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 technique is a procedure
aimed at obtaining experimentally justified evidence of the ability of this tec
hnique to give results characterized by the required accuracy and precision [1 –
7].2 All analytical techniques used for the development of pharmaceuticals and
for the determination of their quality characteristics have to be validated. In
the case of using methods stipulated and described in the State Pharmacopoeia, i
t is not necessary to evaluate their suitability, provided that the analyses are
conducted with strict observation of the text of each particular article. In mo
st other cases, especially in cases of modification of the drug composition, the
scheme of synthesis, or the analytical procedure, it is necessary to re-evaluat
e the suitability of the analytical techniques. This paper is aimed at (i) consi
dering the peculiarities of validation of HPLC techniques for pharmaceutical ana
lysis, (ii) critically assessing the main approaches to evaluation of the valida
tion characteristics, and (iii) providing practical recommendations and criteria
for finding correct solutions. The USSR State Pharmacopoeia (valid in the Russi
an Federation) introduced the section “Statistical Analysis of Biological Test R
esults” in 1968 (Xth Ed.) and the section “ Statistical Processing of Chemical E
xperimental Data” in 1988 (XIth Ed.). These sections are devoted to problems inv
olved in the metrological attestation of analytical techniques. In 1987, the Uni
ted States Food and Drug Administration (FDA) issued practical guides on the mai
n principles of vali1 2
“Akrikhin” Chemico-Pharmaceutical Joint-Stock Company, Staraya Kupavna, Moscow R
egion, Russia. Here and below the terms “accuracy” and “precision” (repeatabilit
y, reproducibility) are treated in accordance with the State Standard GOST R ISO
5725–1–2002. Previously, accuracy had the meaning of correctness, but now corre
ctness is described in terms of “trueness.” In justifying the suitability of tec
hniques related to the qualitative determination of substances, statistical proc
essing of the experimental results is obligatory [8].
dation [5] and on the presentation of samples and analytical data pertaining to
the validation of methods [6]. In 1993, the International Conference on Harmoniz
ation (ICH) developed generalized recommendations on the validation of analytica
l 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 (CD
ER) issued a guide on the validation of chromatographic methods [4]. These docum
ents and some review papers and monographs [9 – 13] provided a basis for extensi
ve implementation of the procedure of validation of analytical methods. The Inte
rnet offers the Laboratory Guide to Method Validation and Related Topics at http
://www.eurachem.ul.pt /. In recent years, new guides have become available from
CDER [15], Waters Company [16], and Labcompliance [17 – 19]. Special sections ar
e devoted to these problems in national and international pharmacopoeias and gui
des on the validation of analytical procedures [7, 20 – 22]. Also available are
computer program packages, such as ELISA Method Validation Templates (Waters) an
d LaChrom 2000 Validation Manager (Merck) representing electronic tables with in
corporated functions of statistical processing of the results of measurements an
d issuing validation certificates, and monographs on the related subjects [23 –
34]. Tables 1 and 2 summarize the most recent recommendations concerning selecti
on of the validation characteristics depending on the type of analytical procedu
res. A comparative analysis of these data shows that, according to the United St
ates Pharmacopoeia (USP-26), methods of dissolution testing have to be validated
only with respect to precision (repeatability, reproducibility), while the othe
r characteristics “may require validation, depending on the specific test nature
.” In contrast, according to the FDA CDER guidelines [15], procedures used for q
uantitative analysis and dissolution testing need the same validation characteri
stics. 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 quantitative analysis have to b
e characterized with respect to robustness (see below). Robustness is not includ
ed in USP-26 [7] because this characteristic has to be studied at the stage of d
evelopment of an analytical procedure, rather than in the course of validation.
Since any analytical situation poses a multifactor problem, validation has to in
clude at least testing of the analytical system as a whole under the conditions
stipulated by the description.3 The second task is evaluation of the stability (
robustness) of the analytical system with respect to small variations of the mai
n factors (for example, the ratio of the mobile phase components, pH, temperatur
e, etc.). This problem is less important than the first one because it is usuall
y solved at the stage of development of a given analytical procedure. For this r
eason, problems pertaining to robustness are only briefly mentioned in this revi
ew. Let us consider the main stages of validation of the HPLC techniques used in
pharmacy. 1. TESTING THE ANALYTICAL PROCEDURE AS A UNIFIED SYSTEM UNDER THE CON
DITIONS STIPULATED IN THE DESCRIPTION Figure 1 shows the general scheme of evalu
ation of the suitability of an analytical procedure, which takes into account sp
ecific features of HPLC. As can be seen, testing an analytical procedure as a wh
ole in the general case allows the following validation characteristics to be de
termined [1 – 7]: (i) specificity; (ii) precision; (iii) linearity; (iv) accurac
y; (v) suitability range; (vi) limit of detection; (vii) limit of quantitation;
and (viii) stability of solutions.4 Depending on a particular type of the analyt
ical procedure (HPLC technique) only a part rather than all of the above charact
eristics may be required (see Tables 1 and 2). 1.1. Confirming the Specificity o
f a Given Analytical Procedure (Separating Power of a Chromatographic System) By
the specificity of a system is meant its ability to detect a given substance un
ambiguously (reliably) in the presence of other components (including impurities
) that may be present in the samples [1 – 4]. The proof of specificity depends o
n the task of a given procedure and on the availability of reference samples of
the main impurities. 1.1.1. The specificity of procedures aimed at determination
of the content of a parent substance, the parameters of solubility, and the hom
ogeneity of dosage. In order to confirm the specificity of these procedures, it
is usually required that peaks of the substances to be determined are sufficient
ly well resolved between themselves and from peaks of the main impurities, the s
ystem components (e.g., of the sample solvent), and the placebo. For this purpos
e, the sepa3
Specificity (Section 1.1) Recommended requirements to the repeatability of sampl
e injections (Section 3.3) 2.3 Reproducibility. Checked in special cases (Sectio
n 1.2.3)
2. Precision (Section 1.2) 2.1 Repeatability (Section 1.2.1) 2.2 Intermediate pr
ecision (Section 1.2.2)
3. Linearity (Section 1.3) 4. Accuracy (Section 1.4) 5. Suitability range (Secti
on 1.5) It is expedient to determine these validation characteristics in the cou
rse of proof of the analytical system accuracy (using statistical parameters det
ermined for the calibration graph) (Sections 1.3; 1.4; 1.6.2; and 1.7.2)
6. Limit of detection (Section 1.6)
7. Limit of quantitation (Section 1.7) 8. Stability of solutions (Section 1.8.)
9. Robustness (stability) of HPLC procedures with respect to small variations in
the main system factors (ratio of the mobile phase components, pH, temperature,
etc.). Robustness is usually studied in the stage of development of the given H
PLC tecnique (Section 2)
Criteria of suitability of a given chromatographic system (Section 3)
Fig. 1. The general scheme of validation of HPLC-based analytical procedures.
4
According to this concept, instrumentation, electronics, analytical procedures,
and analyzed samples constitute a unified analytical system, which can be consid
ered as a whole [7]. Sometimes, the stability of phases and solutions is conside
red within the framework of the problem of robustness.
ration of peaks is confirmed by a set of chromatograms, at least of (a) the test
solution, (b) the reference parent substance solution, (c) the solvent (blank),
(d) the placebo (for filled drugs), and (e) the solution used for the evaluatio
n of suitability of the chromatographic system. The degree of peak separation is
usually described in terms of the separation coefficient Rs. Recommended values
of this coefficient are given below in the Section 3.3. It is recommended to co
nfirm the specificity by investigation of the “purity” of peaks of the parent su
bstance to be determined [4, 27]. This test is usually performed using a diode m
atrix detector and a special program evaluating spectral homogeneity of the meas
ured peak (e.g., Peak Purity Millennium, Waters). The principle of evaluation of
the peak purity is as follows. The sample chromatograms are measured at various
detector wavelengths l (numbered n ). Each point of the peak is characterized b
y 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 wav
elengths (the length of this vector is proportional to the substance concentrati
on 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 Pharmacopoeia
(2003)
Category Characteristic I II Quantita- Limiting tive tests tests III IV
TABLE 2. Validation Characteristics Recommended for Various Tests by the United
States FDA (CDER and CBER) [15]
Type of analytical procedure Tests for impurities Chatacteristic Identification
quantitative limiting Qualitative determination and dissolution tests
Accuracy Precision Specificity Limit of detection Limit of quantitation Linearit
y Suitability range
Yes Yes Yes No No Yes Yes
Yes Yes Yes No Yes Yes Yes
MB No Yes Yes No No MB
MB Yes MB MB MB MB MB
No No Yes No No No No
Notes: Yes = usually studied; No = usually not studied; MB = may be required (de
pending on a specific test nature). Category I includes analytical methods inten
ded for determination of the content of the main component in parent substances
or ready-to-use medicinal forms; category II includes methods of determination o
f impurities and decomposition products; category III includes methods of determ
ination of the parameters of dissolution, drug release, etc.; category IV includ
es identification tests; the terms “accuracy” and “precision” (repeatability, re
producibility) are treated in accordance with the State Standard GOST R ISO 5725
–1-2002.
Accuracy Repeatability Intermediate precision Specificity Limit of detection Lim
it of quantitation Linearity Suitability range Robustness
No No No Yes2 No No No No No
Yes Yes Yes1 Yes No3 Yes Yes Yes Yes
No No No Yes Yes No No No No3
Yes Yes Yes1 Yes4 No No Yes Yes Yes
Notes: Yes = usually studied; No = usually not studied; 1 in cases where the rep
roducibility is studied, there is no need to specially determine the intermediat
e precision; 2 insufficient specificity of a given analytical procedure can be c
ompensated by introducing additional tests; 3 may be required in some cases (e.g
., when the limit of detection is close to the rated limiting content of an impu
rity); 4 insufficient specificity of a given analytical procedure can be compens
ated by determining the content of impurities.
trast angle q). If q = 0, the spectra are considered similar (homogeneous). This
implies that the value of absorption in one spectrum measured at a given wavele
ngth l can be obtained from the value in another spectrum measured at the same w
avelength by multiplying by a certain constant factor. In order to evaluate the
spectral homogeneity of a chromatographic peak, the spectral contrast angles q a
re 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 determined. Two spectra dete
rmined for the same substance can differ, for example, because of the influence
of the baseline noise. In order to take this into account, a threshold spectral
angle qth is determined (i) by determining the maximum spectral angle between pi
nts of the baseline with maximum noise levels or (ii) by taking six chromatogram
s of a standard sample solution, determining the maximum purity angle qp for eac
h chromatogram, and considering the maximum of these values as the threshold spe
ctral angle qth. This threshold angle characterizes the level below which the di
fference between two spectra can be considered insignificant. The obtained qp va
lues are compared to qth. For qp < qth, the peak is considered spectrally homoge
neous; otherwise the peak is influenced by the presence (i.e., additional absorp
tion) of another substance. In practice, it is possible to use a simplified proc
edure, whereby the chromatograms of the same peak are measured at two or three w
avelengths 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 compositi
on (e.g., under gradient HPLC conditions), or by deviations from the Lambert – B
eer law at high levels of absorption. Therefore, negative results of evaluation
of the spectral homogeneity of peaks in the gradient HPLC should be critically a
ssessed and checked for optical densities not exceeding 1 AU. It should be noted
that specificity is rarely checked using chromatomass spectroscopy (HPLC – MS)
— because of the high cost of this procedure — and the chemical and other analys
es of the eluate fractions corresponding to the parent substance, because of ted
ious procedures. 1.1.2. The specificity of procedures aimed at determination of
impurities. In evaluating the specificity of such procedures, it is necessary to
differentiate between two cases. Case 1. The main impurities are known and avai
lable. In order to confirm the specificity of a procedure used for determining t
he impurities in a parent substance, it is necessary to show that (i) this proce
dure allows the peaks of the main products of decomposition of the parent substa
nce to be detected and that (ii) the peaks of impurities are sufficiently well s
eparated between themselves and from peaks of the parent substance and the syste
m components (solvents). Developers of the technology of a parent substance have
to prove additionally that (iii) the proposed procedure allows determining the
main impurities related to the technological process and that (iv) all such impu
rities present at an amount 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 determining impuriti
es 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 p
eaks of these impurities are sufficiently well separated between themselves and
from peaks of the parent substance and the system components (solvents). The spe
cificity 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 a
dding these known impurities or their solutions to the parent compound or its mi
xture with the placebo), (b) the solvent, (c) the placebo (for filled drugs), (d
) the test solution, and (e) the solution used for the evaluation of suitability
of the chromatographic system. In addition, it is recommended to confirm the sp
ecificity 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 un
known (unidentified) or absent. In this case, it is expedient to use special exp
eriments involving modification of the parent compound (sometimes called “stress
testing” [2, 15, 27]), whereby a solution of the given drug (or of the parent c
ompound) is subjected to factors leading to its partial destruction with the for
mation of related identifiable compounds. The degree of decomposition can be rea
dily determined from the decrease in the area of the main peak in the chromatogr
am of the final solution relative to that in the initial solution. The specifici
ty of the given analytical procedure is judged by separation of the peaks of imp
urities between themselves and from the peak of the main component and by the pe
ak purity of the parent compound. In particular, the specificity of HPLC procedu
res can be proved using the following methods of chemical modification. (i) Hydr
olysis with 0.1 N solutions of HCl or NaOH at room temperature or at elevated te
mperatures. 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 a
nd 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 impur
ity [22]), extracted according to the proposed procedure, and analyzed by HPLC.
(iii) Thermal decomposition by heating to 60 – 100°C. Example: a granulate was k
ept for 7 days at 80°C. The resulting solid products of decomposition can be sto
red and used as control substances in the tests for suitability of a chromatogra
phic system. (iv) Photochemical decomposition under illumination (e.g., UV irrad
iation). (v) Chemical addition reactions, for example, drug bromination at multi
ple bonds.
A mixture of the initial substance and the products of its chemical modification
can be used for preparing solutions used for the evaluation of suitability of t
he chromatographic system. The duration of action used for the chemical modifica
tion of drugs is selected taking into account the following factors. (i) The pea
ks of the products of drug modification are to be clearly distinguishable in the
chromatogram. Therefore, the treatment duration must be sufficient to provide f
or a not less than 10% decrease in the main peak height (area), which is proport
ional to the drug content. (ii) The peaks of the products of drug modification h
ave to be sufficiently well separated from the main peak, but the main peak heig
ht (area) must be comparable with that in the initial test solution. According t
o [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 deco
mposition of the parent compound can be compensated by adding it in the necessar
y amount. (iii) It is desired that the percentage “content” of the products of d
rug modification determined by the method of internal normalization would be clo
se to the level of maximum permissible content of a single impurity. Further ste
ps in the proof of specificity of the analytical procedure in the case under con
sideration are the same as in case 1. The validation characteristics additionall
y include chromatograms obtained in the course of experiments on the chemical mo
dification of the parent compound. Data presentation. The proof of the specifici
ty of an analytical procedure is presented in the form of a set of chromatograms
(see above) with discussion of the obtained results. These data are supplemente
d by the results of calculation of (i) the separation coefficient Rs for two pea
ks 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 se
paration of impurities by HPLC is still an art, with the results depending on th
e skill of developers. It is very important to select the optimum chromatographi
c 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) t
o find a certain number of such peaks, a part of which cannot be quantitatively
characterized, or (c) to reveal a very large number of additional measurable pea
ks upon overloading the column with respect to the main drug component. Therefor
e, in developing and validating the methods of impurity determination (especiall
y in the case of parent compounds), it is expedient to check for the possible pr
esence of additional peaks (i.e., impurities) by chromatography of drug solution
s with elevated concentrations providing overloading of the column with respect
to the main component. However, this approach is usually inapplicable in the cas
e of ready-to-use drugs containing large amounts of auxiliary substances.
216
N. A. Épshtein
1.2. Confirming the Precision of a Given Analytical Procedure The precision (rep
eatability, reproducibility) of an analytical procedure characterizes the random
scatter (variation) of the results relative to the mean value. It should be emp
hasized that, in order to obtain reliable results, the sample must have a homoge
neous composition. The results have to be statistically treated. Variants leadin
g to rough errors (e.g., using variation range R or the “three sigma” rule [8])
should be rejected. In evaluating the suitability of HPLC procedures (actually,
in determining the metrological characteristics), it is necessary to use measuri
ng vessels of class A or special calibrated measuring vessels and take all other
possible measures to increase the reproducibility and accuracy of HPLC measurem
ents [27]. The precision (repeatability, reproducibility) of an analytical proce
dure is evaluated in terms of the standard deviation (SD) of the relative standa
rd deviation (percentage RSD) determined in a series of measurements and calcula
ted by the formulas SD =
å( X i - X )2
i =1
m
( m - 1) ,
RSD(%) =
100SD . X
(1)
According to the ICH recommendations [4] on the validation of chromatographic pr
ocedures, the characteristics of precision are considered at three levels: repea
tability, intermediate precision, and reproducibility (Table 3). In [4, 27] and
in many other papers, the set of validation characteristics of HPLC procedures i
ncludes the injection repeatability. However, the author believes that this is i
ncorrect: this parameter characterizes the quality of injector (e.g., syringe) r
ather than the suitability of a proposed procedure.
TABLE 3. Main Precision Characteristics for the Validation of HPLC Procedures Ac
cording to ICH [4]
Precision characteristic Conditions of determination
Repeatability (Rt )
Injection repeatability
Determined for the same sample preparation by the same analyst using the same in
strument (chromatograph) during a short period of time
Intermediate precision (Ip )
Intra-assay precision –
Reproducibility (Rp )
–
Determined for the same sample preparation by different analysts using various i
nstruments (chromatographs) during a prolonged period of time (not less than two
days) The same, in various laboratories
This characteristic is not included in the list of CDER [15] and USP-26 [7]. In
HPLC validation, data on the injection repeatability should be provided as addit
ional material required in cases of search for the “bottleneck” of a proposed me
thod (dispersion analysis). 1.2.1. Injection repeatability and intra-assay preci
sion. In the general case, repeatability (Rp) characterizes the reproducibility
of a given analytical procedure for the same sample preparation, as performed by
the same analyst using the same instrument (chromatograph) during a relatively
short period of time. For evaluating the repeatability in a given laboratory, th
e same analyst prepares samples of a model mixture or the same batch of a parent
compound or a drug: (a) not less that nine samples of solutions covering the ra
ted range of concentrations. For example: a homogenized powder of triturated tab
lets from the same batch is used to prepare drug solutions with concentrations e
qual to 50, 100, and 150% of the reference sample solution concentration accordi
ng to the proposed procedure, or (b) not less than six samples of solutions in t
he region of concentrations close to the nominal value. For example: a homogeniz
ed powder of a parent compound or triturated tablets from the same batch is used
to prepare six solutions with nominal concentration according to the proposed p
rocedure. Note that each sample solution is (i) prepared independently of the ot
her solutions and (ii) chromatographed at least three times. Each solution is ch
aracterized by the drug content X i (i = 1, …, N ), the average value X = S X i
N , the standard deviation SD, the relative standard deviation RSD of particular
measurements, and the confidence interval (for P = 95%) of the average value. I
t is required to show that the average results are statistically equivalent (e.g
., in terms of the Student t-criterion) or, which is more convenient for the pra
ctical analysis, that RSD £ 1.0% for determination of parent compounds, RSD £ 2.
0% for drugs, or RSD £ 10.0% for impurities [13, 27].5 1.2.2. Intermediate preci
sion. This value characterizes the reproducibility of results obtained in the sa
me laboratory by different analysts using various instruments (chromatographs) d
uring a prolonged period of time (not less than two days) for the same homogeneo
us sample or a model drug mixture according to the proposed analytical procedure
. Typically, not less than six solutions are prepared with concentrations close
to the nominal value (see the preceding section). Each sample solution is (i) pr
epared independently of the other solutions and (ii) chromatographed at least th
ree times.
5
For small values of the standard deviation (SD << 1), the t-criterion may give s
tatistically significant differences even for close (almost identical) values of
the compared average concentrations. This is related to the fact that this crit
erion is proportional to the ratio of systematic and random errors.
Validation of HPLC Techniques for Pharmaceutical Analysis
217
These solutions are characterized by the average drug content according to the r
esults obtained by each of the analysts, X i and X j (i, j = 1, …, N ). These da
ta 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, S
Dj ) and relative standard deviations (RSDi, RSDj ) of particular measurements.
First, it is required to show that the proposed procedure of determination of th
e drug content and the impurity coincentraiton provides for the statistically eq
uivalent standard deviations SDi and SDj of the results obtained by different an
alysts (in terms of the Fisher F-criterion). Then, it is necessary to demonstrat
e that the average results of these (for certainty, two) analysts are statistica
lly reliably (P = 95%) identical in terms of the t-criterion calculated as t= |
X 1 - X 2|
2 SD1
m1
+
SD 2 2 m2
=
| X 1 - X 2|
2 SD1 + SD 2 2 m
,
where X 1 , X 2 are the average results of analyses performed by analysts 1 and
2 and SD1, SD2 are the standard deviations in the particular series of m1 and m2
parallel determinations (usually m1 = m2 = m ). This t value is compared to tab
ulated values of the Student criterion t (P = 95%, f = m1 + m2 – 2), where P = 9
5% 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 d
ifference of average values can be considered as statistically insignificant wit
h a 95% confidence probability. Otherwise, the average results differ to a great
er extent than that admitted by random errors in both series [35]. In pactice, v
alidation of the procedures of determination of the content of impurities is som
etimes performed using a less strict method [13], by showing that the scatter (R
SD) of the results of one analyst (characterized by a greater standard deviation
(SDi or SDj ) relative to the average result of another analyst (with a lower S
Di or SDj) does not exceed a certain preset level, for example, so that RSD £ 10
% for impurities with a rated content up to 1%, RSD £ 25% for an impurity conten
t within 0.1 – 1%, and RSD £ 50% for an impurity level below 0.1%. It should be
noted that, according to USP-26 [7], it is in most cases sufficient to determine
only the repeatability for proper validation of an analytical procedure, while
the intermediate precision and reproducibility characteristics should be determi
ned for procedures included in the pharmacopoeial articles. 1.2.3. Reproducibili
ty. This characteristic is determined by comparing the results obtained upon ana
lysis of the same samples in different laboratories using a proposed analytical
procedure. The necessary statistical methods are described in monograph [35, Cha
pter 8.4] and in the State Standard GOST R ISO 5725-2002. It should be noted tha
t for reliable evaluation of the statistical significance of the difference be-
tween the results obtained in such investigation, it is necessary that the round
robin tests involve not less than five laborqtories [35]. In practice, however,
the reproducibility is usually evaluated using two or three laboratories and ch
aracterized by less strict estimates. For example, validation of a procedure pro
posed for the quantitative determination of a parent compound is performed by de
monstrating the statistical equivalence of the standard deviations SDi and SDj o
f the results obtained in different laboratories (in terms of the Fisher F-crite
rion). Then, it is demonstrated that the scatter (RSD) of the results of analyse
s 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 r
eproducibility of analytical procedures is rarely validated because (i) it is ne
cessary to involve certified laboratories capable of reproducing the proposed pr
ocedure with high precision and (ii) this requires high organizational facilitie
s and expenditures. 1.3. Confirming Linearity of the Response to Drug Concentrat
ion Linearity characterizes the ability of a proposed analytical procedure to gi
ve (within the suitability range) a response signal with the magnitude Y (e.g.,
peak height or area) directly proportional to the amount C (concentration) of a
drug to be determined: Y = a + bC. According to ICH recommendations [3], the lin
earity 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 met
hods of regression analysis in terms of the linear equation Y = a + bC. For the
analytical procedures for determining the content of a parent compound, CDER rec
ommends establishing the criterion of linearity at a level of the correlation co
efficient r not lower than 0.999 [4]. However, even such a high level of correla
tion may be accompanied by significant deviations from linearity in the regions
of high and low drug concentrations [13]. For this reason, ICH [3] recommends th
at the linearity be validated by a plot of the difference Y–C showing deviations
(residuals) of the calculated values yi = a + bC from the measured Yi values as
the function of the concentration Ci. The “outbursts” of the points (xi, yi ) r
elative to the regression model can be determined by calculating the parameter t
using the formula [36] t= SD 0 | y i -Y i | (Y i - y ) 2 1 1+ + N ( N - 1) × SD
2 y ,
Here, yi and Yi are the calculated and experimentally measured values of the res
ponse, respectively; y = Syi /N; N is the total number of experimental points (x
i , yi ), and
218
N. A. Épshtein
SD 0 =
S( y i - Y i ) 2 S( y i - y ) 2 , SD y = . N -2 N -1
The calculated t value is compared to tabulated values of the Student criterion
t (P = 95%, f = N - 2). If the calculated parameter is greater than the tabulate
d value, the given point can be considered as deviating from the adopted regress
ion model with a 95% confidence probability. In practice, validation of the proc
edures of determination of the content of impurities with respect to linearity i
s sometimes performed proceeding from a correlation coefficient of r ³ 0.98 [13]
. According to our estimates, use of the calibration graphs with such correlatio
n 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 suitabil
ity range of a proposed analytical procedure [35]. According to ICH recommendati
ons [3], the linearity can be demonstrated directly by using the reference paren
t substance (dilutions of a standard solution) and/or model artificial mixtures
including components of the drug studied. The most adequate approach consists in
taking thoroughly weighed aliquots of the drug components and preparing solutio
ns according to the proposed procedure, since all operations of the analyst shou
ld correspond strictly to those stipulated in the description. In practice, howe
ver, an “intermediate” approach recommended by ICH [3] is frequently employed. A
ccording to this, solutions are partly prepared using weighed aliquots of the dr
ug components and the other are obtained by diluting these stock solutions. It s
hould be emphasized that the linearity of a proposed analytical procedure should
be confirmed in the course of validation of the accuracy, which reduces expendi
tures 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 th
e compound with a concentration at or above the upper limit of the expected conc
entration interval (suitability range of the proposed procedure). Then, a series
of dilutions is prepared so as to cover the entire range. Each solution is stud
ied in a series of two or three injections. (b) For the analysis of ready-to-use
drugs, the linearity is frequently checked in the same way as for the parent co
mpound (i.e., using solutions of the parent compound as described in (a)). Howev
er, it is incorrect to ignore the possibility that auxiliary components (placebo
) may influence the results. Therefore, it is more correct to validate the linea
rity using model mixtures of the parent compound and placebo. (c) For the determ
ination of impurities using the method of internal normalization of the peak are
as or heights, it is possible to evaluate the linearity by preparing dilutions o
f the reference sample solution (with a concentration equivalent to the rated va
lue) in the model impurity solution so as to obtain
drug concentrations in the range from 0.05% (dilution by a factor of 2000!) to 2
.5%. Instead of making some dilutions, it is possible to use a proportional decr
ease in the volume of applied sample (which saves the mobile phase). Data presen
tation. The validation characteristics include (i) a regression equation of the
Si = a + bC type (for example, S = 15536 + 15833969C [mg/ml]), (ii) the correlat
ion coefficient (e.g., r = 0.9998), and (iii) a plot visually confirming the lin
earity of relationship between S and C. 1.4. Confirming the Accuracy of a Given
Analytical Procedure The accuracy characterizes the proximity of the experimenta
l results, obtained using a proposed analytical procedure, to the “true” value i
n the entire suitability range of this procedure. The accuracy represents a comb
ination of the random and systematic error.6 In order to provide for accurate HP
LC determinations, it is recommended to use standard solutions with concentratio
ns close to within 10% of the test solution concentration. The accuracy of analy
tical procedures should be determined using homogeneous samples with exactly kno
wn concentrations of the compounds to be determined. For validation purposes a s
eries of such solutions is prepared using the reference parent compound. Accordi
ng to ICH recommendations and USP-26 [1, 7, 15], the accuracy can be expressed b
oth in the classical form, as the difference X – m between the average experimen
tal value (X ) and the true value (m) with the corresponding confidence interval
DX ,7 (2) ( X - m ) DX , and in an alternative (and more illustrative) form,
in terms of the percentage recovery of the known amount of the compound to be de
termined, ( found content ) (3) R= ´ 100%. ( introduced content ) The author bel
ieves that the content recovery testing should be preferred for evaluating the a
ccuracy. This approach provides a more illustrative characteristic of the reliab
ility of results obtained using a proposed analytical procedure and reveals the
need for additional checks in the case of a significant systematic error (see be
low). On the other hand, the recovery defined by formula (3) incompletely charac
terizes the accuracy, since this quantity is also random and requires knowledge
of the corresponding confidence interval. Thus, it is recommended to determine b
oth the recovery R (%) and the confidence interval at a preset probability (P =
95%), representing the accuracy in a form analogous to expression (2): (4) R D
R . Obviously, the proposed methods should not involve significant systematic er
rors. In the absence of systematic er6 7
Accordiong to the State Standard GOST R ISO 5725-1–2002 the systematic error usu
ally characterizes “trueness.” 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 permissible RSD val
ues (see above), experimental experience, and analysis of the published data [13
, 27], it is possible to draw the conclusion that there is no need to verify a p
roposed analytical procedure in the absence of a significant systematic error, p
rovided that it is established that the recovery with allowance of the confidenc
e interval does not fall outside the following limits: 99.0–101.0%, for the quan
titative analysis of parent substances with a high rated content of the active c
omponent (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 r
eady-to-use drugs; 90.0–110%, for the quantitative determination of impurities w
ith a rated maximum content of up to 1%; 75–125%, for the quantitative determina
tion of impurities with a rated maximum content from -0.1 to 1%; 50.0–150%, for
the quantitative determination of impurities with a rated maximum content below
0.1%. 1.4.1. The procedure of accuracy evaluation. ICH recommends making three d
eterminations (i.e., analyze three model mixtures) for three different concentra
tions. However, this approach does not allow the accuracy to be determined toget
her with linearity and other validation characteristics. The author believes tha
t the accuracy should be evaluated using not less than nine determinations at va
rious concentrations covering the entire range of suitability of the proposed pr
ocedure. This provides for the possibility of determining this characteristic si
multaneously with the calibration graph parameters and their statistical charact
eristics (for evaluating the linearity according to Section 1.3), the limit of q
uantitation (Section 1.7.2), and the limit of detection (Section 1.6.2). It shou
ld be emphasized that these determinations should include all stages of the prop
osed analytical procedure. For parent substances, the accuracy of analysis is us
ually determined by comparison to a reference sample. According to this, the ref
erence sample (or a high-purity substance) is analyzed using the standard proced
ure and the results are compared to data in the certificate of the reference sam
ple 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 act
ive component, the average recovery should be not less than 99 – 101% at each le
vel [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 t
he quantitative determination of impurities, this characteristic is determined b
y the analysis of such mixtures containing known amounts of these impurities. Th
ese analyses are performed using two principal methods. The method of recovery o
f a parent compound introduced into the placebo (matrix). This method, used for
the
analysis of drugs comprising mixtures of parent and auxiliary compounds, is base
d on determining the recovery of a known amount of the parent substance introduc
ed into the placebo. The placebo is prepared separately and then introduced in a
nominal (or proportional) amount into measuring flasks. Then, thoroughly weighe
d amounts of the parent compound or its concentrated solutions are added so that
(upon filling the flasks to the marks) the sample concentrations would cover th
e entire expected suitability range of the proposed analytical procedure. For ex
ample, a parent compound can be introduced into a placebo solution at a level of
80, 100, and 120% of the nominal concentration indicated on the label (or the r
eference solution concentration). The method of standard additives. This method
is generally analogous to that described above but is applied only when it is im
possible 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 biologica
l 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 analy
zed solution. Using the proposed procedure, the amount of the parent compound is
 determined (found content) and compared to the known additive (introduced conte
nt). Alternatively, it is possible to compare the results of analyses performed
using the proposed method and the data obtained for the same samples by validate
d 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 su
bstance for preparing both model mixtures and standard solutions. This eliminate
s errors related to the possible uncertainty of the composition indicated on the
label of the reference sample and allows using commercial samples instead of sp
ecial 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 compou
nd), one may add a known amount of concentrated solution and then fill the measu
ring flask with a solvent stipulated by the proposed analytical procedure. For t
he quantitative determination of impurities, the accuracy of evaluation has cert
ain peculiarities. In this case, the method of recovery of a parent compound int
roduced into the placebo and the method of standard additives have limited appli
cability because these validation procedures require large amounts of identified
impurities. In this case, the accuracy is most frequently checked using the met
hod described below. The method of internal normalization with or without respon
se factors. According to this method, identified impurities are characterized by
the response factors with respect to the parent compounds determined by the ana
lytical procedure under consideration. These coefficients depend on the mobile p
hase composition and the analytical wavelength. For reproduction (or modificatio
n) of the analytical procedure, the detector wavelength is not changed, while th
e mobile phase composition can be corrected for the difference in the
220
N
N. A. Épshtein
parameters of chromatographic columns. In the case of a considerable change in t
his composition (see Section 4), it is necessary to re-determine at least the va
lues of the response factors. For determining unidentified and accidental impuri
ties, these factors are conventionally taken equal to unity (assuming that the s
ensitivity 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 fo
r systematic error. The analytical procedure can be checked for the absence of s
ystematic errors by one of the three methods considered below. Method based on t
he Student t-criterion without regression analysis [8]. Each sample (whose total
number is N ³ 5) with known values m (introduced content) of the component to b
e determined is analyzed in m = 3 – 6 parallel determinations. The total data ar
ray is characterized by the dispersion (SD0) and the Student criterion8 t= | m -
x| m SD 0
SD 2 = 0
k =1
2 å SD k
N
.
Finally, the Student t-criterion is calculated as t = (| m - x |× m ) SD 0 (6)
This t value is compared to tabulated values of the Student criterion t (P, f =
m – 1). If the calculated parameter is greater than the tabulated value for P =
95% and f = m – 1. t > t (P, f ), the results obtained using the proposed method
can be considered as involving a systematic error d. This error is calculated b
y the formula d= | x - m| 100%. m (5)
The standard deviation SD0 in a particular analysis is calculated using the set
of all m parallel determinations performed for N (or g in the notation of [8]) s
amples. This allows the SD0 value to be reduced and the sensitivity of determina
tion of the systematic error to be increased with a simultaneous decrease in the
confidence interval for the results of analysis, DX = t SD0/ m, where t is the
Student criterion for f = m – 1 degrees of freedom. The algorithm of these calcu
lations is as follows. Each kth sample is characterized by the deviation of the
experimental value from average, d i = X i - X , and the dispersion æm ö SD 2 =
ç å d i2 ÷ ( m - 1). Then, the difference between the k è i =1 ø maximum and min
imum values of the dispersion SD 2 is k checked to be insignificant in terms of
the Fisher F-criterion. If this difference is actually insignificant, the SD 2 v
alue is 0 calculated as the sum of square deviations for all samples divided 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.
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 in
significant) systematic error and small random error, the calculated t-criterion
 can be greater than the tabulated value. However, a small systematic error can
be ignored in practice when the analytical problem does not require high accurac
y of determination. This approach is also valid for other methods of evaluation
of the accuracy of a proposed analytical procedure. Method of regression analysi
s with the Student t-criterion [35, 38]. This method seems to be the most effect
ive, since it provides for the possibility of using the calibration graph for th
e accuracy evaluation and determination of some other validation characteristics
(see the generalized scheme in Fig. 1). For simultaneous determination of the c
onstant and variable systematic errors, not less than N = 5 samples with known v
alues of the parent compound are studied and the relationship between the “intro
duced content” (mt ) and the “found content” (mf ) is processed by least squares
in terms of the equation mf = a + bmi. Using these data, the parameters ta = |a
|/SD0 and tb = |1 – b|/SD0 are calculated and compared to the critical (tabulate
d) 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 sys
tematic error, provided that ta £ t (P, f = N – 2), and the absence of a linear
variable systematic error, provided that tb £ t (P, f = N – 2). It is expedient
to perform validation of the accuracy of an analytical procedure together with c
hecking for the linearity of the system response (area or height of the peak) as
a function of the concentration of the parent compound to be determined (in fac
t, linearity of the calibration graph in terms of the criteria described in Sect
ion 1.3) and with finding the limit of detection (Section 1.6.2) and the limit o
f quantitation (Section 1.7.2). Method of regression analysis with the Fisher F-
criterion. This method is based on the assumption that a linear variable systema
tic error can be ignored. This assumption is justified because HPLC in pharmacy
is used in a relatively narrow range of sample solution concentrations. The meas
urements are performed for not less than N ³ 5 samples (model mixtures) with kno
wn values of the parent compound, after which the relationship between the “intr
oduced content” (mt ) and “found content” (mf ) is processed
Validation of HPLC Techniques for Pharmaceutical Analysis
221 be < 3, then DX is expressed by a value with two significant digits. 1.5. Va
lidation of the Suitability Range of a Given Analytical Procedure The range of s
uitability of a given analytical procedure is the interval between minimum and m
aximum concentrations (amounts) of a compound to be determined in which (i) the
linearity is observed, (ii) the characteristics of repeatability fall within per
missible (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 rang
e of suitability of a given analytical procedure is expressed in the same units
as the results of analyses. It should be noted that, in practice, it is not nece
ssary to determine the maximum possible range of suitability for an HPLC procedu
re. If it were necessary, this range could be determined using threshold RSD val
ues obtained in the course of validation of the linearity and precision. For exa
mple, RSD must not exceed 3% for HPLC procedures aimed at determination of the p
arent compounds and 10% for procedures of impurity determination [13]. In practi
ce, it is sufficient to show that a given range of suitability covers “with marg
in” the rated limiting concentrations of the substances to be determined, as ind
icated in the corresponding pharmacopoeial articles. For this reason, it is reco
mmended that the range of suitability of a given analytical procedure be not les
s than the following intervals [3, 7]. (i) For the quantitative determination of
the main component concentration in parent compounds and ready-to-use drugs: fr
om 80 to 120% of the nominal content (i.e., the concentrations of test solutions
should range within 80 – 120% relative to the concentration of a reference samp
le solution used accordsing to the proposed procedure). (ii) For evaluation of t
he homogeneity of dosage: from 70 to 130% of the nominal content, provided that
a wider interval is not required (in special cases such as aerosols). (iii) For
dissolution tests: 20% (absolute percentage) of the rated value of drug releas
e. For example, for monitoring the behavior of a drug with delayed release of a
parent compound, for which the release is rated as 20% within the first hour and
up to 90% within a 24-h period of time, the suitability range must extend from
0 to 110% of the nominal drug content. (iv) For the quantitative determination o
f impurities by the method of external standard: from 50 to 120% of the nominal
content. It should be noted that, in view of the possibility of RSD values amoun
ting 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 biol
ogical activity, toxicity, or unpredictable behavior, the limits of detection an
d 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 com
pound and the detection of impurities are per-
by least squares in terms of the relation mf = a + bmt. The absence of a constan
t systematic error is confirmed by the insignificance of the coefficient a [35]
at a commonly accepted confidence probability level of P = 95%. For this purpose
, the Fisher criterion calculated is as F ( P, f 1 = N – 1, f 2 = N – 2 ) = SD 2
( N – 1) – SD 2 ( N – 2 ) 01 02 SD 2 ( N 02 – 2) (7), ,
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 th
e 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 wi
thin a 95% confidence probability. 1.4.3. Data presentation and evaluation of th
e systematic error. The final judgment about the accuracy of a proposed analytic
al procedure can be made upon validation of its specificity, precision (repeatab
ility, reproducibility), and linearity.9 It is necessary to indicate a particula
r method of normalization of the content of impurities (weight fractions, percen
tage of the area under the peak of the main component, etc.). The validation res
ults are presented by data on the recovery of the amount of introduced parent co
mpound with a confidence interval, R DR , or the difference between the averag
e experimental value X and the true value m with the corresponding confidence in
terval: ( X - m ) DX . The proposed procedure involves no significant systemat
ic error, provided that the recovery with allowance of the confidence interval d
oes not fall outside the following limits: 99.0–101.0%, for the quantitative ana
lysis of parent substances with a high rated content of the active component (98
% and above); 98.0–102%, for the quantitative analysis of parent substances with
a lower rated content of the active component (98% and below) and ready-to-use
drugs; 90.0–110%, for the quantitative determination of impurities with a rated
maximum content of up to 1%; 75–125%, for the quantitative determination of impu
rities with a rated maximum content from – 0.1 to 1%; 50.0–150%, for the quantit
ative determination of impurities with a rated maximum content below 0.1%. The n
umerical result of a particular analysis, X (or R), must contain the last signif
icant 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 v
alue is determined as follows. If the first significant digit in the error is ³
3, then DX is expressed by a value with one significant digit; should the first
significant digit in the error
9
It is expedient to confirm the linearity together with determining the accuracy.
222
N. A. Épshtein
formed jointly and only the reference sample solution of the main component at a
nominal concentration is used, the suitability range must extend from the rated
LPI value (more precisely, from half of this value, see the preceding point) up
to 120% of the nominal concentration of the parent compound. If the LPI value i
s unknown (e.g., during the development of an analytical procedure) the initial
lower limit of the suitability range according to the ICH recommendations should
be established at 0.1% of the nominal concentration of the parent compound for
a daily drug dose below 1 g; should this dose exceed 1 g, the lower limit of the
suitability range should be reduced to 0.05% of the nominal drug concentration.
1.6. Determining the Limit of Detection of a Given Analytical Procedure The lim
it of detection (LOD) is determined as the minimum concentration of analyzed sub
stance in the sample, which (i.e., the corresponding response) can be detected u
nder 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 detecti
on of limiting impurity concentrations. Obviously, the LOD may depend on the HPL
C detectors and pumps. For this reason, this characteristic has to be rated for
various instruments, including those available for potential users. Irrespective
of the method used for evaluation, it is necessary to have a chromatogram showi
ng that a response peak exceeding the baseline noise is actually observed at a c
oncentration 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 a
nalyzed substances (except for analytical procedures intended for monitoring the
cleanness of technological equipment). In most other cases, it is sufficient to
show that the impurity of interest is reliably detected at a preset level (see
Section 1.6.5). 1.6.1. Determining the LOD from the Standard Deviation of the Re
sponse and the Slope of the Calibration Curve. According to this method [3], the
LOD value is calculated by the formula LOD = 3.3(SDa/b ), (8)
(a) Using the calibration curve S = a + bC constructed using the results of anal
yses for a series of the reference sample solutions with decreasing concentratio
ns 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 d
eviation SDa of the free term is determined. Alternatively, the standard deviati
on is determined for the intersections of several regression lines of the calibr
ation curve with the ordinate axis, constructed using several series of referenc
e sample solutions with concentrations in the region of the LOD. (b) Using the s
tandard deviation of the area under the peak in the chromatograms of an impurity
with concentrations within 0.01 – 0.05% of the nominal drug concentration deter
mined using the given analytical procedure, typically for ten sequential injecti
ons. 1.6.2. Determining the LOD Using the Free Term and the Coordinates of the M
idpoint of the Calibration Curve. This is the most convenient way to determine t
he LOD. According to this method, the calibration curve is described by the regr
ession 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 LO
D = 2C × t ( P, f ) × SD 0 Y - a + t ( P, f ) × SD 0 nj nj , (9)
assuming that the response – concentration relation is linear in the range from
the maximum possible concentration of the analyzed compounds down to zero. It is
recommended to determine the slope b of the calibration curve using reference s
ample solutions with concentrations in the vicinity of the LOD (see Section 1.7)
. The value of the standard deviation SD can be determined using one of the two
methods:
10
For the analysis of impurities, it was recommended [27, p. 695] to perform tests
 in the range from the limit of quantitation with respect to the main component
(typically, below 0.1%) up to a 5% concentration in solution.
where C and Y are the coordinates of the midpoint; a is the free term; SD0 is th
e 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 calibrat
ion 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 influe
nce this value. The error has been eliminated in the new edition of this monogra
ph. 1.6.3. Determining the LOD for the Signal-to-Noise Ratio S/N¢ » 3. The metho
d 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 strong
ly depends on the experimental conditions. On a scale comparable with the noise
intensity, the baseline appears as a broken line of complicated shape and admits
only a very subjective estimate of the maximum noise N¢. 1.6.4. Evaluating the
LOD from the Minimum Concentration for Which the RSD is Below a Preset Value. Ac
cording to calculations [39], the relative error of the LOD amounts to at least
20%. For this reason, the LOD can be theoretically evaluated as the minimum solu
tion concentration for which the RSD for the peak area (height) of the analyzed
compound for five sequential determinations does not exceed 20%. However, this a
pproach is not convenient in practice 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 method in accordance wit
h FDA recommendations (defining the LOD as the minimum concentration for which t
he RSD does not exceed 15%) would require 288 determinations. In order to take i
nto account the influence of the drug matrix on the LOD, it was suggested [30] t
o take into account the so-called “specific LOD.” These values are determined us
ing mixtures of a given parent substance with a placebo instead of a solution, w
hich takes into account the effect of asymmetry of the peaks of other components
on the LOD. According to ICH [3], the LOD has to be refined using various colum
ns and instruments because this characteristic depends on the noise level (and,
hence, on the working life of the column, on the properties of the detector, etc
.) and on the peak shape. 1.6.5. Alternative Proof of the Reliable Detection of
Impurities in Parent Substances and Ready-to-Use Drugs. For a test solution conc
entration 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 correspond
s to a response on the order of 100 mV, which is one order of magnitude higher t
han the noise level of modern detectors. For validating the reliable detection o
f impurities, it is sufficient to present the chromatogram of a reference (or te
st) solution diluted D = 2L times, where L is an integer indicating the ratio of
the main component concentration to the minimum rated concentration of the impu
rity. This chromatogram must visually confirm the possibility of reliably detect
ing impurities at the level of about half of their rated content. For example, i
f 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, whic
h corresponds to an individual impurity content of 100/D = 0.05% (the British Ph
armacopoeia does not take into account impurities with concentrations below 0.05
%, except for highly toxic substances). In many cases, this approach eliminates
the need to establish the LOD during the determination of impurities in parent c
ompounds and ready-to-use drugs. Data presentation. According to ICH [3], it is
necessary to validate the LOD value and indicate the method of determination. If
the LOD was calculated from experimental data, it is necessary to present the r
esults of analysis of the required number of samples with the content of a detec
ted component close to the LOD. If the LOD was estimated by visual assessment of
chromatograms, it is necessary to present chromatograms confirming reliable det
ection of the peaks of parent compounds or impurities. The number of significant
digits in the LOD [39] is determined as follows. If the first significant digit
in the LOD value is > 3, then this limit is expressed by a value with one signi
ficant digit and rounded to the closest integer; should the first significant di
git in the LOD be £ 3, then the LOD can be is expressed by values with two signi
ficant digits and the second digit should be rounded to 0 or 5.
1.7. Validation of the Limit of Quantitation of a Given Analytical Procedure The
limit of quantitation (LOQ ) is the minimum concentration of analyzed substance
that can be determined at an acceptable precision (repeatability, reproducibili
ty) and accuracy under rated conditions of analysis by a given method [1, 15]. T
he ICH [3] and USP-26 [7] recommend several methods of determining LOQ for the i
mpurity analysis. 1.7.1. Evaluating the LOQ for the signal-to-noise ratio S/N¢ =
10. This is the most widely used method of determining the LOQ. However, since
this procedure makes use of the peak heights (rather than areas), it is more sui
ted for the HPLC techniques using this measure of the response. This approach do
es not take into account the requirements on reproducibility of the HPLC procedu
re. According to this method, an initial reference solution of the analyzed comp
ound is determined for which the signal-to-noise ratio is at least 30 [27]. Then
, this solution is sequentially diluted until this ratio decreases to S/N¢ » 10.
The corresponding concentration is considered as the LOQ. For this evaluation o
f 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, w
here W0.5 is the full width at half maximum (FWHM) of this peak [22]. For HPLC p
rocedures used in pharmacy, it would be interesting to study the influence of th
e maximum noise N¢ on the relative standard deviation of results for a nearly Ga
ussian peak shape. According to [27], RSD[%] = 50 , S N¢ (10)
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. Eva
luating 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 o
f the response signal. The peculiarities of this approach were considered in Sec
tion 1.6.1. A significant disadvantage of this approach (as well as of some othe
rs) 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 reproduci
bility requirements, can be obtained in the following way [41]. First, the value
s of response Y (area under the peak of the analyzed component) within the limit
s of the calibration curve Y = a + bC are used to calculate (i) the theoretical
values of C = (Y – a)/b and (ii) the standard deviations
224
N. A. Épshtein
(SDc) and relative standard deviations (RSD = SDc/C ´ 100) of the results of ana
lysis. 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 und
er consideration. This permissible concentration Cp is taken to be LOQ with allo
wance for the RSD (i.e., for the reproducibility) requirements. The values of st
andard deviations of the results of analyses are calculated by the formula [35]
SD 0 b 1 1 æ SD b + +ç N mçb è ö ÷ ÷ ø
2
SD c =
æ Y s -Y ç ç SD è 0
ö ÷ ÷ ø
2
(12)
for f = N – 2 degrees of freedom, where SD0 is the standard deviation of the lin
ear 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
parallel (independent) determinations, b is the slope of the calibration curve,
SDb is the corresponding standard deviation, and Y = Yk/N is the average respons
e of all experimental points used for constructing the calibration graph. 1.7.3.
Other methods. Other methods of determining the LOQ are not theoretically justi
fied 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 sequent
ial injections give RSD £ 3.0% [27, p.695] or RSD £ 2.0% [29]. With this approac
h, 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 th
e LOQ. It was suggested to determine the so-called specific LOQ p30]. This chara
cteristic 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 presenta
tion. The LOQ value is presented with indication of the method used for its dete
rmination. 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 b
y calculation or extrapolation, it is necessary to present the results of analys
is of a sufficiently large number of samples with the content of the analyzed co
mpound close to the LOQ level. 1.8. Stability of Solutions. 1.8.1. Confirming th
e stability of solutions using the regression relation for the area under the pe
ak versus the time. As a rule, the analytical solutions are used for a relativel
y short period of time (th ) for which the content of the analyzed substances (p
roportional to the areas under the corresponding peaks) remains practically unch
anged. In order to confirm this statistically, it is sufficient to determine the
coefficient b in the regression relation St = S0 + bt (or St – S0 = bt ) betwee
n the area St under the peak at the time t
(within the interval from 0 to th ) and the area S0 at the initial moment, calcu
late the parameter tb = |b |/SDb, and compare this value with the critical (tabu
lated) 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 p
oints 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, ther
e 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 l
ast experimental point. 1.8.2. Methods of evaluation. The stability of test and
reference solutions ST (%) can be evaluated using the following methods. (i) For
continuous testing within a relatively short period of time — by the ratio of t
he area St under the peak of the analyzed 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 compound (or impurity) at the moment t to the in
itial value m0: ST = 100mt /m0. (14)
The test and reference solutions are prepared according to the given procedure a
nd analyzed with certain time intervals. The maximum possible duration of measur
ements depends on the practical requirements on the possible storage time of the
given solution (which may not necessarily be equal to the maximum rated storage
time). The solutions can be considered stable until the difference |100 – ST |
does not exceed the relative error of determination of the main component (or im
purity) according to the given analytical procedure.11 Data presentation. The st
ability of solutions is confirmed by data on the storage times of solutions and
mobile phases used in the analyses. 2. EVALUATION OF THE STABILITY OF ANALYTICAL
SYSTEMS WITH RESPECT TO SMALL VARIATIONS OF PARAMETERS (ROBUSTNESS) The robustn
ess of an analytical procedure is the characteristic of its stability with respe
ct to small variations of the system parameters possible under real conditions.
This stability is usually evaluated in terms of the RSD of the results of analys
es compared to the analogous data obtained using strictly observed conditions ac
cording to the validated analytical procedure. A more correct procedure based on
evaluation of the statistical significance of the difference between these resu
lts in terms of the Student t-criterion is rarely applied to the HPLC techniques
.
11
The absolute value of |100 – ST | is taken because ST can be greater than 100% d
ue to random errors.
Validation of HPLC Techniques for Pharmaceutical Analysis
225
Robustness is usually studied at the stage of development of the analytical proc
edure. Typical parameters capable of influencing the results of analyses and, he
nce, studied in the course of validation, are as follows:11 (i) the content of t
he organic solvent in the eluent ( 2%); (ii) the amount of additives (salts, i
on-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) influenci
ng the results of analyses should be indicated in the validation report. The des
cription of the given analytical procedure must provide the corresponding warnin
g 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 metho
ds of experiment planning, which are capable of quantitatively following the eff
ect of several parameters (factors) varied simultaneously [32 – 34, 42]. There i
s no need for special additional investigations of robustness if no critical par
ameters were revealed in the course of development and/or implementation of the
proposed analytical procedure. Data presentation. The validation report should i
nclude the chromatograms and tables showing the effects of each critical paramet
er on the results of analyses (in comparison to the normal values) and the absen
ce of such effects for the other most important parameters (see above). It is de
sired that the proposed analytical procedure be robust with respect to all the i
mportant parameters, which makes this procedure suitable for routine laboratory
use. 3. CRITERIA OF SUITABILITY OF A CHROMATOGRAPHIC SYSTEM USP-26 [7] stipulate
s the System Suitability Test (SST) intended to establish that the separating po
wer and reproducibility of a given chromatographic system provide for the adequa
te solution of the task of analysis. It is assumed that the equipment, electroni
cs, 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 suitabil
ity of the given system for implementing the required analyses.
12
Values in parentheses indicate recommended limits of variation relative to the v
alues stipulated by the given procedure.
The additives may include other analyzed components, identified impurities, poss
ible decomposition products, and substances playing the role of internal standar
ds. 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 produc
ts of destruction of the main drug components and/or compounds structurally clos
e to the analyzed parent substances. Such solutions are expediently used in the
tests for suitability of a given chromatographic system and frequently allow one
to reject expensive standard samples of impurities. The author believes that th
e suitability of a given chromatographic system is adequately described by the f
ollowing main criteria. (i) Criteria of the separating power of the chromatograp
hic system. (ii) Criteria of reliable determination of the beginning and end of
a peak (and/or of the peak height) of the analyzed compound on the chromatogram.
(iii) Criteria of reproducibility of the results of measurements (repeatability
of injections) (iv); Additional criteria should be introduced only provided tha
t critical parameters (see above) were established in the course of robustness e
valuation. The SST criteria should not be overloaded by numerous extra parameter
s, 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 proced
ures. 3.1. Separating Power of a Chromatographic System The criteria of separati
ng power include the separation coefficient Rs , the peak-to-valley ratio h/v (s
ee below), and the relative retention times of components. 3.1.1. Relative reten
tion times. For analytical procedures intended for the quantitative analysis of
drugs, homogeneous dosing tests, and determination of the drug’s dissolution cha
racteristics, it is usually sufficient to characterize the separating power by t
he relative retention times of several substances, including the main drug compo
nent, a structurally close compound, and/or an internal standard. The relative r
etention 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 suita
bility of a chromatographic system because these values depend on the eluate flo
w rate, temperature, and dead volume of the system. On the other hand, the reten
tion times do not influence the accuracy and precision of an analytical procedur
e (provided that other suitability requirements are satisfied). It is more corre
ct to indicate the recommended values (or intervals) of retention times of the m
ain components in the description of the analytical procedure and characterize t
he other substances by their relative retention times.
226
N. A. Épshtein
3.1.2. Peak separation coefficient. For analytical procedures intended for the d
etermination of impurities, the system suitability requirements should indicate
the minimum possible values of the peak separation coefficient Rs at least for t
he main (rated) impurities. In the case of HPLC procedures intended for the quan
titative analysis of drugs, homogeneous dosing tests, and determination of the d
rug dissolution characteristics, it may be necessary to introduce the peak separ
ation coefficient into the system suitability requirements if (i) a high content
of some impurity is admitted, (ii) there is a possibility of partial overlap be
tween the peaks of the main components, or (iii) the peaks of the placebo compon
ents occur in the vicinity of the main analytical peaks. The minimum possible va
lues of the separation coefficient Rs are determined proceeding from the followi
ng considerations. If the peaks are not significantly different in heights and p
ossess nearly Gaussian shapes, their complete separation at the baseline level r
equires that Rs ³ 1.5. This condition is recommended by the British Pharmacopoei
a and by the EEC Pharmacopoeia. If the peaks have significantly different height
s (e.g., used in the determination of impurities) and/or exhibit tailing, CDER r
ecommends that Rs ³ 2.0 [4]. It should be noted that this situation is most freq
uently encountered in practice during the analysis of drugs. For separating peak
s with large tailing factors ( > 2.5, see below), it is recommended to restrict
the separation coefficient to Rs ³ 2.5. This situation is quite rare, being only
typical of drugs containing several heteroatoms capable of forming strong hydro
gen bonds with silanol groups of the sorbent. Higher “critical” Rs values are us
ually unnecessary, because the condition Rs > 2.5 ensures good separation virtua
lly at the baseline level. On the other hand, it should be noted that, due to th
e availability of highly effective columns and computer-optimized systems, value
s of Rs < 1.2 cannot be justified, except for (i) the isomer peaks, (ii) the pea
ks of some components in complex multicomponent mixtures (such as extracts of bi
ological objects, antibiotics, alkaloids, and multivitamin preparations), and (i
ii) 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 p
eaks of main components and impurities are incompletely separated), the system s
uitability requirements may describe the peak separation in terms of the peak-to
-valley ratio p/v = hp/hv, where hp is the peak height relative to the extrapola
ted baseline and hv is the height of the lowest point in the valley separating t
he peaks (relative to the same extrapolated baseline). This parameter is rarely
used during the analysis of impurities in drugs: sometimes it is convenient for
the description of impurity peaks occurring immediately in front of the main ana
lytical peak. For suitability of the chromatographic system, it is usually requi
red that hp > hv. For example, in the analysis
of fluoxetin hydrochloride, it is required that hp/hv ³ 1.1/0.1 = 9. 3.2. Criter
ia of Reliable Determination of the Beginning and End (and/or the Height) of a C
hromatographic Peak The peak asymmetry is described in terms of the tailing fgac
tor T0.05 [7]. This parameter, which characterizes the asymmetry at a level of 0
.05% of the peak height, is especially important for the procedures of quantitat
ive analyses. According to BP 2001 [22], the recommended values fall within T0.0
5 = 0.8 – 1.5. Under this condition, the peaks are sufficiently symmetric and ex
hibit no significant tailing, which favors reliable determination of the beginni
ng and end of the peak (and, hence, of the peak area) and reduces the probabilit
y of overlap. An analysis of the corresponding articles of the BP 2001 and USP-2
6 showed that the permissible T0.05 interval extends from 0.75 to 2.5. Asymmetri
c peaks with T0.05 outside the 0.75 – 2.5 range should be avoided: for peaks wit
h T0.05 on the order of 3 and above or 0.7 and below, the boundaries of peaks pr
actically cannot be determined. An additional criterion of peak separation is pr
ovided by the efficiency of a chromatographic column (N ) with respect to the ma
in analytical peak. This parameter characterizes the peak width at half height (
FWHM) and is calculated by the conventional formula [7]. The ICH recommends usin
g columns with efficiencies N ³ 2000 theoretical plates, which can be considered
as the lower limit for the procedures of impurity determination in parent compo
unds. 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 mai
n 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 chromatographic 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 respect to the N value are not absolute: this parameter must only p
rovide for the required accuracy and precision of the proposed analytical proced
ure. 3.3. Criteria of Reproducibility of the Results of HPLC Measurements The cr
iterion of reproducibility of the results of HPLC measurements with respect to t
he repeatability of injections is usually formulated in terms of the relative st
andard deviation RSD of the peak areas or heights. In the quantitative analysis
of drugs, the permissible value is usually restricted at RSD £ 2.0% for the main
components and RSD £ 5.0% for impurities. Higher RSD values should be avoided o
r their validity should be additionally justified. The quantitative analysis of
parent compounds requires increased accuracy because the rated content of the ma
in active 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 sequentially mea
sured chromatograms (three to six) have RSD of the peak areas or heights not exc
eeding 1%. However, even this value does not always provide an acceptable confid
ence interval. For this reason, PR 2001 [22] suggested a formula for determining
the maximum possible RSD values depending on the upper rated limit (B ) of the
drug content and the number of repeated injections m. According to this, the pos
sible RSD (Table 4) is calculated for a series of injections of a reference solu
tion as RSD max = Here, K = 0.349 K = ( 0.6 2 ) × ( t 0.90%,5 BK m , t 90% , m -
1 (15)
TABLE 4. Maximum Permissible Values of the Relative Standard Deviation RSD Depen
ding on the Upper Limiting Content (B) of the Analyzed Compound and the Number o
f Sequential Injections (m)
m=3 B, % Maximum permissible RSD, % m=4 m=5 m=6
1.0 1.5 2.0 2.5 3.0 4.0* 5.0*
*
0.21 0.31 0.41 0.52 0.62 0.83 1.04
0.30 0.44 0.59 0.74 0.89 1.19 1.49
0.37 0.55 0.73 0.92 1.10 1.47 1.83
0.42 0.64 0.85 1.06 1.27 1.70 2.13
Values calculated in this study.
is a constant calculated as 6 ), where 0.6/ 2 is the “required” system. Until no
w, there is no commonly accepted opinion which kinds of modification of the chro
matographic system can be considered insignificant and requiring additional reva
lidation of the entire procedure. Nevertheless, some acceptable limits of variat
ion of the main parameters have been published [22, 31], which can be considered
as a basis for assessing the need for revalidation even of an “insignificantly
modified” analytical procedure. REFERENCES
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C8, C18, etc) and on the grade and even manufacturer of a particular sorbent. Fo
r this reason, it is usual practice in HPLC (and other chromatographic technique
s) to perform an objectively unavoidable modification of the chromatographic con
ditions as compared to those stipulated by the validated procedure, with observa
tion of the criteria of suitability of the chromatographic
228
N. A. Épshtein
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