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To cite this article: Alexander H. Schmidt & Carsten Wess (2014) A QBD WITH DESIGN-OF-
EXPERIMENTS APPROACH FOR DEVELOPMENT OF A STATE-OF-THE-ART UPLC PURITY METHOD FOR
CARBAMAZEPINE, Journal of Liquid Chromatography & Related Technologies, 37:18, 2653-2666, DOI:
10.1080/10826076.2013.853312
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Journal of Liquid Chromatography & Related Technologies, 37:2653–2666, 2014
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2013.853312
3
Waters GmbH, Eschborn, Germany
INTRODUCTION
Carbamazine is widely used as an antiepileptic drug, which is administered
orally in doses, usually 200–1200 mg=day.[1] It is available in different pharma-
ceutical formulations (conventional tablets, extended-release tablets, and
suspensions). Its chemical name is 5H-dibenzo[b,f]azepine-5-carboxamide,
as described in the monograph of the European Pharmacopeia.[2]
The purity testing of carbamazepine was accomplished in accordance to
the monograph of the European Pharmacopeia by using high performance
liquid chromatography (HPLC) with UV-detection on a Thermo Scientific
Hypersil BDS cyano column (4.6 mm x 250 mm, 10 mm particle size) in iso-
cratic mode with an eluent consisting of tetrahydrofuran, methanol, and
water (3:12:85, v=v=v). To 1000 mL of this solution, 0.2 mL anhydrous formic
acid and 0.5 mL triethylamine were added. The flow rate was 2.0 mL=min.
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the developed method is robust. Typical examples for the use of statistic
tools are the widespread use of the ‘‘Plackett-Burman’’ design, a highly
fractionated factorial design recommended for screening experiments only,
or the more advanced Box-Behnken or Central Composite designs.[4–8]
2656 A. H. Schmidt and C. Wess
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FIGURE 2 Typical chromatogram of carbamazepine and its impurities A, B, C, D, E, and G by using the
E.P. method.
Selecting the experimental design is a critical step because not all stat-
istical designs have the same ability to identify which factors are important
and to quantify variable effects. Therefore, software packages for multivari-
ate data analysis (e.g., Fusion AE or Design Expert) are using a two-step
approach: In the first step a screening design identifies the most influential
operating parameters and in the second step response surface designs are
used to select the optimal values for method parameters.[4–8]
The aim of our study is to describe a Quality-by-Design based method
development strategy with Design-of-Experiments to develop a new state-of-
the-art UPLC method for testing of impurities in the active pharmaceutical
ingredient (API) carbamazepine.
EXPERIMENTAL
Chemicals and Eluents
Methanol and acetonitrile were HPLC-gradient grade, all other
chemicals were at least analytical grade and purchased from Sigma
(Taufkirchen, Germany). Water used was purified by a Milli-Q academic
water purification system (Millipore, Eschborn, Germany).
as solvent. This selectivity standard solution was protected from light by use
of amber glass ware and used in all screening and optimization experiments.
Impurity F was excluded from the study because of its unavailability.
Design of Experiments
Our method development strategy was carried out in two phases. The
first phase involved column screening to identify the stationary phase in
2658 A. H. Schmidt and C. Wess
combination with parameters such as pH, gradient time and slope, and
organic solvent type with the best resolution (Rs > 2.0) for all compounds
(carbamazepine and six of its impurities). These factors are known to have
a major effect in column selectivity.[9–11] In the second phase, the chroma-
tographic parameters identified to affect the resolution were studied
with additional instrument settings to provide optimum chromatographic
performance.
mental design ensures that all important study factor effects will be
expressed in the experiment data and taken together can comprehensively
explore a multifactorial design space.[12–14] In the initial scouting, four
different column chemistries (ACQUITY UPLC BEH C18, HSS T3, BEH
RP18 Shield, and CSH C18) were screened against two organic eluents
Optimization Phase
In the second phase the best column, organic solvent type and pH were
used for further method optimization. These parameters were fixed and
the other important chromatographic parameters, which are flow rate, final
% of gradient, concentration of phosphoric acid, and temperature were
studied to characterize their responses effects and establish correct operat-
ing spaces.[12–14]
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1 0.45 95 45 0.1
2 0.65 75.2 45 0.1
3 0.65 95 45 0.2
4 0.45 75.2 45 0.2
5 0.45 95 45 0.4
6 0.65 75.2 45 0.4
7 0.55 95 45 0.4
8 0.65 85.1 45 0.4
9 0.45 85.1 45 0.4
10 0.55 75.2 45 0.4
11 0.55 85.1 45 0.1
12 0.65 95 45 0.2
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FIGURE 4 Design Space for optimization and points of prediction. a.) Graph settings: Flow rate and
Final percnt; Organic.
Flow rate and final % of acetonitrile are varied at (T): 0.600 mL=min, 90.6%, (A): 0.571 mL=min, 88.0%,
(B): 0.571 mL=min, 93.3%, (C): 0.629 mL=min, 88.0% and (D): 0.629 mL=min, 93.3%, while
temperature and % concentration of phosphoric acid are fixed at 65 C and 0.4%, respectively.
as the organic eluent. The white unshaded area highlights the experimental
region where all performance goals are met. Optimal conditions could be
found in a lower pH range (0.1% phosphoric acid) and with gradient time
around 5 min.
The best results (conditions) were used for the optimization phase in
addition to other chromatographic parameters such as flow rate, final % of
gradient, concentration of phosphoric acid, and temperature to determine
the optimum UPLC method. The corresponding DoE plan, which once
again was created by the Fusion software, is shown in Table 2.
After running the experiments and data analysis the results were visua-
lized in final overlay graphs. The primary goal of the purity method for
2664 A. H. Schmidt and C. Wess
FIGURE 5 Chromatogram of the selectivity standard solution containing carbamazepine and its
impurities A, B, C, D, E, and G for conditions at the working point.
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CONCLUSION
A Quality by Design (QbD) approach to define an operating space
within the design space is often based on knowledge gained through
Design-of-Experiments. In this case study, we used the statistic software pack-
age Fusion AE to develop a state-of-the-art purity method for carbamazepine
that is intended to replace the current method in the EP monograph. An
impressive 22-fold reduction in analysis time was achieved. An operating
space within the design space is established and ensures a robust UPLC
method, which increases confidence in the ability to validate that method.
If necessary the UPLC method can be transferred to HPLC conditions
without any further development.
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ACKNOWLEDGMENT
The authors gratefully acknowledge Mijo Stanic for excellent technical
assistance and helpful discussions.
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