Journal of Chromatographic Science, 2020, 1–18

doi: 10.1093/chromsci/bmaa099
Article

Article

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QbD-Based UPLC Method for Quantification
of Brexpiprazole in Presence of Impurities
and Application to In Vitro Dissolution
Prasad Nukendra Nadella1,3,*, Venkata Nadh Ratnakaram2 , and
Srinivasu Navuluri1
1 Science and Humanities Department, Vignan University-VFSTR, Vadlamudi, Guntur 522213, Andhra Pradesh, India,
2 Chemistry Department, GITAM University, Bengaluru 561203, Karnataka, India, and 3 AET Laboratories, Analytical

Research and Development, Gaddapotharam, Hyderabad 502319, Telangana, India

*Address to whom correspondence should be addressed. Email: prasadnn14@gmail.com

Received 24 July 2019; Revised 2 August 2020; Editorial Decision 26 October 2020; Accepted 26 October 2020

Abstract
Quality-by-design-based UPLC method was developed for chromatographic separation to quantify
the antischizophrenic drug brexpiprazole in the presence of impurities. Research findings from pH-
scouting studies were used as control variables which influence the chromatographic separation.
The peak tailing and resolution are the response variables and established the design-space by DoE-
study for selection of suitable chromatographic conditions. Separation was achieved with lower
particle size stationary phase and buffer pH 2.0 in the mobile phase. The present method developed
through C18 50 × 2.1 mm, Ethylene-Bridged-Hybrid technology column with 1.7 µm particles,
mobile phase consists of pH 2.0 buffer and acetonitrile (67:33 v/v), flow rate of 0.5 mL min−1 and
detection wavelength at 215 nm. The retention time of brexpiprazole is 0.6 min and all impurities
were eluted within 2 min. The method linearity ranges were 20.4–61.3 µg mL−1 for assay and
0.88–6.59 µg mL−1 for dissolution with correlation-coefficients of 0.9999 and 0.9998 for assay and
dissolution, respectively. The recovery values were found in between 99.3 and 100.9%. The method
shows stability-indicating on the basis of noninterference of placebo, and impurities from forced-
degradation studies. Method validation was carried out according to ICH guideline Q2 (R1).

Introduction 2.0 to 6.8. The brexpiprazole drug substance is having impurities

Brexpiprazole is an atypical-antipsychotic drug. It is chemically namely impurity-A (7-hydroxy 1H-2-quinolinone), impurity-B (1-
7-{4-[4-(1-benzo-thiophen-4-yl)piperazin-1-yl] butoxy} quinolin 2- benzo[b]-thiophen 4yl-piperazine), impurity-C (7-{4-[4benzo[b]thio
(1H) -one and having chemical formula C25 H27 N3 O2 S (Figure 1). phen-4yl-1oxido-piperazin-1yl]butoxy}1H 2-quinolinone), impurity-
The brexpiprazole drug substance appears as a white to off-white D (7-[4-chlorobutoxy]-1H-2-quinolinone) and impurity-E (7-[4-
crystalline powder (1). bromobutoxy]-1H-2-quinolinone) (Figure 1). The above process
Brexpiprazole is used in an adjunctive treatment of major impurities of drug substance are considered in addition to the
depressive disorders and schizophrenia. The pharmacological action impurities from forced degradation studies for chromatographic
is produced by a partial agonist at 5 HT1A -serotonergic, D2 - separation in the present method.
dopaminergic receptors and antagonist at 5 HT2A -serotonergic, Brexpiprazole is not official in any of the pharmacopoeia such
α 2 , α 1 -noradrenergic receptors. Brexpiprazole is a class-II drug of as USP, JP, BP, IP and Ph. Eur. Analytical methods reported in the
biopharmaceutical classification system due to its high permeability literature are very few for analysis of brexpiprazole, which includes
and lower solubility (1). Brexpiprazole is having a pH-dependent estimation in biological fluids by UPLC–MS/MS (2, 3), estimation
solubility, where the solubility in aqueous buffers at temperature of brexpiprazole in bulk drugs or tablets by high performance
37◦ C decreases from 276 to 0.3 µg mL−1 with a change in pH from liquid chromatography (HPLC) (4–10), high-performance thin-layer

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1
2 Prasad et al.

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Figure 1. Molecular Structures of (a) brexpiprazole (b) impurity-A (c) impurity-B (d) impurity-C (e) impurity-D and (f) impurity-E.

chromatography (HPTLC) (11), identification of process-related understanding and process control, based on sound science and
impurities of brexpiprazole by LC–MS (12), estimation of related quality risk management (16). ICH Q8 (R2) guideline does not
substances in the brexpiprazole API by HPLC (13), spectrophoto- explicitly discuss the analytical development, however, the concepts
metric technique for estimation of brexpiprazole in tablets (14) and can apply. Analytical QbD concepts were used in the present
voltammetry method for determination of brexpiprazole (15). method for UPLC chromatographic separation of analyte from
Most of the analytical methods reported in the literature are given five impurities. The parameters recommended in ICH Q2 (R1) are
for estimation of brexpiprazole in tablet formulation by HPLC and the basic quality attributes to verify the analytical method during
HPTLC techniques. These methods require longer analysis time due QbD approach (17). The analytical quality by design (AQbD) is a
to long chromatographic run times of each sample injection. Faster systematic approach that includes the basic elements such as defining
chromatographic analysis is valuable in addition to the separation analytical target profile (ATP), critical method quality attributes
of impurities from the main analyte for the current pharmaceutical (CMQA), risk assessment, critical material attributes, critical method
industry. When considering product development in the pharma parameters, design of experiments (DoE), control strategy and
industry, one dissolution profile test requires analysis of 42 sample continual improvement (18–23). The ATP of the analytical procedure
injections. The analysis time required for one in vitro dissolution comprises selecting the analyte, impurities with their target technique
testing using reported methods HPLC (5) and HPLC (13) are 7 and for fast analysis, type of method, mode of detection etc., and CMQA
45.5 h, respectively, whereas the present method requires 1.4 h only. are the method verification parameters or characteristics of the
Few of the reported methods are available for estimation of brex- analytical method that should be controlled within the predefined
piprazole in biological fluids by UPLC–MS/MS technique and which acceptance criteria.
are not demonstrated for estimation of drug in the pharmaceutical In this context, the present method is significantly new and first-
dosage forms where demonstration of stability-indicating nature by time report using QbD approach for development of reversed phase
forced degradation studies is the requirement of method suitability stability-indicating UPLC method for estimation of brexpiprazole in
for intended purpose. the presence of impurities in the tablet formulation and validated.
No methods were reported for estimation of brexpiprazole using This method can be used for in vitro drug release profile estimation
quality-by-design (QbD) approach, which is the current key scientific by dissolution testing of 4 mg tablets, where method feasibility
approach to facilitate sound scientific and more efficient method was verified. This is the first time report on fast analysis UPLC
to understand broad knowledge on all corners of the method crit- method with an established design space. The DoE tool was the basis
icalities. Fast analysis by ultra-performance liquid chromatography for the establishment of design-space for systematic selection and
(UPLC) method is not available in the literature for estimation of optimization of UPLC chromatographic parameters. pH-dependant
brexpiprazole in tablet formulation by QbD approach. solubility of brexpiprazole was studied and sample extraction proce-
QbD is a systematic approach to development that begins dure was established for BCS class-2 drug brexpiprazole from the
with predefined objectives and emphasizes product and process tablet matrix in the present work. The stability-indicating nature
QbD-Based UPLC Method 3

of the present method was demonstrated from forced degradation and mixed. PVDF membrane syringe type filter with 0.2 µm pore size
studies. The present method is validated with the knowledge of ICH was used for filtration of solution and discarded the first collected
method validation guideline Q2 (R1). Present method was economic, 5 mL of filtrate followed by collecting the sample solution into HPLC
and developed to speed up the analytical activities by significantly vials for analysis.
reducing the analysis time for the pharmaceutical industries.

In vitro Dissolution for 4 mg Tablets: Standard

Experimental and Test Solutions

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Brexpiprazole working standard (88 mg) was weighed and trans-
Instrumentation and Reagents
ferred into a 200 mL dried volumetric flask. Measured about 160 mL
UPLC system with photodiode-array (PDA) detector connected to of diluent and added into a volumetric flask and sonicated to solubi-
Empower-3 software (Waters) was used for chromatographic sep- lize the substance. The makeup volume of the flask was adjusted with
arations. Design-Expert® 8.0 software (Stat—Ease) was applied to diluent and mixed. Second dilution was done by diluting 2 to 200 mL
DoE studies. Weighing analytical balance (XP205 dual range model, with dissolution media and mixed (final concentration is 4.4 µg mL−1
Mettler Toledo), pH meter (Thermo, Orion star), rotary shaker of brexpiprazole standard).
(RS 24BL, REMI), vacuum oven (Thermolab), dissolution apparatus The in vitro dissolution test was performed using an acetate buffer
(Electrolab, TDT-08 L), water bath (Meta Lab), sonicator (9L250H, solution pH 4.3 as dissolution media with a medium volume of
PCI), filtration unit with vacuum pump (Millivac-Maxi 230 V, Milli- 900 mL and medium temperature of 37◦ C. The dissolution apparatus
pore) and photostability chamber (NEC103RSPSI, Newtronics) were used for analysis consists of a paddle with rotation speed of 50 RPM.
used during method development and validation. The dissolution test samples were collected after 30 min time point
Brexpiprazole working standard and brexpiprazole tablets and samples were filtered using 0.2 µm PVDF-membrane syringe type
were arranged by AET Laboratories, India. Hydrochloric acid, filter and discard the first 5 mL of filtrate followed by collecting
sodium-hydroxide, triethylamine, potassium-dihydrogen-phosphate the samples into HPLC vials for analysis. In case of multipoint
(KH2 PO4 ), hydrogen peroxide, HPLC grade acetonitrile and profile analysis, the amount of drug present in the sample discard
methanol were purchased from Merck, India. Phosphoric acid with volume shall be considered during profile calculation, where the same
HPLC grade was purchased from Sigma-Aldrich, India. Purified volume of media will be replaced to maintain the dissolution volume
water was collected from the PALL Biowater system. 0.2 µm constant.
polyvinylidene fluoride (PVDF) membrane syringe filters were Quantification of brexpiprazole was done using the following
procured from Merck, India. formula. Where, “Aa ” and “As ” are the peak area responses of
brexpiprazole in the sample and standard chromatograms, respec-
Chromatographic Conditions tively, “Wspl ” is mg of sample used for sample solution preparation,
“Ws ” is mg of brexpiprazole used for standard solution preparation,
DoE was used to optimize the UPLC chromatographic conditions.
“AW” represents in mg and determined by average weight of tablets,
The chromatographic separation of impurities from the principle
“P” is the percentage assay of brexpiprazole standard, “LC” is the
peak in the present method was developed on C18, 50 × 2.1 mm,
label-claim of brexpiprazole tablets in mg and “Ad ” “Ads ” are the
Ethylene-Bridged-Hybrid (BEH) technology with 1.7 µm particle size
peak area responses of brexpiprazole in the sample and standard
UPLC column, mobile phase consisting of 67 volumes of buffer
chromatograms, respectively, for the in vitro dissolution test.
(10 mM KH2 PO4 , pH 2.0) and 33 volumes of acetonitrile. The
detection wavelength 215 nm, pump flow rate 0.5 mL min−1 , oven

temperature for column 35◦ C were set as chromatographic condi- Assay of brexpiprazole %Label claim
tions. The injection volume was set at 1 µL for assay and 2 µL for Aa Ws 4 100 AW
= × × × × ×P
in vitro dissolution study. The present method is isocratic with 2 min As 100 50 Wspl LC
of chromatographic run time. Diluent was prepared by mixing 70
volumes of buffer (10 mm KH2 PO4 , pH 2.0) and 30 volumes of

acetonitrile. Dissolution drug release %Labelled amount
Ad Ws 2 900
= × × × ×P
Ads 200 200 LC
Assay: Preparation of Standard and Test Solutions
Brexpiprazole working standard (about 50 mg) was weighed and
transferred into a 100 mL dried volumetric flask. Measured about Method Validation
70 mL of diluent and added into a volumetric flask and sonicated The technical requirements of ICH Q2 (R1) guideline were verified
to dissolve the drug completely. The makeup volume of the flask during method validation of present UPLC method for quantita-
was adjusted with diluent and mixed. Second dilution was done by tive estimation of brexpiprazole in the drug product. The system
diluting 4 mL of this solution to 50 mL with diluent and mixed (final suitability was verified for a brexpiprazole peak in the standard
concentration is 40 µg mL-1 of brexpiprazole). solution chromatogram by checking the system suitability test (SST)
Test solution was prepared using twenty tablets. Tablets were parameters, i.e., plate count not less than 2000, peak symmetry factor
ground to get powdered sample. The powdered sample was weighed not more than 2.0, and the percent relative standard deviation (RSD)
(corresponding to 4 mg of brexpiprazole) into a 100 mL dried of brexpiprazole peak area response from five replicate injections of
volumetric flask. Measured about 70 mL of diluent and added into a standard not more than 2.0 (24, 25).
volumetric flask. Sonication was done for 15 min, followed by flask- Noninterference of placebo, impurity and blank peaks at the
shaking for 10 min on flask shaker for drug extraction. The makeup chromatographic retention time (RT) of brexpiprazole peak was
volume of the flask was adjusted to the 100 mL mark with diluent the evidence for method specificity. Peak purity test was performed
4 Prasad et al.

for brexpiprazole peak during specificity and forced degradation Precision

studies to demonstrate that brexpiprazole peak is spectrally pure Precision test samples (six replicate samples) were prepared at 40
in the present method. Preparations of test and standard solutions and 4.4 µg mL−1 concentrations of brexpiprazole for assay and
were done at 40 µg mL−1 of brexpiprazole. The placebo sample dissolution test methods, respectively, to demonstrate the repeatabil-
was prepared similar to the test solution where placebo was used ity. Intermediate precision was demonstrated by preparing precision
instead of the test sample. Individual impurity (Imp.) preparations samples by another analyst and on different day. The precision
of Imp.-A, Imp.-B, Imp.-C, Imp.-D and Imp.-E were prepared at sample sequence was injected into a UPLC system and calculated
0.8 µg mL−1 concentration i.e., 2% level w.r.t. test solution concentra- the percentage RSD of results obtained from each analyst and also

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tion. Specificity test solutions were injected into a UPLC-PDA system between values obtained from both analysts.
by ensuring a wavelength range of 200 to 400 nm to enable peak
purity test.

Robustness of Method
Forced Degradation Studies The method robustness studies were done by taking into considera-
Sample stress studies were done using acid, alkali, thermal, peroxide, tion of small changes in detector wavelength (215 ± 2 nm), UPLC
water and light. The individual volumetric flasks containing stress pump flow rate (0.5 ± 0.1 mL min−1 ), Oven temperature for UPLC
samples were made up to volume with diluent. Placebo solutions column (35 ± 5◦ C), and buffer, solvent ratio in the mobile phase
were prepared for each stress condition similar to test solutions. We (67:33, 65:65, 69:31 v/v). Filter interference and solution stability
injected the forced degradation study test solutions into a UPLC-PDA were studied.
system by ensuring a wavelength range of 200 to 400 nm to enable
peak purity test. Peak purity was evaluated for brexpiprazole peak.
Specificity of brexpiprazole peak in forced degradation samples was
Results
verified to demonstrate the noninterference of peaks due to placebo
and impurities at the RT of brexpiprazole peak. Method Development
ATP provides the basic requirements of the method and goal to
initiate the method development. ATP includes the objective of the
Linearity method and analytical target requirements along with justification
The linearity test solutions were prepared in the concentration range which was illustrated in Table I. The CMQA are the method veri-
from 20.4 to 61.3 µg mL−1 of brexpiprazole for assay. Weighed fication parameters of the analytical method that mostly covers all
and transferred 20.49 mg of brexpiprazole into a 50 mL dried the method validation parameters including robustness requirements
volumetric flask. Measured about 30 mL diluent and added into (Table I).
a volumetric flask and sonicated to dissolve the drug. The flask The UV spectrum of brexpiprazole was recorded with a solution
final volume was made up to the mark to obtain 409.8 µg mL−1 containing 40 µg mL−1 of brexpiprazole in a diluent composed of
of brexpiprazole. The obtained solution was labeled as linearity 67 volumes of 10 mM KH2 PO4 buffer (pH 2.0) and 33 volumes of
stock. Linearity test solutions were prepared at 50, 80, 100, 120 and acetonitrile. PDA spectral data were collected from 200 to 400 nm
150% levels by dilution of 500, 800, 1000, 1200 and 1500 µL of wavelength range. The wavelength maxima for brexpiprazole peak
linearity stock to 10 mL with diluent, respectively. The linearity test were observed at 214.1, 274.7 and 323.5 nm (Figure 2). Among the
solutions for dissolution method were prepared in the concentration three maxima of wavelengths, the highest absorption is observed at
range from 0.88 to 6.59 µg mL−1 of brexpiprazole using dissolution 214.1 nm. Hence, 215 nm was chosen as wavelength detection for
media as diluent. The linearity test was demonstrated by calculating brexpiprazole peak.
correlation-coefficient (R) and regression-coefficient (R2 ) from the A solubility study of brexpiprazole was performed in aqueous
linearity plot established between brexpiprazole concentration and buffers with a pH range from 2.0 to 6.8. The observed solubilities
chromatographic peak area response. of brexpiprazole are 276, 165.9, 107.5, 15.2, 1.2 and 0.3 µg mL−1 in
0.01 N hydrochloric acid (pH 2.0), acetate-buffer (pH 4.3, pH 4.5,
pH 5.5), water and phosphate-buffer (pH 6.8), respectively. The pH
Accuracy of diluent for sample extraction/solubilization and the pH of mobile
The accuracy test solutions were prepared at 50, 100 and 150% phase buffer were selected based on high solubility of brexpiprazole
level by spiking known amount of brexpiprazole to the placebo at pH 2.0. The pH of diluent plays a major role for extraction of BCS
solution. Weighed and transferred 50.09 mg of brexpiprazole into class 2 drug brexpiprazole from the tablet matrix. The sonication
a 100 mL dried volumetric flask. About 60 mL of diluent was added time of 15 min, followed by flask-shaking for 10 min on flask shaker
and sonicated to solubilize the drug substance. The flask volume were established for complete extraction of brexpiprazole from tablet
was made up to the mark with diluent to obtain 500.9 µg mL−1 matrix.
of brexpiprazole. The obtained solution was labeled as an accuracy- The chromatographic conditions during preliminary method
stock. Placebo was weighed (equal to the weight of one tablet) and development experiments are as follows. Mobile phase-A was 10 mM
transferred into a 100 mL dried volumetric flask. Also, 50, 100 and KH2 PO4 buffer with pH 2.0. Acetonitrile was employed as mobile
150% concentration levels, respectively, were obtained by adding phase-B. C18 50 × 2.1 mm (column dimensions), BEH technology
4, 8 and 12 mL of accuracy-stock solution into a flask containing with 1.7 µm particle size UPLC-column, oven temperature for column
placebo and 60 mL of diluent. Accuracy test solutions were sonicated at 35◦ C, pump flow rate of 0.5 mL min−1 , detection wavelength of
for 15 min and final volume of flask was adjusted with diluent and 215 nm and an injection volume of 1 µL were used. The injection
mixed. Accuracy test solutions were injected into a UPLC system and volume of 2 µL was selected for in vitro dissolution analysis due to
percentage recoveries were calculated to demonstrate the accuracy. the lower peak area response of brexpiprazole.
QbD-Based UPLC Method 5

Table I. Analytical Target Profile and Critical Method Quality Attributes

Analytical Target Profile Target Requirement (25–27) Rational/Justification Status of Present

Study
(Yes/No/Remarks)

Type of method Assay: Quantification of brexpiprazole in Assay: To quantify the brexpipraazole in the Yes
presence of impurities and degradation tablet formulation or bulk.
products Dissolution: To estimate the drug release

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Dissolution: Quantification of drug release from in vitro dissolution of tablet
from tablet dosage form
Instrument UPLC To achieve the fast analysis Yes
Mode of detection and method UV, Isocratic The molecule is having chromophore groups Yes, UV detection
and can be detected by UV. at 215 nm.
Specificity Should comply noninterference of blank, Requirement as per ICH Q2 (R1) guideline. Yes
placebo and impurity peaks at the retention
time of brexpiprazole
Linearity The correlation coefficient should be not Linearity should be established with a Yes
less than 0.99 minimum range of 80 to 120% of the test
sample concentration and as per
requirements of ICH Q2 (R1) guideline.
Precision (Repeatability) Should comply the precision with % RSD Method precision should be established by Yes
of test results not more than 2.0 preparing six replicate samples. Requirement
as per ICH Q2 (R1) guideline.
Accuracy % Recovery should be between 98 to 102 The percentage recovery should be good for Yes
the drug product and established at three
concentration levels with three triplicates
each and as per requirements of ICH
guidelines
Robustness The end results should not be affected by Robustness should be established to provide Yes
small, but deliberate variations in the an indication of method consistency and
method parameters. reliability during regular usage.
Stability-indicating nature The brexpiprazole peak should be pure The stability-indicating nature of the method Yes
(co-elution should be absent, peak purity is suitable for analysis of stability samples.
should pass) even after stability and forced
degradation studies
Filter interference Interference should not be observed with If interference is present with filters, then it No interference
filter membrane. will affect the results. with PVDF filter
Solution stability All the analytical solutions should be stable To get the consistent results within the Yes
for a suitable time period. established time period of solution stability
Green chromatography Should use minimum % of organic phase as To avoid the use of more organic solvents Yes, low solvent
possible and to develop an Enviro-friendly method consumption with
a shorter run time
of 2 min.

Risk Assessment prepared individually by mixing with acetonitrile (70:30 v/v). The
The risk assessment was done for identifying the critical material summary of results for pH-scouting experiments was tabulated in
attributes (e.g., Reagents, chemicals, etc.) and critical method param- Table III. The increase in the pH of mobile phase buffer would result
eters (e.g., Wavelength, pH of diluent, mobile phase ratio, flow rate, in increase of peak tailing and decrease of plate count and resolution.
filter interference, column oven temperature, solution stability, etc.) Based on pH scouting studies, the mobile phase buffer pH 2.0 to 4.0
that are affecting the CMQA or end results. The impact of different is favorable for chromatographic peak separation with acceptable
reagents and method parameters on analytical method quality were system suitability test results. The resolution values at pH 2.0 and
studied by risk assessment process and illustrated in Table II. pH 3.0 were 2.53 and 2.61, respectively. The increase of 0.08 is not
a significant change. Similarly, the plate counts at pH 2.0 and pH 3.0
were 3588 and 3665, respectively. Here also, the increase of 77 plate
pH Scouting Studies count was insignificant. Based on the results, both the pH values
The pH-scouting experiments were executed to find out the optimum found suitable for mobile phase buffer. The sample concentration was
buffer pH suitable for mobile phase with a target requirement of set at 40 µg/mL for assay method. Considering the high solubility
shorter run time with noninterference of impurities and placebo of brexpiprazole at pH 2.0, this buffer was selected in diluent for
peaks at the RT of brexpiprazole peak and also to meet the SST. extraction of drug. To have same pH for both sample extraction
Different pH buffer solutions range from pH 2.0 to pH 6.0 were diluent and in the mobile phase buffer, the pH 2.0 was selected.
prepared. Mobile phases with different pH buffer solutions were Hence, it will avoid the multiple preparations during routine use.
6 Prasad et al.

Table II. Risk Assessment for Identification of Critical Material Attributes and Critical Method Parameters

A. Critical Material Attributes

S.No Material Attributes CMQA Risk assessment Justification
(High/Medium/Low)

1. Potassium dihydrogen
phosphate (KH2 PO4 )
Grade Molar strength Robustness, Specificity, Low It is inorganic and has no effect on

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Chromatography mobile phase buffer pH. Retention time
is not altered by molar strength; hence
it has no impact on CMQA.
2. Ortho phosphoric acid
Purity/Assay Robustness, Specificity, Low pH of the buffer was not altered by minor
Concentration Chromatography variations in the reagent. Concentration
Miscibility remains same as less quantity is added
volumetrically. Miscibility has no effect
due to through mixing. Hence risk is
minimized and not affecting CMQA.
3. Acetonitrile
Grade Composition Robustness, Specificity, Medium Targeted method was isocratic and HPLC
Chromatography grade solvent was used. The organic
solvent composition in the mobile
phase impact on the resolution of
impurity peaks from main analyte.
Hence, it has impact on CMQA and need
to be evaluated for suitable solvent ratio
in the mobile phase.

B. Critical Method Parameters

S.No Method parameters CMQA Risk assessment Justification

(High/Medium/Low)

1. Wavelength Chromatography Low The wavelength was selected based on

spectra of brexpiprazole. At 215 nm +
2 nm, the peak response in both standard
and sample will change, respectively.
Hence, it will not impact on the end
results.
2. pH of diluent Sample extraction Low The pH of diluent plays a major role for
extraction of BCS class 2 drug
brexpiprazole. The pH of diluent was
selected considering high solubility at
pH 2.0. And sample extraction procedure
was established. Hence, the impact of this
factor is low.
3. Flow rate Robustness, Specificity, Low Retention times are also changing
Chromatography proportionately, final results were
evaluated which are not affecting CMQA.
Hence, the impact of this factor is low.
4. Column oven Robustness, Specificity, Medium The column temperature may impact on
temperature Chromatography the resolution of impurity peaks from
main analyte. Hence, it has impact on
CMQA and need to be evaluated.
5. Filter interference Robustness, Accuracy, Medium Filter membranes will have impact on
Specificity the end results due to absorption of drug.
Hence, filter saturation volume needs to
be established.
6. Solution stability Robustness, Accuracy, Medium If sample solution is not stable in the
Specificity selected diluent, then it will impact on
the end results. Hence, solution stability
to be established.
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QbD-Based UPLC Method
Table III. Results of Mobile Phase Buffer pH Scouting Studies and DoE Studies

A. Effect of Mobile Phase Buffer pH on Chromatographic Separation and System Suitability Parameters

Buffer pH Peak Retention Time in Minutes System Suitability Parameters

Imp A Imp B BRE Imp C Imp D Imp E Resolution Between Tailing Theoretical
BRE & Imp-C Factor Plates
pH 2.0 0.272 0.337 0.559 0.651 1.203 1.469 2.53 1.13 3588
pH 3.0 0.271 0.336 0.557 0.652 1.202 1.467 2.61 1.13 3665
pH 4.0 0.270 0.341 0.565 0.663 1.189 1.45 2.68 1.32 3523
pH 5.0 0.272 0.352 0.589 0.66 1.191 1.452 — (Co- Elution) 1.83 2872
pH 6.0 0.273 0.597 1.218 Peak merge Peak merge Peak merge — (Co- Elution) 2.26 1267

B. Design of Experiments and Results of Response Variables

Design of Experiments Results of Response Variables

Run No. A: Mobile Phase B Ratio (%) B: Column Temperature (◦ C) Resolution Between BRE & Imp-C USP Tailing

1 35 25 3.15 1.14
2 33 45 3.62 1.03
3 35 35 3.02 1.12
4 35 45 2.95 1.07
5 37 25 2.55 1.16
6 33 35 3.74 1.09
7 37 35 2.46 1.13
8 33 25 3.83 1.14
9 37 45 2.44 1.09

Observations from DoE studies

Increase of Control Variable Effect on Response Variables

Resolution Between BRE & Imp-C USP Tailing

A Mobile phase B (Organic Solvent) Ratio Significantly decreases Insignificant

B Column Temperature Decreases and insignificant Decreases

7
8 Prasad et al.

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Figure 2. UV Spectrum of Brexpiprazole.

DoE Studies and it was less likely that any of the factors have a significant effect
With the help of Design-Expert software, the DoE were executed to on the response.
select the rugged and robust method parameters within the opera- The model F value was calculated by model mean square value
tional range of established design space. The relationship between divided by residual mean square value. The criteria for selection of
control variables and response variables were described in the design model to make significant was “Prob>F” or “P value” should be
space. The working area in the design-space demonstrated to provide <0.0500, and if the value is >0.1000 then the model terms are not
assurance on a method to produce consistent, accurate and precise significant as per evaluation by Design-Expert software from Stat
results (16, 26, 27). DoE trials were executed considering control ease. The obtained model F-value of 927.82 and P value < 0.0001
variables, i.e., oven temperature for the column and mobile phase indicates the model terms have significant effect on the response.
composition. USP tailing factor and resolution between brexpipra- Based on ANOVA calculations, the terms A (Mobile phase organic
zole and impurity-C were considered as response variables. The DoE solvent ratio), B (Column temperature) and A2 are the significant
parameters such as central-composite (design type), response-surface model terms of quadratic model.
(study type) and quadratic (design model) were selected in the Design- The term “predicted residual error sum of squares (PRESS)”
Expert 8.0 software. Column oven temperature (25 to 45◦ C), mobile indicates a measure of how the model fits in each point of the selected
phase solvent (from 33 to 37%) were chosen as ranges of control design. The “R-square” value indicates a measure of the amount of
variables. The details of data were entered into the software and it variation around the mean described by the model. The “adjusted
has given nine experiments. DoE-based test runs were performed on R-square” was adjusted for the number of terms in the model.
UPLC system and summarized the outcome of the results in Table III. The adjusted R-square decreases as the number of model terms
The target requirements for peak tailing not more than 2.0 and increases if those additional terms do not add value to the model. The
resolution between brexpiprazole and imp-C not less than two, were difference between “adjusted R-square” and “predicted R-square”
fed into the software for multivariate analysis. values should be within 0.2. The obtained difference value (0.9983–
0.9924 = 0.0059) indicates the “predicted R-square” was having
good agreement with the “adjusted R-square” value for measuring
ANOVA Results the amount of variation in new data explained by the model. The
The statistical results of analysis of variance (ANOVA) were eval- evaluation term “Adequate precision” indicates a signal-to-noise ratio
uated for the response parameter resolution between brexpiprazole and it compares the range of predicted values to the mean prediction
and impurity-C using Stat ease Design-Expert software. The best- error. The ratio should be >4 for the best design of model. The
fitting ANOVA model observed for the response parameter “Reso- obtained adequate precision ratio 76.649 indicates adequate signal
lution between brexpiprazole and impurity-C” was response surface for model discrimination. Hence, this model was used to navigate the
quadratic model. The ANOVA statistical evaluation was illustrated in design space.
Table IV. The term “DF” is the degrees of freedom and it was equal to
the number of model terms. The term “mean square” value provides
the information on model variance and it was calculated by model Post ANOVA and prediction equation
sum of squares divided by degrees of freedom. The “F value” was Coefficient estimate represents the expected change in the response
used to test by comparing model variance with residual error. If the per unit change in the experimental parameter. Variation inflation
variances are close to the same values, then ratio will be close to one factor (VIF) indicates a measure of how much variance of the model
QbD-Based UPLC Method 9

Table IV. ANOVA Results for Response Surface Quadratic Model for the Response Parameter “Resolution Between Brexpiprazole and
Impurity-C”

Source Sum of squares DF (Degrees of Mean Square F-Value P-value (Prob > F)
Freedom)

Model 2.38828 5 0.47765556 927.82 <0.0001

A (Mobile phase 2.33127 1 2.33126667 4528.36 <0.0001
organic solvent ratio)

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B (Column 0.04507 1 0.04506667 87.54 0.0026
temperature)
AB 0.00250 1 0.00250000 4.86 0.1148
A2 0.00889 1 0.00888889 17.27 0.0253
B2 0.00056 1 0.00055556 1.08 0.3753
Residual 0.00154 3 0.00051481

Fit statistics of the model: Predicted and adjusted R square values

Standard deviation 0.02269 R-Squared 0.9994
Mean 3.08444 Adjusted R-Square 0.9983
Coefficient of variation (C.V %) 0.73561 Predicted R-Square 0.9925
PRESS 0.01798 Adequate precision 76.649

Post ANOVA predictions: Coefficients in terms of coded factors

Experimental Coefficient Estimate Standard Error 95% Confidence 95% Confidence Variance Inflation
Variables (Factor) Interval-Low Interval-High Factor
Intercept 3.0289 0.0169 2.9751 3.0827 1
A −0.6233 0.0093 −0.6528 −0.0594 1
B −0.0867 0.0093 −0.0116 −0.0572 1
AB 0.025 0.0113 −0.0111 0.0611 1
A2 0.0667 0.0160 0.0156 0.1177 1
B2 0.0167 0.0160 −0.0344 0.0677 1

was inflated by lack of orthogonality in the design. The obtained VIF Contour plot indicates a two-dimensional representation of the
value in the ANOVA was 1, which indicates the factor was orthogonal response plotted against combination of numeric control factors. The
to the all other factors. The quadratic equation derived in terms of contour plots were shaded with colors for understanding effect of
coded factors for quadratic polynomial model was presented below. input parameters on response parameters. The resolution increases
with decrease of organic solvent in the mobile phase (Color indi-
R = C0 + C1 A + C2 B + C12 AB + C11 A2 + C22 B2 cation: blue to red). The effect of column temperature on resolu-
tion was insignificant. Similarly, the tailing factor decreases with
decrease of organic solvent and increase of UPLC column temper-
where R was the measured response associated with each factor, C0
ature (Color indication: red to blue), but the change of peak tailing
was the constant coefficient where the effect of variables was absent,
value was insignificant considering the value <1.2 and change was
C1 was the linear coefficient of the respective factor A, C2 was the
only about 0.1. The 3D plot indicates a three-dimensional projection
linear coefficient of the respective factors B, C12 was the interaction
of contour plot giving shape in addition to the color and contour.
coefficient between two factors A and B, C11, C22 were the quadratic
It shows how the response varies as a function of two factors.
coefficients and A, B were the input variables, i.e., mobile phase
The increase of resolution was observed with decrease of organic
organic solvent ratio and column temperature, respectively.
solvent ratio in the mobile phase. The column temperature does
Effect of control parameters on response parameters and interpre-
not have any significant effect on the resolution. The change of
tation from DoE plots.
tailing factor was insignificant with selected range of temperature
The effect of the change in method control parameters on
and organic solvent ratio. The design-space plot was established and
response parameters was studied using perturbation plot, contour
shown in Figure 4. The optimum method parameters such as the
plot and 3D plots (Figure 3) obtained from Stat ease Design-Expert
mobile phase solvent ratio of 33% and the column oven temperature
software. Perturbation plot shows how the response variable changes
of 35◦ C were selected from the center point of the design space
as each factor of control parameter moves from selected reference
plot.
point coded with zero. Based on information obtained from the
perturbation plot, it was observed that there was a significant
increase of resolution between brexpiprazole and impurity-C with
Discussion
the decrease of organic solvent ratio in the mobile phase, whereas
column temperature did not have significant effect on the resolution. The present stability-indicating UPLC method was developed
The peak tailing was slightly reduced with lower organic solvent through C18 50 × 2.1 mm, BEH technology column with
ratio and higher column temperature, but the change in tailing factor 1.7 µm particle, mobile phase consists of pH 2.0 buffer and
is marginal and it was not a significant change. acetonitrile (67:33 v/v), detection wavelength for brexpiprazole
10 Prasad et al.

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Figure 3. Effect of Control Variables A (Mobile Phase Solvent Ratio), B (Column Oven Temperature) on Response Variables a) Resolution between Brexpiprazole
and Impurity-C, b) USP Tailing by Perturbation, Contour and 3D Plots.

peak at 215 nm and flow rate of 0.5 mL min−1 . The RT of Method Validation
brexpiprazole is 0.6 min and all impurities were eluted within
Specificity
2 min. The method was validated according to ICH guideline
Diluent as blank, placebo, standard, sample, impurity-A, impurity-
Q2 (R1).
B, impurity-C, impurity-D and impurity-E and impurity spiked solu-
QbD-Based UPLC Method 11

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Figure 4. Schematic Representation of Design Space Plot.

tions of the test samples were analyzed by UPLC-PDA system and Filter interference was studied using a PVDF membrane syringe type
extracted the chromatograms at a wavelength of 215 nm. The speci- filter with 5 and 10 mL saturation against centrifuged sample and
ficity test chromatograms were provided in Figures 5 and 6. Forced no interference was observed. The stability of test solutions was
degradation test chromatograms were provided in Figures 7 and 8. established upto 24 h. The obtained results of robustness study were
The specificity data are summarized in Table V and system suitability within the 2% from initial results, hence, the present method was
test details are given in Table VI. Forced degradation stress studies considered robust (Table VI).
reveal that the method was specific, i.e., peak purity test for PDA
extracted brexpiprazole peak was passed and no peak interference
found at RT of brexpiprazole peak.
Precision and Accuracy
The recovery values of brexpiprazole to demonstrate the accuracy test
were found from 99.3 to 100.9% and each of the nine recovery values
Linearity were within the limit i.e., 98.0 to 102.0% (24, 25). Repeatability
Individual linearity test solutions for assay (concentration: 20.4– and intermediate precision test samples were injected to establish
61.3 µg mL−1 ) and for dissolution (concentration: 0.88–6.59 µg mL−1 ) the precision of analytical method. The percent RSD results of
were filled into HPLC vials and injected into a UPLC system. The repeatability, intermediate precision and between two analyst values
obtained correlation-coefficients for assay and in vitro dissolution were 0.53, 0.74 and 0.79, respectively (Table VII). The percent RSD
are 0.9999 and 0.9998, respectively. The correlation between for dissolution method repeatability was 0.77. Since the percent RSD
brexpiprazole concentration and detector response was linear based of six replicates of test results during repeatability and intermediate
on statistical analysis of the regression line (Table VI) for in vitro precision was not more than 2.0 (24, 25) the present method is
dissolution and assay methods. precise.

Robustness Control Strategy and Continual Improvement

Method robustness experiments were studied by considering the A control strategy was designed to ensure that an analytical method
small variations or changes in method parameters such as pH of of required quality will be produced consistently. Based on DoE
mobile phase buffer, oven temperature for the column, detector study, it was observed that the mobile phase solvent ratio was
wavelength, pump flow rate and mobile phase solvent composition. critical and shall be controlled to minimize the risk. Slight change
12 Prasad et al.

Table V. Results of Specificity and Forced Degradation Studies

Sample Name Retention Time (min) Purity Angle Purity Threshold Peak Purity

Impurity-A 0.276 — — —
Impurity-B 0.360 — — —
Impurity-C 0.809 — — —
Impurity-D 1.492 — — —

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Impurity-E 1.853 — — —
Brexpiprazole Standard 0.672 24.760 69.611 Pass
As such sample 0.671 22.380 79.002 Pass
Acid-degradation (0.1 N 0.670 20.453 60.157 Pass
HCl, 70◦ C, 6 h)
Alkali-degradation (0.1 N 0.671 19.496 62.683 Pass
NaOH, 70◦ C, 6 h)
Photodegradation (1.2 0.670 24.615 70.349 Pass
million lux hours, 200 watt
hours per square meter)
Thermal-degradation 0.670 20.915 56.273 Pass
(105◦ C, 6 h)
Water-degradation (Water, 0.670 24.057 65.831 Pass
70◦ C, 6 h)
Oxidation with peroxide 0.677 18.094 50.618 Pass
(3% H2 O2 , 6 h)

Table VI. Results of System Suitability Evaluation, Linearity and Robustness

Parameter Acceptance Criteria (25) Results of the Test Remarks

Specificity Precision

% RSD of Area Not less than 2.0 0.62 0.12 Satisfactory

Response/five injections
Tailing factor Not more than 2.0 1.19 1.05 Satisfactory
Theoretical plates Not less than 2000 5352 4615 Satisfactory
Linearity Results
Type of Test Assay In vitro Dissolution
Test concentration (µg mL−1 ) 20.4 to 61.3 0.88 to 6.59
Correlation coefficient [R] 0.9999 0.9998
Regression coefficient [R2 ] 0.9998 0.9996
Slope 18402.35 37127.85
Intercept −1848.01 −1514.02
Robustness Results
Parameter Type of Change Assay Results Remarks
Solution stability Initial 99.2 Solutions were stable for 24 h
After 24 h 99.5
Wavelength (215 ± 2 nm) 215 99.2 No significant variation in the results
213 99.6
217 100.2
Flow rate 0.5 99.2
(0.5 ± 0.1 mL min−1 ) 0.4 99.0
0.6 99.7
Column oven temperature 35 99.2
(35 ± 5 ◦ C) 30 99.1
40 99.4
Mobile phase ratio (Buffer: 67:33 99.2
Acetonitrile, v/v) 69:31 99.9
65:35 99.5
Filter Study: PVDF Centrifuged Sample 98.9 There was no filter interference. Hence, no variation in the
membrane filter saturation 5 mL Saturation 99.4 results
10 mL Saturation 99.3
QbD-Based UPLC Method 13

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Figure 5. Representative Brexpiprazole Sample Chromatogram.

Figure 6. Representative Impurity Spiked Sample Chromatogram.

or variation in column oven temperature, pH of buffer and pump Comparison with Reported Analytical Methods
flow rate were not significant effect on the end results. A con- The present UPLC method was developed with good chromato-
trol strategy was established by selecting the mobile phase ratio of graphic separation for rapid estimation of brexpiprazole without any
77:33 v/v, system suitability of tailing factor not more than 1.5, interference of placebo and impurities in bulk and drug product.
plate count not less than 2000, percent RSD of brexpiprazole peak In addition, the same method was capable for estimation of drug
area response from five replicate injections of standard not more release from a tablet dosage form in the in vitro dissolution test.
than 2.0, to ensure that the proposed UPLC method will produce The RT of brexpiprazole peak in present UPLC method was 0.6 min
consistent results. As a continual improvement, the method changes and demonstrated forced degradation studies to prove the stability-
and improvements to be made with reference to the working range indicating nature, whereas the minimum RT of brexpiprazole peak in
of the design space to have method performance throughout the life reported UPLC method was 1.95 min (2, 3); this method was applica-
cycle. ble for estimation of drug in biological fluids. The reported analytical
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Prasad et al.

Figure 7. Representative Chromatograms of Acid, Base and Photodegradation.

14
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15

Figure 8. Representative Chromatograms of Thermal, Water, and Peroxide Degradation.

QbD-Based UPLC Method
16 Prasad et al.

Table VII. Results of Accuracy and Precision Parameters

% Spiking w.r.t. Sample Theoretical Concentration (µg mL−1 , Experimental Concentration (µg mL−1 , as % Recovery
Concentration as Brexpiprazole) Brexpiprazole)

50%-Sample-1 19.976 19.828 99.3

50%-Sample-2 19.976 19.875 99.5
50%-Sample-3 19.976 19.911 99.7
100%-Sample-1 39.952 40.131 100.4

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100%-Sample-2 39.952 40.184 100.6
100%-Sample-3 39.952 40.145 100.5
150%-Sample-1 59.928 60.034 100.2
150%-Sample-2 59.928 60.439 100.9
150%-Sample-3 59.928 59.950 100.0
Mean 100.1
%RSD 0.54

Precision Results

Preparation In vitro Dissolution-Precision Assay Precision Assay Intermediate Precision

% Labeled Amount % Label Claim % Label Claim

1 101.5 99.1 99.1

2 101.3 98.8 101.0
3 103.0 99.6 99.8
4 102.6 98.9 100.5
5 101.8 100.2 100.1
6 103.1 99.2 101.0
Mean 102.2 99.3 100.3
% RSD 0.78 0.53 0.74
Mean between two analyst values 99.8
% RSD between two analyst values 0.79

methods for assay of brexpiprazole in tablets are HPLC methods with (BEH technology column with lower particle size stationary phase
a run time of more than 10 min (4, 5). The HPLC method with run with acidic pH mobile phase) were used for UPLC separation. The
time of 5 min (6) was reported, where known impurities are not present study report is the first time report on the fast analysis,
considered during development. The present method was the first stability-indicating UPLC method with established design space for
report based on the QbD-driven UPLC method for pharmaceutical chromatographic separation of impurities from brexpiprazole. Sam-
drug product. DoE study was the basis for the establishment of ple extraction procedure from the tablet matrix was established as
design-space for systematic selection and optimization of chromato- the brexpipazole is a BCS class 2 drug having low solubility. The
graphic parameters in the present method. Stability-indicating nature method has demonstrated the robustness, linearity, range, accuracy
of the present method was demonstrated from forced degradation and precision based on ICH method validation guideline i.e., ICH
studies and this method was suitable for both assay and in vitro Q2 (R1). Noninterference of blank, placebo and impurity peaks for
dissolution analysis of drug product and stability samples. The com- brexpiprazole peak from forced degradation stress studies reveal
parative data of reported analytical methods for quantification of that the present method is stability-indicating. Design-space was
brexpiprazole with the present UPLC method were provided in established by DoE studies and this multivariate analysis will pro-
Table VIII. vide the broad knowledge on the interaction and effect of input
method parameters on system suitability parameters. Present method
was economic, and developed to speed up the analytical activities
by significantly reducing the analysis time for the pharmaceutical
industries. In addition to the quantification of brexpiprazole in bulk
Conclusions
and finished drug product, this method can be used for estimation of
AQbD-approach-based reversed-phase UPLC method was developed drug release from a tablet in the in vitro dissolution analysis of drug
for chromatographic separation of brexpiprazole from impurities. product as an application. For dissolution its suitability verified for
DoE was the basis for the establishment of design-space for system- 4 mg strength and further scope for applicability to all strengths to
atic selection and optimization of UPLC chromatographic parameters be verified and validated.
 Downloaded from https://academic.oup.com/chromsci/advance-article/doi/10.1093/chromsci/bmaa099/6041171 by guest on 19 December 2020
QbD-Based UPLC Method
Table VIII. Comparison of Selected Analytical Methods for Brexpiprazole

S.No. Column, Elution, Mobile Phase, Flow Rate Sample Linear Range, Run Time (RT of Intended Purpose Ref. No.
Detection Brexpiprazole) or any
specific

1 BEH C18 50 × 2.1 mm, 1.7 µm, gradient, Acetonitrile, 5–10000 pg mL−1 , 3 min (1.99 min) Estimation of drug in (2)
0.1% Formic acid in water, 0.4 mL min−1 UPLC–MS/MS rat plasma
2 Inertsil ODS 3 V 150 × 4.6 mm, 5 µm, Isocratic, 20 mM 0.96–71 µg mL−1 , 15 min (6 min) For assay of bulk (3)
KH2 PO4 pH 6.8: Acetonitrile (50:50 v/v), 1.5 mL min−1 HPLC-UV at 220 nm drug.
3 Phenomenex C18, 250 × 4.6 mm, 5 µm, Isocratic, 0.1% 0.1–250 µg mL−1 , 10 min (2.1 min) For assay of drug in (4)
acetic acid: Methanol (65:35 v/v), 0.9 mL min−1 HPLC-UV at 214 nm tablets.
4 Kromasil C18, 250x4.6 mm, 5 µm, Gradient, a) K2 HPO4 HPLC-UV at 254 nm 65 min (28.9 min) For related substances (5)
buffer pH 5.5, b) Acetonitrile: THF (9:1 v/v), c) Methanol, of API
1.2 mL min−1
5 HPTLC aluminium plate (Silica gel 60 F254 ), n-Butanol 200–1600 ng band−1 , Rf value 0.38 For assay of bulk drug (6)
HPTLC, at 215 nm,
6. BEH C18 50x2.1 mm, 1.7 µm, gradient, 10 mM ammonium 1–1000 ng mL−1 , 4 min (1.95 min) For assay of drug in (7)
aetate, methanol, 0.5 mL min−1 UPLC–MS/MS dog plasma
7 Waters C18 150 × 4.6 mm, 5 µm, isocratic, 10 mM 10 to 60 µg mL−1 5 min (2.5 min) For assay of drug in (14)
KH2 PO4 (pH 2.0): Acetonitrile (50:50 v/v), 1 mL min−1 HPLC-UV at 213 nm bulk
8 UPLC BEH-C18 2.1 × 50 mm, 1.7 µm, Isocratic, 10 mM 20.4 to 61.3 µg mL−1 for 2 min (0.6 min) QbD approach for Present
KH2 PO4 (pH 2.0): Acetonitrile (67:33 v/v), 0.5 mL min−1 assay, 0.88 to assay and its method
6.59 µg mL−1 in vitro application to in vitro
dissolution (for 4 mg dissolution of
tablets) UPLC-UV at brexpiprazole in drug
215 nm product.

17
18 Prasad et al.

Acknowledgements 11. Anjali, M.T., Usmangani, K.C., Nikunj, P., Jaineel, V.D., Dimal, A.S.;
Stability indicating TLC method for quantification of Brexpiprazole in
The authors sincerely thank to the running administration of AET Laborato-
bulk and its pharmaceutical dosage form and determination of content
ries, India for providing the opportunity and facilities to perform the present
uniformity; Journal of Chromatographic Science, (2019), 2019); . doi:
research work.
10.1093/chromsci/bmz039.
12. Tyagi, R., Singh, H., Singh, J., Arora, H., Yelmeli, V., Jain, M. et al.; Identifi-
cation, systhesis, and control of process-related impurities in antipsychotic
Funding disclosures drug substance brexpiprazole; Organic Process Research and Develop-
ment, (2018); 22(11): 1471–1480.

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work and/or preparation of this manuscript. phy method for determination of related substances in brexpiprazole API;
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