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A QBD WITH DESIGN-OF-EXPERIMENTS

APPROACH FOR DEVELOPMENT OF
A STATE-OF-THE-ART UPLC PURITY
METHOD FOR CARBAMAZEPINE
a b c
Alexander H. Schmidt & Carsten Wess
a
Chromicent GmbH , Berlin , Germany
b
Freie Universität Berlin, Institute of Pharmacy , Berlin , Germany
c
Waters GmbH , Eschborn , Germany
Accepted author version posted online: 31 Dec 2013.Published
online: 20 May 2014.

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

To link to this article: http://dx.doi.org/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

A QBD WITH DESIGN-OF-EXPERIMENTS APPROACH FOR

DEVELOPMENT OF A STATE-OF-THE-ART UPLC PURITY METHOD
FOR CARBAMAZEPINE

Alexander H. Schmidt1,2 and Carsten Wess3

1
Chromicent GmbH, Berlin, Germany
2
Freie Universität Berlin, Institute of Pharmacy, Berlin, Germany
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3
Waters GmbH, Eschborn, Germany

& A state-of-the-art ultra-performance liquid chromatographic (UPLC) method has been

developed for purity testing of carbamazepine.
Successful chromatographic separation of the active pharmaceutical ingredient (API) from its
impurities was achieved on a WATERS ACQUITY UPLC CSH C18 column with the dimensions
2.1 mm  100 mm and 1.7 lm particle size with gradient elution of 0.2% phosphoric acid and
acetonitrile in only 5 min.
Incorporating Quality by Design (QbD) principles to the method development approach by
using the statistical software package Fusion AE allows the study of the relationship between
chromatographic parameters (factors) and the resolution (response) between the peaks of interest.
In a screening phase, the factors known to have a major effect on column selectivity (stationary
phase, pH of the aqueous eluent, organic eluent type, gradient time, and slope) were studied. In
the second phase, the chromatographic parameters that were identified as affecting the resolution
were studied with additional instrument settings. In both phases, statistical concepts with experi-
mental design plans (Design-of-Experiments) are used as an efficient and fast tool to simul-
taneously gain knowledge regarding the influencing factors and interactions. An operating
space within the design space was established and a verification study confirmed the robustness
of the final method.
Total analysis time was only 5 min, which is an impressive 22-fold increase in productivity in
comparison to the method published in the European Pharmacopeia.

Keywords carbamazepine, design of experiments, design space, fusion AE software,

quality by design, UPLC method development

Address correspondence to Alexander H. Schmidt, Chromicent GmbH, Rudower Chaussee 29,

12489, Berlin, Germany. E-mail: alexander.schmidt@chromicent.de
Color versions of one or more of the figures in the article can be found online at www.tandfonline.
com/ljlc.
 2654 A. H. Schmidt and C. Wess

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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On the basis of the synthetic route, the monograph recommends testing

the impurities A, B, C, D, E, F, and G (chemical structures see Figure 1).
A resolution of at least 1.7 is required between the corresponding peak
of carbamazepine and impurity A. A single run required 110 min and a
typical chromatogram is shown in Figure 2.
The drawbacks of the current purity method are the long runtime, the
use of a complex eluent, and the required resolution especially when used
on a day-to-day basis. It highlights the fact that Pharmacopeia’s methods are
indeed not appropriate for the twenty-first century and, therefore, there is a
need to reconsider and update these methods.
Quality by Design (QbD) is a hot topic in the pharmaceutical industry for
the development of drugs and is promoted by the FDA, EMA, and ICH. The
general concepts are not new, but the tools to implement them have dramati-
cally improved in recent years. Quality-by-Design (QbD), as defined by the ICH
guideline Q8(R2), is ‘‘a systematic approach to development that begins with
predefined objectives and emphasizes product and process understanding
and process control, based on sound science and quality risk management.’’[3]
This approach can be extended to analytical method development and
should replace the obsolete trail-and-error approach in which, for example,
one-factor-at-a-time (OFAT) is varied. A modern Quality-by-Design approach
uses more statistical concepts with experimental design plans (also referred
as Design-of-Experiments) as an efficient and fast tool for method develop-
ment. The Design-of-Experiments (DoE) is defined by the ICH guideline
Q8(R2) as ‘‘A structured, organized method for determining the relation-
ship between factors affecting a process and the output of that process.’’[3]
In a DoE-based QbD approach, a couple of experiments in a full or frac-
tional factorial design are carried out, in which one or more factors are
changed at the same time. Using statistic tools the effect of each factor
on the separation is calculated to define a design space, an area in which
 UPLC Method Development for Carbamazepine 2655
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FIGURE 1 Chemical structures of carbamazepine and its impurities.

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).

Standard and Sample Preparation

A selectivity standard solution containing 0.3 mg=mL carbamazepine
(in-house standard substance) and 0.003 mg=mL of each of the six impuri-
ties A, B, C, D, E, and G (LGC, Wesel, Germany) was prepared with methanol
 UPLC Method Development for Carbamazepine 2657

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.

UPLC Equipment and Chromatographic Conditions

The method development study was carried out on an ACQUITY UPLC
H-Class system consisting of a Quaternary Solvent Manager with build-in Sol-
vent Selection Valve, Sample Manager (flow-through needle technology), a
Column Manager with four-column switch, and Photo-Diode Array detector
with analytical flow cell (10 mm pathlength, 500 nL volume), controlled by
Empower 3 software (Waters, Eschborn, Germany). The dwell volume of the
system was 400 mL.
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Fusion AE software (version 9.6.22, S-Matrix, Eureka, California, USA)

was used for screening and optimization of gradient time and slope, tem-
perature, organic solvent type, and pH to separate a mixture of carbamaze-
pine and its impurities. The Fusion AE software interacts directly with the
chromatography data software Empower 3 such that no further manual
involvement was needed.
The four different ACQUITY UPLC columns BEH C18, HSS T3, BEH
RP18 Shield, and CSH C18 with the dimension 2.1 mm x 50 mm and
1.7 mm particle size were used in the screening study (Waters, Eschborn,
Germany). For the optimization, an ACQUITY UPLC CSH C18 column
with the dimension 2.1 mm  100 mm and 1.7 mm particle size was used.
All chromatographic experiments were performed in the gradient mode.
Eluent A was acetonitrile and eluent B was methanol. Eluent D1 was 0.1%
phosphoric acid in water pH 2.4; D2 was 0.1% acetic acid in water pH 3.7; D3
was 10 mM ammonium bicarbonate buffer pH 7.3; and D4 was 0.1%
ammonium hydroxide in water pH 10.0 (for screening experiments only).
For the optimization studies, eluent A was acetonitrile, eluent B was
water, and eluent C was 10% phosphoric acid (the gradient was performed
in tertiary mode with a final concentration of phosphoric acid between 0.1%
and 0.4%). The gradient was started with 10% acetonitrile, the flow-rate was
set between 0.45 and 0.65 mL=min, and the injection volume was 1.0 mL.
The temperature in the screening experiments was fixed at 40 C and
varied between 45–65 C in the optimization experiments. The UV detection
of the compounds of interest was carried out at 230 nm and the UV spectra
were taken in the range of 200–400 nm.

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.

Scouting Experiments for Selection of Column and Eluent

Combination
The Design-of-Experiment (DoE) for the scouting and the optimization
runs was set up by the Fusion AE software. The use of this statistical experi-
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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

Table 1 Design-of-Experiments for the Scouting

Run Gradient Run Gradient

No. Solvent Time pH Column No. Solvent Time pH Column

1 ACN 6 2.4 BEH C18 25 ACN 3 2.4 BEH Shield RP18

2 ACN 3 2.4 CSH C18 26 ACN 6 2.4 HSS T3
3 ACN 3 2.4 CSH C18 27 ACN 4.5 3.7 HSS T3
4 ACN 4.5 3.7 CSH C18 28 ACN 4.5 3.7 BEH Shield RP18
5 ACN 3.75 3.7 BEH C18 29 ACN 4.5 7.3 BEH Shield RP18
6 ACN 6 7.3 CSH C18 30 ACN 4.5 7.3 HSS T3
7 ACN 4.5 7.3 CSH C18 31 ACN 3 7.3 HSS T3
8 ACN 4.5 7.3 BEH C18 32 ACN 4.5 7.3 BEH Shield RP18
9 ACN 4.5 7.3 BEH C18 33 ACN 4.5 7.3 HSS T3
10 ACN 4.5 7.3 CSH C18 34 ACN 5.25 7.3 BEH Shield RP18
11 ACN 3 10 CSH C18 35 ACN 6 7.3 HSS T3
12 ACN 6 10 BEH C18 36 ACN 3 10 BEH Shield RP18
13 MeOH 6 2.4 CSH C18 37 MeOH 3 2.4 HSS T3
14 MeOH 6 2.4 CSH C18 38 MeOH 4.5 2.4 BEH Shield RP18
15 MeOH 3 2.4 BEH C18 39 MeOH 6 2.4 HSS T3
16 MeOH 4.5 7.3 CSH C18 40 MeOH 3 2.4 HSS T3
17 MeOH 3 7.3 CSH C18 41 MeOH 3.75 7.3 BEH Shield RP18
18 MeOH 4.5 7.3 BEH C18 42 MeOH 4.5 7.3 HSS T3
19 MeOH 6 7.3 BEH C18 43 MeOH 4.5 7.3 BEH Shield RP18
20 MeOH 4.5 7.3 BEH C18 44 MeOH 4.5 7.3 HSS T3
21 MeOH 4.5 7.3 CSH C18 45 MeOH 5.25 7.3 HSS T3
22 MeOH 6 10 CSH C18 46 MeOH 4.5 7.3 BEH Shield RP18
23 MeOH 3 10 BEH C18 47 MeOH 6 10 BEH Shield RP18
24 ACN 6 2.4 BEH Shield RP18 48 MeOH 3 10 BEH Shield RP18
 UPLC Method Development for Carbamazepine 2659

(acetonitrile and methanol) over an aqueous eluent pH range from 2.4 to

10.0. Table 1 presents the experimental design used in the column and
eluent combination scouting phase. For statistical reasons some of the
experiments were performed in replicate.

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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FIGURE 3 Overlay graph of multiple responses for scouting.

 2660 A. H. Schmidt and C. Wess

Table 2 Design-of-Experiments for the Optimization

Flow Rate Final

Run No. ml=min % ACN Temperature C % H3PO3

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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13 0.55 85.1 45 0.1

14 0.6 90.1 50 0.3
15 0.5 90.1 50 0.3
16 0.6 80.2 50 0.3
17 0.5 80.2 50 0.2
18 0.55 85.1 55 0.2
19 0.65 95 55 0.1
20 0.55 95 55 0.2
21 0.65 95 55 0.4
22 0.65 75.2 55 0.4
23 0.45 75.2 55 0.4
24 0.45 85.1 55 0.4
25 0.45 95 55 0.1
26 0.55 85.1 55 0.2
27 0.65 75.2 55 0.1
28 0.45 75.2 55 0.1
29 0.5 80.2 60 0.3
30 0.6 80.2 60 0.2
31 0.5 90.1 60 0.2
32 0.6 80.2 60 0.3
33 0.55 75.2 65 0.1
34 0.65 95 65 0.1
35 0.45 75.2 65 0.1
36 0.65 95 65 0.4
37 0.45 95 65 0.4
38 0.65 75.2 65 0.4
39 0.45 75.2 65 0.4
40 0.65 85.1 65 0.1
41 0.55 85.1 65 0.4
42 0.65 75.2 65 0.2
43 0.55 75.2 65 0.1
44 0.55 95 65 0.2
45 0.45 85.1 65 0.2
 UPLC Method Development for Carbamazepine 2661
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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.

RESULTS AND DISCUSSION

Once the experiments were run and the chromatogram integrated, the
method performance goals (multiple peak responses) of the screening
phase were calculated. These are:

1. number of integrated peaks in each chromatogram

2. number of peaks which are baseline separated (USP resolution Rs > 1.5)
3. USP resolution of carbamazepine
 2662 A. H. Schmidt and C. Wess
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FIGURE 4 Continued. b.) Graph settings: Temperature and Buffer Concentration

The temperature and % concentration of phosphoric acid is varied at (T): 60 C, 0.3%, (A): 56 C, 0.2%,
(B): 56 C, 0.3%, (C): 64 C, 0.2% and (D): 64 C, 0.3%, while the flow rate and final % of organic eluent
was fixed at 0.6 mL=min and 89.7%, respectively.

4. USP resolution of impurity A.

This so-called trend-response approach eliminates the requirement

for time-consuming and error-prone peak tracking. However, it has to be
taken into account that without peak tracking, any knowledge about the
chromatographic behavior (peak movements) of the analytes cannot be
collected.
For each modeled response a contour plot (response surface plot) can
be generated, and these plots can be overlaid to visualize the knowledge
space. Figure 3 shows the overlay graph for the CSH column with acetonitrile
 UPLC Method Development for Carbamazepine 2663
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FIGURE 4 Continued. c.) Graph settings: Temperature and Final % Organic

Final % of acetonitrile and oven temperature are varied at (T): 85%, 60 C, (A): 81%, 56 C, (B): 89%,
56 C, (C): 81%, 64 C and (D): 89%, 64 C, while flow rate and % concentration of phosphoric acid are
fixed at 0.6 mL=min and 0.2%, 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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carbamazepine is, of course, to maximize the resolution of all peaks. Figure 4

shows zoomed-in overlay graphs; each graph highlights the knowledge space
for two variables: (a) flow rate and final % of the organic eluent, (b) tem-
perature and concentration of phosphoric acid, and (c) temperature and
final % of gradient. The white unshaded area in each graph defines the
design space, a robust region of the method, where the resolution of all
peaks is achieved.
The optimum conditions determined by the Fusion software were
achieved with a CSH C18 column with the dimensions 2.1  100 mm,
1.7 mm and a gradient from 10 to 85% acetonitrile in 5 min. The aqueous
eluent was 0.2% phosphoric acid and a temperature of 60 C was applied.
Within the design space, a square region was selected to define the oper-
ating space. In order to confirm the method, a verification and robustness
study at the conditions of the geometric center points (‘‘T’’) and the lower
and upper bounds of the operating space (‘‘A’’, ‘‘B’’, ‘‘C’’ and ‘‘D’’) was
performed. A chromatogram of the final method (at the working point)
for the system suitability standard solution containing carbamazepine and
the six impurities is shown in Figure 5. All compounds are well baseline
separated with a resolution Rs > 2.0: a result that could not be achieved with
the method published in the European Pharmacopeia. Total runtime is
only 5 min.
It should be mentioned that this UPLC method was successfully
transferred to HPLC conditions by using the ACQUITY UPLC Columns
Calculator (free available on www.waters.com). The method was scaled to
the corresponding XSelect CSH C18 column with the dimensions 4.6 mm
x 150 mm, 3.5 mm particle size without any further development.
A formal validation study should be performed before this new method
can replace the existing method.
 UPLC Method Development for Carbamazepine 2665

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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