Research Paper

Development and Validation of a Stability-indicating RP-

HPLC Method Using Quality by Design for Estimating
Captopril
K. VEERUBHOTLA AND R. B. WALKER*
Division of Pharmaceutics, Faculty of Pharmacy, Rhodes University, Grahamstown 6140, South Africa

Veerubhotla and Walker: Stability-indicating RP-HPLC Method for Captopril

The applicability of a quality by design framework for the development of a sensitive, simple and selective,
stability-indicating reversed-phase high-performance liquid chromatography analytical method for the
analysis of captopril was investigated. Design of experiments using a central composite design approach was
used for method development. Twenty experimental runs were performed with acetonitrile content ranging
between 28 and 36 % v/v, pH from 2.8 to 3.6 and temperature between 22° and 32°. The experimental data
obtained was used to derive a quadratic model for the retention time of captopril. The optimized method
produced sharp peaks with good resolution (>2) for captopril and the internal standard with retention times
of 3.1 and 6.2 min, respectively. The experimental data revealed that acetonitrile content in the mobile phase
and pH are significant factors that affect the retention time and resolution of captopril. Normal probability
plots revealed that the residual and predicted data fall approximately on a straight line, indicating that
the experimental error for these studies was evenly distributed suggesting that the model could be used
to navigate the design space. This approach is useful to expedite method development and optimization
activities in analytical laboratories.

Key words: Quality by Design, experimental design, forced degradation studies, stability indicating,
captopril, RP-HPLC

Captopril (CP) is an orally active antihypertensive that successfully developed but no information in respect
acts by inhibiting the angiotensin converting enzyme of degradation studies was included[8]. The ICH Q1A
and preventing the conversion of angiotensin I to (R2)[11] guideline suggested that stress testing of
angiotensin II[1]. Due to the lack of a strong chromophore, an active pharmaceutical ingredient can facilitate
CP does not absorb UV radiation at high wavelengths. identification of likely degradation products, which
It is relatively unstable in solutions of pH >3.8 in in turn permits degradation pathways and the intrinsic
which it oxidizes to form a disulphide dimer[2]. Several stability of the molecule to be elucidated due to the
methods have been reported for the analysis of CP using stability-indicating power of the analytical procedure
spectrophotometry, fluorimetry gas chromatography[3], developed.
high pressure liquid chromatography with UV-
Routine practice when developing HPLC methods
detection[4-7], electro-chemical detection (ECD)[8] and
relies on the change “one factor at a time” approach
capillary zone electrophoresis (CZE)[9]. Reversed-phase
which is time-consuming and does not completely
high performance liquid chromatography (RP-HPLC)
demonstrate the flexibility of an analytical method[12,13].
is a method of choice for analysis in the pharmaceutical
During initial method development studies, parameters
industry. A method using CZE and UV-detection for
CP has been reported[9]. A stability indicating liquid
This is an open access article distributed under the terms of the Creative
chromatography analytical method for the analysis Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
of CP has been reported, however, robustness and allows others to remix, tweak, and build upon the work non-commercially,
as long as the author is credited and the new creations are licensed under
the method optimization approach used were not the identical terms
discussed[10] and a RP-HPLC method with ECD for
Accepted 17 November 2018
CP using a design of experiments (DoE) approach was
Revised 16 April 2018
Received 12 April 2017
*Address for correspondence
E-mail: [email protected] Indian J Pharm Sci 2019;81(1):45-56

January-February 2019 Indian Journal of Pharmaceutical Sciences 45

 www.ijpsonline.com
and interacting terms that affect the quality of an output indicating RP-HPLC method for the quantitation of
are assessed by applying the principles of quality by CP in dosage forms, to optimize the method using
design (QbD)[14]. The main reason for applying this RSM and subsequently validate the stability-indicating
approach is to define a method or product in terms of RP-HPLC method. To our knowledge this is the first
quality profiles and establish critical method parameters report of a stability-indicating RP-HPLC method for
at the commencement of the process. The use of DoE the analysis of CP optimized using RSM in conjunction
facilitates the study of the design space (DS) for a with QbD.
particular process and can be used to establish the
parameters of an analytical method with consistency MATERIALS AND METHODS
and adequate quality. The DS consists of combinations CP was donated by Protea Chemicals (Midrand,
of input variables that will ensure a quality output South Africa), captopril disulphide (CD) USP
and a study of the impact of variables on the quality reference standard was purchased from Sigma-Aldrich
of the output or performance of a method ensures (Darmstadt, Germany) and phenobarbital (PB) was
the development of a next level control strategy to donated by Aspen Pharmacare (Port Elizabeth, South
guarantee a method meets or achieves predefined Africa). HPLC far UV grade acetonitrile (ACN) was
objectives. The application of QbD to the development purchased from Microsep (Port Elizabeth, South
of an analytical method can ensure the robustness and Africa) and 85 % w/v ortho phosphoric acid was
reproducibility of a method when used under different procured from Merck Laboratories (Merck, Wadeville,
input conditions. From a pharmaceutical and regulatory South Africa). HPLC grade water was used for all
perspective, functioning and continuing an operation analyses. Sample solutions were filtered through a
within a DS does not constitute a significant change 0.45 μm HVLP Durapore® membrane filters (Millipore,
and obviates the need for submission of post-approval Bedford, MA, USA). CaptoHexal® 50 (Sandoz SA
change protocols[15]. In a QbD framework, the impact of (Pty) Ltd., South Africa), Mylan Captopril 50 (Mylan
critical material and process variables on the ultimate (Pty) Ltd., South Africa), Adco-Captomax 50 (Adcock
quality of a process, in this case an analytical method, Ingram Ltd., South Africa) were purchased from a
are usually evaluated. In order to evaluate the impact of local pharmacy.
different independent input variables in a process on the
The HPLC system consisted of a Model 2695 Waters
output of that process, multivariate regression models
Alliance separation module equipped with a solvent
such as DoE can be used[16]. DoE is a mathematical
delivery module, auto sampler, online degasser and a
and statistical approach that has been used effectively
Model 2996 photodiode array detector (Waters, Milford,
during the development and optimization of processes.
MA, USA). The stationary phase was a Phenomenex
Response surface methodology (RSM) is a widely
Luna C18 column (150×4.6 mm i.d. 5 μm) and data
used approach for the simultaneous study of multiple
acquisition, processing and reporting were achieved
input factors on a response. QbD methodology using
using Waters Empower 2 software. A Model GLP21
RSM, a full or fractional factorial, central composite
pH-meter (Crison Instruments, Barcelona, Spain) was
(CCD)[17], Box-Behnken[18] or Doehlert[19] design
used in all studies and a Colora ultra-thermostat water
can be used for the identification of experimental
bath (Colora, Lorch, Germany) was used to maintain
conditions for an optimized chromatographic
temperature for stress testing studies. An Atlas Suntest
separation. Identification of the factors that must be
CPS+ (Lisengericht, Germany) was used for photolytic
investigated and that affect key responses is necessary
degradation studies. Experimental design and data
in order to define an experimental matrix that is fit for
analysis calculations were performed using version
purpose so as to ensure that experiments are conducted
8.0.4.1 Design-Expert software (Minneapolis, MN,
in accordance and compliance with a well-defined
USA).
experimental domain. The fitness of the model can
be evaluated using mathematical approaches and Preparation of stock solutions:
polynomial equations and the final stage of analysis
Approximately 10 mg of CP and PB (the internal
includes verification of the optimal input region of an
standard) were accurately weighed into two separate
operation for each of the responses monitored[20,21].
100 ml A-grade volumetric flask and dissolved in
The objective of this study was to develop a simple, mobile phase with the aid of a Branson B12 ultrasonic
sensitive, selective, accurate and precise, stability- bath (Shelton, CN, USA). Appropriate aliquots of the
46 Indian Journal of Pharmaceutical Sciences January-February 2019
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stock solution were then serially diluted with mobile performance were identified, based on knowledge
phase to produce seven working standards of 1, 5, 10, gained during method development activities
20, 40, 60, and 80 μg/ml concentration and PB was and scientific understanding of chromatographic
prepared as a 10 μg/ml solution. The solutions were separations. Information such as the physicochemical
prepared on a daily basis. properties, ultraviolet absorption spectrum, pKa,
solubility and chemical stability of CP were collected
Preparation of mobile phase: prior to commencement of experimental activities.
Milli-Q water (700 ml) and 300 ml of ACN were Operating conditions such as temperature, humidity,
measured separately using a 1000 ml A-grade nature of starting materials and purity of reagents,
measuring cylinder and mixed in a 1000 ml Schott which may influence the quality of an analytical
Duran bottle (Schott Duran GmbH, Wertheim, method were established and closely monitored during
Germany), the pH of mobile phase was adjusted to 3 development of the HPLC separation. A common
using 85 % w/v ortho-phosphoric acid. The mobile approach to screening QA is to undertake a structured
phase was filtered through a Millipore HVLP 0.45 μm risk assessment exercise by constructing a fishbone or
filter membrane (Merck Millipore Ltd, Cork, Ireland) Ishikawa diagram in which traditional knowledge used
with the aid of an Eyela Aspirator A-2S vacuum pump for analytical method development is combined with
(Rikakikai Co. Ltd, Tokyo, Japan) and degassed for current state-of-the-art approaches applied to method
15 min after preparation using a Branson B12 ultrasonic development activities. The use of a fishbone diagram
bath (Shelton, CN, USA). permits categorization and individualisation of input
factors according to the instrument, materials, method,
Chromatographic conditions: measurements and laboratory environment is discussed
Separation of peaks was achieved using a Waters in the results[25,26].
Alliance system fitted with a PDA detector set at
Method validation, linearity:
210 nm and a Phenomenex Luna C18 (150×4.6 mm i.d.
5 μm) stationary phase and mobile phase consisting of A calibration curve was constructed following replicate
a mixture of ACN and water in a 30:70 % v/v ratio. A (n=6) analysis of seven standards of 1, 5, 10, 20, 40, 60
0.8 ml aliquot of the CP (80 μg/ml) together with 0.5 ml and 80 μg/ml concentration. The peak height ratio of CP
PB solution (10 μg/ml) were transferred into an amber to IS was calculated and plotted versus concentration
glass screw thread vial and this mixture was injected after which least squares linear regression analysis of
onto the system that was operated in isocratic mode at data was undertaken to establish the equation for the
a flow rate of 1.0 ml/min and a column temperature of best fit line and the correlation coefficient (R2) was
24.0°. used to confirm linearity.

QbD approach to analysis: Precision:

The application of QbD in HPLC method development Intra-day (repeatability) precision was established
commences with establishing analytical objectives following analysis of replicate samples (n=6) at three
based on sound science to ensure consistent method concentrations indicative of low, medium and high
performance characteristics are achieved[22]. The use levels within the linear range viz., 5, 20, 60 μg/ml.
of QbD for an analytical method commences with Analysis was performed over a short period of time
defining the target analytical profile (TAP) in which on the same day. Inter-day precision or reproducibility
the pre-defined objectives for method performance was assessed at low, medium and high concentration
must be appropriately validated and documented[23,24]. on three consecutive days and the percent relative
Such analytical methods are, in fact, an indicator of standard deviation (% RSD) was used to assess intra-
a quality product and the robustness of that product and inter-day precision. An upper limit of 2 % was
for the duration on the lifecycle of that product. The used to confirm precision in our laboratory.
main goal of any HPLC method is to separate and
Accuracy:
quantitate analyte(s) of interest from any impurity
and/or excipients. Initially it is important to establish A limit for % RSD was set at <2 % for accuracy.
the critical quality attributes (CQA) of a system that Accuracy was established by applying the optimized
may impact the quality of the analytical method. RP-HPLC method to the analysis of a CP standard of
Quality attributes (QA) that may impact method known purity at low, medium and high concentration
January-February 2019 Indian Journal of Pharmaceutical Sciences 47
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and the percent recovery for each concentration 100 ml A-grade volumetric flask. Approximately 70 ml
calculated. mobile phase was added to the volumetric flask and
the contents were sonicated for 15 min to dissolve the
Limits of detection (LOD) and quantitation (LOQ): CP. Following sonication the solution was allowed
Several approaches for the calculation of the LOD and to cool to room temperature (22°) and then made up
LOQ of a method have been suggested in different volume with mobile phase. An aliquot of the solution
guidelines and include visual evaluation, use of signal- was filtered through a 0.45 μm Millipore Millex-HV
to-noise ratio, calculations based on standard deviation hydrophilic PVDF filter membrane and was further
of a response and the slope of the calibration curve[27]. diluted to produce a solution of final concentration
By convention, the LOD is estimated as one third of of 50 μg/ml. Replicates (n=3) of the test sample were
the LOQ. A series of samples of 0.25, 0.5, 1, 1.25, 1.75, analysed using the validated RP-HPLC method.
2.5, and 5 μg/ml was prepared and analysed using the
RESULTS AND DISCUSSION
optimized RP-HPLC method and the peak height ratio
calculated. The LOQ was determined by establishing The use of a TAP is to ensure flexibility following
the lowest concentration of CP that resulted in a method development, so as to facilitate continuous
% RSD value for precision of <2 %. improvement of a product whilst avoiding the need
for costly post-approval changes following market
Specificity:
authorization[23]. The performance characteristics
The specificity of an analytical method is defined as the required to ensure that an analytical method is fit for the
ability of a method to ensure that the peak(s) of interest purpose should be stability indicating, have a short run
elute as distinct responses in the presence of excipients, time, produce sharp peaks that are well-resolved from
impurities or degradation compounds[27]. impurities[28]. The method must conform to regulatory
requirements and include an assessment of specificity,
Forced degradation studies:
linearity, range, accuracy, precision, sensitivity and
Approximately 10 mg of CP was accurately weighed robustness as key parameters addressed during HPLC
and dissolved in 10 ml 1 M HCl, 0.1 M NaOH, water and method validation as suggested in USP and in ICH
3 % v/v H2O2. A known quantity of CP was dispersed guidelines[27,29]. Of these parameters, accuracy and
as a thin layer on a watch glass and placed in an oven precision are important performance characteristics
(Gallenkamp®, Loughborough UK) set at 100° for for the quantitation of materials as is the range of the
6 h to evaluate dry heat degradation and a similar method. Linearity and specificity are not a requirement
sample was also exposed to UV/Vis radiation using an for a TAP as they are not directly implicated in the
Atlas Suntest® CPS+ instrument for 24 h. Degradation understanding of the extent of agreement of a measured
was performed by refluxing 10 ml of the stock solution and true value[24,30]. The set of objectives defined in
at temperatures 60°, 70°, 80°, 90° and 100° for 6 h using the TAP includes critical factors that are necessary to
a Colora® Ultra-Thermostat water bath to maintain establish the quality of a method.
temperature. The peroxide solution was placed in the
In order to meet predefined TAP objectives, outputs or
dark for 1 h at 22°. Aliquots (1 ml) were removed from
responses such as retention time, resolution between
test solutions, diluted with mobile phase and analysed
CP and IS and peak tailing were identified as CQA for
using the RP-HPLC method. Solid samples were
this study. The first branch of the fish bone diagram
prepared for analysis following exposure, as previously (fig. 1) refers to the properties of the materials used
described. such as for example the purity of CP, stability of the
Application of analytical method: IS, age of the column and grade of solvent used. The
purity of CP may result in variable peak height/area
Commercially available CP tablets were used to assess or an interaction between the IS and CP may alter the
the application of this method. Mylan Captopril 50, concentration and/or retention time of the analyte of
Adcock® Captomax 50 and Sandoz CaptHexal® 50 interest. The analytical grade of the solvents used must
tablets were purchased from a local pharmacy. Twenty be considered as any particulate matter in solvents
tablets were weighed and crushed in a mortar to form may block the stationary phase and/or produce an
a fine powder. An aliquot of powder equivalent to the unwanted peak in chromatograms since elution time
average weight of one tablet was weighed directly into a of a compound is dependent on the affinity of that
48 Indian Journal of Pharmaceutical Sciences January-February 2019
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material to C18 chains of the stationary phase packing of three key components viz., ACN composition
material. A new Phenomenex Luna 150×4.6 mm i.d of the mobile phase, pH and column temperature
5 μm C18 column was used throughout the study. To was undertaken followed by optimization of
minimize the effect of solvent impurities all analyses chromatographic conditions using prediction software.
was performed using HPLC grade ACN. Process Experimentally derived data was modelled and a
related variables of HPLC instruments and analytical number of chromatographic conditions predicted
techniques include mobile phase composition and based on that data, were identified and evaluated. This
pH, flow rate, injection volume, column temperature, approach to optimisation was selected after considering
detector wavelength and may have a serious impact on all method attributes and based on the assumption that
HPLC responses. Early method development studies the factors investigated would be reliable, thereby
facilitated identification of initial conditions for the limiting the amount of work required to demonstrate
separation. An injection volume of 20 μl and a flow the robustness of the analytical method.
rate of 1 ml/min and a wavelength of 210 nm were DoE is an approach that enables scientists to evaluate
used for the analysis. Parameters such as flow rate, the effect and interactions of a number of variables on
column, injection volume, instrumentation variables an output simultaneously using a limited number of
were considered controllable and were kept constant experiments. From the data generated in preliminary
within the experimental domain whereas mobile studies when developing a separation for CP, limits
phase composition, pH and column temperature were for experimental levels were identified. The five
identified as critical process parameters that had to be coded levels for the independent variables in the
investigated to establish the robustness of the analytical rotatable CCD were set at –α, –1, 0, +1 and +α and the
method[31]. Environmental factors such as temperature corresponding experimental levels used, are listed in
and relative humidity, proper maintenance and level of Table 1.
knowledge of the analyst when handling instruments The upper and lower limits for ACN content (x1) were
may also interfere with the quality of the analysis. 28 and 36 % v/v, respectively. The axial points were set
An efficient and comprehensive experimental design at 26 and 38 % v/v and for mobile phase pH (x2) were
based on systematic and simultaneous examination 3.6 and 2.8 with the lower and upper limits for the axial

Materials HPLC Man

Stability of Flow rate Operator

Internal Standard handling
Injection volume
Solvents Calculation
analytical grade errors
Column temperature
Purity of
CP Improper
Detector wavelength training
Column specifications Retention
time,
resolution,
peak area,
Temperature peak
Mode of separation tailing
Percentage of acetonitrile Relative Humidity

Degassing and filtration Location

Proper
pH of mobile phase maintenance/
surroundings

Analytical Process Environmental

Fig. 1: Ishikawa diagram showing factors that may impact key method attributes
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points set at 2.5 and 3.8. The minimum and maximum and quadratic terms with positive coefficients were
column temperatures (x3) were 22 and 32° and the directly proportional, whereas the negative terms have
lower and upper axial levels were set at 20 and 35°. The an inverse relationship to the response. The interaction
critical responses monitored were retention time (y1), terms x1x2 and x2x3 had an agonistic effect on the
resolution (y2) and peak tailing (y3). System suitability, retention time of CP whereas x1x3, had an antagonistic
precision, accuracy and specificity were considered effect. The polynomial equations for critical quality
as important method performance characteristics. responses are represented mathematically in Eqn. 1.1,
The experiments were performed randomly in order 1.2 and 1.3, respectively. y1 = 2.84–0.39x1+0.17x2–
to eliminate any possible experimental bias and 0.0070x3+0.068x12+0.11x22-0.0071x32+0.019x1 x2-
the resultant HPLC data from 20 experiments with 0.051x1 x3+0.011x2 x3 (Eqn. 1.1); y2 = 3.23–0.93x1-
8 factorial points, 6 axial and 6 centre points were 0.33x 2 –0.079x 3 +0.18x 1 2 -0.15x 2 2 +0.14x 3 2 -0.077x 1
analysed and are summarized in Table 2. x2+0.14x1 x3-0.11x2 x3 (Eqn. 1.2); y3 = 1.07+0.21x1+0
.11x2+0.077x 3+0.059x 12+0.066x 22+0.038x 32+0.099x 1
Statistical analyses were used to generate second-order
x2+0.069x1 x3-0.016x2 x3 (Eqn. 1.3). ANOVA results
quadratic equations that best fitted the experimental data
suggest that the response surface quadratic model for
in addition to prediction of responses. The polynomial
the three responses was significant and adequate. The
equation that was generated following fitting of
data for retention time, resolution and peak tailing are
experimental data represents the quantitative values
summarized in Table 3. The accurate response factors
for coefficients of independent variables, first order
with a low standard deviation can be predicted by
main effects, higher order effects and their interactions
the model as the correlation coefficient R2 is close to
for retention time. The HPLC variables x1, x2 and x3
unity. The results of modelling the data reveal that
TABLE 1: ACTUAL AND CODED LEVELS FOR the R2 value was >0.9 indicating that there is a good
HPLC VARIABLES INVESTIGATED correlation between the experimental and predicted
Real values for the coded levels responses for this model[32].
Variables -α +α
-1 0 +1
(1.68) (1.68) The R2 values for responses y1, y2 and y3 were 0.9852,
ACN content (x1) % v/v 26 28 32 36 38 0.9259 and 0.7107, and the predicted and experimental
pH (x2) 2.5 2.8 3.2 3.6 3.8 values were found to be in good agreement for y1 as
Temperature (x3)° 20 22 27 32 35 the predicted R2 value was 0.8776, thereby indicating
TABLE 2: CODED LEVELS FOR THE CCD EXPERIMENTAL DOMAIN AND RESULTANT RESPONSES
ACN pH Temp Retention time minutes Resolution Peak tailing
Run % v/v (x2) (°) (y1) (y2) (y3)
Standard Point
number
(x1) (x3)
14 1 Axial 0 0 1.68 2.79 3.08 1.25
16 2 Centre 0 0 0 2.81 3.24 1
10 3 Axial 1.68 0 0 2.36 2.15 1.25
5 4 Factorial -1 -1 1 3.27 4.94 0.87
1 5 Factorial -1 -1 -1 3.31 4.8 0.83
18 6 Centre 0 0 0 2.86 3.09 1
6 7 Factorial 1 -1 1 2.46 3.08 1.5
2 8 Factorial 1 -1 -1 2.48 2.84 1.16
9 9 Axial -1.68 0 0 3.69 4.77 1.12
15 10 Centre 0 0 0 2.84 3.4 1.16
20 11 Centre 0 0 0 2.85 3.18 1.25
17 12 Centre 0 0 0 2.85 3.17 1
7 13 Factorial -1 1 1 3.66 3.98 1
19 14 Centre 0 0 0 2.86 3.37 1
4 15 Factorial 1 1 -1 2.9 2.02 1.75
12 16 Axial 0 1.68 0 3.5 2 1.25
8 17 Factorial 1 1 1 2.7 2.27 2
13 18 Axial 0 0 -1.68 2.83 3.64 1
11 19 Axial 0 -1.68 0 2.8 3.08 1.16
3 20 Factorial -1 1 -1 3.43 4.75 1
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reasonably good agreement with the adjusted R value 2
significant effect on the retention time of CP. The 2D
of 0.9720. However, the predicted R2 value of 0.4573 contour depicted in fig. 2 reveals that an increase in
for response y2 is not close in value to the adjusted ACN content in the mobile phase whilst maintaining
R2 value of 0.8592 and may be indicative of a large a constant pH and temperature results in a decrease
block effect for the prediction of this response when in the retention time of CP whereas an increase in
using this model. A negative predicted R2 value of the pH of mobile phase at constant temperature and
–0.9615 was observed for response y3 implying that ACN content results in longer retention times for the
the overall mean value of 1.18 is a better predictor of drug. A higher proportion of ACN in the mobile phase
the model responses than the coefficient of regression. results in a high elution strength with a consequent
Further, adequate precision measures the signal-to- reduction in the retention time of CP due to a change
noise ratio and a desirable ratio to produce an adequate in the polarity of the mobile phase. CP is a weak acid
signal should be >4. All responses exhibited adequate with a pKa of 2.8 and therefore a change in the pH has
precision values >4, indicating that the model is a significant effect on the retention of CP and elution
adequate to navigate the DS. The relatively low value is therefore pH-dependent[33]. The normal probability
for the coefficient of variation indicates the precision plot of residuals for retention time reveals that the
and reliability of the experiments. ANOVA was used to data points were distributed along a straight line
estimate the quality of the quadratic regression models (fig. 3A) indicating that the error was equally distributed
and to validate the response surface models[32]. across each individual point. The plot of actual versus
The F-values of 507.12 and 97.01 and p-values predicted values (fig. 3B) reveals that all points fall
<0.001 for x1 and x2 indicate that these factors have a nearly in the same region, confirming that the model
could be used to predict response values for specific
TABLE 3: ANOVA DATA FOR RESPONSE SURFACE and independent input values.
QUADRATIC MODEL FOR RETENTION TIME
Retention The resolution (Rs) between CP and PB was considered
Statistical Resolution Peak tailing
parameter
time
(y2) (y3)
an important factor for the development of this method.
minutes (y1) The 2D contour plot depicted in fig. 2 reveals that
F-value 507.12 99.65 13.83
an increase in ACN content resulted in a significant
P-value <0.0001 <0.0001 0.0040
Regression decrease in resolution between the compounds. An
0.9852 0.9259 0.7107
coefficient(R2) increase in the pH of the mobile phase only, resulted
Predicted R2 0.8776 0.4573 -0.9615 in a slight decrease in resolution as pH influences
Adjusted R2 0.9720 0.8592 0.4503 the degree of ionization of CP and ionic species are
Mean 2.96 3.34 1.18
favourably bound to silica sites on the stationary phase,
Standard
deviation
0.064 0.34 0.21 reducing the resolution between the peaks yet resulting
Adequate in acceptable resolution with Rs >2 for all studies. The
29.259 13.482 6.054
precision addition of an organic solvent in RP-HPLC results
C.V. % 2.18 10.27 18.10 in a low Rs due to interactions of the mobile phase
A. B. C.
Design-Expert® Software
Retention Time
Factor Coding: Actual ResolutionFactor
Design-Expert® Software
Coding: Actual Peak tailing
1.00 Resolution 1.00 Peak tailing 1.00
Design Points Design Points
4.94 2
3.4
2 0.83
1.4

0.50 X1 = A: ACN composition 0.50 X1 = A: ACN2.5

composition 0.50
X2 = B: pH X2 = B: pH

Actual Factor Actual Factor

3.2 3 2.8
C: Column temperature = 0.00 C: Column temperature = 0.00
1 1.2
3.5 3
B: pH

B: pH

B: pH

0.00 6 0.00
4 6 6
0.00

2.6

-0.50 -0.50 -0.50

-1.00 -1.00 -1.00

-1.00 -0.50 0.00 0.50 1.00 -1.00 -0.50 0.00 0.50 1.00 -1.00 -0.50 0.00 0.50 1.00

A: ACN composition A: ACN composition A: ACN composition

Fig. 2: Contour plot depicting the impact of ACN content and pH on (A) the retention time of CP, (B) the resolution factor, (C) peak
tailing of CP
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components with the surfaces of the stationary phase A. Normal Plot of Residuals
in competition with analyte molecules for these sites.
The resolution for the optimized chromatographic 99

conditions was established as 2.7, which was considered 95

90

as acceptable for the purposes of quantitation of CP.

Normal % Probability
80
70

Peak tailing was calculated using the USP approach 50

and was significantly affected by an increase in ACN 30

20

content in the mobile phase as depicted in the 2D 10

5

contour plot (fig. 2). Based on these data, it is clear 1

that column temperature and pH have only a minor

effect on peak shape. It also reveals that an increase -3.00 -2.00 -1.00 0.00 1.00 2.00 3.00

in ACN content results in an increase in peak tailing

of CP due to preferential partitioning of the API into Internally Studentized Residuals

the stationary phase. The optimized method resulted in B. Predicted vs. Actual

an acceptable peak tailing factor of 1.0 when the ACN 3.80

content was 30 % v/v and pH was 2.8. 3.60

3.40

The optimized conditions for the quantitative analysis

3.20
of CP were predicted using Design-Expert software

Predicted
and resulted in the improvement of conditions to
3.00

22
ensure a superior response within a set of targeted 2.80

experimental parameters. A typical chromatogram 2.60

of a separation using these conditions is depicted in 2.40

fig. 4. The predicted responses for the optimized 2.20

method generated using Design-Expert software were 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

found to be in close agreement with the experimental Actual

responses and the low percent predicted error suggests Fig. 3: Normal plot (A) of residuals for retention time of CP and
that the model is robust and furthermore indicates (B) plot of predicted vs. actual data for retention time of CP
that RSM is an efficient tool for the optimization of
analytical processes.
CP
The results of linearity studies conducted on three
consecutive days as summarized in Table 4 reveal that
the method is linear over the ranges studied with high
R2 values and low intercepts. The results for intra-day
precision were all within the 2 % limit, indicating that
the method is precise. Intra-day and inter-day precision
results are summarized in Table 5. The data for both, intra
and inter-day precision indicate the method is precise.
The accuracy of the method established by evaluating
low, medium and high level concentrations of CP in
replicate (n=6) resulted in a mean percent recovery of IS

99.18, 100.15 and 99.93 %, respectively with % RSD

Injection Point

values ranging from 0.13-0.20 % indicating that the

method is accurate. The LOQ of the method established
as described in the ICH Q2A guideline was 1 μg/ml
with an associated % RSD of 1.79. By convention, the
LOD was 0.3 μg/ml. The method was considered to be 0 1 2 3 4 5 6 7 8 9 10
Minutes
specific as no interfering peaks were evident during the Fig. 4: Typical chromatogram depicting the separation of CP
analysis of commercially available tablets and during and PB using the optimized separation conditions
forced degradation studies. CP: 80 μg/l; PB: 10 μg/ml
46 Indian Journal of Pharmaceutical Sciences January-February 2019
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One of the major impurities of CP is CD, which products labelled A and CD (fig. 5E) and in an alkaline
can be detected for almost every stress condition medium, degradation peaks B and CD were observed
evaluated[7,8,34]. The presence of CD was confirmed and were well separated from the approximately
by including the USP reference standard of CD in 30.6 % CP recovered (fig. 5F). The degradation product
testing and was resolved with a peak at retention time CD was observed when CP was exposed to a neutral
of 4.2 min, as depicted in fig. 5J. HPLC analysis of hydrolytic conditions (fig. 5G) and also during solid
degradation (wet heat) samples revealed that no state studies (fig. 5H). Following exposure to dry heat
degradation of CP was observed at temperatures of at 100° only 3 % CP degraded and the chromatogram
60°, 70°, 80° and 90°, however, approximately 3 % CP depicted in fig. 5I reveals the presence of CD.
degraded at a temperature of 100° and a peak for CD
The data from analysis of commercially available CP
was observed. The resultant chromatogram is depicted
tablets are summarized in Table 6. The average amount
in fig. 5A. The amount of CP remaining in the mixture
of CP in each product was 48.51 to 50.50 mg equivalent
following acid degradation was 91.4 % and the resultant
to 97-101 % of the label claim. The percent recovery
chromatogram (fig. 5B) revealed the presence of three
degradation peaks represented as A, CD and D that was calculated and falls within the specifications for
were well resolved from the peak for CP. The amount assay in the USP.
of CP remaining following degradation under basic The use of a DoE and QbD approach facilitated the rapid
condition was 16.4 % and the resultant chromatogram development and optimization of an analytical method
is depicted in fig. 5C. As CP contains a thiol functional for the analysis of CP in raw material and dosage forms
group, it readily oxidizes in the presence of 3 % v/v that was established as robust during the early stages
H2O2 and approximately 43.5 % CP was recovered of development. The use of the statistical tools ensured
following oxidation with three degradation products sufficient scientific knowledge about the impact of
observed viz., C, E and CD in the chromatogram input variables on the critical quality parameters of
(fig. 5D). The results of photolytic degradation in an the method was generated and interactions between
acidic medium resulted in the formation of degradation input variables on a response are better understood. In

TABLE 4: LINEARITY DATA FOR CP ON THREE CONSECUTIVE DAYS

Day
Parameter Day1 Day2 Day3
n=6 n=6 n=6
Concentration range (µg/ml) 1.01-80.6 1.1-80.5 1.1-80.6
Correlation coefficient (R2) 0.9997 0.9998 0.9998
Regression equation y = 0.0355x–0.0117 y = 0.0355x–0.0117 y = 0.0355x–0.0117
%RSD < 1.8 < 1.5 < 1.7

TABLE 5: INTRA-DAY AND INTER-DAY PRECISION STUDIES FOR ANALYSIS OF CP

Intra-day
Mean concentration ±SD
CP level Theoretical concentration µg/ml % RSD
µg/ml
Low 5.05 5.12±0.001 0.47
Medium 20.01 19.43±0.002 0.28
High level 60.03 59.73±0.008 0.39
Inter-day
Theoretical concentration Mean concentration ±SD
CP Level Day % RSD
µg/ml µg/ml
1 5.01 5.02±0.023 0.46
Low
2 5.01 5.10±0.050 0.95
n=6
3 5.01 4.98±0.030 0.64
1 19.85 19.60±0.074 0.38
Medium
2 20.02 20.07±0.040 0.20
n=6
3 20.01 19.88±0.090 0.44
1 60.60 60.77±0.170 0.28
High
2 60.06 59.96±0.070 0.12
n=6
3 60.30 60.34±0.130 0.22
January-February 2019 Indian Journal of Pharmaceutical Sciences 47
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Fig. 5: Chromatograms
Captopril disulphide (CD) (J), degradation of CP in water at 100° (A), 1 M HCl at 80° (B), 0.1 M NaOH at 60° (C), 3 % v/v H2O2
(D) following photolysis for 24 h in acidic medium (E), alkaline medium (F), in neutral medium (G), in the solid state (H) and
exposure to dry heat at 100° (I)

TABLE 6: ASSAY RESULTS FOR COMMERCIALLY AVAILABLE CP TABLETS

Product Label claim Mean CP±SD mg Mean recovery ±SD % % RSD
Mylan Captopril 50 50 mg 50.10±0.21 100.17±0.42 0.42
Adcock Captomax 50 50 mg 48.53±0.11 97.03±0.22 0.23
Sandoz CaptHexal®50 50 mg 50.49±0.20 100.91±0.49 0.40

addition the method, when operated in the defined DS, of CP, confirming a rapid, stability indicating method
will ensure the generation of high quality analytical had been developed. The use of DoE and QbD ensured
data from which informed formulation development the identification of robust ranges for the operating
decisions can be made. CP was found to be stable under conditions thereby ensuring the possibility of efficient
acidic conditions but undergoes significant degradation application of the method in a regulatory context. In
under photolytic conditions in the solid state and in conclusion a new, rapid and comprehensive RP-HPLC
solution. This approach to method development was method that is simple, precise, accurate, selective,
selected by considering all method attributes and specific and stability-indicating for the analysis of
assuming that several equipment and process factors CP has been successfully developed using a QbD
would be reliable so as to limit the amount of work approach. The method could be operated effectively
required to demonstrate the robustness of the analytical within the defined DS with the assurance that the data
method. Whilst CP is an official USP compound and generated are reliable and valid.
the analytical method reported in the USP is suitable
Acknowledgments:
for the analysis of the molecule, the retention time is
4.0 min[35] whereas this method exhibits an elution time The authors thank the Rhodes University Research
for CP of 3.0 min. Furthermore the results of forced Committee (RBW) and the National Research
degradation studies revealed no shift in the elution time Foundation (NRF) of South Africa (KV) for funding.
48 Indian Journal of Pharmaceutical Sciences January-February 2019
 www.ijpsonline.com

Conflict of interest: 17. Pebdan, AA, Shabani AMH, Dadfarnia S, Talebianpoor MS,
Khodadoust S. Preconcentration of valsartan by dispersive
liquid-liquid microextraction based on solidification of
None. floating organic drop and its determination in urine sample:
Central composite design. J Sep Sci 2016;39:1935-44.
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50 Indian Journal of Pharmaceutical Sciences January-February 2019