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N. A. �pshtein1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 38, No. 4, pp. 40 � 56,
April, 2004.
1
�Akrikhin� Chemico-Pharmaceutical Joint-Stock Company, Staraya
Kupavna, Moscow Region, Russia.
2
Here and below the terms �accuracy� and �precision� (repeatability,
reproducibility) are treated in accordance with the State Standard GOST R
ISO 5725�1�2002. Previously, accuracy had the meaning of correctness,
but now correctness is described in terms of �trueness.� In justifying the
suitability of techniques related to the qualitative determination of substances,
statistical processing of the experimental results is obligatory [8].
212
2.3Reproducibility. Checked
in special cases
(Section 1.2.3)
3.Linearity (Section 1.3)
4.Accuracy (Section 1.4)
5.Suitability range (Section 1.5)
6.Limit of detection (Section 1.6)
7.Limit of quantitation
(Section 1.7)
8.Stability of solutions
(Section 1.8.)
It is expedient to determine
these validation characteristics
in the course of proof of the
analytical system accuracy
(using statistical parameters
determined for the calibration
graph) (Sections 1.3; 1.4;
1.6.2; and 1.7.2)
9.
Robustness (stability) of HPLC
procedures with respect to small
Criteria of suitability of a given
chromatographic system
factors (ratio of the mobile
(Section 3)
Category
Characteristic
II
I Quantita-Limiting III IV
tive tests tests
Accuracy Yes Yes MB MB No
Precision Yes Yes No Yes No
Specificity Yes Yes Yes MB Yes
Limit of detection No No Yes MB No
Limit of quantitation No Yes No MB No
Linearity Yes Yes No MB No
Suitability range Yes Yes MB MB No
p
thpth
N. A. �pshtein
TABLE 2. Validation Characteristics Recommended for Various
Tests by the United States FDA (CDER and CBER) [15]
Chatacteristic
Identifica
tion
quantitative limiting
solution
tests
Case
2. The main impurities are unknown (unidentified)
or absent. In this case, it is expedient to use special experiments
involving modification of the parent compound
(sometimes called �stress testing� [2, 15, 27]), whereby a solution
of the given drug (or of the parent compound) is subjected
to factors leading to its partial destruction with the formation
of related identifiable compounds. The degree of decomposition
can be readily determined from the decrease in
the area of the main peak in the chromatogram of the final
solution relative to that in the initial solution. The specificity
of the given analytical procedure is judged by separation of
the peaks of impurities between themselves and from the
peak of the main component and by the peak purity of the
parent compound.
SD .
#( Xi #X )2 (m #1) ,
i1
100SD
RSD(%) .
.
(1)
Intra-assay
precision
Intermediate � Determined for the same sample
precision (Ip )
preparation by different analysts
using various instruments
(chromatographs) during a prolonged
period of time (not less
than two days)
N. A. �pshtein
This characteristic is not included in the list of CDER [15]
and USP-26 [7]. In HPLC validation, data on the injection
repeatability should be provided as additional material required
in cases of search for the �bottleneck� of a proposed
method (dispersion analysis).
N , the standard
deviation SD, the relative standard deviation RSD of particular
measurements, and the confidence interval (for P = 95%)
of the average value.
It is required to show that the average results are statistically
equivalent (e.g., in terms of the Student t-criterion) or,
which is more convenient for the practical analysis, that
RSD .
1.0% for determination of parent compounds,
RSD .
2.0% for drugs, or RSD .
10.0% for impurities
[13, 27].5
5
For small values of the standard deviation (SD << 1), the t-criterion may
give statistically significant differences even for close (almost identical)
values of the compared average concentrations. This is related to the fact
that this criterion is proportional to the ratio of systematic and random errors.
Validation of HPLC Techniques for Pharmaceutical Analysis
ij
t .
X X#| |1 2 .
X X#| |1 2 ,
#1
2
2
2SD SD #1
2
2
2SD SD
m1 m2 m
ij
ij
ij
ij
ii
| yi #Yi |
,
1(Y .
y)2
t .
SD 1##
N (N #
1) SD
i
#( y #Y )2
#( yi .
y)2
ii
SD0 .
, SD .
N #2
N #1
( X ##) ##X ,
(2)
(found content )
R .
100%. (3)
(introduced content )
R ##R.
(4)
6
Accordiong to the State Standard GOST R ISO 5725-1�2002 the systematic
error usually characterizes �trueness.�
#.
xm
||
t .
SD
| x ##|
#.
100%.
(5)
.
m .
SD2
k .
.
d 2 .
(m #1). Then, the difference between the
.
i1 .
maximum and minimum values of the dispersion SDk
2 is
checked to be insignificant in terms of the Fisher F-criterion.
If this difference is actually insignificant, the SD02 value is
calculated as the sum of square deviations for all samples di
8
This t value is essentially the ratio of the systematic error |x ##| and the
random error SD.
m.
0
N. A. �pshtein
N
SD2.
k
SD0
2 .
1k
N
.
t .
(|#.
x|.
m) SD0 (6)
a 0 b 0
FPf 1 N �, f 2 N �2
(,1 ) .
2 2 (7),
N 1 SD N 2)
SD (�)� (�
01 02
02 N 2)
SD2(�
01 02
for the above relations without the free term (mf = bm) and
N. A. �pshtein
(a) Using the calibration curve S = a + bC constructed
using the results of analyses for a series of the reference sample
solutions with decreasing concentrations in the region of
the LOD. Using these data, a regression equation S = a + bC
of the area under peak S versus concentration C is calculated
and the standard deviation SD of the free term is deter-
a
LOD .
, (9)
##tP f ) .
0
Ya (, SD
0.5,
where W0.5 is the full width at half maximum (FWHM) of
this peak [22].
For HPLC procedures used in pharmacy, it would be interesting
to study the influence of the maximum noise N
on
the relative standard deviation of results for a nearly Gaussian
peak shape. According to [27],
50
RSD[%] .
, (10)
SN .
cc
SD
11 .
SDb .
2
.
Ys #Y .
2
SD .
####.
.
(12)
Nm .
b #.
SD0 .
N. A. �pshtein
(within the interval from 0 to th ) and the area S0 at the initial
moment, calculate the parameter tb =|b |/SDb, and compare
this value with the critical (tabulated) Student criterion
t (P = 99%, f = N � 1) for the confidence probability
P = 99% and f = N � 1 degrees of freedom, where N is the
number of experimental points on the above interval. If tb is
smaller than the tabulated t (P = 99%, f = N � 1) value, the
coefficient b is statistically insignificant and, hence, there is
no significant difference between the areas under the peak
(and, hence, the solution concentration) at the initial time and
at any other moment up to the last experimental point.
ST = 100S#S0. (13)
ST = 100m#m0. (14)
3. CRITERIA OF SUITABILITY
OF A CHROMATOGRAPHIC SYSTEM
USP-26 [7] stipulates the System Suitability Test (SST)
intended to establish that the separating power and
reproducibility of a given chromatographic system provide
for the adequate solution of the task of analysis. It is assumed
that the equipment, electronics, analytical procedures, and
samples comprise a unified system investigated as a whole.
Upon chromatography of a special SST solution, the results
are checked for obeying the SST requirements (see below).
The SST solution must contain the analyzed compound and
all other additives necessary for evaluating the suitability of
the given system for implementing the required analyses.
considerations.
For separating peaks with large tailing factors ( > 2.5, see
below), it is recommended to restrict the separation coefficient
to R .
2.5. This situation is quite rare, being only typi
be justified, except for (i) the isomer peaks, (ii) the peaks of
some components in complex multicomponent mixtures
(such as extracts of biological objects, antibiotics, alkaloids,
and multivitamin preparations), and (iii) the peaks of minor
components (impurities, aromatic and color additives, etc.).
pv
BK m
RSD.
, (15)
max
t
%, m #1
90
(.K .
06 2)( .
t 090 6 ), where 0.6.
2 is the �required�
.%, 5
m =3 m =4 m =5 m =6
B,%
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