Karumuru Durga Vara Prasad Reddy ve Dr.

Harbeer Singh

38(2) – 2023 47-79 ISSN:1307-3311

Validation of an HPLC Method for Accurate Quantification of

Impurities in Brivaracetam to Ensure Drug Quality and Safety
1
Karumuru Durga Vara Prasad Reddy, 2Dr. Harbeer Singh
1
Research Scholar, School of science , Glocal University, Saharanpur, U.P.
2
Department of Science, Associate Professor, School of science, Glocal University, Saharanpur,
U.P.

Abstract:

Brivaracetam (BRV) represents a significant advancement in the treatment of epilepsy, offering

improved efficacy and safety over traditional antiepileptic drugs (AEDs). Despite these benefits,
the presence of impurities in BRV formulations can impact drug quality and patient safety. This
study presents the development and validation of a high-performance liquid chromatography
(HPLC) method for the accurate quantification of key impurities in BRV. Specifically, the
method targets impurities such as (R)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide and (S)-
2-((S)-2-oxo-4-propylpyrrolidin-1-yl)butanamide. The method's robustness and sensitivity are
validated in accordance with International Council for Harmonization (ICH) guidelines. This
analytical approach ensures the high purity of BRV, contributing to its efficacy and safety profile
and supporting its continued clinical use.

Keywords Brivaracetam, Epilepsy, Antiepileptic drugs (AEDs),High-performance liquid

chromatography (HPLC),Impurity analysis ,Analytical method validation, ICH guidelines.
1. Introduction:
Epilepsy is defined as a condition of the central nervous system (CNS) prone to seizure activity,
affecting 70 million people worldwide [1]. Contemporary antiepileptic drugs (AEDs) are effective
in treating many forms of epilepsy; nonetheless, a third of patients do not respond to treatment
intervention. In addition, side effects from the current AEDs affect about half of the patients
undergoing treatment [2]. Brivaracetam (BRV), an AED that was just licensed, binds to synaptic
vesicle protein 2A (SV2A) with great selectivity and affinity. Though SV2A is widely distributed
throughout the central nervous system, it is believed to have a part in regulating synaptic vesicle
exocytosis and neurotransmitter release. Levetiracetam (LEV) and BRV are comparable
chemically, BRV, on the other hand, demonstrates stronger selectivity, a higher affinity for the

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SV2A binding site, and more selectivity . BRV is more potent and effective in animal models of
epilepsy [3].
Brivaracetam, chemically known as [(S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide], is a
highly potent anticonvulsant employed in the management of partial-onset seizures in epilepsy
patients. Its mechanism of action involves binding to the synaptic vesicle protein 2A (SV2A), a
crucial modulator of neurotransmitter release, which stabilizes neuronal activity and helps in
preventing seizures. Available in various formulations, including tablets, oral solutions, and
injectables, brivaracetam is rapidly absorbed, reaching peak plasma concentrations approximately
one hour after oral administration. Its molecular formula is C11H20N2O2, with a molecular
weight of 212.29 g/mol. The drug undergoes hepatic metabolism primarily through hydrolysis
and hydroxylation, with renal excretion contributing to an elimination half-life of 8-9 hours.

Fig No.1. Structure of Brivaracetam

The chemical synthesis of brivaracetam, however, can introduce isomeric impurities, such as (R)-
2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide and (S)-2-((S)-2-oxo-4-propylpyrrolidin-1-
yl)butanamide, along with related substances like (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-
yl)butanoic acid and (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butane nitrile(4). The analysis of
related substances in brivaracetam formulations by HPLC with specific acceptance criteria for
each impurity. The impurity BRV-SS should not exceed 1.0% area, while BRV-RR and BRV-RS
should each not exceed 0.15% area. These stringent criteria ensure the purity of brivaracetam,
minimizing potential adverse effects and maintaining its efficacy and safety for clinical use. These
impurities, which can result from synthesis or degradation processes, are critical to identify and
quantify due to their potential impact on the drug's efficacy and safety, potentially causing
adverse effects ranging from mild somnolence to severe psychiatric symptoms(5).
To ensure the quality and safety of brivaracetam formulations for clinical use, this study aims to
validate a high-performance liquid chromatography (HPLC) method for the accurate

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quantification of these impurities. Adhering to International Council for Harmonization (ICH)
guidelines, this validation process will ensure that brivaracetam products meet stringent quality
standards, ultimately safeguarding patient health.
2. Materials and Method:
2.1. Materials Used:
The study follows Standard Test Procedure (STP) No. AFPSTP-008, Revision No. 03, utilizing
high-performance liquid chromatography (HPLC) equipped with a UV/PDA detector and
appropriate software. The reagents and solvents used include methanol (HPLC grade,
Spectrochem or equivalent), acetonitrile (HPLC grade, Merck or equivalent), and trifluoroacetic
acid (HPLC grade, Merck or equivalent). The mobile phase is prepared by mixing methanol,
acetonitrile, and trifluoroacetic acid in a ratio of 90:10:0.1 (v/v), filtering through a 0.45 µm
membrane filter, and sonication to degas. The diluent consists of methanol and acetonitrile in a
90:10 (v/v) ratio, which is also used for needle wash. The seal wash solution is composed of water
and acetonitrile in a 90:10 (v/v) ratio. Chromatographic separation is achieved using a Chiralpak
IG column (250 x 4.6 mm, 5 µm, Part No: 87325A). The detector wavelength is set at 210 nm,
with an injection volume of 15.0 µL and a flow rate of 0.50 mL/min. The column temperature is
maintained at 30°C, and the sample temperature is set at 25°C. The total run time for the analysis
is 30 minutes.
2.2. Method:
[Link] Used:
The study utilized various compounds for the analysis of related substances in brivaracetam
formulations. The primary active pharmaceutical ingredient (API), (S)-2-((R)-2-oxo-4-
propylpyrrolidin-1-yl)butanamide (BRV-API), was obtained from TCG Life sciences Pvt. Ltd.,
batch number CR494-15468-78-BRV, with a potency of 99.8% and Additional impurities and
related substances included (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl) butanoic acid (BRV-IMP-
1) from batch CR494-16260-36-BRV-I-ACID-Imp, with a potency of 95.4%, and (S)-2-((R)-2-
oxo-4-propylpyrrolidin-1-yl) butanenitrile (BRV-IMP-2) from batch CR494-15882-61-BRV Im-
3-P, with a potency of 99.5% and Other compounds included (S)-N-((S)-1Amino-1-Oxobutan-2-
yl)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide (BRV-IMP-3), batch CR494-16260-37-
BRV-diam-P, with a potency of 93.6% and (3R)-N-((1S)-1-Amino-1-oxobutan-2-yl)-3-
(Hydroxymethyl)Hexanamide (BRV-I), batch CR494-16471-I-BRV-I, with a potency of 99.0%

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and (3R)-N-((1S)-1-Amino-1-oxobutan-2-yl)-3-(chloromethyl) hexanamide (BRV-II), batch
CR494-16260-57-BRV-II-F, with a potency of 98.4%.Additionally, (R)-2-((S)-2-oxo-4-
propylpyrrolidin-1-yl) butanamide (BRV-RS) from batch CR494-16042-89-BRV-API-RS-Imp,
with a potency of 98.0% and, (R)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl) butanamide (BRV-RR)
from batch CR494-15882-94-BRV-III-RR-P, with a potency of 97.4% and (S)-2-((S)-2-oxo-4-
propylpyrrolidin-1-yl) butanamide (BRV-SS) from batch CR494-15883-79-BRV-III-SS-P, with a
potency of 97.7% were also included.
2.2.2. Sample:
The primary active pharmaceutical ingredient, (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)
butanamide (brivaracetam), used in this study was obtained from TCG Life sciences Pvt. Ltd. The
specific batch number for this compound is CR494-16260-66-BRV, and it was manufactured in
May 2021
Preparation of standard solution:
Weigh about 10 mg of BRV standard and transfer in a 10 mL volumetric flask. Add 8 mL of
diluents and sonicate to dissolve completely and make up the volume up to the mark with diluent
and mix well. [Concentration: BRV: 1.0 mg/mL]
Preparation of Sample solution:
Weigh about 50 mg of BRV sample and transfer in a 50 mL volumetric flask. Add about 40 mL
of diluents and sonicate to dissolve completely and make up the volume up to the mark with
diluents and mix well.
Procedure:
After equilibrating the column, separately inject diluents as blank and standard solution. If the
system suitability criteria pass then inject sample solutions as per the sequence given below.
Table-1: Injection sequence
S. No. Solution details No. of Injections
1. Blank (diluent) solution 1 (at least)
2. Standard solution 1
3. Blank (diluent) solution 1 (at least)
4. Sample solution-1 1
5. Sample solution-2 1

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6. Standard solution bracketing 1

2.2.3EXPERIMENTAL PLAN AND DATA EVALUATION:

The analytical method validation will be executed as per the following plan:
 The experiments may be done as sequential or simultaneous operations.
 Sample sequence of each experiment may be run together or independently with necessary alteration of
sample sequence.
 The system suitability parameters should be monitored throughout the validation study.
 Pre evaluation sequence shall be conducted by injecting blank standard preparation, system suitability
preparation etc. (If applicable).
 Unless otherwise specified in test method, bracketing standard to be injected after every six sample
injections or at the end of the sequence, whichever is earlier.
 There is no restriction for number of blank injection.
[Link]:
Experiment:
Prepare and analyze the blank solution, standard solution and sample solution as per analytical method.
Prepare selectivity solutions and spiked sample solution as below:

Table-2: Selectivity solution

Pipett
Pipett
Dilut e
Weig Diluted e Final % w/w
S. ed to volu Dilute
Sample ht to mL volum conc. w.r.t
No mL me d to
Name taken (Stock- e from (in sample
. (Stoc from mL
(mg) 1) stock- ppm) conc.
k-2) stock-
1
2
1. BRV-IMP-1 5.0 20 3.0 50 5.0 50 1.5 0.15
2. BRV-IMP-2 5.0 20 3.0 50 5.0 50 1.5 0.15
3. BRV-IMP-3 5.0 20 3.0 50 5.0 50 1.5 0.15
4. BRV-I 5.0 20 3.0 50 5.0 50 1.5 0.15

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5. BRV-SS 10.0 10 5.0 50 5.0 50 10.0 1.0
6. BRV-RR 5.0 20 3.0 50 5.0 50 1.5 0.15
7. BRV-RS 5.0 20 3.0 50 5.0 50 1.5 0.15
0.0007
8. BRV-II 5.0 200 2.5 250 1.5 50 7.5#
5
#
Final concentration for BRV-II has been calculated in µg/g unlike other impurities in µg/mL.

Preparation of Spiked sample solution:

Weigh about 50 mg of BRV sample and transfer in a 50 mL volumetric flask. Add 5.0 mL each of
Selectivity stock-2 solution of BRV-IMP-1, BRV-IMP-2, BRV-IMP-3, BRV-I, BRV-SS, BRV-RR,
BRV-RS, and 1.5 mL of selectivity stock-2 of BRV-II into the same volumetric flask and sonicate to
dissolve completely. Make up the volume up to the mark with diluent and mix well [Concentration is
about 1000 ppm of BRV and 1.5 ppm of BRV-IMP-1, BRV-IMP-2, BRV-IMP-3, BRV-I, BRV-RR, BRV-
RS, 10.0 ppm of BRV-SS and 7.5 µg/g(ppm) of BRV-II]
[Link] and LOQ:
Determination of LOD and LOQ:
To prepare the LOD-LOQ solutions, start by weighing about 10 mg each of BRV and BRV-SS
standards in separate 10 mL volumetric flasks. Add about 8 mL of diluent, sonicate to dissolve,
then dilute to the mark with diluents (each about 1000 ppm). For BRV-RR and BRV-RS
standards, weigh about 5 mg each in separate 20 mL volumetric flasks, add about 10 mL of
diluents, sonicate to dissolve, then dilute to the mark with diluents (each about 250 ppm).
Combine 0.5 mL each of BRV and BRV-SS stock solutions with 2.0 mL each of BRV-RR and
BRV-RS stock solutions in a 20 mL volumetric flask, dilute to the mark with diluents (about 25
ppm each). Finally, pipette 1.0 mL of this mixture into a 50 mL volumetric flask, dilute to the
mark with diluents, achieving a final concentration of about 0.5 ppm for BRV, BRV-SS, BRV-
RR, and BRV-RS.

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2.2. LOQ Precision:
Experiment:
Prepare and analyze the blank solution and standard solution as per analytical method.
[Link] Verification:
Experiment:
Prepare and analyze the blank solution and standard solution as per analytical method.
Prepare a solution containing the analytes at LOD Level concentration from above experiment
and analyze LOD level concentration thrice as per the analytical method.
Preparation of LOD Level solution:
Pipette out 3.0 mL of LOQ Level solution into a 10 mL volumetric flask and make the volume up
to the mark with diluents and mix well.

[Link]:
Experiment:
Prepare and analyze the blank solution and standard solution as per analytical method.
Prepare a series of BRV preparations over a range starting from 50 to 150% of nominal sample
concentration (i.e. 1000 ppm); another series of specified impurities concentrations ranging from
LOQ to 200% of specification limit and BRV for single maximum unknown impurity.
Preparation of linearity solution from LOQ to 200% of the specification limit for each
specified impurities and single maximum unknown impurity:

To prepare the linearity stock solutions, begin by weighing about 10 mg each of BRV and BRV-
SS standards in separate 10 mL volumetric flasks. Add about 8 mL of diluent, sonicate to
dissolve, then dilute to the mark with diluents (each about 1000 ppm). For BRV-RR and BRV-RS
standards, weigh about 5 mg each in separate 20 mL volumetric flasks, add about 10 mL of
diluents, sonicate to dissolve, then dilute to the mark with diluents (each about 250 ppm).
Combine 5.0 mL each of BRV and BRV-SS stock solutions with 3.0 mL each of BRV-RR and
BRV-RS stock solutions in a 50 mL volumetric flask, dilute to the mark with diluents, achieving
final concentrations of about 100 ppm each for BRV and BRV-SS, and about 15 ppm each for
BRV-RR and BRV-RS.

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Preparation of linearity solution from 50% to 150% of sample concentration (i.e. 1000 ppm)
of BRV:
Weigh about 50 mg of BRV standard in a 20 mL volumetric flask. Add about 15 mL of diluent
and sonicate to dissolve. Dilute up to the mark with diluents and mix well (Concentration is about
2500 ppm of BRV).

[Link] (% Recovery):
Experiment:
Prepare and analyze the Blank (Diluents) and standard solution as per analytical method.
The accuracy is to be determined by injecting the solutions containing analytes ranging from LOQ to
200% of specified limit concentration of specified impurities of BRV.
To prepare the accuracy stock solutions, weigh about 10 mg each of BRV and BRV-SS standards
in separate 10 mL volumetric flasks, add about 8 mL of diluents, sonicate to dissolve, then dilute
to the mark with diluents (each about 1000 ppm). For BRV-RR and BRV-RS standards, weigh
about 5 mg each in separate 20 mL volumetric flasks, add about 10 mL of diluents, sonicate to
dissolve, then dilute to the mark with diluent (each about 250 ppm). Combine 5.0 mL of BRV-SS
stock solution, 3.0 mL each of BRV-RR and BRV-RS stock solutions in a 50 mL volumetric
flask, dilute to the mark with diluents (about 100 ppm BRV-SS, 15 ppm BRV-RR, and 15 ppm
BRV-RS). For another mixture, combine 0.5 mL of BRV-SS stock solution, 2.0 mL each of
BRV-RR and BRV-RS stock solutions in a 20 mL volumetric flask, dilute to the mark with
diluents (about 25 ppm each of BRV-SS, BRV-RR, and BRV-RS).

Preparation of Accuracy standard solution:

Further pipette out 5.0 mL each of Accuracy stock solution-5and 0.5 mL of Accuracy stock solution-1
transfer into a 50 mL volumetric flask. Make up the volume up to the mark with diluents and mix well
(Concentration is about 10 ppm each of BRV, BRV-SS and 1.5 ppm of each of BRV-RR and BRV-RS).

Preparation of Accuracy samples:

Prepare the sample for accuracy as unspiked sample and spiked at LOQ Level, 100% and 200 % level of
specification limit of known impurities in triplicate.

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Preparation of unspiked sample solution: (3 preparations)

Weigh accurately about 50 mg of BRV sample and transfer in a 50 mL volumetric flask. Add about 40
mL of diluent and sonicate to dissolve completely and make up the volume up to the mark with diluent
and mix well. [Concentration is about 1000 ppm of BRV)
Accuracy – LOQ: (3 preparations)
Weigh accurately about 50 mg of BRV sample and transfer in a 50 mL volumetric flask. Add about 40
mL of diluent and sonicate to dissolve completely. Then add 1.0 mL of Accuracy stock solution-6 and
make up the volume up to the mark with diluents and mix well. [Concentration is about 1000 ppm of
BRV and 0.5 ppm of each of BRV-SS, BRV-RR, BRV-RS]
Accuracy – 100%: (3 preparations)
Weigh accurately about 50 mg of BRV sample and transfer in a 50 mL volumetric flask. Add about 40
mL of diluents and sonicate to dissolve completely. Then add 5.0 mL of Accuracy stock solution-5 and
make up the volume up to the mark with diluents and mix well. [Concentration is about 1000 ppm of
BRV and 10 ppm of BRV-SS and 1.5 ppm each of BRV-RR, BRV-RS].
Accuracy – 200%: (3 preparations)
Weigh accurately about 50 mg of BRV sample and transfer in a 50 mL volumetric flask. Add about 30
mL of diluents and sonicate to dissolve completely. Then add 10.0 mL of Accuracy stock solution-5 and
make up the volume up to the mark with diluents and mix well. [Concentration is about 1000 ppm of
BRV and 20 ppm of BRV-SS and 3 ppm each of BRV-RR, BRV-RS].
[Link]:
[Link] Precision :
Experiment :
Prepare and analyze the blank solution, standard solution and sample solution (6 preparations) as
per the analytical method.
[Link] Precision:
Experiment:
Intermediate precision express within laboratory precision by different analysts, different instruments,
and different columns of same make with different serial numbers by using the same sample and method
as described in method precision on a different day as per the following matrix.

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Prepare and analyze the blank solution, standard solution and sample solution (6 preparations) as per the
analytical method.
[Link]:
Experiment:
The range shall be determined from Linearity, Precision and Accuracy study for related substances .
[Link] of analytical solution:
Experiment:
Prepare blank solution, standard solution and sample solution as per the analytical method.
Analyze the Blank, Sample Solution initially, after 12 hours, after 24 hours and after 48 hours at room
temperature.
[Link]:
Experiment:
Prepare and analyze the blank solution, standard solution and sample solution (duplicate preparations) as
per the analytical method and inject using different chromatographic conditions as shown below:
Change in Flow rate ( 0.1 mL).
Change in detection wavelength (±2 nm).
Change in volume of Trifluoroacetic acid (± 10%) in Mobile phase.
Change in volume of Acetonitrile (± 10%) in Mobile phase.
Change in column oven temperature ( 2°C).

3. Results and Discussion:

1. Specificity:
 No interfering peak was observed at the retention time of BRV and its specified impurity peak in
blank.
 The peak due to BRV and its isomeric impurities were well resolved from each other and also
from other specified impurities and any other peaks (Refer table 3).
 Purity angle found to be less than purity threshold for BRV and its specified impurities (Refer
table 3).

Table 3: Selectivity data (Purity angle and Purity threshold)

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Purity angle and Purity threshold

BRV BRV-SS BRV-RR BRV-RS
Sample name
Purity Purity Purity Purity Purity Purity Purity Purity
angle thresh angle threshol angle threshol angle thresho
Standard1 0.198 old
0.269 NA d
NA NA d
NA NA ld
NA

Unspiked sample NA NA 4.809 6.914 NA NA NA NA

Spiked sample NA NA 0.792 1.259 4.416 6.936 3.761 5.608

Diluted Unspiked
0.175 0.269 NA NA NA NA NA NA
sample
Diluted Spiked
0.187 0.267 NA NA NA NA NA NA
sample
BRV-SS selectivity
NA NA 0.809 1.366 NA NA NA NA
solution
BRV-RR
NA NA NA NA 4.995 6.568 NA NA
selectivity solution
BRV-RS
NA NA NA NA NA NA 3.077 5.765
selectivity solution

Figure 2: Specificity – Diluent (Blank)

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Figure 3: Specificity – Standard solution

Figure 4: Specificity – BRV-IMP-1 selectivity solution

Figure 5: Specificity – BRV-IMP-2 selectivity solution

Figure 6: Specificity – BRV-IMP-3 selectivity solution

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Figure 7: Specificity – BRV-SS selectivity solution

Figure 8: Specificity – BRV-RR selectivity solution

Figure 9: Specificity – BRV-RS selectivity solution

Figure 10: Specificity – Unspiked sample solution

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Figure 11: Specificity – Spiked sample solution

Figure 12: Specificity – Diluted standard solution

Figure 13: Specificity – Diluted unspiked sample solution

Figure 14: Specificity – Diluted Spiked sample solution

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Figure 15: Specificity – Peak purity plot of diluted standard

Figure 16: Specificity – Peak purity plot of BRV-SS selectivity solution

Figure 17: Specificity – Peak purity plot of BRV-RS selectivity solution

Figure 18: Selectivity – Peak purity plot of BRV-RR selectivity solution

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Figure 19: Specificity – Peak purity plot of Unspiked sample

Figure 20: Specificity – Peak purity plot of Spiked sample

Figure 21: Specificity – Peak purity plot of Diluted standard

Figure 22: Specificity – Peak purity plot of Diluted Unspiked sample

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Figure 23: Specificity – Peak purity plot of Diluted Spiked sample

2. LOD and LOQ:

Table 4: LOD-LOQ Determination
Determined LOQ Expected LOD
Conc. (%w.r.t Conc.
Component Area USP S/N
sample (%w.r.t sample
concentration) concentration)
BRV 15531 134 0.05 0.02
BRV-SS 15184 79 0.05 0.02
BRV-RR 14511 100 0.05 0.02
BRV-RS 14601 187 0.05 0.01

2.2. LOQ Precision:

Table 5: Results of LOQ Precision
BRV BRV-SS BRV-RR BRV-RS
LOQ Level US
USP USP USP
injections Area P Area Area Area
S/N S/N S/N
S/N
LOQ Conc. %
0.05 0.05 0.05 0.05
w.r.t. test
LOQ Solution,
15669 133 15294 78 14650 99 14532 187
Inj.-1
LOQ Solution, 15492 134 15345 78 14262 99 14609 183

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Inj.-2
LOQ Solution,
15180 134 15249 76 14552 102 14564 183
Inj.-3
LOQ Solution,
15633 138 15667 78 15576 105 15275 170
Inj.-4
LOQ Solution,
15591 139 15099 76 15185 104 15044 166
Inj.-5
LOQ Solution,
15965 141 15308 77 15316 105 15389 148
Inj.-6
Mean 15588 15327 14923 14902
255.4030 187.2502 382.8799
SD NA NA 509.77143 NA NA
3 0 5
% RSD 1.6 1.2 3.4 2.6

2.3. LOD Verification:

Table 6: Results of LOD verification
BRV BRV-SS BRV-RR BRV-RS
LOD Level
USP USP USP USP
injections Area Area Area Area
S/N S/N S/N S/N
LOD Conc. %
0.02 0.02 0.02 0.01
w.r.t. test
LOD Solution,
4836 41 4614 23 5064 32 4916 50
Inj.-1
LOD Solution,
4786 41 4735 23 4368 29 4642 51
Inj.-2
LOD Solution,
5102 42 4752 24 4385 31 4686 52
Inj.-3

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Figure 24: LOD Verification – 1st injection

3. Linearity:
Table 7A: Linearity data of BRV
(LOQ to 200% of specification limit)

Concentration
Linearity Percent (%) Mean area
(in % w.r.t. sample
Level (w.r.t spec conc.) (n = 2)
concentration)
Level – 1 LOQ 0.051 15780
Level – 2 50 0.507 153076
Level – 3 100 1.013 305438
Level – 4 150 1.520 463218
Level – 5 200 2.027 616120
Correlation coefficient 0.9999
Y-Intercept -710.034
Slope 304254.896
Residual standard deviation 1646.777451
Residual sum of squares 8135627.917

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Figure 25: Linearity graph of BRV (LOQ to 200% of specification limit)

Table 7B: Linearity data of BRV-SS

(LOQ to 200% of specification limit)
Concentration
Linearity Percent (%) Mean area
(in % w.r.t. sample
Level (w.r.t spec conc.) (n = 2)
concentration)
Level – 1 LOQ 0.050 15194
Level – 2 50 0.505 148504
Level – 3 100 1.009 294324
Level – 4 150 1.514 446274
Level – 5 200 2.018 594381
Correlation coefficient 0.9999
Y-Intercept -424.208
Slope 294466.565
Residual standard deviation 1598.303855
Residual sum of squares 7663725.634

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Figure 26: Linearity graph of BRV-SS (LOQ to 200% of specification limit)

Table 7C: Linearity data of BRV-RR
(LOQ to 200% of specification limit)
Concentration
Linearity Percent (%) Mean area
(in % w.r.t. sample
Level (w.r.t spec conc.) (n = 2)
concentration)
Level – 1 LOQ 0.051 14729
Level – 2 50 0.076 21830
Level – 3 100 0.152 43068
Level – 4 150 0.228 65107
Level – 5 200 0.304 87225
Correlation coefficient 0.9999
Y-Intercept 12.017
Slope 285944.986
Residual standard deviation 324.3004016
Residual sum of squares 315512.2514

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Figure 27: Linearity graph of BRV-RR (LOQ to 200% of specification limit)

Table 7D: Linearity data of BRV-RS
(LOQ to 200% of specification limit)
Concentration
Linearity Percent (%) Mean area
(in % w.r.t. sample
Level (w.r.t spec conc.) (n = 2)
concentration)
Level – 1 LOQ 0.050 15280
Level – 2 50 0.074 22308
Level – 3 100 0.149 42849
Level – 4 150 0.223 65128
Level – 5 200 0.298 86767
Correlation coefficient 0.9998
Y-Intercept 685.202
Slope 288054.384
Residual standard deviation 497.920361
Residual sum of squares 743774.0578

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Figure 28: Linearity graph of BRV-RS(LOQ to 200% of specification limit)

Accuracy:
Mean recovery and % RSD for accuracy sample preparations were within the acceptance
criteria (Refer table 8 to 9).
Table 8: % Recovery data of BRV-SS

Theoretical Amount Amount found % Recovery

Level No. Test ID
(% w/w) (% w/w) (Accuracy)

Test-1 0.21232 0.21144 99.585

Recovery level-1
Test-2 0.21294 0.21232 99.708
(LOQ)
Test-3 0.21303 0.21186 99.449
Mean recovery 100
SD 0.129537
% RSD 0.1
Test-1 1.16863 1.15600 98.919
Recovery level-2
Test-2 1.17014 1.15729 98.901
(100%)
Test-3 1.16979 1.15435 98.680
Mean recovery 99
SD 0.133332
% RSD 0.1
Recovery level-3 Test-1 2.17397 2.15075 98.932

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(200%) Test-2 2.17390 2.15928 99.327
Test-3 2.17529 2.15930 99.265
Mean recovery 99
SD 0.212586
% RSD 0.2
Table 9: % Recovery data of Recovery data of BRV-RR

Theoretical Amount Amount found % Recovery

Level No. Test ID
(% w/w) (% w/w) (Accuracy)

Test-1 0.05071 0.04940 97.422

Recovery level-1
Test-2 0.05071 0.04920 97.036
(LOQ)
Test-3 0.05071 0.04890 96.428
Mean recovery 97
SD 0.501090
% RSD 0.5
Test-1 0.15212 0.14602 95.993
Recovery level-2
Test-2 0.15212 0.14666 96.410
(100%)
Test-3 0.15212 0.14481 95.195
Mean recovery 96
SD 0.617390
% RSD 0.6
Test-1 0.30424 0.29587 97.251
Recovery level-3
Test-2 0.30424 0.29791 97.920
(200%)
Test-3 0.30424 0.29717 97.675
Mean recovery 98
SD 0.338780
% RSD 0.3

Method Precision:
%RSD of the content of % area for the isomeric impurities and total impurities results from six
sample preparations were within the acceptance criteria. (Refer table 10A & 11).

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Table 10A: Method precision data for Related substances (Unspiked sample)

Sample % Content (w/w)

preparations BRV-SS BRV-RR BRV-RS Total impurities

Preparation-
0.18035 <RL <RL 0.18035
1
Preparation-
0.18086 <RL <RL 0.18086
2
Preparation-
0.18014 <RL <RL 0.18014
3
Preparation-
0.18214 <RL <RL 0.18214
4
Preparation-
0.18332 <RL <RL 0.18332
5
Preparation-
0.18039 <RL <RL 0.18039
6
Mean 0.18 <RL <RL 0.18
Std.
0.001264 NA NA 0.001264
Deviation
%RSD 0.7 NA NA 0.7

Table 10B: Method precision data for Related substances (Spiked sample)

Sample % Content (w/w)

preparations BRV-SS BRV-RR BRV-RS

Preparation-
1.27338 0.16110 0.16716
1
Preparation-
1.26387 0.16042 0.16575
2
Preparation- 1.26597 0.15907 0.16644

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3
Preparation-
1.27590 0.16130 0.16781
4
Preparation-
1.26179 0.15997 0.16626
5
Preparation-
1.27492 0.16172 0.16788
6
Mean 1.3 0.16 0.17
Std.
0.006144 0.000976 0.000871
Deviation
%RSD 0.5 0.6 0.5

Table 11: % Recovery data of BRV-RS

Theoretical Amount Amount found % Recovery

Level No. Test ID
(% w/w) (% w/w) (Accuracy)

Test-1 0.05030 0.05368 106.714

Recovery level-1
Test-2 0.05030 0.05396 107.268
(LOQ)
Test-3 0.05030 0.05356 106.464
Mean recovery 107
SD 0.411515
% RSD 0.4
Test-1 0.15091 0.14870 98.534
Recovery level-2
Test-2 0.15091 0.14871 98.545
(100%)
Test-3 0.15091 0.14871 98.540
Mean recovery 99
SD 0.005668
% RSD 0.0
Recovery level-3 Test-1 0.30182 0.29792 98.708
(200%) Test-2 0.30182 0.29882 99.004

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Test-3 0.30182 0.29888 99.026
Mean recovery 99
SD 0.177501
% RSD 0.2

Intermediate Precision:
1. %RSD of the content of % area for the isomeric impurities result from six sample solutions
were within the acceptance criteria (Refer table 13A for unspiked sample and 13B for spiked
sample).
2. %RSD of the content of % area for the isomeric impurities result from twelve spiked sample
preparations (six from method precision and six from intermediate precision) were within the
acceptance criteria (Refer table 12A for unspiked sample and 12B for spiked sample).
Table 12A: Intermediate precision data for Related substances (Unspiked sample) [6
preparations]

Sample % Content (w/w)

preparations BRV-SS BRV-RR BRV-RS Total impurities

Preparation-
0.16892 <RL <RL 0.16892
1
Preparation-
0.16360 <RL <RL 0.16360
2
Preparation-
0.14735 <RL <RL 0.14735
3
Preparation-
0.17727 <RL <RL 0.17727
4
Preparation-
0.15583 <RL <RL 0.15583
5
Preparation-
0.17176 <RL <RL 0.17176
6
Mean 0.16 <RL <RL 0.16
Std. 0.010995 NA NA 0.010995

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Deviation
%RSD 6.7 NA NA 6.7
Table 12B: Intermediate precision data for Related substances (Spiked sample) [6
preparations]

Sample % Content (w/w)

preparations BRV-SS BRV-RR BRV-RS

Preparation-
1.26604 0.14215 0.16414
1
Preparation-
1.25801 0.14359 0.16386
2
Preparation-
1.26092 0.15502 0.14809
3
Preparation-
1.27217 0.13244 0.14655
4
Preparation-
1.21617 0.15520 0.12331
5
Preparation-
1.28262 0.14312 0.15883
6
Mean 1.3 0.15 0.15
Std.
0.022888 0.008665 0.015453
Deviation
%RSD 1.8 6.0 10.2

Stability of Analytical Solution:

Table 13: Stability of analytical solution: Standard preparation – at room temperature
Time points USP Tailing USP Theoretical
(in hours) factor plates
Initial 1.4 12503

After 12 hours 1.4 12523

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After 24 hours 1.4 12510

After 48 hours
1.4 12477
(48 Hours and 32 min.)

Robustness:
 System suitability criteria were within the acceptance criteria.
 Overall %RSD with two results of same sample with the standard condition: n=8 (2+6) i.e. two
data from changed condition and six data from method precision was as per the acceptance
criteria (Refer table 14).
Table 14: Robustness [Change in flow rate (0.40 mL/minute)] for Spiked sample
% Content (w/w)
Sample preparations
BRV-SS BRV-RR BRV-RS
Method precision
1.27338 0.16110 0.16716
preparation-1
Method precision
1.26387 0.16042 0.16575
preparation-2
Method precision
1.26597 0.15907 0.16644
preparation-3
Method precision
1.27590 0.16130 0.16781
preparation-4
Method precision
1.26179 0.15997 0.16626
preparation-5
Method precision
1.27492 0.16172 0.16788
preparation-6
Robustness preparation-1 1.26735 0.16445 0.16543
Robustness preparation-2 1.26461 0.15842 0.16041
Mean 1.3 0.16 0.17
Std. Deviation 0.005465 0.001851 0.002389
%RSD 0.4 1.2 1.4

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4. Conclusion:
The validation of the HPLC method for analyzing impurities in brivaracetam formulations
confirms that the method adheres to the highest quality standards. It demonstrated specificity by
effectively distinguishing brivaracetam from its impurities and other substances. The method's
limits of detection (LOD) and quantitation (LOQ) are sufficiently low, ensuring accurate
detection and measurement of impurities. Linearity, accuracy, and precision were all validated,
showing reliable performance across a broad range of concentrations and varying conditions.
Additionally, the method proved robust against changes in chromatographic parameters and
exhibited stability in analytical solutions over a 48-hour period. Overall, this validated HPLC
method ensures precise, accurate, and consistent impurity analysis, supporting the quality and
safety of brivaracetam formulations.

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