Marini Electro 2006

Roland Djang’eing’a Marini1
Carl Groom2
Franc¸ ois R. Doucet2
Jalal Hawari2
Yaser Bitar3
Ulrike Holzgrabe3
Roberto Gotti4
Julie Schappler5
Serge Rudaz5
Jean-Luc Veuthey5
Roelof Mol6
Govert W. Somsen6
Gerhardus J. de Jong6
Pham Thi Thanh Ha7
Jie Zhang7
Ann Van Schepdael7
Jos Hoogmartens7
Willy Briône8
Attilio Ceccato8
Bruno Boulanger9
Debby Mangelings10
Yvan Vander Heyden10
Willy Van Ael11
Ilias Jimidar11
,
Matteo Pedrini12
Anne-Catherine Servais12
Marianne Fillet12
Jacques Crommen12
Eric Rozet1
Philippe Hubert1
Received November 8, 2005
Revised February 15, 2006
Accepted February 16, 2006
Research Article
Interlaboratory study of a NACE method for
the determination of R-timolol content in
S-timolol maleate: Assessment of uncertainty
Analyses of statistical variance were applied to evaluate the precision and practicality
of a CD-based NACE assay for R-timolol after enantiomeric separation of R- and
S-timolol. Data were collected in an interlaboratory study by 11 participating laboratories
located in Europe and North America. General qualitative method performance was
examined using suitability descriptors (i.e. resolution, selectivity, migration times and
S/N), while precision was determined by quantification of variances in the determina-
tion of R-timolol at four different impurity levels in S-timolol maleate samples. The
interlaboratory trials were designed in accordance with the ISO guideline 5725-2. This
allowed estimating for each sample, the different variances, i.e. between-laboratory
(s2
Laboratories), between-day (s2
Days) and between-replicate (s2
Replicates). The variances of
repeatability (s2
r) and reproducibility (s2
R) were then calculated. The estimated uncer-
tainty, derived from the precision estimates, seems to be concentration-dependent
above a given threshold. This example of R-timolol illustrates how a laboratory can
evaluate uncertainty in general.
Keywords: Interlaboratory study / Nonaqueous capillary electrophoresis / Reproduci-
bility / R-Timolol / Uncertainty estimation DOI 10.1002/elps.200500832
2386 Electrophoresis 2006, 27, 2386–2399
1
Laboratory of Analytical Chemistry, Institute of Pharmacy, University of Liège, Liège, Belgium
2
Environmental Analytical Laboratory, Biotechnology Research Institute, National Research Council, Montreal, Canada
3
Institute of Pharmacy and Food Chemistry, University of Würzburg, Würzburg, Germany
4
Laboratory of Pharmaceutical Analysis, Department of Pharmaceutical Science, Faculty of Pharmacy, University of Bologna, Bologna,
Italy
5
Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences – EPGL, University of Geneva, Geneva,
Switzerland
6
Department of Biomedical Analysis, Utrecht University, Utrecht, The Netherlands
7
Laboratorium voor Farmaceutische Chemie en Analyse van Geneesmiddelen, K.U.Leuven, Leuven, Belgium
8
Lilly Development Centre, Analytical Sciences R&D, Mont-Saint-Guibert, Belgium
9
Lilly Development Centre, Statistical & Mathematical Sciences, Mont-Saint-Guibert, Belgium
10
Analytical Chemistry and Pharmaceutical Technology, VUB, Brussels, Belgium
11
Johnson & Johnson Pharmaceutical, Research and Development Analytical Development – Method Development, Beerse, Belgium
12
Department of Analytical Pharmaceutical Chemistry, Institute of Pharmacy, University of Liège, Liège, Belgium
1 Introduction
To meet fit-for-purpose requirements, the data obtained
from a given analytical method must be reported with a
well-defined estimate of precision, i.e. result uncertainty,
in order to facilitate result comparison and interpretation
[1, 2]. The advantages of such practices are already
Correspondence: Professor Philippe Hubert, Laboratory of Analyti-
cal Chemistry, Institute of Pharmacy, University of Liege, CHU, B36,
B-4000 Liege 1, Belgium
E-mail: Ph.Hubert@ulg.ac.be
Fax: 132-4-3664317
Abbreviations: HDMS-â-CD, heptakis(2,3-di-O-methyl-6-sulfo)-b-
CD; SST, system suitability test
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2006, 27, 2386–2399 CE and CEC 2387
described [3], and the reporting of result uncertainty is
increasingly requisite in matters related to government
regulation, industrial production, international trade and
for a multitude of applications carried out according to
fixed norms [4]. This puts chemists/analysts under
increasing pressure in order to demonstrate the quality of
their results. The definition of uncertainty can be found in
the literature [1, 5]. In order to evaluate this performance
parameter, three strategies are frequently applied
depending on the presented situation: (i) the intralabora-
tory one, which includes the ISO approach published in
several guidelines or articles [1, 6–9] and the approach
using the validation [9–17] or the robustness [9, 18] data,
(ii) the interlaboratory strategy developed by the Analyti-
cal Method Committee of the Royal Chemistry Society
[19] or (iii) the mixed strategies that combine data
obtained from both intra- and interlaboratory data. These
latter procedures include that proposed by Barwick and
Ellison [20] and that developed by the Technical Commit-
tee ISO/TS 69 [21].
The recent development of several capillary electropho-
retic methods using acidic nonaqueous media and hep-
takis(2,3-di-O-methyl-6-O-sulfo)-b-CD (HDMS-b-CD) as
chiral selector has allowed the enantioseparation of basic
drugs, among them timolol [22]. The active molecule
(S-timolol) is a nonselective b-adrenergic blocker used as
a single enantiomer against hypertension, arrhythmias
and angina pectoris. Other medical indications are the
secondary prevention of myocardial infarcts [23, 24] and
the topical treatment of increasing intraocular pressure
[25, 26]. The NACE method developed for timolol enan-
tioseparation was successfully validated and applied for
the determination of R-timolol in S-timolol maleate sam-
ples in the presence of pyridoxine used as internal
standard [27]. While the uncertainty of the R-timolol con-
tent was reported using intralaboratory validation data
[27], an examination of interlaboratory variance and
uncertainty will assist in the scale up of method usage,
and uncover issues related to the transfer of this method
to a wider range of users. To the best of our knowledge,
no report exists on the collaborative evaluation of uncer-
tainty in CE techniques using interlaboratory data. Colla-
borative CE studies related to method development are
reported [28–33], but no relevant conclusions regarding
the measurement uncertainty are presented.
The objective of this study was to evaluate, by means of
an interlaboratory study, the ability of the developed and
validated NACE method to be transferred. For that pur-
pose, qualitative and quantitative responses were exam-
ined with regard to the performance of the method within
a single laboratory, and to the method performances of
any given laboratory compared to another. The repro-
ducibility of the quantitative results, namely the content of
R-timolol, was determined according to the ISO 5725-2
guidelines related to interlaboratory studies [34].
2 Materials and methods
2.1 Chemicals and reagents
Four homogeneous S-timolol maleate samples were
subjected to analysis: one (batch No. 107200204) kindly
provided by Prosintex Industrie Chimiche Italiane (Milan,
Italy) and three others (batches No. 11484, 11483 and
11351) obtained from the European Pharmacopoeia
Secretariat (Strasbourg, France). Chemical reference
substance of R-timolol maleate (batch No. 11381) was
kindly provided by Merck (Rahway, NJ, USA) and pyrido-
xine hydrochloride by SMB Technology (Marche-En-
Famenne, Belgium).
HDMS-b-CD, potassium formate, ammonium formate
(1R) or (1S)-(-)-10-camphorsulphonic acid, formic acid
(98–100%) and methanol were purchased from different
suppliers depending on each laboratory. However, all
materials satisfied the requirements prescribed for NACE
(i.e. the quality requirement for methanol was liquid chro-
matographic grade whereas an analytical grade of formic
acid was sufficient).
2.2 Electrophoretic conditions
The CE conditions [27], applied by the different participat-
ing laboratories, include the use of uncoated fused-silica
capillaries having 50 mm internal diameter and 48.5 cm
length (40 cm to the detector). The electrophoretic
separations were carried out using 30 mM HDMS-b-CD in
combination with 30 mM potassium camphorsulfonate
(camphorSO3
2
) in methanol acidified with 0.75 M formic
acid. Hydrodynamic injection was made by applying a
pressure of 50 mbar for a period of 8 s and UV detection
was performed at 295 nm. The capillary was thermostated
at 157C. Under these conditions, by applying a voltage of
25 kV, the last migrating peak was expected to be detect-
ed at the cathodic end within 14 min.
A new capillary was conditioned at 157C with methanol for
15 min. Before running the experiments, the capillary was
conditioned at 157C successively with the BGE and BGE-
CD solutions for 5 min each. Between runs, the capillary
was rinsed at 157C with methanol for 2 min and condi-
tioned at 157C with BGE-CD solution for 4 min. Both
BGE-CD and BGE were renewed after about 70 min of
analysis to avoid the phenomenon of buffer depletion
which causes loss of separation efficiency. At the end of

2388 R. Djang’eing’a Marini et al. Electrophoresis 2006, 27, 2386–2399
each working day, the capillary was rinsed at 257C with
methanol for 30 min, with 20 mM ammonium formate so-
lution for 20 min and finally with methanol for 30 min.
Capillary wash cycles were performed at a pressure of
approximately 1 bar.
2.3 Preparation of the solutions
Repeated vortexing and ultrasonication were specified to
ensure complete dissolution of the substances and
homogeneity of solutions. Prior to their use in the CE
system, all solutions were filtered directly in suitable CE
vials through filters resistant to organic solvents (i.e.
polypure propylene membrane filter, 0.2 mm).
2.3.1 Solutions for CE separation
The BGE, which consisted of a camphorSO3
2
solution at
the required concentration, was prepared by dissolving
simultaneously the corresponding amount of 30 mM
(1R)- or (1S)-(2)-10-camphorsulfonic acid and the cor-
responding amount of 30 mM potassium formate in
methanol acidified with the corresponding quantity of
0.75 M formic acid. In solution, the camphorsulphonic
acid is converted into the potassium salt. The use of an
ultrasonic bath was necessary to obtain complete dis-
solution.
The BGE-CD solution was prepared by dissolving 30 mM
of HDMS-b-CD in the BGE and was placed for 5 min in
the ultrasonic bath. During the CE separation, the BGE
was placed at the outlet of the capillary and the BGE-CD
at the inlet.
2.3.2 Solutions for system suitability tests (SST)
To assess the suitability of the different CE systems,
three reference solutions were defined: (i) a reference
mixture solution (a) containing pyridoxine (5 mg/mL),
S-timolol (10 mg/mL) and R-timolol (5 mg/mL), (ii) a refer-
ence solution (b) containing R-timolol maleate (20 mg/mL)
and pyridoxine (5 mg/mL), and (iii) a reference solution (c)
which corresponds to a 0.1% solution (referred to as
R-timolol maleate), prepared by diluting ten times the
reference solution (b). The chemical structures of R-
timolol maleate, S-timolol maleate and pyridoxine are
given in Fig. 1.
The reference mixture solution (a) was used in a first SST
to identify the different peaks in the electropherogram as
well as to test the CE system’s enantioresolution. Indeed,
the three analyte concentrations were selected to allow
peak identification on the basis of peak height. Under the
Figure 1. Chemical structures of timolol maleate (A) and
pyridoxine (B).
prescribed CE conditions, the enantiomeric resolution
was not allowed to be less than 5.5. The resolution be-
tween the internal standard and S-timolol had also to be
reported. Its value had to be at least 9. In case of an
unsuitable test result, i.e. the enantioresolution below 5.5,
the protocol stipulated to adjust the voltage. A decrease
may improve the enantioseparation. In all cases, the
migration times of analytes and the selectivity (ratio of
migration times) between consecutive peaks had to be
reported. The resolution (Rs) was calculated according to
the European Pharmacopoeia [34]:
RsAB ¼ 1:18 Â
tM;B À tM;A
À Á
wAð0:5Þ þ wBð0:5Þ
À Á
" #
(1)
with tM,A and tM,B, the migration times of the peaks corre-
sponding to the first (A) and second (B) analyte, respec-
tively, and wA(0.5) and wB(0.5), their corresponding peak
widths at half of the signal height, respectively.
In a second SST, the reference solution (c) was used to
check the sensitivity of the detector by determining the
S/N. That ratio was calculated according to the European
Pharmacopoeia [35] specification:
S=N ¼
2H
hn
(2)
where H is the height corresponding to the R-timolol
peak in the electropherogram obtained with the refer-
ence solution (c) (= 0.1% solution) and hn is the absolute
value of the largest noise fluctuation from the baseline in
the same electropherogram and observed over a dis-
tance equal to 20 times the width at half height of the
peak obtained with reference solution (b) and situated
equally around the place where R-timolol was found. If
the ratio is at least 10, then the 0.1% concentration
level is considered to be above the quantitation limit
and all solutions containing the analyzed substance at
concentrations above that level can be quantified. If not,

the detector should be checked or changed. Since the
reference solution (c) contained the internal standard, the
S/N value related to that analyte also had to be reported.
In the third SST, the repeatability of the injection was
evaluated by injecting six times the reference solution (b).
The repeatability of the migration times for each analyte
was also evaluated as well as the relative migration time
which is the ratio of R-timolol migration time over that of
the internal standard.
2.3.3 Solutions for quantitation
2.3.3.1 Internal standard solution
The internal standard solution was prepared by dissolving
about 12.5 mg of pyridoxine hydrochloride in 25.0 mL
methanol. Then the solution was diluted 50-fold with the
same solvent to obtain the stock solution of the internal
standard (10 mg/mL).
2.3.3.2 Reference solution
A stock solution of R-timolol was prepared by dissolving in
10.0 mL methanol an accurately weighed amount of ap-
proximately 20 mg of R-timolol maleate. This stock solu-
tion was then diluted 100-fold to obtain a reference solu-
tion representing 1.0% (20 mg/mL) of impurity level and
containing the internal standard at 5 mg/mL. This reference
solution was used for the determination of R-timolol ma-
leate content in the different S-timolol maleate samples.
2.3.3.3 Test solutions
Four different test samples (A, B, C and D) containing
R- and S-timolol were provided in powder form. Two
separate independent solutions for each sample were then
prepared to contain 2.0 mg/mL S-timolol and 5 mg/mL
pyridoxine internal standard. Each solution was then an-
alysed under identical conditions as in the repeatability
trials. The test solution was prepared by dissolving to
10.0 mL methanol an accurately weighed amount of ap-
proximately 20 mg of the test sample and adding the
corresponding volume of the internal standard stock so-
lution to obtain 5 mg/mL.
2.4 Instrumentation
In the framework of this collaborative study, instruments
from different manufacturers must meet the minimum
requirements to run the NACE method. They must there-
fore ensure sufficient cooling to maintain a capillary at
157C, and be fitted with uncoated fused-silica capillaries
with internal diameter, total length and length to detector
of 50 mm, 48.5 cm and 40 cm, respectively. However, for
Beckman equipments, the total length was adapted
according to the capillary handling device (50.2 cm).
Three laboratories used P/ACE MDQ systems from
Beckman Coulter (Fullerton, USA) and eight used HP3D
CE
ones from Agilent Technologies (Waldbronn, Germany);
however, for the latter, the data managers were different.
The capillaries were from different suppliers such as BGB
Analytik, Polymicro technologies, Beckman Coulter and
Composite Metal Services.
The statistical calculations were performed using the JMP
software version 5.1 for Windows (SAS Institute, Cary,
NC, USA).
2.5 Setup of the study
The study included 11 laboratories from pharmaceutical
companies, private and governmental institutes as well as
from universities, located in Europe and in North America.
The number of laboratories for the study fitted with the
requirements of the ISO guideline 5725-2 [34]. The setup of
the study is shown in Fig. 2. Each laboratory had to exe-
cute the whole analysis on two different days (c = 2). For
each day, the four S-timolol maleate samples had to be
analysed twice meaning that two sample solutions have to
be prepared independently and analyzed (g = 2). The con-
tent of R-timolol was determined by comparison to the
reference solution. Commonly, in interlaboratory studies,
e.g. in [36, 37], only the top and bottom levels of the
experimental design are performed, i.e. different labora-
tories and within each laboratory two replicates. In this
study, the setup represents a more elaborate collaborative
study since it allows evaluation of the intermediate preci-
sion time-dependence for each laboratory, which is the
sum of the repeatability and interday (s2
Interday) variances.
This can be done only if the contribution of each variance
component (laboratory, time and replicate) is estimated.
Figure 2. Setup of the collaborative study for each
sample: r = 8 laboratories, c = 2 days and g = 2 replicate
measurements.

2.6 Statistical analysis of the results
Prior to any further analysis, the data issued from the study
were first submitted to a critical examination foroutliers and
stragglers. Two approaches defined by the ISO guideline
[34] were used, namely graphical consistency techniques
and numerical outlier tests. Both within- and between-lab-
oratoryvariabilitieswereevaluated.Mandel’sk statisticwas
applied for the graphical evaluation of the within-laboratory
variability, while the Cochran’s test was applied as a
numerical test. Mandel’s h statistic and Grubbs’ tests were
applied for graphical and numerical evaluations of the be-
tween-laboratory variability, respectively.
Different Grubbs’ tests [34] were used for the numerical
evaluation: G1 for one small suspected outlier, Gr for one
large suspected outlier, G1,2 for two small suspected out-
liers and Gr,r 2 1 for two large suspected outliers. A G test
for a polar pair (one low and one high result) was also used
[38]. The different formulas applied in Mandel’s, Cochran
and Grubbs’ tests are detailed elsewhere [36, 37, 39].
After the test for outliers, the mean squares between-
laboratories (MSLaboratories), between-days (MSDays) and
between-replicates (MSReplicates) were calculated by
applying the ANOVA indicated in Table 1. The laboratories
are ordered in an arbitrary way. From the mean-squares
data, the repeatability (s2
r) and the between-laboratory
(s2
Laboratories) variances as well as the time-dependent
intermediate precision (s2
T) were calculated [34].
3 Results and discussion
3.1 System suitability checking and qualitative
responses
Before undertaking the study, all laboratories were
requested to maintain their instruments as described in
CE user’s guides. Improper vial prepuncher and electrode
maintenance in particular were observed to have negative
effects on separation efficiency. The maintenance proce-
dure was completed prior to operation for each day of the
study.
A detailed protocol was elaborated for the execution of
the study. First, a training round was organised to allow
laboratories to get familiarised with the method and to
verify the adequacy of the protocol. It involved the evalu-
ation of some qualitative (resolution and selectivity of dif-
ferent peak analytes, their migration times and S/N) and
quantitative (content of R-timolol in one S-timolol maleate
sample) aspects. Afterwards, on the basis of the reported
results, the laboratories were allowed to perform the final
study. In the latter, for the quantitative aspect, the content
of R-timolol was determined independently in four differ-
ent S-timolol maleate samples containing the R-enantio-
mer at four different impurity concentration levels.
The training revealed several problems with laboratories
using Beckman equipment: no peak observed within
14 min for some laboratories, low current and even loss of
current in some cases. A closer analysis of these situa-
tions indicated that the problems might be due to the
cooling system of the capillary. In the Agilent equipments,
an air cooling system maintains the capillary at low tem-
peratures whereas the Beckman equipment uses a cool-
ing liquid system which seems to be more efficient [33,
40]. In this NACE method, a temperature of 157C was
prescribed for analytical separation. With the Beckman
equipment, a very efficient capillary cooling seems to
occur; thus one observes a lower current, as it is propor-
tional to the temperature and consequently the peak
analytes will have slower migration than Agilent equip-
ment [33]. Another more dramatic outcome, that can
occur related to the cooling system, is the fact that the
temperature attained causes a crystallisation and a pre-
cipitation of the BGE-CD inside the capillary or at its ends
leading to the absence of current. This situation was fre-
Table 1. ANOVA components
Sources of variability Mean squares Estimated variances
Laboratories MSLaboratories ¼
cg
P
"xi À "xð Þ2
r À 1
s2
Lab ¼
MSLab À MSDays
cg
Days MSDays ¼
g
P P
"xij À "xi
À Á2
rðc À 1Þ
s2
Days ¼
MSDays À MSReplicates
g
Replicates MSReplicates ¼
P P P
ðxijk À "xijÞ2
rcðg À 1Þ
s2
Replicates = MSReplicates
g = Number of replicates per day.
c = Number of days per laboratory.
r = Number of laboratories.

quently reported by the laboratories using Beckman
equipments. However, two of them reported some results
about the reference mixture solution (a). The migration
times of pyridoxine, S-timolol and R-timolol obtained with
that solution were 13.3, 16.4 and 18.8 min, respectively,
for the first laboratory and 12.4, 13.0 and 14.7 min,
respectively, for the second one. Due toproblems reported
above, those laboratories were not able to continue to
provide sufficient results. On the other hand, the labora-
tories using Agilent equipment did not encounter such
problems and reported sufficient results for data analysis.
On the basis of this training, only the laboratories using
Agilent equipment were allowed to continue the inter-
laboratory study. Since their number (r = 8) was still in
agreement with the ISO norms, we were able to continue
the study in the framework of the reproducibility evalua-
tion. The study started with the evaluation of the SSTs.
In the first and the second SSTs, the adequacy of the CE
system had to be checked by running the reference solu-
tions (a) and (c). If the prescribed requirements were
achieved, then the repeatability SST was performed and
the R-timolol quantifications were made as outlined in
Fig. 3. A typical electropherogram of the reference mix-
ture solution (a) is presented in Fig. 4. The electrophoretic
separation was suitably and efficiently achieved by all
laboratories with corresponding currents ranging from 14
to 20 mA. It can be noticed that, according to the differ-
ence in peak heights, all laboratories identified the three
peaks and their migration order. Results of the SSTs are
summarised in Table 2. All laboratories achieved the
requirement concerning enantioresolution since all values
were above 5.5. The expected resolution between pyr-
idoxine and S-timolol (Rs1 2 2 = 9) was also achieved by
almost all laboratories except laboratory 7 which
obtained a lower value (Rs1 2 2 = 5.3). In addition, that
laboratory had low migration times for all peaks com-
pared to other laboratories although the reported current
was acceptable. No particular problem was reported by
that laboratory which, presented satisfactory results dur-
ing the training round. Since the results obtained on the
second day were better, it was suspected that insufficient
capillary conditioning may have been the problem [33, 41,
42]. Laboratory 8 performed the analysis by applying a
separation voltage below 25 kV. Indeed, using 25 kV dur-
ing the training round, the resolution of the enantiomeric
peak pair was sufficient, but that for S-timolol and the
pyridoxine was not (Rs1 2 2 = 7.5). A tailing of the parent
peak that is S-timolol, was also observed. Therefore, a
value of 23.5 kV allowed to improve that resolution
(Rs1 2 2 = 10.7). The parent peak became more symme-
trical. Moreover, the tailing of the parent peak was
decreased in such a way that the detection, integration
and quantitation of the R-timolol peak in the S-timolol
maleate samples were allowed. This phenomenon might
be due to the capillary batch-to-batch differences [41]
which are known to affect the reproducibility of the capil-
lary electrophoretic separations.
The detectability of the CE system was checked by eval-
uating the S/N values for R-timolol and pyridoxine. All
laboratories achieved the prescribed requirements for R-
timolol (Table 2). However, for pyridoxine, S/N values
were never above 10. Particularly, laboratory 5 presented
a strikingly low value (S/N = 2). Moreover, the ratio of
Figure 3. Setup of the injection scheme and determined responses.

Table 2. Results of SST-1 and SST-2 for day 1
Laboratory
no.
Migration time (min) Resolution (Rs) Selectivity (a) S/N
Peak 1 Peak 2 Peak 3 Rs 1–2 Rs 2–3 a 1–2 a 2–3 S/N peak 1 S/N peak 3
1 10.2 11.2 12.5 8.6 10.6 1.12 1.10 5 10
2 9.2 10.2 11.1 10.0 10.0 1.10 1.10 6 11
3 10.0 11.0 12.0 9.3 9.3 1.10 1.09 4 11
4 10.7 12.5 13.5 11.9 5.7 1.17 1.08 8 22
5 10.9 11.5 12.5 9.2 6.5 1.13 1.09 2 15
6 10.0 11.5 12.4 12.9 6.8 1.07 1.15 7 16
7 (day 1) 8.2 8.7 9.6 5.3 8.6 1.07 1.11 5 11
7 (day 2) 9.3 10.2 11.0 8.9 7.9 1.10 1.08 5 10
8 10.4 11.6 12.9 10.7 10.0 1.11 1.11 5 15
(1) = Pyridoxine, (2) = S-timolol, (3) = R-timolol.
Figure 4. Typical electropherogram of a reference mix-
ture solution obtained by carrying out the CE separation
using a BGE containing 30 mM HDMS-b-CD and apply-
ing a voltage of 25 kV (for other conditions, see Section 2).
Peaks and concentrations: (1) pyridoxine (5 mg/mL),
(2) S-timolol (10 mg/mL) and (3) R-timolol (5 mg/mL).
S/NR-timolol over S/Npyridoxine was about 7.5 for that labora-
tory while in general it was between 1.8 and 3.0. This
clearly indicates for laboratory 5 an increased uncertainty
when quantifying R-timolol near the LOQ (0.1%) [27].
In the third SST, the precision of the CE systems was
examined in terms of variations in migration times and
areas for pyridoxine and R-timolol peaks. Results are
summarised in Table 3. The RSD values of the migration
times (tM’s) were not above 1% in all laboratories except
for laboratories 3 and 7 (for pyridoxine and R-timolol). An
improvement was observed in the second day data (not
shown). However, when considering the relative migration
times (tM, R-timolol/tM, Pyridoxine), RSD values were below 1%
for all laboratories. Considering the 2 days, the RSD
values for the relative migration times were below 1.2%,
except for laboratory 5 (RSD = 2%). RSD value for the
overall study was 2.2% (N = 96 runs), demonstrating very
stable relative migration times for the NACE method
under the repeatability, time-different intermediate preci-
sion and reproducibility conditions.
Concerning the peak areas, high variabilities were
observed for both pyridoxine and R-timolol peaks with
higher RSD values (i.e. 12.1% for laboratory 3) which
were not really improved by correcting the peak area with
the corresponding peak migration time. However, accep-
table values (RSD below 3%) could be achieved by at
least 4 laboratories for the ratio of corrected peak areas of
R-timolol over that of pyridoxine (Table 3). In the light of
these observations and those reported in the S/N data,
the variations observed for peak areas seem to be affect-
ed by sources other than the corresponding migration
times (i.e. conditions of UV source, data acquisition rate,
etc.). For the other four laboratories, the RSD values were
higher for the results calculated with internal standard than
without. Higher RSD values for the pyridoxine peak area
than for the timolol peak were also noticed. This may have
an influence on the quantitative results.
3.2 Quantitative responses
3.2.1 Content of R-timolol
It can be observed from typical electropherograms of test
sample solutions A–D (Fig. 5) that in these conditions,
only one impurity peak corresponding to R-timolol could
be observed.
The content of R-timolol in those samples was deter-
mined by comparing the normalised ratio of R-timolol

Table 3. Results of SST-3; repeatability (n = 6 injections)
Labora-
tory
No.
Pyridoxine R-Timolol RMTa)
RSD
(%)
Ratiob)
(R-Timolol/
pyridoxine)
M.T. Area Corr. area M.T. Area Corr. area
Mean RSD
(%)
Mean RSD
(%)
Mean RSD
(%)
Mean RSD
(%)
Mean RSD
(%)
Mean RSD
(%)
Mean RSD (%)
(day 1/
day 2)
1 10.3 0.3 10.85 1.1 1.05 1.3 12.6 0.4 25.71 1.9 2.04 2.1 0.1 1.935 1.1/1.9
2 9.3 0.9 8.06 4.8 0.87 4.9 11.1 1.0 23.12 0.5 2.08 1.2 0.2 2.400 4.4/6.2
3 9.9 1.9 8.64 12.1 0.87 12.7 11.7 1.8 28.80 5.3 2.47 6.4 0.7 2.842 8.2/6.2
4 10.6 0.8 8.52 6.5 0.80 7.2 13.1 0.9 25.52 3.1 1.95 3.8 0.3 2.425 5.2/2.7
5 10.3 0.2 8.03 3.9 0.78 3.9 12.5 0.2 28.17 4.0 2.26 4.1 0.3 2.894 6.3/3.5
6 9.8 0.3 7.28 1.6 0.74 1.6 12.2 0.3 24.98 1.8 2.05 1.9 0.1 2.773 1.8/1.6
7 8.3 2.1 11.13 1.2 1.34 1.6 9.8 2.2 26.41 0.9 2.70 2.6 0.2 2.008 2.1/2.7
8 10.4 0.6 5.69 3.4 0.55 3.3 12.8 0.5 20.63 2.8 1.61 2.4 0.2 2.9539 2.1/2.6
M.T = migration time (in minutes).
Corr. = corrected.
a) Relative migration times (MTR-timolol/MTPyridoxine).
b) Ratio of corrected areas.
(NRT) and the normalized ratio of R-timolol from reference
solution (a) (NRR) using the following equation:
Content of R-timolol ð%Þ ¼
NRT
NRR
Â 1% (3)
The ratio of R-timolol in the test or reference samples was
normalised as follows:
Normalized ratio ¼
ratio of test or reference sample Â 20
weighed mass mgð Þ
(4)
The ratio of test or reference samples was obtained using
the following calculation:
Ratio ¼
corrected area of R-timolol in test or reference sample
corrected area of pyridoxine in test or reference sample
(5)
the corrected area being obtained by dividing the peak
area over the peak migration time.
The results presented in Table 4 show that all the labora-
tories were able to quantify R-timolol in all samples, even
in sample D which contained a R-timolol content near the
LOQ (0.1%) [27]. The reported contents were in the range
of 0.49–0.78, 0.26–0.41, 0.19–0.34 and 0.06–0.20% for
samples A, B, C and D, respectively. The RSD values in
sample A (RSD value of 12.4%), sample B (RSD value
11.7%) and sample C (RSD value 15.5%) were lower than
that obtained in sample D (RSD of 34.9%).
3.2.2 Critical examination of data
The within-consistency evaluation was carried out using
Mandel’s k formula which allowed construction of the cor-
responding plot, presented in Fig. 6. In order to evaluate
whether a problem exists in a certain laboratory or with a
certain sample, the k values were plotted per laboratory
and per sample. As illustrated in Fig. 6A, laboratory 2
showed some large k values suggesting a large intrala-
boratory variance compared to the other participants. For
that laboratory, the largest variability was attributed to
sample D. In contrast, laboratory 1 presented small k va-
lues suggesting that the variability observed in this labora-
tory was very low. From Fig. 6B, it can be deduced that the
problems of within-laboratory variability were not related to
the S-timolol sample. Cochran’s test was used to evaluate
whether the variability observed in laboratory 2 for sam-
ple D was numerically inconsistent. If a calculated Cochran
value is found to be above the critical 5% limit (C = 0.68)
and below the critical 1% limit (C = 0.79), this indicates a
straggler, while a value above the critical 1% limit indicates
an outlier. The calculated Cochran-value for sample D in
laboratory 2 was close (C = 0.65) to but below the critical
5% limit (C = 0.68). Therefore neither outliers nor stragglers
were found in the data. No analytical problem during the
execution of the study was reported by laboratory 2 either.
The Cochran-values for the other samples were below the
critical 5% limit (C = 0.24, 0.41 and 0.37, for samples A, B
and C, respectively). It can be concluded that the within-
laboratory variabilities observed for the four samples are
similar for the participants.

Table 4. Contents (in%) of R-timolol in test samples A, B, C and D
Laboratory
no.
Sample A Sample B Sample C Sample D
Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2
1 0.63 0.64 0.40 0.39 0.34 0.33 0.19a)
0.20a)
0.63 0.64 0.40 0.40 0.34 0.33 0.19a)
0.20a)
2 0.64 0.78 0.35 0.37 0.24 0.31 0.09 0.18
0.69 0.66 0.30 0.41 0.26 0.23 0.07 0.11
3 0.71 0.76 0.33 0.33 0.25 0.27 0.15 0.14
0.61 0.65 0.36 0.32 0.24 0.26 0.11 0.12
4 0.69 0.60 0.34 0.31 0.24 0.24 0.09 0.10
0.70 0.60 0.34 0.33 0.26 0.26 0.14 0.11
5 0.51 0.61 0.30 0.37 0.27 0.28 0.10 0.10
0.65 0.67 0.35 0.35 0.27 0.30 0.10 0.10
6 0.57 0.50 0.31 0.29 0.23 0.22 0.10 0.08
0.61 0.49 0.30 0.26 0.24 0.22 0.06 0.07
7 0.66 0.69 0.37 0.36 0.29 0.33 0.09 0.09
0.64 0.71 0.35 0.32 0.28 0.27 0.09 0.08
8 0.51 0.50 0.30 0.35 0.23 0.20 0.10 0.10
0.51 0.51 0.28 0.28 0.24 0.19 0.09 0.08
General mean 0.62 0.34 0.26 0.11
a) Straggler for between-lab variability.
To evaluate graphically the between-laboratory variability,
Mandel’s h plots (Fig. 7) were used. In a normal result, the
h values are randomly distributed around zero. Either
positive or negative distribution of all (or most) h results is
an indication of an abnormal situation. As illustrated in
Fig. 7A, laboratories 2–5 and 7 have a random distribution
of h values around zero, whereas laboratory 1 showed
only positive values and laboratories 6 and 8 only nega-
tive. Again the variability was not related to the samples
(Fig. 7B). This suggests the occurrence of a systematic
error in these laboratories. An explanation related to this
observation might be the differences in data-sampling
rate of the detectors (a factor not evaluated during meth-
od development), which can affect the low R-timolol
content estimation even in the presence of an internal
standard [41, 43]. The numerical Grubb’s tests were used
to detect statistically outlying values. The results pre-
sented in Table 5 indicate the presence of a straggler in
laboratory 1 for sample D. The stragglers were marked in
Table 4 but were maintained for the determination of
uncertainty.
3.2.3 Calculation of the variance estimates
From the ANOVA equations (Table 1), the mean squares
for the laboratories (MSLaboratories), days (MSDays) and
replicates (MSReplicates) were calculated. The estimated
variance for laboratories (s2
Laboratories), days (s2
Days) and
replicates (s2
Replicates) were also determined. According to
ISO norms [33], calculations of repeatability (s2
r), and re-
producibility (s2
R) estimates are performed using the fol-
lowing equations:
s2
r = s2
Replicates (6)
s2
R = s2
Replicates 1 s2
Laboratories (7)
In the present study and taking into account the setup of
the study in Fig. 2, the analytical method execution was
performed in 2 days allowing calculation of the estimated
variance for replicates (s2
Replicates) and laboratories
(s2
Laboratories) as well as for intermediate precision (s2
T):
s2
r = s2
Replicates (8)
s2
T = s2
Days 1 s2
Replicates (9)
s2
R = s2
T 1s2
Laboratories = s2
Replicates 1 s2
Days 1 s2
Laboratories (10)
The results of the calculations which were done by means
of JMP software are presented in Table 6. It can be noted
that the contribution to the total variability observed in the
R-timolol content results for all samples is mainlydue to the
component “laboratory” (57, 53, 74 and 72% for sam-
ples A, B, C and D, respectively). This indicates that the
deviations for the R-timolol content observed from one
laboratorytoanother are thehighestones.Accordingtothe
set-up of Fig. 2, this result was expected since the condi-

Figure 5. Typical electropherograms of test sample
solution A (A), test sample solution B (B), test sample
solution C (C) and test sample solution D (D) obtained by
carrying out the CE separation using a BGE containing
30 mM HDMS-b-CD and applying a voltage of 25 kV (for
other conditions, see Section 2). Peaks and concentra-
tions (1) pyridoxine (5 mg/mL), (2) S-timolol (about 2.0 mg/
mL) and (3) R-timolol; (A) = 12.4, (B) = 6.8, (C) = 5.2 and
(D) = 2.2 mg/mL).
tions applied in one laboratory differ from that applied in
another, i.e. different equipments, operators and labora-
tory conditions. However, the variability contribution of
the component “day” was the lowest (12, 17, 3 and 5%
for samples A, B, C and D, respectively), whereas that of
component “replicate” was of second importance. This
pattern, which was not expected according to the set-up
of Fig. 2, indicates that the contents of R-timolol in a lab-
oratory were not much deviating from 1 day to another in
comparison to the deviation of values observed between-
laboratories. The fact that the between-day variance is
lower than the within-day variance is a good indication of
the stability and reliability of the NACE method in a labo-
ratory. On the other hand, the variance between-repli-
cates was found to be illogically of second importance.
Some values, such as those observed in the laboratories
3 and 5 for sample A, are explaining this tendency which
was already reported in a previous study [39]. According
to Horwitz [44], it is often expected to have the reproduc-
ibility variance about two to four times higher than the
repeatability variance. This means that the between-
laboratories variance is similar in magnitude or larger than
the repeatability variance. The ratio values were 3.2, 3.3,
4.4 and 4.3 for samples A, B, C and D, respectively (see
Table 6). This situation is often observed when dealing
with impurities at very low concentrations as it is the case
here [37]. The expectations were satisfied in samples A
and B. According to ISO norms [34], one has to check
whether there is a relationship between RSD and con-
centration. In this study, the relationship between uncer-
tainty and concentration was checked.
The reproducibility of variance allowed the calculation of
the standard uncertainty "u"x using the equation:
"u"x ¼
ffiffiffiffiffi
s2
R
q
(11)
The standard uncertainty was ux = sR = 0.0799, 0.0407,
0.0422 and 0.0410 for R-timolol content in samples A, B,
C and D, respectively. The expanded uncertainty Ux
defines an interval around the measurement result which
contains the unknown true value with a defined prob-
ability level. Ux is obtained by multiplying the standard
uncertainty ux by a coverage factor k. In this study, a value
of k = 2 [1, 3, 19] was considered, meaning that the true
value is expected to be within the interval x 6 2ux with a
probability of 95%. The values of Ux were 0.1597,
0.08142, 0.08446 and 0.08206 for R-timolol content in
samples A, B, C and D, respectively. The nominal content
of R-timolol in those samples was 0.62, 0.34, 0.26 and
0.11%, respectively (Table 4). Uncertainty was found to
be constant for samples B, C and D, while it was higher
for sample A. Indeed, this indicates that the uncertainty
seems to be affected not only by R-timolol content but

Figure 6. Plots of Mandel’s
k statistics grouped per labora-
tory (A) and per sample (B) for
the graphical evaluation of
within-laboratory consistency.
Figure 7. Plots of Mandel’s
h statistics grouped per labora-
tory (A) and per sample (B) for
the graphical evaluation of be-
tween-laboratory consistency.

Table 5. Results of the Grubbs’ tests
Grubbs’
tests
Calculated values Critical values r = 8
Sample A
0.66%
Sample B
0.35%
Sample C
0.29%
Sample D
0.10% 5% 1%
G1 1.78 1.85 1.88 1.31 2.13 2.27
Gr 2.08 1.90 1.98 2.16 2.13 2.27
G1,2 0.81 0.80 0.81 0.89 0.11 0.06
Gr,r 2 1 0.75 0.78 0.73 0.68 0.11 0.06
G 9.93 9.04 0.84 8.19 64.5 74.9
Outliers – – – Straggler in
laboratory 1
– = No outliers nor stragglers.
Table 6. Estimation of the variance components
Sources of variability Sample A 0.66% Sample B 0.35% Sample C 0.29% Sample D 0.10%
Variance
component
Laboratories (s2
Laboratories)
Days (s2
Days)
Replicates (s2
Replicates)
36.1161024
7.6461024
20.0461024
8.7661024
2.8161024
5.0161024
13.2761024
0.4761024
4.0961024
12.0961024
0.8161024
3.9461024
Repeatability variance (s2
r)
Reproducibility variance (s2
R)
Ratio reproducibility/repeatability
20.0461024
63.7861024
3.2
5.0161024
16.5761024
3.3
4.0961024
17.8361024
4.4
3.9461024
16.8361024
4.3
also up to a certain level by other uncertainty contribu-
tions. In a collaborative study applying an LC method and
analyzing similar S-timolol maleate samples to those of
this study [39], the uncertainty was found to be con-
centration dependent as observed for samples A and B.
In addition, the uncertainty values found were very similar
in magnitude to those obtained using the present NACE
method. However, in our previous work [27], it was found
that the uncertainty of the R-timolol content obtained
from validation data of only one laboratory was con-
centration dependent.
Finally, for a single result x obtained when applying the
NACE method, the result for R-timolol content in samples
with concentrations similar to sample A (i.e. about 0.62%)
is expected to be x 6 0.16% while for samples containing
R-timolol at about half concentration (i.e. 0.34% in sam-
ple B), it is expected to be about half (x 6 0.081%); i.e.
95% of the reported values will be within 0.500–0.819%
and 0.265–0.428%, respectively. The result for R-timolol
content in samples with concentrations less than 0.35%,
i.e. similar to samples C and D (contents 0.26 and 0.11%,
respectively), the result is expected to be x 6 0.08% for
both, i.e. 95% of the reported values will be within 0.206–
0.375% and 0.018–0.182%, respectively. The measured
uncertainty was found to be rather high compared to the
contents of R-timolol (Table 4). This situation was not
abnormal as far as the impurities are concerned. In a
previous interlaboratory study dealing also with impurities
and applying an LC method [39], similar situation was
observed. As can be seen in Table 4, the measurements
fulfiled this expectation since measurements outside of
these ranges occurred only in 1 out of 32 times for the
R-timolol concentration of samples A and B, and 2 out of
32 times for sample C. However, for sample D (R-timolol
impurity content near the LOQ) measurements outside
the expected range of variability occurred 4 out of
32 times.
It can be concluded that the method is very useful to
determine R-timolol impurity in S-timolol maleate sam-
ples but at concentrations above 0.3%. The method is
still useful when considering the maximum tolerated
content (1.0%) of R-timolol in S-timolol maleate as
reported in the European Pharmacopoeia [35].
If a laboratory wants to evaluate its uncertainty from three
replicates under repeatability conditions when determin-
ing the content of R-timolol in a S-timolol maleate sample
with a concentration similar to sample A (0.62%), the
standard uncertainty of the mean result will be

u"x ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
s2
replicates
3
þ s2
T þ s2
laboratories
s
¼ 0:0710 (12)
Then, the expanded uncertainty will be Ux = 0.142 and
the mean result written as x 6 0.14%. On the other hand,
if the same laboratory performs three replicate analyses in
3 days, then, the standard uncertainty of the mean result
becomes
u"x ¼
s2
replicates
3 Â 3
þ
s2
T
3
þ s2
laboratories
s
¼ 0:0639 (13)
with an expanded uncertainty of Ux = 0.128. The mean
result will be written as x 6 0.13%.
On the other hand, when two laboratories perform the
analysis in triplicate under repeatability conditions, the
standard uncertainty of the mean result will be
u"x ¼
s2
replicates
3 Â 2
þ s2
T þ
s2
laboratories
2
s
¼ 0:0539 (14)
The expanded uncertainty is reported as Ux = 0.108 and
the mean result as x 6 0.11%. It can be remarked that, in
the three cases, the observed uncertainties were not
considerably improved.
In the same manner, the standard and the expanded
uncertainties can be calculated for R-timolol content in
samples with concentration similar to B, C and D.
4 Concluding remarks
An interlaboratory uncertainty assessment was success-
fully applied to a CD based NACE method for the enan-
tiomeric separation and determination of R-timolol
impurity in S-timolol maleate samples. As far as we know,
it is the first time that an interlaboratory study has been
completely achieved in CE allowing to report uncertainty
measurement results. The method was successfully
transferred between equipments from the same manu-
facturer but not to other instruments. The qualitative per-
formance and the suitability of the method were judged to
be acceptable when analysis were conducted using rela-
tive migration times and normalised peak areas. In an
interlaboratory study, on the 11 participating laboratories,
only eight laboratories reported the contents of R-timolol
at four different levels of impurity in S-timolol samples.
The estimated uncertainty of R-timolol seemed to be
concentration dependent at high enantiomeric impurity
contents, i.e. above a given concentration, while at low
impurity contents, such a dependence was not observed.
However, the method is very useful to determine R-timolol
impurity in S-timolol maleate samples but at concentra-
tions above 0.3%. The method is still useful when con-
sidering the maximum tolerated content (1.0%) of R-
timolol in the S-timolol maleate as reported in the Euro-
pean Pharmacopoeia.
The authors wish to thank the Belgian Government (The
Prime Minister Services – Federal Office for Scientific,
Technical and Cultural Affairs, Standardisation Pro-
gramme) for the financial support of this project (No.NM/
12/23). Research grants from the Belgium National Fund
for Scientific Research (FNRS) to A.-C. Servais and
M. Fillet are gratefully acknowledged. Many thanks are
also due to FNRS and to the Léon Fredericq foundation
for financial support. A research grant from the Walloon
Region and the European Social Fund to E. Rozet is
gratefully acknowledged (First Europe Objective 3 project
no. 215269). The authors are also grateful to the European
Pharmacopoeia Secretariat, Prosintex Industrie Chimiche
Italiane (Milan, Italy) and Merck for kindly providing the
samples.
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