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CHAPTER 13
LEVETIRACETAM
332
13.1. DRUG PROFILE
Levetiracetam is an anticonvulsant (antiepileptic) drug used to treat epilepsy.[1]
The FDA approved Levetiracetam in November 1999. Levetiracetam is used in
combination with other antiseizure like gabapentin medications to treat neuropathic pain,
myoclonic, partial onset, or tonic clonic seizures [2]
in children and adults. It is also used
in veterinary medicine for similar purposes. Levetiracetam has potential benefits for other
psychiatric and neurologic conditions such as Tourette syndrome, autism, bipolar
disorder and anxiety disorder, but its most serious adverse effects are behavioral and its
benefit-risk ratio in these conditions is not well understood.[3]
+
Figure 13.A: Structure of Levetiracetam
Systematic (IUPAC) name : (S)-2-(2-oxopyrrolidin-1-yl) butanamide
Formula : C8H14N2O2
Mol. Mass : 170.209 g/mol
Routes : Oral, intravenous
Excretion : Urinary
Levetiracetam is the S-enantiomer of etiracetam, structurally similar to the
prototypical nootropic drug piracetam. Levetiracetam available as a solution (liquid) and
a tablet to take by mouth. Its mechanism of action is unknown, but it inhibits the spread
of seizure activity in the brain. However, the drug binds to a synaptic vesicle protein,
http://en.wikipedia.org/wiki/Anticonvulsanthttp://en.wikipedia.org/wiki/Epilepsyhttp://en.wikipedia.org/wiki/Levetiracetam#cite_note-pmid18830435-0http://en.wikipedia.org/wiki/Gabapentinhttp://en.wikipedia.org/wiki/Neuropathyhttp://en.wikipedia.org/wiki/Levetiracetam#cite_note-1http://en.wikipedia.org/wiki/Tourette_syndromehttp://en.wikipedia.org/wiki/Autismhttp://en.wikipedia.org/wiki/Bipolar_disorderhttp://en.wikipedia.org/wiki/Bipolar_disorderhttp://en.wikipedia.org/wiki/Bipolar_disorderhttp://en.wikipedia.org/wiki/Anxiety_disorderhttp://en.wikipedia.org/wiki/Levetiracetam#cite_note-2http://en.wikipedia.org/wiki/International_Union_of_Pure_and_Applied_Chemistry_nomenclaturehttp://en.wikipedia.org/wiki/Chemical_formulahttp://en.wikipedia.org/wiki/Carbonhttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Nitrogenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Route_of_administrationhttp://en.wikipedia.org/wiki/Excretionhttp://en.wikipedia.org/wiki/Enantiomerhttp://en.wikipedia.org/wiki/Etiracetamhttp://en.wikipedia.org/wiki/Nootropichttp://en.wikipedia.org/wiki/Piracetamhttp://en.wikipedia.org/wiki/File:Levetiracetam.svg
333
SV2A, [4]
which is believed to impede nerve conduction across synapses.[5]
In studies,
addition of Levetiracetam to other antiseizure drugs reduced the frequency of seizures
more than placebo.
Common side effects associated with Levetiracetam include headache,
sleepiness, weakness, dizziness, and infection. Difficulty walking or moving, hostility,
irritability, mood swings, anxiety, hallucinations, and delusions also have been associated
with Levetiracetam. Some serious side effects can be depression, hallucinating (hearing
voices or seeing visions that do not exist), suicidal thoughts, seizures that are worse or
different, fever, sore throat, and other signs of infection, double vision, itching, rash,
swelling of the face.
List of brand names of Levetiracetam
S.No.
BRAND NAME
FORMULATION
AVAILABLE
STRENGTH
mg
MANUFACTURER
1 Evetiracetam Tab 250 UCB Pharma
2 Tab 500
3 Tab 750
4 Tab 1000
5 Keppra Tab 250
6 Tab 500
7 Tab 750
8 Tab 1000
9 Keppra XR Oral solution 100mg/ml
10 Injection solution 100mg/ml
Table 13.1
http://en.wikipedia.org/wiki/SV2Ahttp://en.wikipedia.org/wiki/Levetiracetam#cite_note-Lynch-3http://en.wikipedia.org/wiki/Levetiracetam#cite_note-Rogawski-4http://www.medicinenet.com/script/main/art.asp?articlekey=472http://www.medicinenet.com/script/main/art.asp?articlekey=20628http://www.medicinenet.com/script/main/art.asp?articlekey=64119http://www.medicinenet.com/script/main/art.asp?articlekey=42985http://en.wikipedia.org/wiki/Depression_%28mood%29
334
13.2. LITREATURE SURVEY
Several analytical methods have been reported for the determination of
Levetiracetam in pure drug, pharmaceutical dosage forms and in biological samples using
spcetrophotometry liquid chromatography, electro kinetic chromatography high
performance thin layer chromatography either in single or in combined forms.
J Valarmathy et al [6]
described a simple, reproducible and efficient reverse
phase high performance liquid chromatography (RP-HPLC) method for estimation of
Levetiracetam in its tablet dosage form. Separation was done by using mobile phase
consists of buffer solution (pH 2.8) and acetonitrile in the ratio of 90:10. Chromatography
separations were carried out on prontosil C18 column (150X4.6mn; 5µm) at a flow rate of
1.2 ml/min and UV detection at 215nm and the retention time for Levetiracetam is
4minutes. The linear dynamic response was found to be in the concentration of 45µg-
270µg/ml. The slope, intercept and correlation coefficient were found to be 0.9999
respectively. The percentage recovery of Levetiracetam was found to be 99.08%.
Proposed methods were found to be simple, accurate, precise and rapid and could be used
for routine analysis. This condition is applied only for tablet dosage form. The statistical
parameters and recovery studies were carried out and reported.
A.Lakshmana rao et al [7]
developed a rapid and sensitive high performance
liquid chromatographic method for the estimation of Levetiracetam in bulk and
pharmaceutical formulations. Levetiracetam was chromatographed on a reverse phase
C18 column in a mobile phase consisting of 0.05 M KH buffer (pH 3.0 adjusted with
orthophosphoric acid) and methanol in the ratio 70:30 v/v. The mobile phase was
pumped at a flow rate of 1.2 mL/min. with detection at 210 nm. The detector response
was linear in the concentration of 20-120 µg/mL. The limit of detection and limit of
quantification was found to be 0.0104 and 0.0317 µg/mL, respectively. The intra and
inter day variation was found to be less than 1%. The mean recovery of the drug from the
solution containing 100 µg/mL was 100.038 µg/mL. The proposed method is simple, fast,
accurate, precise and reproducible hence can be applied for routine quality control
analysis of Levetiracetam in bulk and pharmaceutical formulations.
N.Appala raju et al [8]
has been developed a simple, precise, rapid and accurate
reverse phase HPLC method for the estimation of Levetiracetam in tablet dosage form. A
335
Sun Fire C18, 250 x 4.6 mm, 5 µm partical size, with mobile phase consisting of
acetonitrile and 0.03 M potassium dihydrogen phosphate (pH adjusted to 3.0 with
orthophosphoric acid) in the ratio of 15:85 v/v was used. The flow rate was 1 mL/min
and the effluents were monitored at 210 nm. The retention time was 5.53 min. The
detector response was linear in the concentration of 20-240 µg/mL. The respective linear
regression equation being Y= 22119.684 x 6829.3428. The limit of detection and limit of
quantification was 0.16 and 0.5 µg/mL respectively. The percentage assay of
Levetiracetam was 99.87%. The method was validated by determining its accuracy,
precision and system suitability. The results of the study showed that the proposed RP-
HPLC method is simple, rapid, precise and accurate, which is useful for the routine
determination of Levetiracetam in bulk drug and in its pharmaceutical dosage form.
Jens Martens-Lobenhoffer et al [9]
has developed a method for the routine
quantification of the novel antiepileptic drug Levetiracetam in human serum by HPLC–
UV. The procedure is very easy, quick, inexpensive and rugged. The sample preparation
consists only in the precipitation of serum proteins by perchloric acid and extraction of
unpolar components by cyclohexane. The aqueous phase containing the analyte
Levetiracetam is injected onto a porous graphitic carbon analytical HPLC-column and
separated by gradient elution with diluted phosphoric acid/acetonitrile. Detection is
carried out at a wavelength of 205 nm. The calibration function is linear in the range of
1–75 _g/ml. The detection limit is 0.1 _g/ml. using four quality control sample
concentrations, the inter-day relative standard deviations (R.S.D.) are lower than 3% and
the accuracies are better than 6%. The respective inter-day values are: R.S.D. < 4% and
accuracies better than 2%. Frequently co-administered antiepileptic drugs do not interfere
with the assay. The method has been successfully applied to patient samples.
Manuela Contin et al [10]
has been described a simple, fast and validated method
for the determination of the new generation antiepileptic drug (AED) Levetiracetam
(LEV) in plasma of patients with epilepsy using high performance liquid chromatography
(HPLC) with UV detection. Plasma sample (500 μL) pretreatment was based on simple
deproteinization by methanol spiked with the internal standard (I.S.). HPLC analysis was
carried out on a Synergi 4-μm Hydro-RP, 150 mm × 4 mm I.D. column. The mobile
phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5) and
336
acetonitrile (94:6, v/v) at an isocratic flow rate of 1.5 mL/min. The UV detector was set
at 205 nm. Calibration curves were linear (mean correlation coefficient = 0.999) over a
range of 4–80 μg/mL. The quantization limit was 2 μg/mL and the absolute recovery was
>90% for LEV and the I.S. Both intra and interassay precision and accuracy were lower
than 7.5%. The chromatographic run lasted 13 min. The present procedure omitting
expensive solid phase or time-consuming liquid–liquid extraction and drying steps is
cheaper, faster and simpler than mostly published analytical methods for Levetiracetam.
Applied to a large population of patients with epilepsy this assay proved very practical in
our therapeutic drug monitoring setting (TDM).
G. Saravanan et al [11]
has developed a LC Method for the Determination of the
Stability of Levetiracetam Drug Substance under Stressing Conditions. Levetiracetam is
used in combination with other medications to treat certain types of seizures in people
with epilepsy. Levetiracetam is in a class of medications called anticonvulsants and it
works by decreasing abnormal excitement in the brain. This chromatographic separation
was achieved on a YMC pack ODS AQ, 250 mm × 4.6 mm, 5 μm column using diluted
phosphoric acid and acetonitrile in the ratio 85:15 v/v. Forced degradation studies were
performed on the Levetiracetam drug substance. The drug substance was degraded to
Imp-B during acid and base hydrolysis. When the stress samples were assayed, the mass
balance was matching. The sample solution and mobile phase was found to be stable up
to 48 h at 25 °C. The developed method was validated with respect to linearity, accuracy,
precision and robustness.
Z. K. Shihabi et al
[12] were developed a simple and rapid method for
determination of the new antiepileptic drug keppra (Levetiracetam) by capillary
electrophoresis in borate buffer containing sodium dodecyl sulfate. The serum was
injected without any treatment. The method compared well to high performance liquid
chromatography. The mean of keppra in the serum of 35 patients was 25 mg/l (range 7–
77 mg/l).
Can NO et al [13]
emphasized the Developed and validated a RP-HPLC method
for determination of Levetiracetam in pharmaceutical tablets. The separation and
quantification of Levetiracetam and caffeine (internal standard) were performed using a
single analytical procedure with two different types of stationary phases, conventional
http://www.springerlink.com/content/?Author=G.+Saravananhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Can%20NO%22%5BAuthor%5D
337
Phenomenex Gemini C18 (100 x 4.6 mm, 5 micron) and Merck Chromolith Performance
RP18e (100 x 4.6 mm, macropore size 2 mm, micropore size 13 nm) monolithic silica.
Five-micro liter aliquots of samples were injected into the system and eluted using water-
acetonitrile (90 + 10, v/v) mobile phase pumped at the rate of 1 mL/min. The analyte
peaks were detected at 200 nm using a diode array detector with adequate resolution.
Validation studies were performed using the method recommended by the International
Conference on Harmonization, the U.S. Pharmacopeia, and AOAC INTERNATIONAL,
which includes accuracy, precision, range, limits, robustness, and system suitability
parameters. Levetiracetam and caffeine were detected in about 7 min using the
conventional column, whereas less than 5 min was required when the monolithic column
was used. Calibration plots had r values close to unity in the range of 0.8-8.0 microg/mL.
Assay of Levetiracetam in a tablet formulation was demonstrated as an application to real
samples.
Prafulla Kumar Sahu et al [14]
has developed and validated a RP-HPLC
analytical method for estimation of Levetiracetam in pharmaceutical dosage forms. A
Hypersil ODS C18, 4.6mm, 250 mm, 5m column from Supelco (India), with mobile phase
comprised of methanol and ammonium acetate buffer (pH-4) (80:20) with a total run time
of 10 min was used and the wavelength of the detector was set at 240 nm. Ritonavir is
used as Internal Standard. The retention times were 6.20 min and 5.24 min for
Levetiracetam and I.S respectively. The extraction recovery of Levetiracetam from
pharmaceutical dosage form (tablets) was >99.8% and the calibration curve was linear (r2
= 0.999) over Levetiracetam concentrations ranging from 5 to 350g/ml. The method had
an accuracy of >99% and LOD and LOQ of 0.438g/ml and 1.462g/ml respectively. The
method reported is simple, reliable, precise, and accurate and has the capability of being
used for determination of Levetiracetam in bulk and pharmaceutical dosage forms.
Reineks et al [15]
has described the Performance Characteristics of a New
Levetiracetam Immunoassay and Method Comparison with a High-Performance Liquid
Chromatography. The immunoassay was established on the Siemens ADVIA 1200
automated chemistry analyzer. The intraday precision was assessed by 10 replicates of
two levels of quality control materials in a batch, whereas interday precision was
338
estimated by assaying the same materials one set per day for 20 days. Linearity was
evaluated by serially diluting the highest calibrator and a high patient specimen run in
triplicate, whereas the lower limit of quantification was confirmed by 10 measurements
of a low-level specimen diluted from a calibrator and another from a diluted patient
specimen. This method was compared with a commercial high-performance liquid
chromatography method (Chromsystems) using 63 specimens from patients who were on
Levetiracetam therapy. The assay cycle was 10 minutes with a theoretical throughput of
800 per hour. The intra- (n = 10) and interday (n = 20) coefficients of variation were
8.1% or less for the two levels tested. The manufacturer-claimed analytical measurable
range (2.0-100.0 μg/mL) was confirmed by serial dilution and lower limit of
quantification experiments. Among the 63 patient samples studies, four showed
Levetiracetam levels below 2.0 μg/mL by both methods. Deming regression using the
remaining 59 paired patient results by ARK immunoassay and the high-performance
liquid chromatography method showed a correlation coefficient of 0.9962, a linear
regression slope of 0.98, and an intercept of 0.61 with a mean bias of 0.04%.
T. A. C. Vermeij et al [16]
described a method for monitoring drug levels of the
new potential anticonvulsant drug Levetiracetam (ucb L059) in human seru. Two assay
methods were developed and compared. A solid-phase extraction procedure was followed
by either reversed-phase HPLC separation or UV-detection or GLC separation using cold
on-column injection on a megabore column and nitrogen-phosphorous detection.
Absolute recovery of the drug exceeded 97%. Precision and accuracy values for the 16.0
μg/ml quality control sample were 2.4% and 101 ± 5% (n = 10), respectively, for the
GLC method. Precision and accuracy values for the 12.1 μg/ml quality control sample
were 1.0% and 100 ± 1% (n = 7), respectively, for the HPLC method. Agreement
between both methods was excellent (r = 0.993). Both methods are suitable for
pharmacokinetic studies and therapeutic drug monitoring as well. Serum level data for
Levetiracetam in a patient on chronic antiepileptic medication are presented.
339
13.3. EXPERIMENTAL
13.3.1. Instrumentation
Peak HPLC containing LC 20AT pump and variable wavelength programmable
UV-Visible detector and Rheodyne injector was employed for investigation. The
chromatographic analysis was performed on a Chromosil C18 column (250 mm × 4.6
mm, 5µm). Degassing of the mobile phase was done using a Loba ultrasonic bath
sonicator. A Denwar Analytical balance was used for weighing the materials.
13.3.2. Chemicals and Solvents
The reference sample of Levetiracetam (API) was obtained from UCB Pharma.
The Formulation KEPPRA (Levetiracetam l) was purchased from the local market.
Methanols, Acetonitrile, Water used were of HPLC grade and purchased from Merck
Specialties Private Limited, Mumbai, India. And orthophosphoric acid is AR Grade
purchased from Local market.
13.3.3. The mobile phase
A mixture of Methanol: Acetontrile in the ratio of 80:20 v/v was prepared and
used as mobile phase. PH was Adjust to 3.68 with 1% Orthophosphoric acid and then
filtered through Ultipor N66 Nylon 6, 6 membrane sample filter paper.
13.3.4. Standard solution of the drug
For analysis 100ppm standard solution was prepared, required concentrations
were obtained from 100ppm solution by appropriate dilution.
13.3.5. Sample (Tablet) solution
The formulation tablets of Levetiracetam (KEPPRA-250mg) were crushed to give
finely powdered material. From the Powder prepared a 12ppm solution with mobile
phase and then filtered through Ultipor N66 Nylon 6, 6 membrane sample filter paper.
13.3.6. The buffer solution
About 1.0 mL of orthophosphoric acid was diluted to 1000 ml with water makes
the 1% orthophosphoric acid solution. 1ml of this solution was diluted with 99ml water to
prepare 0.1% orthophosphoric acid and filtered through 0.45μ nylon filter.
340
13.4. METHOD DEVELOPMENT
For developing the method (as described in chapter 1 and 2) a systematic study
of the effect of various factors was undertaken by varying one parameter at a time and
keeping all other conditions constant. Method development consists of selecting the
appropriate wave length and choice of stationary and mobile phases. The following
studies were conducted for this purpose.
13.4.1. Detection wavelength
The spectrum of 10ppm solution of the Levetiracetam in methanol was recorded
separately on UV spectrophotometer. The peak of maximum absorbance wavelength was
observed. The spectra of Levetiracetam were showed maximum absorbance at 208nm.
13.4.2. Choice of stationary phase
Preliminary development trials have performed with octadecyl columns with
different types, configurations and from different manufacturers. Finally the expected
separation and peak shapes were obtained on Chromosil C18 (250 mm x 4.6 mm, 5μm)
column.
13.4.3. Selection of the mobile phase
In order to get sharp peak, low tailing factor and base line separation of the
separation of the components, a number of experiments were carried out by varying the
composition of various solvents and flow rate. To have an ideal separation of the drug
under isocratic conditions, mixtures of solvents like methanol, water and Acetonitrile
with or without different buffers indifferent combinations were tested as mobile phases
on a Chromosil C18 column. A mixture of Methanol : Acetonitrile in the ratio of 80:20
v/v with PH adjusted with 0.1% OrthoPhosphoric acid 3.5 was proved to be the most
suitable of all the combinations since the chromatographic peak obtained was better
defined and resolved and almost free from tailing.
13.4.4. Flow rate
Flow rates of the mobile phase were changed from 0.5 – 1.5 mL/min for optimum
separation. A minimum flow rate as well as minimum run time gives the maximum
saving on the usage of solvents. It was found from the experiments that 1.0 mL/min flow
rate was ideal for the successful elution of the analyte.
341
13.4.5. Optimized chromatographic conditions
Chromatographic conditions as optimized above were shown in Table.13.2 these
optimized conditions were followed for the determination of Levetiracetam in bulk
samples and in its Formulations. The chromatogram of standard (50ppm) shown in
Figure 13.B.
Mobile phase Methanol :Acetonitrile 80:20 v/v
Pump mode Isocratic
Mobile phase PH 3.58
Diluent Mobile phase
Column chromosil C18 column (250 mm x 4.6 mm,
5μ)
Column Temp Ambient
Wavelength 208 nm
Injection Volume 20 μl
Flow rate 1.0 mL/min
Run time 5min
Retention Time 2.58 min
Table 13.2: Optimized chromatographic conditions
342
Figure 13.B: Chromatogram of standard solution
343
13.5. VALIDATION OF THE PROPOSED METHOD
The proposed method was validated (as described in chapter 1 and 2) as per ICH
guidelines. The parameters studied for validation were specificity, linearity, precision,
accuracy, robustness, system suitability, limit of detection, limit of quantification, and
solution stability.
13.5.1. Specificity
The specificity of method was performed by comparing the chromatograms of
blank, standard and sample (Prepared from Formulation). It was found that there is no
interference due to excipients in the tablet formulation and also found good correlation
between the retention times of standard and sample. The specificity results are shown in
Table 13.3.
Name of the solution Retention Time in Minutes
Blank No peaks
Levetiracetam (Standard) 2.58
Levetiracetam (Sample) 2.47
Table 13.3: Specificity study
13.5.2 Linearity
Linearity was performed by preparing mixed standard solutions of Levetiracetam
at different concentration levels including working concentration mentioned in
experimental condition i.e. 12ppm. Twenty micro liters of each concentration was
injected in duplicate into the HPLC system. The response was read at 208 nm and the
corresponding chromatograms were recorded. From these chromatograms, the mean peak
areas were calculated and linearity plots of concentration over the mean peak areas were
constructed individually. The regressions of the plots were computed by least square
regression method. Linearity results were presented in Table 13.4.
344
Level Concentration of
Levetiracetam In ppm
Mean peak area
Level -1 2 71593.2
Level -2 4 134100.1
Level -3 6 186327.8
Level -4 8 255167.1
Level -5 10 326742.1
Level-6 12 375845.2
Range: 2-12ppm
Slope
Intercept
Correlation coefficient
30971.79
8160
0.998
Table 13.4: Linearity Results
Figure 13.C: On X axis concentration of sample, On Y axis peak area response
0
50000
100000
150000
200000
250000
300000
350000
400000
0 2 4 6 8 10 12 14
345
13.5.3. Precision
Precision is the degree of repeatability of an analytical method under normal
Operational conditions. Precision of the method was performed as intraday precision,
Inter day precision.
13.5.3.1. Intraday precision
To study the intraday precision, six replicate standard solutions (10ppm) of
Levetiracetam were injected. The percent relative standard deviation (% RSD) was
calculated and it was found to be 0.368, which are well within the acceptable criteria of
not more than 2.0. Results of system precision studies are shown in Table 13.5.
SAMPLE CONC
(PPM)
INJECTION
No.
PEAKS
AREA
R.S.D
(Acceptance
criteria≤2.0%)
Levetiracetam
10
1 325841.3
0.368
2 326047.3
3 325423.9
4 325117.3
5 326856.9
6 328416.2
Table 13.5: System Precision (Intra Day)
13.5.3.2. Inter Day precision
To study the interday precision, six replicate standard solutions (10ppm) of
Levetiracetam was injected on third day of sample preparation. The percent relative
standard deviation (% RSD) was calculated and it was found to be 0.259, which are well
within the acceptable criteria of not more than 2.0. Results of system precision studies are
shown in Table 13.6.
346
SAMPLE
CONC (PPM) INJECTION No. PEAKS AREA R.S.D
(Acceptance
criteria ≤ 2.0%)
Levetiracetam
10
1 327482.3
0.259 2 327841.6
3 328631.6
4 329108.4
5 328246.1
6 329810.6
Table 13.6: System Precision (Inter Day)
13.5.4. Accuracy
The accuracy of the method was determined by standard addition method. A
known amount of standard drug was added to the fixed amount of pre-analyzed tablet
solution. Percent recovery was calculated by comparing the area before and after the
addition of the standard drug. The standard addition method was performed at 50%,
100% and 150% level of 4ppm. The solutions were analyzed in triplicate at each level as
per the proposed method. The percent recovery and % RSD was calculated and results
are presented in Table 13.7. Satisfactory recoveries ranging from 99.0 to 102.0 were
obtained by the proposed method. This indicates that the proposed method was accurate.
347
Level Amount of
Levetiracetam
spiked (ppm)
Amount of
Levetiracetam
recovered(ppm)
% Recovery
%RSD
50 %
6 5.96 99.33
0.387 6 5.98 99.66
6 6.01 100.1
100%
8 7.92 99.0
0.383 8 7.98 99.75
8 7.94 99.25
150%
10 9.92 99.2
0.209 10 9.96 99.6
10 9.93 99.3
Mean % of
recovery
99.46
Mean RSD =
0.326
Table 13.7: Percentage Recovery and %RSD
13.5.5. Robustness
The robustness study was performed by slight modification in flow rate of Mobile
phase, pH of the buffer and composition of the mobile phase. Levetiracetam at 6ppm
concentration was analyzed under these changed experimental conditions. It was
observed that there were no marked changes in chromatograms, which demonstrated that
the developed method was robust in nature. The results of robustness study are shown in
Table 13.8.
348
Condition Mean area % assay % difference
Unaltered 186327.8 100.0 0.0
Flow rate at 0.8 mL/min
Flow rate at 1.2mL/min
186112.6
185904.9
99.88
99.77
0.12
0.33
Mobile phase:
MEOH : Acetonitrile
75% 25%
85% 15%
187005.7
186942.6
100.3
100.3
0.3
0.3
pH of mobile phase at
3.4
186452.9 100.06 0.06
pH of mobile phase at
3.6
186263.4 99.96 0.04
Table 13.8: Robustness
13.5.6. System suitability
System suitability was studied under each validation parameters by injecting six
replicates of the standard solution 12 ppm). The results obtained were within acceptable
limits (Tailing factor ≤2 and Theoretical plate’s ≥2000) and are represented in Table
13.9. Thus, the system meets suitable criteria.
Parameter Tailing factor Theoretical plates
Specificity study 1.87 2629
Linearity study 1.01 2965.3
Precision study 1.52 3054.5
Table 13.9: System Suitability
13.5.7. Limit of detection and Limit of quantification
Limit of detection (LOD) is defined as the lowest concentration of analyte that
gives a detectable response. Limit of quantification (LOQ) is defined as the lowest
Concentration that can be quantified reliably with a specified level of accuracy and
349
Precision. For this sample was dissolved by using Mobile Phase and injected until peak
was disappeared. After 0.1ppm dilution, Peak was not clearly observed. So it confirms
that 0.1ppm is limit of Detection and 0.33ppm dilution is Limit of Quantification. For
this study six replicates of the analyte at lowest concentration were Measured and
quantified. The LOD and LOQ of Levetiracetam are given in Table 13.10.
parameter Measured volume
Limit of Quantification 0.33ppm
Limit of Detection 0.1ppm
Table 13.10: LOQ and LOD
Formulation:
For assay Levetiracetam (KEPPRA-250mg) 20 tablets were weigh and calculate
the average weight. Accurately weigh and transfer the sample equivalent to 10mg of
Levetiracetam in to a 10ml volumetric flask. Add diluent and sonicate to dissolve it
completely and make volume up to the mark with diluents. Mix well and filter through
0.45um filter. Further pipette 1ml of the above stock solution into a 10ml volumetric
flask and dilute up to mark with diluents and finally 12ppm were prepared. Mix well and
filter through 0.45um filter. An aliquot of this solution was injected into HPLC system.
Peak area of Levetiracetam was measured for the determination.
350
13.6. RESULTS AND DISCUSSION
To optimize the RP-HPLC parameters, several mobile phase compositions were
tried. A satisfactory separation and good peak symmetry was found in a mixture of
Methanol :Acetonitrile in the ratio of 80:20 v/v, PH Adjusted to 3.5 with 0.1% Ortho
phosphoric acid and 1.0 mL/min flow rate proved to be better than the other mixtures in
terms of resolution and peak shape. The optimum wavelength for detection was set at
208nm at which much better detector responses for drug was obtained. As it was shown
in Fig. 13.B the retention times were 2.58 min for Levetiracetam. The number of
theoretical plates was found to be 2629, which indicates efficient performance of the
column. A system suitability test was applied to representative chromatograms for
various parameters. The results obtained were within acceptable limits and are
represented in Table 13.9. Thus, the system meets suitable criteria.
Standard curves were constructed using six standard concentrations in a range
of 2, 4, 6, 8, 10, 12ppm for Levetiracetam. The linearity of peak area responses versus
concentrations was demonstrated by linear least square regression analysis. The linear
regression equation was y = 8160 + 30971.79 x (r= 0.998). Linearity values were shown
in Table: 13.4. Method precision was demonstrated by the assay of a series of six samples
(10ppm), prepared as described above on two consecutive days. The inter - (RSD-0.368)
and intra-(RSD-0.259) day means and relative standard deviation (R.S.D.) were
calculated (Table 13.5 and 13.6). The assay method precision acceptance criteria set in
the validation were a R.S.D. ≤ 2.0% for each data. The data of Precision meet these
acceptance criteria.
The accuracy of the method was evaluated by determination of the recovery of
Levetiracetam at three levels concentration. Tablets and capsules sample solutions were
spiked with Levetiracetam standard solution, corresponding to 50 to 150% of the nominal
analytical concentration (4ppm). The results showed good recoveries ranging from 99.0
to 101.45%. The mean recovery data obtained for each level as well as for all levels
combined (Table 13.6) were within 2.0% of the label claim for the active substance with
an R.S.D. < 2.0%, which satisfied the acceptance criteria set for the study.
351
The sample was dissolved by using Mobile Phase and injected until peak was
diapered. After 0.1ppm dilution Peak was not clearly observed. So it confirms that
0.1ppm limit of Detection and Limit of Quantification is 0.33ppm. Typical variations in
liquid chromatography conditions were used to evaluate the robustness of the assay
method. In this study, the chromatographic parameters monitored were retention time,
area, tailing factor and theoretical plates. The robustness acceptance criteria set in the
validation were the same established on system suitability test describe above.
The validated method was applied for the assay of commercial tablets
(KEPPRA-250mg) containing Levetiracetam. Sample was analyzed for five times after
extracting the drug as mentioned in assay sample preparation of the experimental section.
The results presented good agreement with the labeled content.
The proposed method for the assay of Levetiracetam in tablets or
capsules is very simple and rapid. It should be emphasized it is isocratic and the mobile
phase do not contain any buffer. The method was validated for specificity, linearity,
precision, accuracy and robustness. It could be used for the rapid and reliable
determination of Levetiracetam in tablet formulation.
352
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