05% of the nominal drug con- (b) Using the standard deviation of the area under the
centration. peak in the chromatograms of an impurity with concentra-
1.6. Determining the Limit of Detection of a Given Ana- tions within 0.01 – 0.05% of the nominal drug concentration
lytical Procedure determined using the given analytical procedure, typically
The limit of detection (LOD) is determined as the mini- for ten sequential injections.
mum concentration of analyzed substance in the sample, 1.6.2. Determining the LOD Using the Free Term and
which (i.e., the corresponding response) can be detected un- the Coordinates of the Midpoint of the Calibration
der preset conditions [3, 4, 7]. For HPLC procedures used for Curve. This is the most convenient way to determine the
the determination of parent compounds and ready-to-use LOD. According to this method, the calibration curve is de-
drugs, the LOD is required for the detection of limiting impu- scribed by the regression equation Y = a + bC, where Y is the
rity concentrations. Obviously, the LOD may depend on the response (peak area of height) and C is the concentration. For
HPLC detectors and pumps. For this reason, this characteris- this relation, the LOD is calculated by the formula
tic has to be rated for various instruments, including those
2C × t ( P, f ) × SD 0 nj
available for potential users. Irrespective of the method used LOD = , (9)
for evaluation, it is necessary to have a chromatogram show- Y - a + t ( P, f ) × SD 0 nj
ing that a response peak exceeding the baseline noise is actu-
ally observed at a concentration corresponding to the LOD of
where C and Y are the coordinates of the midpoint; a is the
the substance to be detected.
free term; SD0 is the standard deviation of the experimental
According to USP-26 [7], it is usually not necessary to
values from the calculated ones; nj is the number of parallel
determine the actual LOD of the analyzed substances (except
determinations for each experimental point (typically,
for analytical procedures intended for monitoring the clean-
nh = 2 – 3); t (P, f = N – 2) is the Student criterion (F = 99%
ness of technological equipment). In most other cases, it is
[35]) for f = N – 2 degrees of freedom; and N is the number
sufficient to show that the impurity of interest is reliably de-
of experimental points on the calibration graph.
tected at a preset level (see Section 1.6.5).
It should be noted that a different formula for the LOD
1.6.1. Determining the LOD from the Standard Devi-
was presented in [35], according to which the background
ation of the Response and the Slope of the Calibration
(i.e., the free term) did not influence this value. The error has
Curve. According to this method [3], the LOD value is cal-
been eliminated in the new edition of this monograph.
culated by the formula
1.6.3. Determining the LOD for the Signal-to-Noise
LOD = 3.3(SDa/b ), (8) 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
assuming that the response – concentration relation is linear validation of HPLC procedures because the baseline noise
in the range from the maximum possible concentration of the strongly depends on the experimental conditions. On a scale
analyzed compounds down to zero. It is recommended to de- comparable with the noise intensity, the baseline appears as a
termine the slope b of the calibration curve using reference broken line of complicated shape and admits only a very sub-
sample solutions with concentrations in the vicinity of the jective estimate of the maximum noise N¢.
LOD (see Section 1.7). 1.6.4. Evaluating the LOD from the Minimum Con-
The value of the standard deviation SD can be deter- centration for Which the RSD is Below a Preset Value.
mined using one of the two methods: 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
10
For the analysis of impurities, it was recommended [27, p. 695] to per- for which the RSD for the peak area (height) of the analyzed
form tests in the range from the limit of quantitation with respect to the compound for five sequential determinations does not ex-
main component (typically, below 0.1%) up to a 5% concentration in solu- ceed 20%. However, this approach is not convenient in prac-
tion. tice because it requires numerous analyses. For example, it
Validation of HPLC Techniques for Pharmaceutical Analysis 223

was pointed out [40] that evaluation of the LOD by this 1.7. Validation of the Limit of Quantitation of a Given
method in accordance with FDA recommendations (defining Analytical Procedure
the LOD as the minimum concentration for which the RSD The limit of quantitation (LOQ ) is the minimum concen-
does not exceed 15%) would require 288 determinations. tration of analyzed substance that can be determined at an ac-
In order to take into account the influence of the drug ceptable precision (repeatability, reproducibility) and accu-
matrix on the LOD, it was suggested [30] to take into ac- racy under rated conditions of analysis by a given method
count the so-called “specific LOD.” These values are deter- [1, 15]. The ICH [3] and USP-26 [7] recommend several
mined using mixtures of a given parent substance with a pla- methods of determining LOQ for the impurity analysis.
cebo instead of a solution, which takes into account the effect 1.7.1. Evaluating the LOQ for the signal-to-noise ra-
of asymmetry of the peaks of other components on the LOD. tio S/N¢ = 10. This is the most widely used method of deter-
According to ICH [3], the LOD has to be refined using vari- mining the LOQ. However, since this procedure makes use
ous columns and instruments because this characteristic de- of the peak heights (rather than areas), it is more suited for
pends on the noise level (and, hence, on the working life of the HPLC techniques using this measure of the response.
the column, on the properties of the detector, etc.) and on the This approach does not take into account the requirements on
peak shape. reproducibility of the HPLC procedure.
1.6.5. Alternative Proof of the Reliable Detection of According to this method, an initial reference solution of
Impurities in Parent Substances and Ready-to-Use the analyzed compound is determined for which the sig-
Drugs. For a test solution concentration of about 1 mg/ml, nal-to-noise ratio is at least 30 [27]. Then, this solution is se-
the response signal at a level of not less than 1 V can be quentially diluted until this ratio decreases to S/N¢ » 10. The
readily obtained. For an impurity concentration of 0.01%, corresponding concentration is considered as the LOQ. For
this corresponds to a response on the order of 100 mV, which this evaluation of the LOQ, it is recommended to determine
is one order of magnitude higher than the noise level of mod- the maximum baseline noise near a peak of the analyzed
ern detectors. For validating the reliable detection of impuri- compound, in the interval of retention times within ± 10W0.5,
ties, it is sufficient to present the chromatogram of a refer- where W0.5 is the full width at half maximum (FWHM) of
ence (or test) solution diluted D = 2L times, where L is an in- this peak [22].
teger indicating the ratio of the main component For HPLC procedures used in pharmacy, it would be in-
concentration to the minimum rated concentration of the im- teresting to study the influence of the maximum noise N¢ on
purity. This chromatogram must visually confirm the possi- the relative standard deviation of results for a nearly Gaussi-
bility of reliably detecting impurities at the level of about an peak shape. According to [27],
half of their rated content. For example, if the content of an
individual impurity must not exceed 0.5%, the above ratio is 50
RSD[%] = , (10)
D = 2(100/0.5) = 400. For the normalized total impurity S N¢
content, D = 2000, which corresponds to an individual impu-
rity content of 100/D = 0.05% (the British Pharmacopoeia where S is the detector signal intensity. A simple calculation
does not take into account impurities with concentrations be- shows that, at S/N¢ = 10, the noise influence alone can make
low 0.05%, except for highly toxic substances). In many RSD as large as » 5% (!).
cases, this approach eliminates the need to establish the LOD 1.7.2. Evaluating the LOQ using the calibration
during the determination of impurities in parent compounds curve. The ICH [3] recommends determining the LOQ by
and ready-to-use drugs. the formula
Data presentation. According to ICH [3], it is necessary
to validate the LOD value and indicate the method of deter- LOQ = 10(SD/b ), (11)
mination. If the LOD was calculated from experimental data,
it is necessary to present the results of analysis of the re- where b is the slope of the calibration curve and SD is the
quired number of samples with the content of a detected standard deviation of the response signal. The peculiarities of
component close to the LOD. If the LOD was estimated by this approach were considered in Section 1.6.1. A significant
visual assessment of chromatograms, it is necessary to pres- disadvantage of this approach (as well as of some others)
ent chromatograms confirming reliable detection of the recommended by ICH is the inability to take into account the
peaks of parent compounds or impurities. requirement of acceptable reproducibility (see above).
The number of significant digits in the LOD [39] is de- The author believes that a more correct estimate of the
termined as follows. If the first significant digit in the LOD LOQ from the calibration graph, with allowance for the
value is > 3, then this limit is expressed by a value with one reproducibility requirements, can be obtained in the follow-
significant digit and rounded to the closest integer; should ing way [41]. First, the values of response Y (area under the
the first significant digit in the LOD be £ 3, then the LOD peak of the analyzed component) within the limits of the cal-
can be is expressed by values with two significant digits and ibration curve Y = a + bC are used to calculate (i) the theoret-
the second digit should be rounded to 0 or 5. ical values of C = (Y – a)/b and (ii) the standard deviations
224 N. A. Épshtein

(SDc) and relative standard deviations (RSD = SDc/C ´ 100) (within the interval from 0 to th ) and the area S0 at the initial
of the results of analysis. Then, the plot of RSD versus C is moment, calculate the parameter tb = |b |/SDb, and compare
used to determine the concentration Cp corresponding to a this value with the critical (tabulated) Student criterion
permissible RSD value preset for the analytical procedure t (P = 99%, f = N – 1) for the confidence probability
under consideration. This permissible concentration Cp is P = 99% and f = N – 1 degrees of freedom, where N is the
taken to be LOQ with allowance for the RSD (i.e., for the number of experimental points on the above interval. If tb is
reproducibility) requirements. smaller than the tabulated t (P = 99%, f = N – 1) value, the
The values of standard deviations of the results of analy- coefficient b is statistically insignificant and, hence, there is
ses are calculated by the formula [35] no significant difference between the areas under the peak
(and, hence, the solution concentration) at the initial time and
2 2 at any other moment up to the last experimental point.
SD 0 1 1 æ SD b ö æ Y s -Y ö
SD c = + +ç ÷÷ ç ÷ (12) 1.8.2. Methods of evaluation. The stability of test and
b N m çè b ø
ç SD
è 0
÷
ø reference solutions ST (%) can be evaluated using the fol-
lowing methods.
(i) For continuous testing within a relatively short period
for f = N – 2 degrees of freedom, where SD0 is the standard
of time — by the ratio of the area St under the peak of the an-
deviation of the linear relation (calibration curve), N is the
alyzed compound (or impurity) at the moment t to the initial
number of experimental points on the calibration curve, Ys is
area S0:
the response (area under the peak), m is the number of paral-
lel (independent) determinations, b is the slope of the calibra- ST = 100St /S0. (13)
tion curve, SDb is the corresponding standard deviation, and
Y = Yk/N is the average response of all experimental points (ii) Irrespective of the duration of experiment — by the
used for constructing the calibration graph. ratio of the amount mt (concentration) of the analyzed com-
1.7.3. Other methods. Other methods of determining the pound (or impurity) at the moment t to the initial value m0:
LOQ are not theoretically justified and did not find wide use.
In particular, it was suggested to determine the LOQ as the ST = 100mt /m0. (14)
minimum concentration of the analyzed compound for which
six sequential injections give RSD £ 3.0% [27, p.695] or The test and reference solutions are prepared according
RSD £ 2.0% [29]. With this approach, the LOQ is essentially to the given procedure and analyzed with certain time inter-
defined as the concentration at which the RSD becomes vals. The maximum possible duration of measurements de-
greater than a certain preset value. pends on the practical requirements on the possible storage
1.7.4. Allowance of the matrix effect on the LOQ. It time of the given solution (which may not necessarily be
was suggested to determine the so-called specific LOQ p30]. equal to the maximum rated storage time). The solutions can
This characteristic is determined under the same conditions be considered stable until the difference |100 – ST | does not
as described above for the LOD, but in a real test solution, exceed the relative error of determination of the main com-
and therefore takes into account the influence of asymmetry ponent (or impurity) according to the given analytical proce-
of the other peaks on the LOQ of the analyzed compound. dure.11
Data presentation. The LOQ value is presented with in- Data presentation. The stability of solutions is con-
dication of the method used for its determination. If this firmed by data on the storage times of solutions and mobile
characteristic is evaluated using the signal-to-noise ratio, it is phases used in the analyses.
necessary to present chromatograms showing the reliability 2. EVALUATION OF THE STABILITY OF ANALYTI-
of detection of the peak due to the analyzed component or CAL SYSTEMS WITH RESPECT TO SMALL VARIA-
impurity. If the LOQ was determined by calculation or ex- TIONS OF PARAMETERS (ROBUSTNESS)
trapolation, it is necessary to present the results of analysis of The robustness of an analytical procedure is the charac-
a sufficiently large number of samples with the content of the teristic of its stability with respect to small variations of the
analyzed compound close to the LOQ level. system parameters possible under real conditions. This sta-
1.8. Stability of Solutions. bility is usually evaluated in terms of the RSD of the results
1.8.1. Confirming the stability of solutions using the of analyses compared to the analogous data obtained using
regression relation for the area under the peak versus the strictly observed conditions according to the validated ana-
time. As a rule, the analytical solutions are used for a rela- lytical procedure. A more correct procedure based on evalua-
tively short period of time (th ) for which the content of the tion of the statistical significance of the difference between
analyzed substances (proportional to the areas under the cor- these results in terms of the Student t-criterion is rarely ap-
responding peaks) remains practically unchanged. In order to plied to the HPLC techniques.
confirm this statistically, it is sufficient to determine the coef-
11
ficient b in the regression relation St = S0 + bt (or The absolute value of |100 – ST | is taken because ST can be greater than
St – S0 = bt ) between the area St under the peak at the time t 100% due to random errors.
Validation of HPLC Techniques for Pharmaceutical Analysis 225

Robustness is usually studied at the stage of development The additives may include other analyzed components, iden-
of the analytical procedure. Typical parameters capable of in- tified impurities, possible decomposition products, and
fluencing the results of analyses and, hence, studied in the substances playing the role of internal standards.
course of validation, are as follows:11 Effective means for finding SST solutions is provided by
(i) the content of the organic solvent in the eluent experiments on the chemical modification of a particular
( ± 2%); drug under consideration (see Section 1.1.2). The solutions
(ii) the amount of additives (salts, ion-pair reagents, etc.) and substances obtained in such experiments contain the
in the eluent ( ± 10%); products of destruction of the main drug components and/or
(iii) pH of the buffer solution ( ± 0.5); compounds structurally close to the analyzed parent sub-
(iv) HPLC column temperature ( ± 5 °C); stances. Such solutions are expediently used in the tests for
(v) time of extraction of the analyzed compound from a suitability of a given chromatographic system and frequently
drug eluent ( ± 20%); allow one to reject expensive standard samples of impurities.
(vi) extractant composition ( ± 5%); The author believes that the suitability of a given chro-
(vii) eluent concentration gradient ( ± 2%); matographic system is adequately described by the following
(viii) mobile phase flow rate; main criteria.
(ix) column type and/or manufactirer. (i) Criteria of the separating power of the chromato-
The parameters (called critical) influencing the results of graphic system.
analyses should be indicated in the validation report. The de- (ii) Criteria of reliable determination of the beginning
scription of the given analytical procedure must provide the and end of a peak (and/or of the peak height) of the analyzed
corresponding warning remarks. In order to decrease the compound on the chromatogram.
number of tests necessary for the validation of robustness of (iii) Criteria of reproducibility of the results of measure-
a given analytical procedure, it is expedient to use the meth- ments (repeatability of injections) (iv);
ods of experiment planning, which are capable of quantita- Additional criteria should be introduced only provided
tively following the effect of several parameters (factors) that critical parameters (see above) were established in the
varied simultaneously [32 – 34, 42]. course of robustness evaluation.
There is no need for special additional investigations of The SST criteria should not be overloaded by numerous
robustness if no critical parameters were revealed in the extra parameters, since this may lead to a situation where
course of development and/or implementation of the pro- only HPLC columns and equipment used for the validation
posed analytical procedure. purposes will be formally suitable for all analytical proce-
Data presentation. The validation report should include dures.
the chromatograms and tables showing the effects of each 3.1. Separating Power of a Chromatographic System
critical parameter on the results of analyses (in comparison
to the normal values) and the absence of such effects for the The criteria of separating power include the separation
other most important parameters (see above). It is desired coefficient Rs , the peak-to-valley ratio h/v (see below), and
that the proposed analytical procedure be robust with respect the relative retention times of components.
to all the important parameters, which makes this procedure 3.1.1. Relative retention times. For analytical proce-
suitable for routine laboratory use. dures intended for the quantitative analysis of drugs, homo-
geneous dosing tests, and determination of the drug’s disso-
3. CRITERIA OF SUITABILITY lution characteristics, it is usually sufficient to characterize
OF A CHROMATOGRAPHIC SYSTEM the separating power by the relative retention times of sev-
eral substances, including the main drug component, a struc-
USP-26 [7] stipulates the System Suitability Test (SST) turally close compound, and/or an internal standard. The rel-
intended to establish that the separating power and ative retention time of the main component is usually taken
reproducibility of a given chromatographic system provide equal to unity.
for the adequate solution of the task of analysis. It is assumed The author believes that the retention times should not be
that the equipment, electronics, analytical procedures, and used as characteristics of suitability of a chromatographic
samples comprise a unified system investigated as a whole. system because these values depend on the eluate flow rate,
Upon chromatography of a special SST solution, the results temperature, and dead volume of the system. On the other
are checked for obeying the SST requirements (see below). hand, the retention times do not influence the accuracy and
The SST solution must contain the analyzed compound and precision of an analytical procedure (provided that other suit-
all other additives necessary for evaluating the suitability of ability requirements are satisfied). It is more correct to indi-
the given system for implementing the required analyses. cate the recommended values (or intervals) of retention times
of the main components in the description of the analytical
12
Values in parentheses indicate recommended limits of variation relative procedure and characterize the other substances by their rela-
to the values stipulated by the given procedure. tive retention times.
226 N. A. Épshtein

3.1.2. Peak separation coefficient. For analytical proce- of fluoxetin hydrochloride, it is required that
dures intended for the determination of impurities, the sys- hp/hv ³ 1.1/0.1 = 9.
tem suitability requirements should indicate the minimum 3.2. Criteria of Reliable Determination of the Beginning
possible values of the peak separation coefficient Rs at least and End (and/or the Height) of a Chromatographic Peak
for the main (rated) impurities. In the case of HPLC proce-
The peak asymmetry is described in terms of the tailing
dures intended for the quantitative analysis of drugs, homo-
fgactor T0.05 [7]. This parameter, which characterizes the
geneous dosing tests, and determination of the drug dissolu-
asymmetry at a level of 0.05% of the peak height, is espe-
tion characteristics, it may be necessary to introduce the peak
cially important for the procedures of quantitative analyses.
separation coefficient into the system suitability require-
According to BP 2001 [22], the recommended values fall
ments if (i) a high content of some impurity is admitted, (ii)
within T0.05 = 0.8 – 1.5. Under this condition, the peaks are
there is a possibility of partial overlap between the peaks of
sufficiently symmetric and exhibit no significant tailing,
the main components, or (iii) the peaks of the placebo com-
which favors reliable determination of the beginning and end
ponents occur in the vicinity of the main analytical peaks.
of the peak (and, hence, of the peak area) and reduces the
The minimum possible values of the separation coeffi-
probability of overlap. An analysis of the corresponding arti-
cient Rs are determined proceeding from the following
cles of the BP 2001 and USP-26 showed that the permissible
considerations.
T0.05 interval extends from 0.75 to 2.5. Asymmetric peaks
If the peaks are not significantly different in heights and
with T0.05 outside the 0.75 – 2.5 range should be avoided: for
possess nearly Gaussian shapes, their complete separation at
peaks with T0.05 on the order of 3 and above or 0.7 and be-
the baseline level requires that Rs ³ 1.5. This condition is rec- low, the boundaries of peaks practically cannot be deter-
ommended by the British Pharmacopoeia and by the EEC mined.
Pharmacopoeia. An additional criterion of peak separation is provided
If the peaks have significantly different heights (e.g., by the efficiency of a chromatographic column (N ) with
used in the determination of impurities) and/or exhibit tail- respect to the main analytical peak. This parameter charac-
ing, CDER recommends that Rs ³ 2.0 [4]. It should be noted terizes the peak width at half height (FWHM) and is calcu-
that this situation is most frequently encountered in practice lated by the conventional formula [7]. The ICH recommends
during the analysis of drugs. using columns with efficiencies N ³ 2000 theoretical plates,
For separating peaks with large tailing factors ( > 2.5, see which can be considered as the lower limit for the procedures
below), it is recommended to restrict the separation coeffi- of impurity determination in parent compounds. According
cient to Rs ³ 2.5. This situation is quite rare, being only typi- to practical experience it is usually sufficient to require that
cal of drugs containing several heteroatoms capable of form- N ³ 1000 theoretical plates.
ing strong hydrogen bonds with silanol groups of the sorbent. In some cases in the HPLC determination of the main
Higher “critical” Rs values are usually unnecessary, be-
drug components, USP-26 admits N ³ 400 theoretical plates.
cause the condition Rs > 2.5 ensures good separation virtu-
However, even this is not the minimum possible level: rapid
ally at the baseline level. On the other hand, it should be
analyses are sometimes performed using short chromato-
noted that, due to the availability of highly effective columns
graphic columns with 3.5-mm (and smaller) sorbent particles,
and computer-optimized systems, values of Rs < 1.2 cannot
which provide for a relatively low error of determination
be justified, except for (i) the isomer peaks, (ii) the peaks of
some components in complex multicomponent mixtures (< 1.5%) even at N » 250 – 300 theoretical plates. The above
(such as extracts of biological objects, antibiotics, alkaloids, criteria of suitability of the chromatographic system with re-
and multivitamin preparations), and (iii) the peaks of minor spect to the N value are not absolute: this parameter must
components (impurities, aromatic and color additives, etc.). only provide for the required accuracy and precision of the
It should be noted that the British Pharmacopoeia (BP proposed analytical procedure.
2001, Appendix III, p. A143) indicates that, in tests for the 3.3. Criteria of Reproducibility of the Results of HPLC
content of related compounds (when the peaks of main com- Measurements
ponents and impurities are incompletely separated), the sys- The criterion of reproducibility of the results of HPLC
tem suitability requirements may describe the peak separa- measurements with respect to the repeatability of injections
tion in terms of the peak-to-valley ratio p/v = hp/hv, where hp is usually formulated in terms of the relative standard devia-
is the peak height relative to the extrapolated baseline and hv tion RSD of the peak areas or heights. In the quantitative
is the height of the lowest point in the valley separating the analysis of drugs, the permissible value is usually restricted
peaks (relative to the same extrapolated baseline). This pa- at RSD £ 2.0% for the main components and RSD £ 5.0%
rameter is rarely used during the analysis of impurities in for impurities. Higher RSD values should be avoided or their
drugs: sometimes it is convenient for the description of im- validity should be additionally justified.
purity peaks occurring immediately in front of the main ana- The quantitative analysis of parent compounds requires
lytical peak. For suitability of the chromatographic system, it increased accuracy because the rated content of the main ac-
is usually required that hp > hv. For example, in the analysis 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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