ANALYTICA

CHIMICA
ACTA
ELSEVIER Analytica Chimica Acta 345 (1997) 185-196

Investigation of sources of variance which contribute to

NIR-spectroscopic measurement of pharmaceutical formulations
A. Candolfi”, D.L. Massarta3*, S. Heuerdingb
“ChemoAC, Pharmaceuiical Institute, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium
?andoz Pharma, Pharmaceutical Development, CH-4002 Basel, Switzerland

Received 17 October 1996; received in revised form 17 October 1996; accepted 31 December 1996

Abstract

The goal of this study is to improve NIR-spectroscopic measurement of pharmaceutical formulations. The variance
contributions of the following factors to the NIR-measurement of tablets, hard and soft gelatine capsules are estimated:
measurement repeatability, positioning, time, different samples of one batch and different batches. The data analysis is
performed on the one hand with PCA (Principal Component Analysis) as a display method and on the other hand numerically
with a nested ANOVA design.

Keywords; NIR; ANOVA; PCA; Tablets; Capsules

1. Introduction properties, such as temperature and others, lead to a

large variation in spectra if they are not controlled.
Near-infrared (NIR) spectroscopy is becoming a Some other properties, for instance particle size, are
widespread technique in the pharmaceutical industry. more difficult to standardise. Certain transformations
Different fields of application are already established or algorithms correct for this. However, some pro-
such as identification of excipients and raw material, blems must not be solved by chemometrics, but by
blending control, drug product identification and blis- better measurement. The aim of this article is therefore
ter pack identification [l-6]. The main advantage of not to improve those algorithms, but po investigate the
NIR-spectroscopy compared to the usually applied quality of the data itself. The attention is focused on
wet chemistry techniques like chromatography is its some manipulation parameters which might be
rapidity and the fact that it is a non-destructive sources of variation for the measurement.
method. The following sources of variation are studied:
NIR-spectra contain chemical information (bands
which are often associated with hydrogen vibrations), l the measurement repeatability;
as well as physical information (particle size, tem- l the positioning of a sample;
perature, molecular weight) [7]. Therefore physical l the day to day variability;
l the object to object within one batch variability;
*Corresponding author. Fax: +32 2 477 4735. l the batch-to-batch variability.

0003.2670/97/$17.00 ;(” 1997 Elsevier Science B.V. All rights reserved

PI1 S0003-2670(97)00059-7
186 A. Candolji et al./Analytica
These error sources are studied on three different
drug delivery systems, namely on tablets, hard and soft
gelatine capsules. The final aim of the study is to
develop procedures for identification of clinical study
lots by pattern recognition techniques.
2. Theory
2.1. Pre-processing
The operation preceding all statistical computations
is pre-processing for the purpose of decreasing the
variance within a group and increasing the variance
between groups. This is necessary to optimise dis-
crimination between groups.
In this article standard normal variate (SNV) trans-
formation was applied for all drug delivery systems. In
one example the effects of SNV and second derivative
were compared with raw data. Before Principal Com-
ponent Analysis (PCA) the data were additionally
transformed with column centering.
2.1.1. Column centering
Column centering is the subtraction of the corre-
sponding column mean mj from each element xii of the
jth column of the absorbance matrix X.
zij=Xq-_j fori= l,..., nandj= I ,..., p,
where mj = (l/n) 2.x~.
I i=l
2.1.2. Standard normal variate transformation (SNV)
This method is used to remove the multiplicative
interferences of scatter and particle size. The trans-
formation of the log (l/R) spectra by calculation of the
standard normal variate at each wavelength removes
slope variation on an individual spectrum basis by use
of the following equation [8]:
x(&j - mi)2
&jSNV = (Xij - mi)/
p-l ’
d posed in the different levels. For e.g. a two-level
where Xijs,v is the ith element of the jth column of
matrix with the SNV transformed spectra, x the log
R) value for the wavelengths, mi the row mean of
log (l/R) values and p is the number of variables in
spectrum.
Chimica Acta 345 (1997) 185-196
2. I .3. Second derivative
Second derivative eliminates baseline shift and
emphasises small features, such as shoulders on a
band. Therefore this pre-processing was selected
and applied as described by Gorry [9].
2.2. Wavenumber selection
The variables are selected according to high peak
loadings obtained after PCA. For each drug delivery
system PCA is performed on the data matrix contain-
ing the spectra collected when investigating the
sources of variation. Prior to PCA the spectra are
pre-processed with SNV and then column centered.
In this study several peak loadings on the first three
PCs are selected. The position of the resulting features
is compared to the original spectrum to check if the
performed selection is reasonable (i.e. corresponds
with a peak in the spectrum of the acrive drug or
main excipient).
2.3. Nested ANOVA
The nested ANOVA is a special case of ANOVA. It
is called nested (or hierarchical) ANOVA because the
subordinate classification is nested within the higher
level of classification. This method is used to deter-
mine the magnitude of error at various stages of an
(1)
experiment or of an industrial process.
For each level of the hierarchy one can determine
the variance. This helps to point out which level of the
experiment is critical and will therefore need the
greatest amount of replication or will need better
experimental control.
Within nested ANOVA there are two variants,
model I and model II, Model II is a pure random
effect nested ANOVA: all effects at the different levels
are random. Model I is a mixed ANOVA, where the
first level has fixed effects, for instance different drugs
or treatments. Here we are working with a model II,
since the effects at all levels are random.
(2) For the computation, the total variance is decom-
the nested ANOVA the following equation is obtained:
(I/
Yijk = p + Ai + Bij $ cijk, (3)
the
the where Y$ is the kth observation in the jth subgroup of
the ith group, ,U the mean of the population, Ai the
 A. Cundolj et ~1./Analytica Chimicrr Actcc 34.5 (I9971 IX-I96 IX7

random contribution to the total variance for the ith optical window
position for tablet
group of (the higher) level A, and B, is the random
contribution for the jth subgroup (level B) of the ith
group; t,,k is the error term of the kth item in the ,jth
subgroup of the ith group [ IO,11 1. sample fixture

3. Experimental i 1-&-- optical fibre

I

All data were collected with a FT-NIR instrument

IFS2WNIR from Bruker with a connected optical
fibre. Two accessories, combined fibre- and sample-
hii
holders, one for tablets and one for capsules, were
developed in house. They fix the position of the optical
fibre and the samples during the measurement and
additionally do not allow light to enter the measuring 1 -t- -- - optical tihrc

cell.
For tablets (Fig. l(a)) the optical fibre is directed to
the top and the sample is lying directly on the optical
window. The underside of the tablet is scanned. For
capsules (Fig. 1(b)) the fibre is directed to the bottom.
The sample is lying below the optical window in a
special cavity with the exact dimension of the capsule
size so that the capsule cannot move. The upper side of
the capsule is scanned.
The data are NIR-spectra of log (l/R) of tablets,
hard and soft gelatine capsules, where R is the reflec-
tance of the sample versus the reflectance of a white
ceramic standard. The standard is measured at the
beginning of each measurement series and directly
subtracted from all spectra of the corresponding mea-
surement series. The spectra are obtained over the
range 10 0004000 cm ’ (l OOO-2500 nm), with 778
measuring points. The measurements were performed
with a resolution of 16 cm ’ and a number of scans of
10. The resulting spectrum is the average spectrum of
these IO scans.
The spectra were measured by using the optical
tibre. The optical properties of the fibre do not allow to
measure accurately in the spectral range 4600-
40()0 cm ‘. For this reason 78 measurement points
were discarded at the end of the spectra. The resulting
spectra contain 700 variables (measuring points).
The tablets contain 4% of an active drug and eight
excipients. The main excipient is lactose. They are
coloured, uncoated, plain tablets with a diameter of
Fig. I. (a) Fihre- and sample-holder for tablets. (bl Fibre- and
sample-holder for capsules.
7.1 mm. The filling of the hard gelatine capsules
consists of four excipients, the main one being cellu-
lose, and 4.5% of the active drug. The capsule shell is
yellow and the capsule size is #4. The tilling of the soft
gelatine capsules contains the active drug in 10%
concentration and five excipients. The pure gelatine
shell takes 29% of the total weight of the capsule and
is red-brown in colour. The soft gelatine capsules are
oblong, and 20 mm in length. It is known from the
hard and soft gelatine capsules analysed in this study
that no cross-linking [ 12,131 occurs, if they are stored
at room conditions. The three drug delivery systems
are completely different drugs, with different active
components.
Definitions of the investigated factors:
Replicures. Replicate measurements of one sample
without moving it between the measurements.
188 A. Candolj et ul./Analyticn
Positioning. After a measurement the sample is
removed from the position and put back for the next
measurement.
Different samples. Different samples of one produc-
tion batch are measured.
Different batches. Samples of different batches of
one product are measured.
The measurements were performed for following
three measuring schemes: A - 30 replicates of one
sample, 30 measurements for positioning, 30 measure-
ments of different samples of one batch; B - on five
consecutive days one sample is measured in three
positions, for each position three replicates were
collected; C - measurement of eight samples taking
into account positioning, time, different samples of
one batch and different batches. The scheme considers
data for four similar batches of one product. It is built
as follows (see Fig. 2).
These measurements were repeated for each drug
delivery system.
3.3. Computations of the variance contributions of
the sources of variance
For each drug delivery system 90 spectra were
collected with scheme A, 45 spectra with scheme B
and 120 spectra with scheme C. The spectra obtained
for each scheme were investigated for outliers by
visual inspection of the plotted spectra and by PC
score plots.
Since nested ANOVA is a univariate method, the
first task was to select appropriate wavenumbers for
each drug delivery system for further computations. It
batch 1 batch 2
Fig. 2. Measuring scheme C: positioning,
Chimica Actu 345 (1997) 185-196
was decided to perform PCA on the data matrix
containing the spectra including the most part of
the investigated sources of variation (scheme C),
and select the wavenumbers according to high peak
loadings on the first three PCs. The full wavenumber
range was used in a first step. SNV and column
centering was carried out prior to PCA. The data were
pre-processed with SNV in order to remove uncon-
trolled variations of the baseline due to the different
particle sizes in solid dosage forms [8]. It turned out
that high peak loadings were especially obtained for
the two main spectral ranges where water is absorbing
(6896 cm-’ res. 1450 nm, 5154 cm-’ res. 1940 nm
[7]). To prevent making wrong decisions based on
computations carried out in the range of the water
peaks, the following ranges for water were defined and
eliminated from the spectra: 7263-6600 cm-’ (74
measuring points) for the first water peak and
535 l-4989 cm-’ (48 measuring points) for the second
water peak. They were removed from the spectra as
well as 15 measuring points at the beginning of the
spectra. The resulting spectra contain 563 variables. In
a second step SNV was applied on the reduced original
data matrix, and the wavenumber selection was car-
ried out again according to high peak loadings in the
first three PCs after PCA. In some of the cases high
peak loadings were obtained for the same variable on
PC 1, PC2 and PC3. This procedure was repeated for
each drug delivery system.
Four wavenumbers were finally selected for tablets,
five for hard gelatine capsules and five for soft gelatine
capsules. In order to make this univariate approach
more robust, the sum of the absorbance at the selected
batch 3 batch 4
days, tablets and batches
 A. Candolji et al./Analytica
value and that at the two neighbouring wavenumbers
on both sides, i.e. the sum of five wavenumbers, was
computed and used as input value for all the computa-
tions.
3.4. Influence of different pre-processing methods
The influence of two pre-processing methods,
namely SNV and second derivative transformation,
was studied and compared with the results obtained
from raw data.
The reduced matrix, without the two water ranges of
spectra of measuring scheme C for hard gelatine
capsules was used once as original data, once after
SNV and once after second derivative transformation.
Again variables with high loadings were selected after
PCA for the corresponding type of pre-processing and
for the original data. The sum of the absorbance at the
selected values and that at the two neighbouring
variables on both sides was computed and used as
input for the nested ANOVA.
3.5. Computer programs
The data collected with the Bruker instrument were
transformed from OPUS format to JCAMP format by
the OPUS V2.0 [ 14] software and exported from the
NIR system. The files were later transformed into
ASCII-code by a program written in Visual basic
3.0 [15] and imported into MATLAB [16], which
was used to perform most of the computations. The
computations for the nested ANOVA were carried out
with Statgraphics Plus V.6.1 [17].
4. Results and discussion
4.1. Wavenumber selection
The features for the analysis with nested ANOVA
were selected with PCA, carried out on the full data
matrix ( 120 x 700) of measuring scheme C. Scheme C
includes measurements for several batches of one
product, different samples of one batch, measurements
over a certain time and for different positions. The
loadings plots for the first three PCs show, that for the
tablets the highest peak loading is found at 5 166 cm-‘,
for hard gelatine capsules at 5143 cm-’ and for soft
Chimica Acta 345 (1997) 185-196 189
gelatine capsules at 5 120 cm- ‘. The selected variables
correspond to the water peak at 5154 cm-’ [7]. There-
fore this spectral band contains the largest variation in
this measuring scheme. Since it was not the goal of
this work to investigate the water variation. this factor
was not controlled while performing the measure-
ments. As a result it was decided to eliminate the
two spectral ranges where water is mainly absorbing
from the spectra. However, that water can affect the
entire baseline of NIR-spectra [7] has to be taken into
account. Our results also show that, when carrying out
pattern recognition, one should take care to avoid
regions where the absorption of water can play a role
or that one should correct for it.
The procedure for variable selection was performed
again on the reduced data matrix (120x563). It
seemed reasonable to select variables lying between
the two water peaks, because the absorbance values
are very low at high wavenumbers ( 10 000 cm-~‘j and
the noise level is increased at low wavenumbers
(4600 cm-‘). The chosen variables correspond to
peaks in the spectrum of the active drug or of the
main excipient.
Fig. 3(a)-(c) show the loading plots on PC I to PC3
for the three drug delivery systems with the selected
features. The indicated wavenumbers represent the
selected variables. They are representative for peaks
of the active drug or the main excipient.
4.2. Measuring scheme A
The variance was computed from the univariate
values for the 30 replicates, for the 30 measurements
for positioning and for the 30 different samples of one
batch. Table 1 shows the results for the three drug
delivery systems.
The computations were carried out on all selected
variables for the respective drug delivery systems. The
results for each investigated factor for the individual
drug delivery systems are similar. The variance
obtained for positioning is significantly higher for
all three systems than for replicates. It is especially
increased for hard gelatine capsules. This is an indica-
tion that for this drug delivery system the inhomo-
geneity of the sample seems to be higher than for the
other two. This is expected, since the tilling of a hard
gelatine capsule is loose and therefore moving by new
positioning of the sample. On the other hand tablets
190 A. Candolfi et al./Anal~tica Chimica Acta 345 (1997) 185-196

loadings on PC1 loadings on PC1

.____

wavenumbers

loadlngs on PC2 loadmgs on PC2

wavenumbers

loadngs on PC3 loadings on PC3

wavenumber

loadlngs on PC1

wavenumbers

loadings on PC2

loadings on PC3

Fig. 3. Loading plots on PCI, PC2 and PC3 from measuring scheme C for (a) tablets, (b) hard gelatine capsules and (c) soft gelatine capsules.

and soft gelatine capsules are fixed systems. From One can see on the score plot for the tablets
positioning to different samples the variance is Fig. 4(a) that the three scores for replicates lay close
increased only a little for tablets; for the capsules it to each other. Each day, nine measurements were
is slightly decreased. The differences for tablets prob- performed, three replicates at three positions. The
ably are due to additional physical parameters, i.e. scores representing positioning are already more dis-
hardness, thickness, surface. tant. The measurements of different days are separated
and placed along the diagonal of the plot. The mea-
4.3. Measuring scheme B surements of day 1 are located in the upper left comer
of the PC space, the measurements of the last (5th) day
The data of measuring scheme B taking into are located in the lower right comer. This effect can be
account replicates, positioning and time are analysed random or can be an indication for a drift in time. It
with PCA for the three drug delivery systems. shows that carrying out measurements on different
Fig. 4(a)-(c) show the corresponding PCl-PC2 score days is a not negligible source of variance in this
plots. measuring scheme.
 A. Candolfi et al. /Anulytica Chimica Arta 34.5 (1997) 185-l 96 191

Table I
Measuring scheme A: The variance obtained from 30 replicates. 30 times positioning and 30 different samples for each drug dehvery system :It
each selected variable

Variable (cm ~‘) Replicates Positioning Samples

72birr.s
6484 7.29x IO-’ 1.54x IO 1 3.62x IO ~’
6276 6.25x10-’ 0.85x10 1 2.81x10 1
6006 2.89x IWh 0.44x IO- 0.70x IO ’
5783 3.24x 10 h 0.90x lo- 3.41 xl0 1
Hard capsulr~
6122 4.84x IOmh 4.86x IO-’ 2.81 x 10~ ’
6037 5.76x10-” 0.02 8.08x IO i
5914 6.25x10 ’ 0.01 9.98~10 ’
5790 9.00x10-h 0.0 I 0.0 1
572 I 4.41 x10-h 0.01 8.56< IO-
SoftcqJ.wlr.s
5945 1.44x10 5 7.13x10 ’ 5.3Xx IO i
5891 1.85x10-5 5.38x lo-~ 3.13x IO ’
5852 2.30x10 ’ 7.02x IO- 3.69x10 a
5767 2.21x10-5 4.28~10 -I 2.37x IO J
542x 1.94x10 5 5.23x10 ’ 5.70x10 q

For the hard gelatine capsules the main variance on
PC 1 is related to positioning, mainly occurring on day
1. The variance for positioning on the other four days
is much smaller. This large variance for positioning
leads to the appearance of tightly clustered replicates.
PC2 more or less separates the measurements per-
formed on day 2, 3,4 and 5. The samples are ordered
according to time, which might be again an indication
of drift in time.
The PCl-PC2 score plot for the soft gelatine cap-
sules (Fig. 4(c)) shows, that the replicates contain
somewhat more variance, compared to the tablets
and hard gelatine capsules. One can see the clusters
for the positions of one day. Again day 1 includes a
large variance, which is observed on PC 1. PC2 sepa-
rates more or less the measurements performed
between days 2 and 5, but does not order them
according to time.
For the same data a nested ANOVA was computed
with the univariate data described above. The results
are summarised in Table 2 and numerically show the
results already displayed by PCA.
The results obtained for the tablets show that the
variance contribution of the replicates is very small.
The rest of the total variance is approximately divided
into equal parts for the two remaining factors, days
and positioning. In the case of the hard gelatine
capsules the variance contribution for the replicates
can be neglected. From the two other factors it seems
that the factor time tends to have a somewhat higher
part of the total variance. This is also valid for the soft
gelatine capsules. Investigating this drug delivery
system one observes, that the replicates have a larger
variance contribution. The high variance contribution
for time can be an indication for a drift. An internal
reference was periodically measured at the beginning
of each measuring series and directly subtracted from
those measurements. However, this standard might not
correct for all changes in the measuring conditions
(instrument, surroundings). Appropriate measure-
ments were performed over a time period of six
months to investigate the long time stability of the
instrument performance. A small drift in time was
observed.
The nested ANOVA results correspond with the
results obtained by the interpretation of the PC score
plots as well as with the results obtained with measur-
ing scheme A.
4.4. Measuring scheme C
The data of measuring scheme C taking into
account positioning, time, different samples of one
batch and different batches are analysed with PCA for
192 A. Candolfi et al. /Analytica Chimica Acta 345 (1997) 185-196

0.04,

Fig. 4. Score plots of the data obtained from measuring scheme B for (a) tablets, (b) hard gelatine capsules and (c) soft gelatine capsules. 1, 2,
3: sample measured after repositioning on day 1; 4, 5, 6: sample measured after repositioning on day 2 etc.

Table 2
Measuring scheme B: The variance contribution obtained with nested ANOVA for different sources of variance for each drug delivery system
at the corresponding selected variables

Sel. variable (cm-‘)

Tablets 6484 6276 6006 5783

Time 51.2% 46.1% 49.9% 45.5%
Positioning 48.1% 52.9% 45.6% 49.0%
Replicates 0.7% 1.O% 4.5% 5.5%
Hard geIatine capsules 6122 6037 5914 5790 5721
Time 62.3% 67.6% 54.2% 49.7% 54.9%
Positioning 37.7% 32.4% 45.8% 50.3% 45.1%
Replicates 0.01% 0.02% 0.02% 0.03% 0.02%
Soft gelatine capsules 5945 5891 5852 5767 5428
Time 49.0% 58.2% 60.9% 53.3% 47.5%
Positioning 45.8% 38.1% 34.4% 37.2% 44.8%
Replicates 5.2% 3.7% 4.7% 9.5% 7.7%
 A. Cundolfi et ul. /Analytica Chimica Acta 345 (I 997) 185-l 96

Fig. 5. Score plots of the data obtained from measuring scheme C for (a) tablets, (b) hard gelatine capsules and (c) soft gelatine capsules. I. 2:
measurements of samples I and 2, for batch 1: 3, 4: measurements of samples I and 2. for batch 2 etc.

the three drug delivery systems. Fig. 5(a)-(c) show the
PC 1-PC2 score plots.
On the score plot for the tablets presented in
Fig. 5(a) one can see that the major variation along
PCI, which represents 74% of all variation, is due to
differences between batch 4 (scorenumbers 7 and 8)
and the others. This is probably due to physical
parameters of the tablets, which are different for this
production batch compared with the other batches
(another tablet machine, another person, who was
producing this batch etc.). PC1 also separates the
two tablets of each batch. PC2 separates the three
other batches from each other. The two remaining
factors, positioning and time, seem to be less impor-
tant.
On the score plot for the hard gelatine capsules
(Fig. 5(b)) no clusters for the batches and for the
different capsules can be seen. These factors will
contain a smaller part of the total variance and there-
fore the factor positioning and time affect to a higher
extent the measurement. A separation along PC1 can
be recognised. The measurements performed between
days 1 and 3 are found on the left and the measure-
ments for days 4 and 5 on the right.
The scores for the soft gelatine capsules (see
Fig. 5(c) for batch 1. 3 and 4 are randomly distributed
194 A. Ccrndolfi et al. /Analytica Chimica Acta 345 (1997) 185-196

Table 3
Measuring scheme C: The variance contribution obtained with nested ANOVA for different sources of variance for each drug delivery system
at the corresponding selected variables

Sel. variable (cm-‘)

Tablets 6484 6276 6006 5783

Batch 65.5% 50.4% 67.8% 64.0%
Tablet 26.6% 40.6% 29.2% 30.0%
Day 1.7% 2.5% 1.1% 2.7%
Position 6.2% 6.5% 1.9% 3.3%
Hurd g&tine capsules 6122 6037 5914 5790 5721
Batch 0.3% 0.5% 2.7% 3.74 2.5%
Capsule 13.3% 17.4% I I .4% 9.4% 1 13.1%
Day 57.2% 53.6% 57.6% 56.8% 56.3%
Position 29.2% 28.5% 28.3% 30.1% 28.1%
Softgelatine capsules 5945 589 1 5852 5767 5428
Batch 21.8% 30.3% 23.1% 28.4% 17.5%
Capsule 26.1% 17.5% 20.4% 18.7% 42.8%
Day 29.1% 33.3% 35.9% 33.5% 9.9%
Position 23.0% 18.9% 20.6% 19.4% 29.8%

over the whole PC space. Batch 2 is somewhat sepa-
rated along PC2 (25% of variance).
For the same data the nested ANOVA computations
were carried out for the selected variables. The results
are summarised in Table 3. The computations were
additionally performed for the selected variables of
the water peak (see wavenumber selection) before
dropping the two spectral regions for humidity.
Results similar to those presented in Table 3 for
non-water features are obtained for the water features.
The main individual parts of variance for hard
gelatine capsules are positioning and time. As
explained earlier, if the capsules are not completely
filled with powder, granules or pellets, the content
moves when the position of the sample is changed.
Another reason for the effect of positioning could be
the round shape of the capsule and the smooth and
brilliant shell. During the measurement the light is
scattered over the full range of reflection angles. Due
to these two reasons positioning is especially proble-
matic. It is not possible to reproduce the same position
twice. The factor time expresses the variance due to
alterations in the sample, surrounding conditions and
the instability of the instrument. As described before a
small drift was observed over a long time period for
the measurements. Alterations of the capsule might be
due to small changes in the water content of the
gelatine shell. Nevertheless, this does not lead to
the cross-linking phenomenon described by Digenis
et al. [ 131 in the investigated capsules. Small altera-
tions in the humidity can, however, lead to background
effects, since the entire baseline of NIR-spectra is
influenced by water and not only the two main spectral
regions [7]. The variance due to the two other factors is
rather small.
For the soft gelatine capsules not all results obtained
at the different selected variables are similar. The
selected variable at 5428 cm-’ leads to a different
repartition of the total variance for all four sources of
variance. This might occur because this variable is
situated close to the water band. For the other features
a clear trend is shown. The total variance is divided
into approximately equal parts. These capsules are
filled with a solution and therefore the filling is more
homogeneous than for hard gelatine capsules. Addi-
tionally they are completely filled. However, the cap-
sule shell is a source of inhomogeneity, and therefore
positioning is rather important. It is also possible that
the water content of the gelatine shell is changing. For
this drug delivery system the variance due to different
capsules is large because the capsule shape varies for
each sample. The production process for soft gelatine
capsules is very complicated and leads to the high
variance contribution for different batches.
The factor positioning and time are less important
for tablets. As long as the diameter of a tablet is larger
 A. Candolj et al. /Analytica Chimica Acta 345 (I 997) 185-196 195

Table 4
Measuring scheme C of the hard gelatine capsules: The variance contribution obtained with nested ANOVA for the different sources of
variance for raw data. SNV data and second derivative data

Sel. variable (cm-‘)

Raw data 6060 6006 5783

Batch 7.7% 8.4% 7.1%
Capsule 5.9% 6.3% 7.0%
Day 47.8% 45.9% 48.1%
Position 38.6% 39.4% 37.8%
SW 6122 6037 5914 5790 5721
Batch 0.3% 0.5% 2.7% 3.7% 2.5%
Capsule 13.3% 17.4% I 1.4% 9.4% 13.1%
Day 57.2% 53.6% 57.6% 56.8% 56.3%
Position 29.2% 28.5% 28.3% 30.1% 28.14
Second derivative 5991 5914 5852 5775 5626
Batch 8.8% 12.0% 9.3% 5.3% 3.1%
Capwle 15.6% 18.6% 20.6% 14.7% 1 I .5%
Day 41.6% 36.2% 40.8% 51.1% 54.9%
Position 34.0% 33.2% 29.3% 28.9% 30.5%

than the diameter of the optical window, positioning
will be less problematic. This drug delivery system
seems to be more stable than capsules since time
affects the measurement less. The two other factors,
different tablets and different batches, are important.
The physical complexity of the sample, i.e. hardness,
thickness, compression force etc. is expressed in the
variance due to tablets. For the production process of
tablets such physical parameters are given in a range,
wherein all samples are normally distributed. There-
fore each tablet is a little different. This is true also for
several batches. However, more parameters can influ-
ence a batch, e.g. the use of different tableting
machines, other batches of active drug and excipients,
temperature, humidity etc. As a result the variance
obtained for this level is the highest for tablets.
4.5. InjluenccJ of d$ferent pre-processing methods
All data of this study are pre-processed with SNV.
We wanted to investigate if the variance contribution
found for the different factors depends on a specific
type of pre-processing, in this situation, the SNV.
Therefore the influence of SNV and second derivative
as pre-processing methods is studied and compared
with the results obtained from original data. The
results are summarised in Table 4.
The variable selection is performed as discussed
earlier for original data, for SNVand second derivative
pre-processed data. For the original data three vari-
ables are selected, 5 after SNV and second derivative
transformation. This means that after applying pre-
processing more variables are selected. The reason for
this in the case of SNV is, that the variance included in
the original peaks is distributed to the corresponding
peaks and some neighbouring variables. Second deri-
vative transformation emphasises small features and
as a result more variables are selected.
For the selected variables of one type of pre-pro-
cessed data, as well as for all three types of data, the
repartition of the total variance is similar. The highest
variance part is obtained for the factor time followed
by positioning. The factors in batch to batch variability
and capsule to capsule variability contain a smaller
part of the total variance. This indicates that the results
obtained from these experiments are in this case rather
independent of pre-processing.
If one compares the results obtained with original
data and with SNV data more in detail, one can see for
SNV, that the individual variance contribution of batch
and of positioning are decreased. SNV removes linear
slope variations of the individual spectra [g]. This
phenomenon depends on sample positioning, and this
contribution is therefore somewhat corrected with
SNV. Second derivative also corrects for baseline drift
which explains why the variance contribution of the
factor positioning is decreased, compared to the
results of the raw data.
196 A. Candolji et al./Annlytica
5. Conclusion
As presented above the investigated error sources
have a different influence on the measurement of the
three drug delivery systems.
The drug delivery system tablet is from a physical
point of view the most complicated. As a result
different samples of one batch as well as different
batches lead to a large variance in the spectra. The
factors positioning and time seem to be less important.
This indicates that the investigated tablets are homo-
geneous and quite stable. For the application of NIR-
spectroscopy as quantitative or qualitative method it is
useful to include a large amount of samples in the
training set. It seems also necessary to take into
consideration each production batch to have a repre-
sentative data set.
For hard gelatine capsules especially the factors
positioning and time include a large variation part. As
already explained, the filling of hard gelatine capsules
is loose and therefore positioning is critical. For this
reason, replicates including new positioning of each
capsule should be measured. The reason for the high
amount of variance for time is the instability of the
instrument and the sample itself. It is known that NIR-
spectroscopy is sensitive to humidity and temperature
modifications 171 and therefore the instrument should
be located in a thermostated room, where such para-
meters are controlled. Moreover, the same parameters
must be controlled for the samples. Especially the
moisture content of gelatine can vary with the humid-
ity of the atmosphere to which it is exposed [18]. It
became clear, that appropriate experiments have to be
carried out to monitor the instrument stability. If there
occurs a drift or shift in the measurements, instrument
standardisation is necessary [ 19,201. The other factors
are less important for this drug delivery system.
For soft gelatine capsules the variance contribution
is approximately the same for the studied factors. Such
capsules usually have a thick coloured gelatine layer.
It is doubtful if the content of the capsule is really
Chimica Acta 345 (1997) 185-196
measured. Positioning is important because the cap-
sule shape is never exactly the same (size and
weightf8%). This affects, of course, also the different
capsules of one batch and different batches. The
variance introduced by the factor time might be again
due to the gelatine and indicates that a careful instru-
ment monitoring is necessary.
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