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

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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

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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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