Research J. Pharm. and Tech.

18(3): March 2025

ISSN 0974-3618 (Print) [Link]

0974-360X (Online)

RESEARCH ARTICLE

LC–ESI-MS Characterization of Degradents of Brivaracetam;

Development and Validation of a Stability-Indicating RP-HPLC Method
Sanjay D. Sawant1, Shital D. Godse2
Department of Pharmaceutical Chemistry, Sinhgad Technical Education Society’s,
1,2

Smt. Kashibai Navale College of Pharmacy, Pune, Maharashtra, India.

1,2
Department of Pharmaceutical Quality Assurance, Vishwakarma University, Pune-411048, Maharashtra, India.
*Corresponding Author E-mail: drsdsawant69@[Link], [Link]@[Link],
shitalgodse10@[Link]

ABSTRACT:
The planned study demonstrates the validation of the developed simple, precise, and robust stability-indicating
assay method by high-pressure liquid chromatography and the identification and characterization of
Brivaracetam (BRV) degradants by High-Performance Liquid Chromatography-Tandem Mass Spectrometry.
The drug was exposed to a few stressors i.e. acidic, basic, oxidative, thermal, and UV light to study the stability-
indicating behavior of the planned method and observed sufficient degradation in the acidic stressor. All
separations were done on Hi-Q SiliC-18 (I250 x 4.6mm, 5µm particle size) by isocratic elution flow at 1ml/min.
Detection was set at 210nm. All obtained results indicate that the present method is helpful in the quality control
(QC) laboratory to the day-to-day investigation of (BRV) for the assay and degradation products. HPLC method
development and validation have been done per the current ICH guidelines. The m/z value obtained in LC-MS
analysis of the degradant was used to study further fragmentation patterns, structural elucidation of the
degradant, and its pathway.

KEYWORDS: Brivaracetam, LC-MS/MS, Stability indicating Assay method, Degradation, Characterization.

INTRODUCTION: Degradation products directly affect drug products'

Brivaracetam (BRV), chemically called as i(2S)-2- efficacy, quality, and safety. Stability indicating, helps
[(4R)-2-oxo-4-propyl pyrrolidin-1-yl]ibutanamide is in determining the degradants and assessing the stability
used in the treatment of convulsions. (BRV) is available of the drug, which has a huge impact on impurity profile
as a crystalline powder.1-3 It shows a clear solution with studies. Stability studies are also necessary for
buffer (pH 1.2, 4.5, and 7.4), water, acetic acid, and validating the stability indicating the influence of
alcohol. It is an orally administered drug used with or analytical procedures.12-18 Further, to establish a
without other medications to regulate partial-onset degradant pathway, deciding on store condition and
seizures.4-5 Few HPLC techniques have been reported retest period characterization of degradants is a
for the quantity of (BRV) in formulation and powder. 6-8 must.19-21 The International Conference on
UPLC-MS/MS and HPLC-tandem mass spectrometry Harmonization (ICH) guidelines Q1A (R2) mandate the
procedures for quantitation of (BRV) in plasma matrix utilization of a validated stability-indicating assay
have been reported.9-11 method for the stability testing of new drug substances
or products. According to these guidelines, it is
recommended that the drug be subjected to stress
Received on 23.04.2024 Revised on 17.08.2024 conditions such as hydrolysis, oxidation, thermal stress,
Accepted on 05.10.2024 Published on 27.03.2025 and photolysis. This exposure helps to generate
Available online from March 27, 2025 information on the degradation products that may form
Research J. Pharmacy and Technology. 2025;18(3):1218-1223. under these conditions.22-28
DOI: 10.52711/0974-360X.2025.00176
© RJPT All right reserved
In existing work, forced degradation studies mainly
This work is licensed under a Creative Commons focused on characterization and in silico toxicity
Attribution-NonCommercial-ShareAlike 4.0
International License. Creative Commons License. estimation of the degradants of (BRV) in alkaline
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conditions and not on other stress conditions.12 Solution preparation:

Furthermore, none of the studies have established a Preparation of standard solutions:
validated force degradation study for the separation and To prepare 1000µg/ml solution strength,transferred
identification of the potential degradants of (BRV). In accurately weighted (BRV) 10mg in a 10ml volumetric
the present research study, the degradation of (BRV) flask, volum was made by using methanol: water
was under various stress conditions and the drug was (70:30v/v) mixture, the prepared solution was further
investigated under different stress conditions. This diluted to get 40µg/ml in a volumetric flask. The
article focuses solely on the identification and solution was filtered bya a 0.45µm membrane filter for
characterization of the acidic DP structure, which has HPLC and LC-MS analyses.
not been reported so far for (BRV). HPLC-ESI mass
spectroscopy elucidated the chemical structure (Figure Mobile phase preparation:
1). Based on the identified structures, degradation HPLC-grade methanol and double distilled water passed
mechanisms of (BRV) have been proposed. In addition, through 0.45µm filter paper and taken in 70:30 v/v
an effective HPLC method for isolating and proportion.
identifyingDegradation product (DP) in (BRV) has been
developed and implemented for routine clinical and Preparation of sample solution from tablet:
safety studies. Twenty tablets weighing equivalent to100mgwere
transferred into a 100ml volumetric flask. Dissolved in
O the mobile phaseand supernatant collected after 30 mins
sonication. The solution was further diluted to get 40
µg/ml. 20µL solution was introduced six times to the
N system.
H2N
Method Validation:
O Specificity:
Fig. 1: Chemical structure of (BRV) specificity was recognized by injecting the degradation
solution to LC-MS, it displayed an outstanding mass
MATERIALS AND METHODS: purity for (BRV) and DP.
Chemicals:
Methanol HPLC grade was procured from Sigma Linearity:
Aldrich Chemicals Pvt. Ltd. AR grade HCl, NaOH, and Five replicates of five concentrations over of 20-100
H2O2 (30%) were procured from SD Fine Chemicals µg/mL range were used for linearity study. The (BRV)
Pvt. Ltd, (Mumbai, Maharashtra). (BRV) (potency achieved a linear response.
certified 99.99%) was a gift sample from Zenvision
Pharma (Mumbai, Maharashtra). Precision:
For precision, the method was considered at level
Instruments: (repeatability and Inter-day) by analyzing three
The instrument was equipped with a quad pump with concentrations at three replicates of the drug, (BRV)
High-Performance Liquid Chromatography (HPLC) (40, 60, and 80µg/mL), the result was expressed in RSD
(Jasco HPLC PU-2080 Plus System), a multi- %.
wavelength photodiode array detector, and controlled by
ChromNAV software. HPLC column: Hi-Q Sil C-18 Accuracy:
(250mm x 4.6mm, 5µm) (Thermoscientific, Japan) This parameter was assessed employing replicated at
taken for separation and mobile phase (methanol: three different levels, 80% (32iµg/mL), 100%
water,70:30v/v) at 1ml/ min flow. A pH was monitored (40iµg/mL), and 120% (48µg/mL) of concentration by
by pH meter (Equip-tronics, Mumbai, India). weighing addition of the standard solution in (BRV) solution. At
balance (ME 204, Mettler Toledo, India) and each level, the percentage of drug recovery was
Ultrasonicator (UCB40, Spectra Lab, Mumbai, India) considered.
were used in this study. UV wavelength was set at
210nm using a photodiode array (PDA) detector. An Quantification:
Impact II UHR-TOF mass spectrometer in LC-MS/MS LOD and LOQ according to the signal-to-noise ratio and
iswith an ESI. Separation was done on HypersiliGold C- were calculated according to the chromatographic
18 (i4.6mm x 250imm, 5iµm). The system is controlled condition.
by HighStar 3. MS scan mode is a good ESI mode, and
the measurement frequency is 20-1200m/z.

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Robustness: found. The LOD and LOQ were 0.0751 and 0.2276
Method robustness was examined by purposefully µg/mL obtained respectively.
changing experimental parameters. (BRV) (20µg/mL)
solution was analysed at various flow rates (0.9, 1, and Concentration Area
1.1mL/min) and wavelengths (208, 210, and 212 nm). 20 5855
40 10526
60 15509
System Suitability Test:
80 19911
For SST, three replicates of (BRV) solution were 100 25295
studied, and calculated parameters such as peak area,
theoretical plates (NTP), capacity factor resolution, and
peak asymmetry.

Forced Degradation Solution preparation:

Forced Degradation Solution preparation: Forced
degradation studies of (BRV) were done by exposing
acidic, alkali, water, oxidative, thermal, and photolytic
stress conditions to drug solution. In acid & base
condition, each 2ml of 20µg/mL solution of (BRV) was
treated with 2ml of 0.1N HCl, and 0.1N NaOH solution
in 10ml volumetric flasks and kept in a hot air oven for
30 min at 80°C. For oxidation, 30% H2O2 solution and
water for neutral hydrolysis was done by dilution of 2ml
of 20µg/mL of stock solution of (BRV) kept for 3hr at Precision: It was studied in different levels of
RT. A heat degradation study was done by spreading repeatability and intermediate precision. For this study,
powder of (BRV) in a 1mm thickness Petri dish and three (40,60 and100µg/mL) concentrations in six times
activated in the dry oven at 130°C for 4 hr. within a day, and for Intermediate precision the process
was done on three different days. Obtained values of
Characterization of degradation products: precision at different levels are shown in Table 1. The
LC-PDA analysis of thestressed solutions, showed a method shows good precision as the RSD values were
sufficient amount of degradation products and further within a limit of < 1.5.
was followed by LC-MS.
Table no 1. Precision studies
RESULTS AND DISCUSSION: Concentration Repeatability Intermediatei
Evaluation of Method Validation Parameters: (µg/ml) (n=i6) precisioni (n=i3)
40 40. i15±0.040,0.100 40.41±0.025,0.062
Specificity: It was determined by the stress degradation 60 60.20±0.030,0.050 60.47±0.041,0.068
action of (BRV) in different stress conditions. The 80 80.17±0.045,0.057 80.44±0.141,0.176
degradants were well resolved. LC-MS analysis showed
a good massipurityof (BRV) and degradants, It also Accuracy: This parameter was studied by spiking
presented the specificity. The resultant chromatogram is standard solutions at three concentrations of the known
shown in Figure 2 drug. The prepared mixture was injected in triplicate and
accurate recovery of (BRV) was found to be 99.90 -
100.11%. The results of the accuracy experiments are
shown in Table 2.
Table no 2. Accuracy studies
Drug Spiked Total Meas % SD %
conc. conc. amount ured Recove RSD
Taken (μg/ml) conc. ry
(μg/ml)
40 32 72 71.93 99.90
40 40 80 80.31 100.38 0.2 0.24
40 48 88 88.10 100.11 40 03
6
Fig.2. Chromatogram of Brivaracetam (BRV)
Linearity: Robustness:
The linearity parameter was checked by subjecting the This parameter was studied purposefully by changing
known concentration of (BRV) over the range 20- experimental conditions. Two parameters, flow rate, and
100µg/mL. A good linear response was recorded of wavelength were selected. The flow rate was altered
(BRV) and the correlation coefficient (r2) was 0.9992 from 0.9, 1, and 1.1mL/min and the retention time was
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noted. The effect of change in wavelength was analyzed

Table no 3. Robustness studies
to 208, 210 and 212nm. The mobile phase ratio was
Factors Level Retention time % RSD
changed to 65:35, 70:30 and 75:25 v/v and the effect on Flow Rate (ml/min)
Rt was studied. In triplicate, the sample was analyzed. 0.9 -0.1 4.49 0.76
The resultant chromatogram of a variable on one 1 0 4.05 0.79
condition is tested while the other conditions are kept 1.1 + 0.1 3.68 0.63
constant at the optimum levels. In all chromatographic Wavelength (nm)
208 -2 4.04 0.93
conditions retention time and the tailing factor of (BRV) 210 0 4.05 0.96
were revealed in (Table 3). 212 +2 4.04 0.92
Mobileiphase- Methanoli: Water (70: 30iv/v)
System suitability testing parameters: 65:35 v/v -5 4.10 0.24
The SST parameters of the method were calculated and 70:30 v/v 0 4.05 0.24
75:25 v/v +5 4.00 0.23
found in the ideal range for theoretical plates, the
asymmetry factor, capacity factor and the resolution
factor are shown in Table 4.
Table no 4. Results of System Suitability Parameter (SST)
Code Retention time (Rt) Capacity factor (K’) Resolution (Rs) Theoreticaliplate Tailingi factor
(NTP) (Tf)
Degradant 6.208 1.0 1.2 4662 0.897
BCT 4.567 1.4 5.6 6763 0.981

LC-MS characterization of (BRV) and its

degradants:
The mixture of stressed solutions was analyzed on
Liquid chromatography-Tendom mass spectrometry to
characterize the degradant. The resulting chromatogram
is revealed in Fig. 4 and 5.

Mass fragmentation pathway:

(BRV) fragmentation pathway was studied by the results
of the LC-ESI-MS in +ve modes (Fig 5). The line
spectrum of [M-H]+ ion at m/z 235 shows adduct
formation with sodium at m/z 235.44 and potassium at
m/z 251.11 abundant fragmentiions at m/z 198 ( lossiof
CH3), m/z 169 (loss of C2H5) and low abundance ions at
Fig. 3. HPLC chromatogram acid stress degradation productof
127 (CH3NO) Fig 6.
(BRV) tablets (n =i3)

Degradation behavior:
(BRV) shows sufficient degradation in acidic stressors
to 5% to form a degradation product. The chromatogram
of the acid solution is depicted in Fig. 3. (BRV) has
shown stability in all stress conditions such as alkaline,
water, H2O2, and exposure of the BRV powder to light
and dry heating at 130°C.

Table no 5. Assay of marketed formulation.

Tablet Period Taken Found %Labeliclaim %
in (μg/ml) (μg/ml) (±SD) RSD
month
(BRV) 1 40 40.33 100.82± 0.01 0.024
25 mg 2 40 40.21 100.52± 0.01 0.025
3 40 40.22 100.55± 0.02 0.021 Fig. 4. LC-MS spectra of control drug sample of (BRV) in + ESI
mode

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Fig. 5. LC-MSispectra of degradation productsof (BRV) in +ESI mode.

Postulated structures of the degradation products

Fig 6. Mass Fragmentation pathway of (BRV) and structure prediction of acid degradant

CONCLUSION: followed ICH guidelines by testing its system suitability,

An acid degradation product was formed during a specificity, precision, linearity, accuracy, LOD, LOQ,
stability study on (BRV). The product was isolated by andirobustness. At the last specific method was
isocratic elution HPLC and as per the ICH guidelines developed to separate the peaks of the drug from its
method was validated. Time-saving, accurate, precise, degradants with accurate resolution, number of
specific, cost effective and robust method performed. It theoretical plates, and capacity factor of the drug. Thus,
has been successfully used to identify marketed stability studies show the efficiency of the projected
formulations kept for upto three months in various stability indicating RP-HPLC and LC-MS/MS method,
temperature and humidity-accelerated conditions. One which can be accepted in routine analysis of tablets.
degradation product was characterized through HPLC
analyses and the study of mass fragmentation patterns in CONFLICT OF INTEREST:
+ESI modes. The Acid hydrolyte product was proposed Authors don’t have conflicts of interest.
to be 1-propyl pyrrolidine-2-one. The projected method
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