DOI 10.

1007/s11094-020-02290-z
Pharmaceutical Chemistry Journal, Vol. 54, No. 8, November, 2020 (Russian Original Vol. 54, No. 8, August, 2020)

DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING HPLC

METHOD FOR THE QUANTIFICATION OF LEVETIRACETAM IN BULK
AND ORAL SOLUTION: APPLICATION TO CHEMICAL KINETICS

Sandeep S. Sonawane,1,* Santosh S. Chhajed,2 Dipika R. Jadhav,1

Nilima A. Thombre,3 and Sanjay J. Kshirsagar3

Original article submitted October 11, 2019.

In the present study, a simple, accurate, precise and specific stability-indicating LC method was developed
and validated for the quantification of levetiracetam (LEV) in bulk and oral solution. LEV and its degradation
products were separated and resolved successfully on C18 column using a methanol:water (30:70 %, v/v)
mixture in the isocratic mode at a flow rate of 1 mL/min. All eluents were detected at 205 nm. In forced degra-
dation experiments, LEV was found to degrade significantly in acid, alkali, wet heat, peroxide mediated oxi-
dation, and photolytic conditions, and found stable under dry heat conditions. Validation experiments showed
acceptable accuracy, precision, and specificity of the developed method. Assay of the oral solution was in
good agreement with the amount of LEV as per the label claim. The method was applied to investigate chemi-
cal kinetics under acid and alkali hydrolysis and the pH rate profile of LEV within a range of pH 2 – 12.
Keywords: levetiracetam; HPLC; Briton – Robinson buffer; validation.

1. INTRODUCTION
The purpose of stability testing is to provide evidence on
how the quality of a drug substance or drug product varies
with time under the influence of a variety of environmental
factors such as temperature, humidity, and light, and to estab-
lish a retest period for the drug substance or a shelf life of the
drug product and recommended storage conditions [1].
Chemical kinetics is concerned with the mechanism by
which a chemical process gets to a final state from its initial
state and the rate in which this reaction proceeds [2]. The re-
action rate is influenced by the concentration of reactants,
products and other chemical species that may be present as
well by other factors such as solvent, pressure, temperature,
pH, radiation energy, presence of a catalyst, etc. [3].
Chemically, levetiracetam (LEV) is (aS)-a-ethyl-2-
oxo-1-pyrrolidine-acetamide (Fig. 1) [4]. It is a second-gen-
eration anti-epileptic drug (AED) which is most commonly
prescribed as adjunct therapy in adult patients with partial
1
Department of Pharmaceutical Analysis, MET’s Institute of Pharmacy,
Bhujbal Knowledge City, Nashik 422 003, India.
2
Department of Pharmaceutical Chemistry, MET’s Institute of Pharmacy,
Bhujbal Knowledge City, Nashik 422 003, India.
3
Department of Pharmaceutics, MET’s Institute of Pharmacy, Bhujbal
Knowledge City, Nashik 422 003, India.
*
e-mail: sandeeps.iop@gmail.com
onset seizures [5]. Various theories have been proposed that
LEV appears to act via binding to synaptic vesical protein 2A
rather than binding to GABA or benzodiazepine receptors
like other AEDs. This synaptic vesical protein is an integral
membrane protein present on synaptic vesicles and some
neuroendocrine cells. This inhibits the release of calcium
from intraneuronal stores, opposing the activity of negative
modulators of GABA and glycin-gated currents and inhibit-
ing excessive synchronized activity between neurons. In ad-
dition, LEV inhibits N-type calcium channels [6, 7].
The literature survey revealed several analytical methods
for the estimation of LEV in tablets [8, 9], stability-indicat-
ing methods for the estimation of LEV in tablets with its deg-
radation products [10 – 14], and estimation of LEV in vari-
ous biological fluids [15 – 17]. In the present work, a simple,
accurate, precise and selective stability-indicating HPLC
method was developed and validated for the quantitation of
LEV in bulk and oral solution. The method was examined for
its specificity and stability-indicating properties by resolving
the drug from its forced degradation products. The developed
method was applied to a detailed study of the chemical kinet-
ics of LEV under acid and alkali hydrolysis conditions. The
reaction rate constant (K) and half-life (t1/2), shelf-life (t90)
were calculated in each case and the Arrhenius plot was ob-
tained to estimate the energy of activation. In addition, the

870
0091-150X/20/5408-0870 © 2020 Springer Science+Business Media, LLC
Development and Validation of Stability-Indicating HPLC Method
pH rate profile was analyzed using the Britton – Robinson
buffer solution in the pH range of 2 – 12.
2. EXPERIMENTAL
2.1. Materials, Chemicals, and Equipment
Pharmaceutical grade LEV was obtained as gratis sample
from Lupin Pharmaceutical Ltd., Palghar, India. HPLC grade
methanol and other chemicals used in the analysis were of
analytical reagent grade. All chemicals were purchased from
S. D. Fine-Chem Ltd., Mumbai, India. Freshly prepared dou-
ble distilled water was obtained from All Glass Double Dis-
tillation Assembly, Borosil, Mumbai, India, and prior to use
it was filtered through Durapore membrane filters
(0.45 mm ´ 47 mm), purchased from Millipore (India) Pvt.
Ltd., Bengaluru, India. Oral solution containing 100 mg/mL
LEV was procured from a local pharmacy store.
The HPLC system consisted of dual pump PU-2080 plus
(JASCO Corporation, Japan) fitted with 20 µL Rheodyne
loop injector (7725i). Detection was carried out on
multi-wavelength UV-2075 detector (JASCO Corporation,
Japan) and the data acquisition was controlled by BORWIN
(Version 1.50) chromatography software. The other equip-
ment used in analyses included weighing balance (AUX 220,
Shimadzu Corporation, Japan), Hot Air Oven (MSI-66,
Meta-Lab Scientific Industries, Mumbai, India), Ultra
Sonicator Bath (PCI analytics Pvt. Ltd., Mumbai, India) and
pH meter (Type: 362, Systronics India Ltd., Ahmedabad, In-
dia).
2.2. Chromatographic Conditions
The chromatographic separation was carried out on
Phenomenex Hyperclone C18 column (250 ´ 4.6 mm, 5 mm)
Fig. 2. Representative chromatograms of LEV: (A) unstressed; (B) acid hydrolysis;(C) alkali hydrolysis; (D) oxidation; (E) photolysis.
871
Fig. 1. Chemical structure of levetiracetam (LEV).
using mobile phase mixture of methanol: water (30: 70 %,
v/v) at a constant flow rate of 1 mL/min. All eluents were de-
tected at 205 nm at ambient temperature and the injection
volume was 20 mL.
2.3. Forced Degradation Studies
Forced degradation experiments were executed at an ini-
tial drug concentration of 1 mg/mL under ICH Q1A (R2)
prescribed conditions [18]. Degradation studies under acid
and alkali stress were performed by heating LEV at 70°C in
0.1 N HCl for 30 min and in 0.1 N NaOH for 20 min, respec-
tively. Degradation under wet heat condition was studied by
heating LEV in doubly distilled water at 70°C for 4 h. Perox-
ide mediated oxidation was performed by exposing LEV to
3% v/v hydrogen peroxide for 24 h at room temperature in
the dark. Photolytic degradation was executed by exposing
LEV spread as a thin film of about 2 mm in quartz Petri plate
to the direct sunlight for 24 h. Likewise, for dry heat condi-
tion, LEV was kept in hot air at 70°C for 4 h. The objective
was to obtain 5 – 20% degradation of LEV.
At the end of exposure, each sample was quenched and
diluted with the mobile phase to obtain a final concentration
of 10 mg/mL and subjected to analyses under optimized chro-
872
Fig. 3. First-order plot of acid hydrolysis of LEV.
matographic conditions. The observation of secondary peaks
and/or reduction in the area of LEV peak was considered as
an indication of degradation. Suitable blanks and controls
were used each time to impede the errors.
2.4. Chemical Kinetics Study
Aliquots equivalent to 10 mg LEV were transferred to
three round-bottom flasks. Then, 10 mL of 0.1 N HCl or 0.1
NaOH was added into each flask and the resulting solutions
were heated at 50, 60C, and 70(C, respectively. The effect of
variable pH on the degradation of LEV was studied using
Britton – Robinson buffer in the pH range of 2 – 12 at 70(C.
The Britton – Robinson buffer was prepared as per the proce-
dure described in [19], where ten solutions with various pH
(pH 2.0, 3.0, 4.0, 5.0, 5.7, 6.8, 8.3, 9.1, 10.8 and 11.9) were
prepared. To maintain the constant ionic strength of 0.1 M in
each buffer solution, sufficient quantity of KCl was added to
each solution. Then, aliquots of 10 mg LEV were taken in
ten round bottom flasks, 10 mL of Britton – Robinson buffer
was added to each sample, and the resulting mixtures were
heated to 70(C.
Sufficient aliquots were removed from each flask at the
specified time intervals, diluted with appropriate volume of
mobile phase, and the resulting 10 mg/mL solutions were
subjected to chromatographic analyses. The percentage of
drug remaining was calculated relative to the initial concen-
tration. The rate constant (K), half-life (t1/2), and shelf-life
(t90) were calculated for each temperature and each pH value.
In addition, the energy of activation (Ea) was determined us-
ing the Arrhenius plots for acid and alkali hydrolysis.
2.5. Calibration Curve and Estimation of LEV
in Pharmaceutical Formulation
From the standard stock solution of 1 mg/mL LEV pre-
pared in methanol, six standard solutions with concentrations
of 5, 10, 15, 20, 25 and 30 mg/mL were prepared in the mo-
bile phase. These CC standards were evaluated in triplicate
Sandeep S. Sonawane et al.
Fig. 4. First-order plot of alkali hydrolysis of LEV.
and the regression coefficient (r2), slope of the straight line
(b) and y-axis intercept (a) were determined.
To estimate the LEV content in oral solution, an aliquot
equivalent to the weight of LEV in oral solution was trans-
ferred to 100 mL volumetric flask and diluted up to the mark
with methanol. From the resulting methanolic solution of
LEV, sufficient aliquot was removed and diluted with the
mobile phase. The final solution was injected into the HPLC
system in triplicate and the corresponding concentration was
determined by extrapolating the obtained AUC on the
straight-line equation (y = bx + a) obtained in calibration
curve measurements.
2.6. Validation Studies
The developed method was validated for accuracy (stan-
dard addition method), precision (intra-day, inter-day and in-
termediate precision) and specificity as per ICH Q2 R1
guidelines [20].
The accuracy and precision of the method were deter-
mined at 80, 100, and 120% for three successive days across
the obtained calibration curve range. The closeness of the %
recovery obtained to the true added values were considered
as an indication of an accuracy and the low % RSD values, as
an indication of the precision. To determine the intermediate
precision, the obtained data were subjected to the analysis of
variance and F(theoretical) was compared with F(observed).
To prove the specificity, the blank LEV solution was
chromatographed before and after subjecting to the afore-
mentioned forced degradation factors. The complete separa-
tion of LEV from its degradation products and the absence of
peaks in the blank runs at the retention times of LEV were
considered the achievement of specificity.
3. RESULTS AND DISCUSSION
3.1. Optimization of Chromatographic Conditions
Different mobile phases were tried to get adequate reten-
tion of LEV and appropriate resolution from its formed deg-
Development and Validation of Stability-Indicating HPLC Method 873

Fig. 5. Arrhenius plot for acid hydrolysis of LEV.

Fig. 6. Arrhenius plot for alkali hydrolysis of LEV.

radation products. It was observed that using Phenomenex

the rate constant (K), half-life (t1/2) and shelf life (t90) were
Hyperclone C18 column (250 ´ 4.6 mm, 5 mm) with a mo- calculated using the following formulas:
bile phase of methanol:water (30:70 %, v/v) mixture, the ac-
ceptable peak asymmetry (1.26), sufficient number of theo- 2.303 C
retical plates (6899), and appropriate resolution of LEV from K = log 0 (1)
t c
its degradation products were obtained in a run time of less
than 10 min in an isocratic mode at a flow rate of 1 ml/min. 0.693
The average retention time of LEV observed was t1 2 = (2)
k
4.38 ± 0.008 min at a wavelength of 205 nm.
0104
.
3.2. Forced Degradation Studies t 90 = (3)
k
In forced degradation experiments, LEV was found to
degrade under acid, alkali, wet heat, peroxide induced oxida- Additionally, the Arrhenius plots for acid and alkali hy-
tion, and photolytic conditions. However, LEV was found drolysis were constructed as the obtained logK values for
stable under dry heat conditions. The degradation behavior acid and alkali hydrolysis against the corresponding recipro-
of LEV under different conditions is summarized in Table 1 cal temperature (1/T). The energy of activation (Ea) was cal-
and corresponding chromatograms are depicted in Fig. 2. culated from the slope of the Arrhenius plot in accordance
with equation
3.3. Chemical Kinetics Studies
From the values of correlation coefficient (r2) obtained log K = log A -
Ea
(4)
for each rate of reaction (Table 2), it is concluded that the 2.303RT
first-order reaction was followed for both acid and alkali hy-
drolysis of LEV, and the first-order plots of time versus The Arrhenius plots of acid and alkali hydrolysis are de-
logCon. (drug concentration remaining upon acid and alkali picted in Figs. 5 and 6, respectively, and the acid and alkali
hydrolysis) are presented in Figs. 3 and 4, respectively. Then, hydrolysis data are presented in Table 3.

TABLE 1. Summary of Data on LEV Degradation under Various Conditions

RT of degradation products
Degradation conditions % Degradation RT of drug (min)
(min)
Acid (0.1 N at 70(C for 30 min) 7.77 4.39 1.99, 2.59
Alkali (0.1 N at 70(C for 20 min) 27.26 4.38 1.70, 2.56
Wet heat (70(C for 4 h) 12.13 4.38 1.69, 2.56
Oxidation (3 % v/v hydrogen peroxide for 24 h in the dark 5.01 4.37 1.78, 2.70
at room temperature) (peroxide blank)
Photolytic degradation (exposure to direct sunlight for 24 h) 20.05 4.39 1.63, 2.64
874 Sandeep S. Sonawane et al.

matographic conditions with regression equation

Fig. 7. pH-Rate profile for LEV.
3.4. Study of pH-Rate Profiles
The first-order degradation rate constant (K), t1/2, and t90
values calculated at each pH are presented in Table 4 and the
pH-rate profile curve of logK versus pH is presented in
Fig. 7. It was found that the LEV oral solution was stable in a
pH range of 4 – 5 and susceptible to degradation at alkaline
pH more than at acidic pH.
3.5. Calibration Curve and Estimation of LEV
in Pharmaceutical Formulation
From the calibration experiments, LEV was found linear
in a range of 5 – 30 µg/mL under the aforementioned chro-
y = 32096.43x + 32154.07 (r2 = 0.9904). When the assay of
oral solution was performed, LEV content was found to be
99.37 % of the label claim.
3.6. Validation Studies
The results of accuracy and precision studies are summa-
rized in Table 5. The developed method was accurate and
precise, as the amount found was close to the amount added
and the low values of % RSD at each level for each day, re-
spectively. To prove the intermediate precision of the
method, one-way ANOVA was performed on the data ob-
tained from intra-day and inter-day precision. The obtained F
values at each level were lower than the theoretical F value,
which evidences the intermediate precision of the method in
the tested calibration curve range.
The method was found specific as there was no interfer-
ence of peaks at the retention time of LEV as well as com-
plete separation of LEV from its degradation products.
4. SUMMARY AND CONCLUSION
In the present work, a simple, accurate, precise and spe-
cific stability-indicating HPLC method was developed for
the estimation of LEV in oral solution. In forced degradation
experiments, LEV was found to degrade under acid, alkali,
wet heat, peroxide mediated oxidation and to photolytic con-
ditions and found stable to dry heat conditions. LEV and its
formed degradation products were well separated and re-
solved on C18 column using methanol: water (30:70 %, v/v)

2
TABLE 2. Correlation Coefficients (r ) for Different Orders of Reaction
Conditions Temperature ((C) r2 (zero-order reaction) r2 (first-order reaction) r2 (second-order reaction)
Acid hydrolysis 50 0.8915 0.9456 0.9295
60 0.9302 0.9549 0.9089
70 0.9334 0.9594 0.9373
Alkali hydrolysis 50 0.8865 0.9662 0.9407
60 0.9200 0.9743 0.9609
70 0.9449 0.9935 0.9381

TABLE 3. Acid and Alkali Hydrolysis behavior of LEV

Conditions Temperature ((C) Rate constant (K) t1/2 t90 Ea (kJ/mol)
Acid hydrolysis 50 0.04499 15.40 2.31 17.19
60 0.05029 13.78 2.06
70 0.06475 10.70 1.60
Alkali hydrolysis 50 0.05780 11.98 1.79 12.71
60 0.06185 11.20 1.68
70 0.07628 9.08 1.36
Development and Validation of Stability-Indicating HPLC Method 875

as mobile phase in an isocratic mode at flow rate of TABLE 4. Summary of pH Rate Profile curves for LEV in
1 ml/min. All eluents were detected at 205 nm. When LEV Britton – Robinson Buffer
was subjected to acid and alkali hydrolysis in the tempera- pH K Log K t1/2 t90
ture range of 50 – 70°C, showed first order reaction rate for
2.0 0.03013 -1.5210 23 3.45
both acid and alkali hydrolysis, respectively. From the
Arrhenius plot, the energy of activation for acid and alkali 3.0 0.04202 -1.3765 16.49 2.47
hydrolysis obtained was 17.19 KJ/mol and 12.71 KJ/mol, re- 4.0 0.03312 -1.4799 20.87 3.14
spectively. The pH-rate profile study conducted for LEV us- 5.0 0.03326 -1.4780 20.83 3.12
ing the Britton – Robinson buffer in a pH range of 2 – 12 in- 5.7 0.04966 -1.3039 13.95 2.09
dicated that the drug was stable in a pH range of 4 – 5 and 6.8 0.05014 -1.2998 13.82 2.07
susceptible to degrade at alkaline pH conditions more than at
8.3 0.05306 -1.2847 13.85 2.0
acidic conditions. Validation experiments proved the accu-
9.1 0.05306 -1.2752 13.06 1.96
racy, precision and specificity of the developed method
within the calibration curve range, and the method was found 10.8 0.05650 -1.2479 12.26 1.84
suitable for the estimation of LEV in oral solution. 11.9 0.06523 -1.1855 10.65 1.59

ACKNOWLEDGEMENTS
The authors are grateful to the management and trustees
of Mumbai Educational Trust for providing necessary analyt-
ical facilities to conduct this work and to Lupin Pharmaceuti-
cals Ltd., Palghar, India, for providing gift sample of LEV.
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TABLE 5. Accuracy and Precision of LEV Determination

Amount added Amount found (mg/mL) Within mean Between mean

F value
(mg/mL) Day 1 Day 2 Day 3 square (WMS) square (BMS)

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17.82 17.45 17.95
17.90 18.28 18.03
0.0648 0.0128 0.1970
Mean 17.94 17.87 18.00
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% RSD 0.77 2.32 0.26
20 19.82 19.48 19.95
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19.98 19.90 20.02
0.0558 0.0511 0.9164
Mean 19.95 19.69 19.80
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% RSD 0.62 1.08 1.64
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Mean 21.98 21.99 22.00
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