Accepted Manuscript

Title: A Simultaneous Determination of Related Substances

by High Performance Liquid Chromatography in a Drug
Product Using Quality by Design Approach

Author: Trupti Tol Nilesh Kadam Nilesh Raotole Anita Desai

Gautam Samanta

PII: S0021-9673(15)01878-6
DOI: http://dx.doi.org/doi:10.1016/j.chroma.2015.12.080
Reference: CHROMA 357182

To appear in: Journal of Chromatography A

Received date: 9-11-2015

Revised date: 23-12-2015
Accepted date: 28-12-2015

Please cite this article as: Trupti Tol, Nilesh Kadam, Nilesh Raotole,
Anita Desai, Gautam Samanta, A Simultaneous Determination of Related
Substances by High Performance Liquid Chromatography in a Drug
Product Using Quality by Design Approach, Journal of Chromatography A
http://dx.doi.org/10.1016/j.chroma.2015.12.080

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A Simultaneous Determination of Related Substances by High Performance Liquid
Chromatography in a Drug Product Using Quality by Design Approach

Trupti Tola, Nilesh Kadamb, Nilesh Raotoleb, Anita Desaib, Gautam Samantaa*
gautam.samanta@cipla.com
a
Department of Quality by Design, Cipla Ltd, Vikhroli (West), Mumbai 400083, India
b
Formulation Analytical Development Laboratories, Cipla Ltd, Vikhroli (West), Mumbai 400083,
India
*
Corresponding author. Tel.: +91 2225761900; Cell: +91 8879117221.

1
Highlights
 Simultaneous determination of related substances in a drug product comprising of three APIs;
Abacavir, Lamivudine, and Dolutegravir using QbD principles
 Establishment of design space and normal operating range
 Robustness study through process capability using Monte Carlo simulation
 Improvement of time and resource efficiency

2
ABSTRACT

The combination of Abacavir, Lamivudine and Dolutegravir is an anti-retroviral formulation that

displays high efficacy and superiority in comparison to other anti-retroviral combinations. Analysis
of related substances in this combination drug product was very challenging due to the presence of
nearly thirty peaks including the three active pharmaceutical ingredients (APIs), eleven known
impurities and other pharmaceutical excipients. Objective of this study was to develop a single,
selective, and robust high performance liquid chromatography method for the efficient separation of
all peaks. Initially, one-factor-at-a-time (OFAT) approach was adopted to develop the method. But, it
could not resolve all the critical peaks in such complex matrix. This led to the advent of two different
HPLC methods for the determination of related substances, one for Abacavir and Lamivudine and the
other for Dolutegravir. But, since analysis of a single sample using two methods instead of one is
time and resource consuming and thus expensive, an attempt was made to develop a single and robust
method by adopting quality by design (QbD) principles. Design of Experiments (DoE) was applied as
a tool to achieve the optimum conditions through Response surface methodology with three method
variables, pH, temperature, and mobile phase composition. As the study progressed, it was
discovered that establishment of the design space was not viable due to the completely distant pH
requirements of the two responses, i.e. i) retention time for Lamivudine carboxylic acid and ii)
resolution between Abacavir impurity B and unknown impurity. Eventually, neglecting one of these
two responses each time, two distinguished design spaces have been established and verified. Edge of
failures at both design spaces indicate high probability of failure. It therefore, becomes very
important to identify the most robust zone or normal operating range (NOR) within the design space
with low risk of failure and high quality assurance. For NOR establishment, Monte Carlo simulation
was performed on the basis of which process capability index (Cpk) was derived. Finally, the
selectivity issue problem faced due to the pH dependency and the dissimilar pH needs of the two
critical responses was resolved by introducing pH gradient into the program. This new ternary
gradient program has provided a single robust method. Thus, two HPLC methods for the analysis of
the combination drug product have been replaced with a selective, robust, and cost effective single
method.

Keywords: Abacavir; Lamivudine; Dolutegravir; Quality by Design; Antiretroviral; Monte Carlo

simulation; Normal operating range; Robustness.

3
1. Introduction
In recent times the number of pharmaceutical drug products containing combination drug
components has increased considerably. Though this results in a complex formulation, it helps in
attaining multiple targets in a single dose. As can be seen in the antiretrovirals, where the therapy
regimen complexity e.g. pill burden, dose frequency etc. is known to influence treatment adherence,
which is a key factor in determining the successful long-term management of the HIV-infected
patients. To reduce the complexity, a simple, once daily, single tablet regimen containing three
antiretroviral agents has been developed [1]. The work presented here deals with a similar
formulation, comprising of the integrase strand transfer inhibitor (INSTI) Dolutegravir and the
nucleos(t)ide reverse transcriptase inhibitors (NRTIs) Abacavir and Lamivudine. The provision of
multicomponent drug product eases the need to take several medicines but, it adds complications
during the development of analytical method for separation of all the components in a single run.
Many analytical methods are available in literature for the determination of related substances
and degradation products for either Abacavir [2,3] or Lamivudine [4] or Abacavir and Lamivudine
[5,6], but to the best of our knowledge there is no published method for either Dolutegravir or for the
simultaneous determination of Abacavir, Lamivudine and Dolutegravir with their impurities and
other pharmaceutical ingredients. Therefore, to analyse the said product an analytical method needs
to be developed. An attempt was made to simultaneously determine all the three molecules on a
single method by OFAT approach. But, the conventional technique was futile in establishing the
selectivity and the failure to develop a single method eventually gave birth to two different HPLC
methods, one for Abacavir and Lamivudine and the other for Dolutegravir. These methods were
practised during routine experimentation of the development batches. But, as it is not practical and
efficient to analyse a single dosage form by using two different HPLC methods, QbD principles were
adopted to resolve the complexity.
QbD is a dynamic approach that has gained paramount importance in diverse pharmaceutical
fields [7-11]. As mentioned in the International Conference of Harmonisation (ICH) Guidelines Q8
(R2) for pharmaceutical development [12], ‘It is important to recognise that quality cannot be tested
into the products; i.e. quality should be built in by design’. Designing the product with infusion of
quality helps the product to triumph over all odds during its lifecycle. As QbD is a ubiquitous
approach, it has been successfully extended for the development of analytical method and can be
termed as Analytical QbD (AQbD) [13-25]. One of the key elements of QbD is DoE. DoE is a
structured, organized method for determining the relationship between factors affecting a process and the
output of that process [26]. DoE is superior to OFAT owing to its ability to predict interactive effects
of parameters on performance of the method.

4
 There are many benefits to developing analytical methods in an AQbD framework e.g. methods
will be built upon the understanding of the measurement and performance requirements. The
resultant method development knowledge, method robustness and transferability will result in more
robust methods with fewer method failures and transfer issues.
The AQbD work flow starts with understanding the method needs specified in the Analytical
Target Profile (ATP), followed by selection of the initial chromatographic parameters and refinement
of these conditions, risk assessments to identify and DoEs to alleviate experimental risk factors [27].
The end result is the robust analytical method with a well understood design space and method
optima.
Ideally, for selection of the initial chromatographic parameters, screening designs should be
employed. But, in this case ample development data were available. The knowledge obtained from
this development data was utilised to fix some of the factors at certain level and to identify the
critical factors for further evaluation through DoE.
As mentioned earlier, DoE is of utmost importance in QbD. Designs are available for screening
variables or optimization of variables. Response surface methodology is widely used for optimization
purpose. The responses obtained from the DoE experiments are statistically analysed to provide the
model relationship between the responses and the independent variables. Based on this model, the
best operating conditions are determined as a part of the design space. Further, NOR is identified to
establish the robustness of the method. Often, Monte Carlo simulations are used as a powerful tool to
derive and/or improve process capability by moving into a region of the design space which is more
robust to process input variables [28-31].
In this study, the key objective was to develop a single method for the determination of related
substances for the drug product composed of three active pharmaceutical ingredients (Abacavir,
Lamivudine and Dolutegravir). The drug product matrix consisted of about thirty peaks including
eleven known impurities and other pharmaceutical ingredients. The idea was to embrace the QbD
principles coupled with DoE to improve the resolution between the critical peaks, to reduce the time
and resource consumption and to establish the robustness of the method on the basis of process
capability indices using Monte Carlo simulation.

2. Material and Methods

2.1. Chemicals
The chemicals and reagents used for experimentation were: Potassium di-hydrogen phosphate
(AR Grade, SDFCL), Ortho-phosphoric acid (LR Grade, SDFCL), Acetonitrile (HPLC Grade,
SDFCL), Methanol (HPLC Grade, SDFCL) and Purified water (HPLC Grade, Millipore). The tablets

5
used for the sample preparation were in-housed Cipla manufactured as well the APIs Abacavir
sulphate, Lamivudine and Dolutegravir. Of the known impurities the Lamivudine Impurity G,
Lamivudine Impurity H, Abacavir Cyclopropyldiaminopurine impurity, Abacavir impurity A,
Abacavir impurity B, Abacavir impurity C, DTV ether, 2- Fluoro dolutegravir and 4 – Fluoro
Dolutegravir were Cipla manufactured whereas Lamivudine carboxylic acid impurity and
Lamivudine impurity J were manufactured at Sitec labs (Mumbai, India).

2.2. Sample preparation

For the sample preparation 20 tablets were finely ground to a powder, the powder equivalent to 5
tablets was weighed accurately into a 100-mL standard flask. Twenty mL acetonitrile and 20 mL
water were added with the help of 25-mL glass cylinder and sonicated for 15 minutes in a sonicator
(Ultrasonic Bath, PCI Analytics) maintained at room temperature (25 °C). Thirty mL of diluent
(50:50 mixture of 0.1% ortho-phosphoric acid and MeOH) was added to it with the help of a 50-mL
glass cylinder and again sonicated for 15 mins. The contents were diluted up to the mark with diluent
and filtered through 0.45 µm Polytetrafluoroethylene (PTFE, mdi) filter. Two mL of this filtrate was
transferred to a 20-mL standard flask, to the same flask, 0.9 mL of 200 ppm Imp G, 0.9 mL of 200
ppm Imp H, 0.9 mL of 200 ppm Lamivudine Carboxylic acid impurity, 0.9 mL of 200 ppm
Lamivudine Impurity J, 1.0 mL of 240 ppm Abacavir Cyclopropyldiaminopurine impurity, 1.2 mL of
200 ppm Abacavir impurity A, 1.2 mL of 200 ppm Abacavir impurity B, 1.2 mL of 200 ppm
Abacavir impurity C, 1.5 mL of 10 ppm DTV ether, 1.5 mL of 10 ppm 2- Fluoro dolutegravir and
1.5 mL of 10 ppm 4 – Fluoro Dolutegravir were added. It was mixed well and made up to volume 20
mL with diluent.

2.3. Instrumentation and chromatographic conditions

Analyses were performed by injecting 5 µL of the sample matrix in a HPLC Agilent 1100 series
system (USA) equipped with an auto-sampler, a quaternary gradient pump, a column thermostat, a
photodiode array detector operating at 260 nm and the Durashell C18, 150 mm x 4.6 mm, 5 µ column
(Bonna-Agela technologies, China) maintained at 25°C. The mobile phase buffer was prepared by
dissolving 1.361 g of potassium di-hydrogen phosphate in 1000 mL of purified water to obtain 0.01M
solution. pH was adjusted with ortho-phosphoric acid to 3.0 and then filtered through 0.45 µ nylon
membrane filter (Millipore). Binary solvent gradient was applied at varying flow rates of 0.7 mL min-
1
and 1.0 mL min-1 programmed as follows: 99% buffer and 1% ACN at 0 min, progressing linearly at
0.7 mL min-1 to 85 % buffer and 15 % ACN at 30 min, followed by the decrease in the buffer to 60%
and rise in ACN to 40 % with the change in the flow rate to 1.0 mL min-1 at 40 min. Subsequently,

6
the gradient was kept constant till 50 min, finally returning to the initial gradient and flow at 53 min
and maintained at this composition and flow for 12 min in the total time of 65 min of analysis.

2.4. Peak integration

Data acquisition was performed on Chromeleon chromatography software (Dionex, USA). The
identity of the three APIs and their respective known impurities were confirmed from the individual
identification solutions injected during the preliminary development. Threshold limits were adjusted
to give accurate peak integration and thus resolution. PDA (Photodiode array detector) scans too
were generated to verify the peaks in the case of change of selectivity during the experiments.

2.5. Analytical target profile, risk identification, risk evaluation and statistical data analysis
As given in the USP Stimuli article the concept of an ATP parallels the concept of Quality
Target Product Profile described and defined in ICH Q8. The ATP defines the objective of the
method and the quality requirements [32]. Selectivity is the chief motive in this case and hence the
spotlight is on resolution between the peaks. Since, resolution was to be measured as the reportable
result, on the basis of scientific rationale and the development data, the factors that pose maximum
risk to resolution were considered as critical during risk identification. Thus, temperature, mobile
phase B (MP B) composition (to vary the gradient slope) and buffer pH were selected as critical
variables/factors. The risk was evaluated through statistically designed experiments generated
through Design expert 9.0.3.1 software (Stat Ease Inc., Minneapolis, USA). Initially, a full factorial
design was employed, but due to the presence of curvature the design was augmented to central
composite design (CCD) to better understand the surface. Graphical tools such as half-normal
probability plot, pareto chart etc. were used to help assess which factor is highly important and which
is comparatively unimportant. Diagnostic tools such as normal plots, residual vs predicted plots etc.
were used to evaluate the data. The right graphs, plots or visual displays of a dataset can uncover
anomalies or provide insights that go beyond what most quantitative techniques are capable of
discovering. Further, the relationship between the variables and the responses was estimated through
regression. Analysis of variance (ANOVA) was used to assess the importance of one or more factors
by comparing the response variable means at the different factor levels. The model, F-value, p-value,
Lack of Fit (LoF), the R- squared values etc. revealed significance and predictability of the
established model. Model graphs were used to understand the interaction between the factors and
their relationship with the responses.

2.6. Design space and optimum condition establishment

7
 Design space has been established by overlaying contours of all responses each having a
predefined acceptance criteria. The objective of design space is to present a region where the method
will be fit for purpose. However, since the design space is based on prediction, experimental
verification was performed at random conditions within the design space to prove the agreement
between the predicted and observed values. The optimum condition for each factor within the design
space was identified and with the help of Monte Carlo Simulation and process capability the NOR
was established.

2.7. Sensitivity analysis and robustness study

Even after the establishment of design space it is necessary to demonstrate the robustness of the
method given the possibility of variation in the method parameters about their standard deviation.
The robustness at the optimum condition was demonstrated here by generating large amount of data
with the help of Monte Carle simulation (as given in the steps below) and deriving the measure of
capability analysis i.e. Cpk values for responses. The analytical method robustness is strongly
dependent on the sensitivity of method parameters. Hence, to find the most sensitive parameter per
response, sensitivity analysis was performed using Monte Carlo simulation and the sensitivity plots
were drawn using Devize software (Minitab, USA). Also, as the Cpk values are largely dependent on
the standard deviation of the method variables, special attention was given to the most sensitive
method variables.

Monte Carlo simulation method follows the steps as given below [28-31]:

1. Define the relationship between the method variables and response output as a response
surface model [f(y) = f(x1, x2, x3,…..xn)].
2. Analyse the method variables’ distributions and define means and standard deviations and
simulate 100,000 random variable data.
3. Generate 100,000 data for the output variable and its distribution by the model and the
distribution of method variables.

The capability analysis provides a better understanding of how much variability in the output can be
expected at the normal operating conditions. Finally, the optimum values and corresponding standard
deviations of the factors producing data with acceptable Cpk values were utilised for the
establishment of the NOR.

8
3. Results and Discussion
3.1. Identification of critical attributes and variables

The drug matrix was chromatographed on HPLC system by adopting the tentative method
developed through OFAT approach. The resulting chromatogram shown in Figure 1, exhibits 3 API
peaks, 11 known impurities and other pharmaceutical ingredient peaks amongst which reproducible
and good resolution is desired.
The attributes from ATP identified as critical are given below:
1) Retention time of the Lamivudine carboxylic acid impurity (peak 3)
2) Difference between retention time of Lamivudine carboxylic acid impurity (peak 3) and
Lamivudine (peak 5)
3) Resolution between an unknown impurity (peak 4) and Lamivudine (peak 5)
4) Resolution between Lamivudine impurity J (peak 8) and Abacavir cyclopropyldiaminopurine
impurity (peak 7)
5) Resolution between Abacavir impurity B (peak 12) and the unknown impurity (peak 11)
6) Retention time of Abacavir (peak 14)
7) Difference between retention time of Abacavir (peak 14) and Abacavir Imp C (peak 16)
8) Resolution between DTV ether (peak 23) and 2-Fluoro Dolutegravir (peak 24)
9) Resolution between 2-Fluoro Dolutegravir (peak 24) and 4-Fluoro Dolutegravir (peak 25)
10) Resolution between 4-Fluoro Dolutegravir (peak 25) and Dolutegravir (peak 26)
Of these 10 attributes, two were highly critical, the first was the retention time for Lamivudine
carboxylic acid impurity (peak 3) and the second was the resolution between unknown impurity
(peak 11) and Abacavir impurity B (peak 12). In the case of Lamivudine carboxylic acid impurity,
the retention time was found to be unstable, during random OFAT trials this impurity was observed
to be co-eluting with the Lamivudine impurity G and H peaks (peak 1 and 2, respectively) making
individual identification and quantification difficult. In the second case, the unknown impurity seen
at the fronting of Abacavir impurity B was many a times found to be co-eluting with Impurity B,
causing interference in the determination of the known impurity. Both the above mentioned
criticalities were unresolved during the development of the method through conventional approach.
Hence, the QbD approach was adopted with an intention of solving these issues and attaining a robust
method with the help of DoE.
Next, the listing of critical attributes was followed by identification of the variables using
Fishbone diagram (Figure 2) as a risk assessment tool. Since, it is not feasible to experimentally
investigate each variable, the scouting of the critical variables is essential which demands rigorous

9
brain storming. As mentioned before, already two HPLC methods were developed for the analysis of
a sample, hence sufficient data was available to determine the critical variables. Thus, on the basis of
the development data paired with scientific rationale and previous experience, the trivial factors were
neglected and the factors posing maximum risk (temperature, gradient slope, and buffer pH) were
chosen as critical. The high and low levels for the critical factors were finalised for experimentation.
The justification for neglecting and selecting the factors is given in Table S1 in the Supplementary
data.

3.2. Design of experiments

A 23 full factorial design was adopted to evaluate the three independent variables buffer pH, MP
B composition (for varying the gradient slope) and temperature. Each variable was investigated at
two levels: low and high along with the centre points as given in Table 1. Total eleven experiments
were performed including eight factorial and three centre points. The centre point experiments depict
curvature if present, and replicates serve in demonstrating system suitability. The runs were
randomised to avoid systematic error [33].Factorial design points and the resulting dataset are given
in Table S2 in the Supplementary data. Responses were analysed statistically by using Design expert
software. The multivariate analysis proves that all the three variables studied through DoE are
significant and the regression analysis provides evidence for their contribution in variation in the
responses. From the main effects plots as well as from the information given in Table 2, it can be
realised that most of the responses unveiled curvature effects, i.e. the centre point data is not in line
with the high and the low values, hinting the existence of a non-linear relationship between the
factors and the response.
The data indicate that pH is the most important factor in maximum number of cases followed by
temperature and MP B composition, curvature contribution is as high as 50% in a couple of cases
[Resolution between an unknown impurity (peak 4) and Lamivudine (peak 5) and Resolution
between Abacavir Impurity B (peak 12) and the unknown impurity (peak 11)]. Though the full
factorial design offered some valuable information, incorporation of a response surface design was
necessary to model the design space adequately. To serve the purpose additional axial points were
studied through CCD (α = 1.47), with wider range of responses to get more information along with
two additional centre points. The complete CCD design (factorial, centre and axial points) along with
the responses is given in Table S2 in the Supplementary data.

3.3. Statistical and graphical interpretation

10
 After experimentation as per the design, all the responses were analysed statistically through
Design Expert software. Out of ten responses, the two most critical responses [R1: retention time for
Lamivudine carboxylic acid impurity (peak 3) and R2: resolution between Abacavir impurity B (peak
12) and the unknown impurity (peak 11)] are being discussed extensively here. In the case of R1, the
response data was not normally distributed which violates the basic assumptions of most of the
statistical tests. To retain the normality of the data it was subjected to power transformation. The
resultant regression equations given in (3) and (4) provide the two responses (R1 and R2) as the
function of studied variables, respectively.

(R1)-1.64 = (-0.32673) + 0.19589 pH + 1.64804e-3 Temperature - 0.024888 pH2 (3)

R2 = (- 47.03137) + 26.09172 pH + 0.24680 MP B - 3.53441 pH2 (4)
The equations were tested and the significance of the model was checked through the analysis of
variance, (ANOVA), as shown in Table 3 and 4. For both responses p-values lower than 0.05 indicate
statistically significant models. The model F-value (calculated by dividing model mean square by
residual mean square)for both the responses, imply that the models are significant. Lack of fit (LoF)
expresses the model adequacy to describe the data. This parameter is achieved by comparing the
variability of the current model residuals to the variability between observations in replicate settings
of the factors. In case of R1, LoF is found to be non-significant, suggesting sufficient predictability of
the model. Whereas in case of R2, LoF is observed to be significant. This can be seen in cases where
the pure error is so small that even though the response surface fits the model points well (providing
significant model fit) the differences between the actual data points and the response plane are greater
than the differences between the centre points. This leads to significant LoF. Similar issue was
encountered in the case of R2 and therefore, the model predictability was not questioned. Further, the
established model is an estimate of the true model, hence to prove its proximity to the actual, the
model is investigated with the help of elements such as R-squared, Adjusted R-squared and Predicted
R-squared and adequate precision. These values indicate how well the model explains the variation in
the response and how well it predicts the response. The R-squared, Adjusted R- squared and
Predicted R-squared value for the responses R1 and R2 are 0.9647, 0.9577, 0.9238 and 0.8883,
0.8643 and 0.7295, respectively, thus indicating models with good reliability and predictability.
Thus, the model can be used to navigate the design space [7]. The R-squared, Adjusted R- squared
and Predicted R-squared values for all other responses are given in Table S3 in the Supplementary
data indicating models with good predictability.

3.4. Contour and three dimensional (3D) plots

11
 Contour and the 3D plots exhibit a quick, easy, and an impressive way of interpreting the
relationship between the variables and responses. While evaluating which variables are the most
influential, the plots prove that pH has significant impact on both the responses as individual term as
well as higher order term thus explaining the curvature observed during the factorial experiments.
Temperature too has some impact on the retention time for Lamivudine carboxylic acid (peak 3)
while mobile phase B composition is identified as insignificant and therefore can be disregarded.
Contrary to this, for the resolution between Abacavir impurity B (peak 12) and unknown impurity
(peak 11), mobile phase B composition has an impact while temperature is not critical. The plots
given in Figure 3, indicate that there is a considerable increase in the retention time of Lamivudine
carboxylic acid (peak 3) as the pH is varied from 2.1 to 3.2, but the impact wanes with further
increase in the pH. The contour lines denote the response values. Since sufficient separation is
required between Lamivudine carboxylic acid and the initial impurity peaks the desired retention
time is about 7 to 8 minutes. As demonstrated by the plots (Figure 3), this aspiration demands pH of
about 2.5 or below and temperature below 40°C. The low pH requisite is also supported by the fact
that carboxylic acid is in the ionised form at pH greater than 3.0, and since ionised functionalities
exhibit extreme case of polarities the retention time is shorter at pH 3.0 and above, while the desired
retention is achievable at a pH lower than 3.0[34]. Whereas, for the resolution between Abacavir
impurity B (peak 12) and unknown impurity (peak 11), the desired resolution of more than 2.0 is
achievable at pH between 3.2 and 4.2 (Figure 4). Thus, the contour and the 3D plots highlight the
contrary requisites of the responses. For all the other responses, the contour and 3D graphs are given
in Figure S1 in the Supplementary data and the model equations for the same are given in Table S4 in
the Supplementary data.

3.5. Establishment of design space

As mentioned in the ICH guideline Q8 (R2) for Pharmaceutical Development [12], design
space is the multidimensional combination and interaction of input variables. Any set point within
the (regulatory approved) design space will produce acceptable product and changes within the
design space are (regulatory) acceptable [27]. The design space is the integral and conclusive part of
any study through the QbD approach.
The acceptance criteria for all the responses as given in Table S5 in the Supplementary data are
used to obtain a design space. But, due to the completely distant pH requirements for the two
responses discussed earlier (retention time for Lamivudine carboxylic acid and resolution between
Abacavir impurity B and unknown impurity) establishment of design space is challenging. An

12
alternative approach was adopted to establish the design space by ignoring these two pH dependent
responses one at a time. Figure 5 and Figure 6 exhibit the design space neglecting the retention time
of Lamivudine carboxylic acid and the design space neglecting the resolution between the Abacavir
impurity B and the unknown impurity, respectively. Obviously, the establishment of two different
design spaces greatly disturbs our motive of achieving a single method. However, to authenticate
the predictions, it’s important to verify the design spaces experimentally. For the design space
verification, experimentation was performed at one point in each of the design spaces. The M.P B
composition was fixed to an optimum of 21%. For verification of the design space obtained by
excluding resolution between Abacavir impurity B and unknown impurity, the pH value at the
centre i.e. 2.5 was selected. Similarly, for the verification of the design space obtained by excluding
retention time for Lamivudine carboxylic acid the centre pH value i.e. 3.35 was chosen. In both the
conditions the column oven temperature was set at the room temperature (23 ± 2°C). Thus, the two
experimental conditions were i) pH: 2.5, Temperature: 22°C, % M.P.B: 21 and ii) pH: 3.35,
Temperature: 24°C, % M.P.B: 21. The predicted and observed values are given in Table 5a and
Table 5b. The results indicate that all the responses are within the prediction interval (PI) and close
to the 95% confidence interval (CI). The small difference between the predicted and observed
values can be attributed to the fact that during the DoE experimentation, the critical factors e.g. pH
were not strictly controlled and were allowed to be within their natural variability. But, during the
robustness study it was observed that few responses were impacted by the small change within the
natural variation of the factors. Therefore, it is quite possible that minor variations in the sensitive
factors during DoE have most likely contributed to this variation. Once the method is optimised,
stringent controls on variables are required as a control strategy.

3.6. Final optimisation

From the above discussion it is clearly evident that it was not possible to achieve both the
responses within the specified acceptance criteria on the same method using a single buffer pH. To
resolve this issue the gradient program was modified by changing the gradient from binary to ternary
by introducing an additional buffer of pH 2.5, thus the gradient now comprised of three mobile phase
components viz. buffer pH 2.5 (M.P A), ACN (M.P B) and buffer pH 3.5 (M.P C). The gradient was
reconstructed to introduce buffer pH 2.5 at the beginning so that it governed the retention of
Lamivudine Carboxylic acid peak. After a few minutes, the gradient was linearly switched to buffer
pH 3.5 to control the resolution between Abacavir Impurity B and the unknown impurity. This
finalised gradient was experimentally evaluated and slight changes were made in the factors for the

13
better separation of the minor peaks. These changes were within the boundary of the design space.
The modifications consisted of revision of optimum temperature to 18°C and M.P B composition to
18 % (in the third step of the gradient). The modified gradient program is given in Table 6.

3.7. Sensitivity analysis and robustness study

Design space has been established and the method has been optimised, but how robust is the
output response due to method input variables, pH, temperature, and mobile phase composition?
Even with the best controls in place, it becomes important to study the effect of process input
variations on the output. It was observed that the probability of failure for retention time of
Lamivudine carboxylic acid at the edge of the design space, under the experimental conditions pH
2.7, temperature 20°C, and MP B 18% is 61% (Figure 7A) and for difference in retention time of
Abacavir (peak 14) and Abacavir impurity C (peak 16) at pH 3.6, Temperature 20°C, and MP B 18%
is 50% (Figure 7 B). This makes it clear that at the edge of the design space the assurance of quality,
for the retention time of Lamivudine carboxylic acid and the difference in retention time of Abacavir
and Abacavir impurity C is only 39% and 50%, respectively. Thus, robustness study and the
identification of the robust region called as NOR within the design space becomes crucial to gain
assurance in quality within the random variation of the method parameters. To assess this robustness,
Monte Carlo simulation was performed at the optimum condition. Based on the sensitivity analysis
the retention time of Lamivudine carboxylic acid impurity was found to be highly pH sensitive
(Figure 8). To exhibit the impact of standard deviation of pH on the robustness of the method, Monte
Carlo simulation for the retention time of Lamivudine carboxylic acid impurity was performed at pH
2.5 (with standard deviation of 0.05, 0.04, 0.03), Temperature 18°C (with standard deviation of 1°C)
and M.P B composition 18% (with the standard deviation 0.9%) as given in Figure 9A, 9B, and 9C,
respectively. The process capability values (Cpk) have been calculated at these conditions. The Cpk
value for Lamivudine carboxylic acid impurity with standard deviation 0.05 is 0.99 which is lower
than the desired Cpk value of 1.33 necessary for assurance of the robustness of the method. There is
an increase in the Cpk value with the decrease of standard deviation of pH. At standard deviation
0.03, the Cpk value has been improved to 1.46. Thus, the robustness for the retention time of
Lamivudine carboxylic acid impurity lays within the pH range 2.41 to 2.59. To restrict the variability
a control strategy is a must. For all responses, Monte Carlo simulation has been performed to identify
the sensitivity of the method variables and to calculate the process capabilities as given in Figure S2
in the Supplementary data. Based on this analysis, the NOR within the design space has been

14
established as a control strategy and given in Table 7 where the method is highly robust (Cpk about
1.33).

3.8. Ruggedness

The final chromatogram with the new gradient program is given in Figure 10, which clearly
indicates that all the peaks are well separated and also the issues with the retention time of
Lamivudine carboxylic acid and the resolution between the Abacavir impurity B and the unknown
impurity have been resolved in a single chromatographic run. The above study provided the
assurance of robustness of the method. Further, reproducible results were obtained when analysis
was performed on the optimised method on three different days, by two different analysts and on two
different systems, hereby proving ruggedness. Thus, the objective of the development of a single
method for the determination of related substances for the Abacavir, Lamivudine and Dolutegravir
tablets has been achieved maintaining the selectivity for all peaks and thereby substantially reducing
the time and resource consumption.

4. Conclusion
A single, selective, and robust analytical method for the determination of related substances in a
complex drug product combination of three drug substances, Abacavir, Lamivudine, and
Dolutegravir has been developed using QbD principles. About thirty peaks are well separated by this
method. DoE was used to establish the relationship between the critical response and method
variables. It also helped to create the design space where the global optima exists. pH plays the most
critical role on the retention time of Lamivudine carboxylic acid impurity and the resolution between
Abacavir impurity B impurity and the unknown impurity. Ternary gradient program was introduced
to resolve the pH sensitivity issue. To establish the NOR, Monte Carlo simulation was done and
robustness was evaluated within the NOR and at the edge of the design space. Finally, this method
has reduced about 50 % analysis time, resources, and solvent consumption in comparison to the two
separate methods of analysis.

Acknowledgement
This work was performed at Cipla R &D Center and financially supported by Cipla Ltd,
Vikhroli, Mumbai, India. The authors specially thank Ms. Geena Malhotra, Head, R & D, Cipla Ltd.,
for her continuous encouragement and also acknowledge Mr. Arshad Alam, QbD department, Cipla
Ltd. for his support during writing the manuscript and Mr. Avinash Kasbale and Mr. Amol Kulkarni
for their support during experimentation.

15
Appendix A. Supplementary data
Supplementary data associated with this article has been submitted along with this manuscript.

16
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Figure Captions

Figure 1: Chromatogram for the sample matrix spiked with all known impurities using existing
tentative method (experimental conditions are given in the Instrumentation and
Chromatographic conditions section) 1: Lamivudine Impurity G, 2: Lamivudine Impurity
H, 3: Lamivudine carboxylic acid impurity, 4: unknown impurity, 5: Lamivudine, 6:
unknown impurity, 7: Abacavir Cyclopropyldiaminopurine impurity, 8: Lamivudine
impurity J, 9: unknown impurity, 10: Abacavir impurity A, 11: unknown impurity, 12:
Abacavir impurity B, 13: unknown impurity, 14: Abacavir, 15: unknown impurity, 16:
Abacavir impurity C, 17: unknown impurity, 18: unknown impurity , 19: unknown
impurity, 20: unknown impurity, 21: unknown impurity, 22: unknown impurity, 23: DTV
ether, 24: 2- Fluoro dolutegravir, 25: 4 – Fluoro Dolutegravir, 26: Dolutegravir, 27:
unknown impurity.

20
Figure 2: Fishbone diagram with method variables for the critical attributes.

21
Figure 3: Contour and 3D graph for retention time of Lamivudine Carboxylic acid (Peak 3, MP.B
composition: 21%).

22
Figure 4: Contour and 3D graph for the resolution between the Abacavir impurity B (peak 12) and the
unknown impurity (peak 11) at Temperature 25°C.

23
Figure 5: Design space established neglecting the response for retention time of Lamivudine
Carboxylic acid (peak 3) (MP B Composition = 18%).

24
Figure 6: Design Space established neglecting only the response for retention time of Abacavir
impurity B (peak 12) and the unknown impurity (peak 11) (MP B Composition = 18%).

25
Figure 7. A: Distribution plot for Retention time of Lamivudine carboxylic acid (peak 3) exhibiting
the area for failure at pH 2.7, Temperature 20°C and MP.B 18%.
Figure 7. B: Distribution plot for the difference in retention time of Abacavir (peak 14) and Abacavir
impurity C (peak 16) exhibiting the area for failure at pH 3.6, Temperature 20°C and MP
B 18%.

26
Figure 8: Sensitivity graph for retention time of Lamivudine carboxylic acid impurity (peak 3).

27
Figure 9. A, B, C: Process capability for retention time of Lamivudine carboxylic acid impurity (peak
3) with standard deviation of pH 0.05, 0.04, and 0.03, respectively.

28
Figure 10. Final Chromatogram with the new gradient programme as given in Table 7 with column
oven temperature at 18°C, 1: unknown impurity, 2: Lamivudine Impurity G, 3:
Lamivudine Impurity H, 4: unknown impurity, 5: Lamivudine carboxylic acid impurity, 6:
unknown impurity, 7: Lamivudine, 8: Abacavir Cyclopropyldiaminopurine impurity, 9:
Lamivudine impurity J, 10: unknown impurity, 11: Abacavir impurity A, 12: unknown
impurity, 13: Abacavir impurity B, 14: unknown impurity, 15: Abacavir, 16: unknown
impurity, 17: unknown impurity, 18: unknown impurity, 19: Abacavir impurity C, 20:
unknown impurity , 21: unknown impurity, 22: unknown impurity, 23: unknown impurity,
24: unknown impurity, 25: unknown impurity, 26: unknown impurity, 27: DTV ether, 28:
2- Fluoro dolutegravir, 29: 4 – Fluoro Dolutegravir, 30: Dolutegravir.

29
Tables

Table 1. Critical factors identified and their ranges for DoE runs.

Critical Factors Low Centre High

Buffer pH 2.5 3.5 4.5
Temperature(°C) 20 32.5 45
Mobile Phase 10 15 20
Composition B (%)

30
Table 2. Contribution of factor(s) on each response and curvature.
Sr Response Significant Contribution Curvature
No. Factor (%) Contribution
(%)
1 Retention time of the Lamivudine Carboxylic Temperature 39.7 18.67
acid impurity (peak 3)
pH 35.2
2 Difference between retention time of pH 83.6 0.013
Lamivudine Carboxylic acid impurity (peak 3)
and Lamivudine (peak 5)
3 Resolution between an unknown impurity (peak pH 40.4 59.05
4) and Lamivudine (peak 5)
4 Resolution between Lamivudine Impurity J pH 73.8 11.75
(peak 8) and Abacavir
Cyclopropyldiaminopurine impurity (peak 7)
5 Resolution between Abacavir Impurity B (peak pH 27.3 53.76
12) and the unknown impurity (peak 11)
MP B 15.4
6 Retention time of Abacavir (peak 14) MP B 70.0 11.66
pH 14.2
7 Difference between retention time of Abacavir pH 78.7 10.53
(peak 14) and Abacavir Imp C (peak 16)
8 Resolution between DTV ether (peak 23) and 2- Temperature 94.29 2.86
Fluoro Dolutegravir (peak 24)
9 Resolution between 2-Fluoro Dolutegravir (peak Temperature 52.59 15.15
24) and 4-Fluoro Dolutegravir (peak 25)
10 Resolution between 4-Fluoro Dolutegravir (peak MP B 60.21 0.76
25) and Dolutegravir (peak 26)
Temperature 25.21

31
Table 3. ANOVA table for the retention time of Lamivudine Carboxylic acid (R1)
Analysis of variance table
Source Sum of df Mean F p-value
Squares Square Value Prob > F
Model 0.017 3 5.609E-003 136.74 < 0.0001 Significant
A-pH 5.791E-003 1 5.791E-003 141.18 < 0.0001
B-Temperature 5.232E-003 1 5.232E-003 127.56 < 0.0001
A^2 5.804E-003 1 5.804E-003 141.49 < 0.0001
Residual 6.153E-004 15 4.102E-005
Lack of Fit 5.751E-004 11 5.228E-005 5.20 0.0626 not significant
Pure Error 4.020E-005 4 1.005E-005
Cor Total 0.017 18
df = degree of freedom

32
Table 4. ANOVA table for the Resolution between Abacavir impurity B and the unknown impurity
(R2)
Analysis of variance table
Sum of Mean F p-value
Source Squares df Square Value Prob > F
Model 146.33 3 48.78 37.10 < 0.0001 significant
A-pH 16.87 1 16.87 12.84 0.0030
C-MP B 18.77 1 18.77 14.28 0.0020
A^2 79.87 1 79.87 60.75 < 0.0001
Residual 18.40 14 1.31
Lack of Fit 18.35 10 1.84 141.17 0.0001 significant
Pure Error 0.052 4 0.013
Cor Total 164.74 17
df = degree of freedom

33
Table 5a. Design Space verification data at pH: 2.5, Temperature: 22°C, %M.P.B: 21
Response Mean Observed 95% CI low 95% CI high

Retention time of the Lamivudine 6.8 8.0 6.3 7.5

Carboxylic acid impurity

Difference between retention time of 1.9 1.2 0.8 3.0

Lamivudine Carboxylic acid impurity
and Lamivudine
Reso between unknown impurity and 1.7 1.0 1.3 2.0
Lamivudine
Lamivudine impurity J and Abacavir 4.5 3.4 3.7 5.2
Cyclopropyldiaminopurine impurity

Retention time of Abacavir 20.2 20.4 18.8 21.7

Difference between retention time of 3.9 4.0 3.2 4.6
Abacavir and Abacavir impurity C
Resolution between DTV ether and 2- 1.7 1.6 1.6 1.9
Fluoro Dolutegravir
Resolution between 2-Fluoro 2.0 1.7 1.9 2.1
Dolutegravir and 4-Fluoro Dolutegravir
Resolution between 4-Fluoro 3.8 3.1 3.7 3.9
Dolutegravir and Dolutegravir

34
Table 5b. Design Space Verification data at pH: 3.35, Temperature: 24°C, %M.P.B: 21
Response Mean Observed 95% CI low 95% CI high
Reso between unknown impurity and 2.1 1.3 1.8 2.4
Lamivudine
Lamivudine impurity J and Abacavir 3.8 2.5 3.2 4.4
Cyclopropyldiaminopurine impurity
Resolution between Abacavir impurity 5.9 2.9 4.7 7.1
B and the unknown impurity
Retention time of Abacavir 19.9 20.8 18.6 21.2
Difference between retention time of 3.5 3.6 2.9 4.1
Abacavir and Abacavir impurity C
Resolution between DTV ether and 2- 1.5 1.5 1.4 1.7
Fluoro Dolutegravir
Resolution between 2-Fluoro 2.0 1.7 1.9 2.1
Dolutegravir and 4-Fluoro Dolutegravir
Resolution between 4-Fluoro 3.9 3.2 3.7 4.0
Dolutegravir and Dolutegravir

35
Table 6. Final gradient program for the analysis of the combination drug product.
Time (mins) Flow rate (mL min -1) MP.A (%) MP.B (%) MP.C (%)
0.0 0.7 99.0 1.0 0.0
8.0 0.7 0.0 5.6 94.4
30.0 0.7 0.0 18.0 82.0
40.0 1.0 60.0 40.0 0.0
47.0 1.0 60.0 40.0 0.0
51.0 0.7 99.0 1.0 0.0
60.0 0.7 99.0 1.0 0.0

36
Table 7. Normal Operating Range within the design space for robust chromatographic separation.
Factors Unit Optimum Low High
pH 1 ---- 2.5 2.41 2.59
pH 2 ---- 3.5 3.425 3.575
Temperature °C 18 15 21
M.P B % 18 15.3 20.7

37