INTRODUCTION:

Levetiracetam (LVT) is from anti-epileptic category and chemically it is (S)-2-(2-oxopyrrolidin-1-yl) butanamide (Figure 1) with a molecular formula (C8 H14N2 O2). LVT is very soluble in water and freely soluble in methanol and chloroform¹.

Figure 1: Structure of Levetiracetam

The U.S. Food and Drug Administration approved this antiepileptic medication as a monotherapy treatment for epilepsy in cases of partial seizures or as an adjuvant therapy for juvenile myoclonic epilepsy and tonic-clonic seizures.^2-4

The aim of the analytical studies is to obtain quantitative and qualitative information about the compounds of interest (analyte) in a sample⁵.

A literature survey revealed that various analytical methods include spectrophotometry^6-9 HPLC^10-17, UPLC ¹⁸, UHPLC¹⁹, HPTLC²⁰, LC-MS/MS^21,22 and UPLC-MS/MS²³ for the estimation of LVT in pharmaceutical formulations and biological fluids such as human plasma and human serum. Analytical methods like first order spectrophotometric and HPTLC have been not studied yet for LVT.

Due to its advantages of low operating costs, high sample throughput, and minimal sample cleaning requirements, HPTLC is becoming a common analytical method. In contrast to HPLC, a minimal amount of mobile phase can be used to analyse multiple samples at once with HPTLC, which reduces analysis time and cost per analysis²⁴.

So, in this present investigation an attempt has been made to develop HPTLC method (Method A) and first derivative spectrophotometric method (Method B) which may be simple, accurate, precise and economically viable for the estimation of LVT in bulk drug and in tablet dosage form and validate as per ICH guidelines²⁵.

MATERIALS AND METHODS:

Instrumentation:

For Method A, ALUGRAM Xtra TLC plates precoated with silica gel 60 F₂₅₄of dimensions 10cm ×10cm with 250mm thickness was used. Application of samples was done using 100µl sample syringe as a band of width 6 mm using a Camag Linomat V applicator. Plate was developed in twin trough glass chamber of dimension 20cm x 10cm by linear ascending mode. Densitometric scanning was performed on Camag TLC scanner 3 at 218nm for all measurements and operated by Wincats software (V 1.4.2, Camag).

Method B was performed on UV-Visible Double beam Spectrophotometer (Shimadzu 2450, Japan) attached to a computer software UV probe 2.0, with a spectral width of 2nm, wavelength accuracy of 0.5nm and pair of 1cm matched quartz cells.

Reagents and Solvents:

LVT was obtained as a gift sample from INTAS pharmaceuticals, Ahmadabad (Gujarat). Methanol (AR Grade), Ethyl acetate (AR Grade), Toluene (AR Grade) purchased from Modern science and Bansal Pvt. Ltd chemical lab, Nasik, Maharashtra (India). Tablet Formulation was purchased from local drug store. (Brand Name: Levera, INTAS Pharmaceutical, India Pvt. Ltd., labeled claim: 500mg LVT per tablet)

Analytical Conditions:

Method A:

Solution of LVT was applied to silica gel 60F254 HPTLC plates (10cm × 10cm) by means of a Linomat V automatic spotter equipped with a 100µL syringe and operated with settings of band length 6mm; distance from the plate edge 10mm; and distance from the bottom of the plate 10mm. The plate was developed in a twin trough chamber previously saturated for 20min with the mobile phase, toluene: methanol: Ethyl acetate (4:2:4v/v/v). The spots on the air-dried plate were scanned with the scanner III at 218nm using the deuterium source.

Method B:

The standard solution of LVT (10µg/mL) was scanned separately in the UV range of 200-400nm. The zero order spectra thus obtained was then processed to obtain first derivative spectrum (N = 4, ∆λ = 3.0nm).

Preparation of standard stock solution:

Method A:

Accurately weighed 10mg of Levetiracetam was dissolved in 10ml of methanol to obtain 1000µg/mL of standard solution. Stock solution of Levetiracetam was further diluted in methanol to get the working standard solutions of concentration 100µg/mL. From these solutions specific volume (µL) was spotted to yield 400, 800, 1200, 1600, 2000, 2400, 2800, 3200ng/spot.

Method B:

Standard stock solution was prepared by accurately weighing 10mg of LVT and dissolved in 10mL of methanol to get concentration of 1000µg/mL in volumetric flask. This solution was used as standard working solution. Aliquot 0.8, 1.2, 1.6, 2, 2.4 and 2.8 mL were pipetted out from working standard mixture solution into a series of 10mL volumetric flasks. The volume was made up to the mark with methanol to get 80-280µg/mL concentration of LVT.

Study of Resolution of LVT:

Stock solution of 1000µg/mL of LVT was prepared. 400-3200ng/spot of LVT were spotted on the plate using 100µL Camag syringe. The development chamber was saturated with developing phase for 20 min. The spotted plate was placed in the saturated chamber and developed up to 70mm. The plate was air dried and scanned up to 70mm. Densitogram of LVT is shown in Figure 2.

Method validation:

Specificity:

Specificity involves quantitative detection of an analyte in the presence of those components that may be expected to be part of the sample matrix²⁶. The specificity of the developed methods was established by spiking of Magnesium stearate (MS)in standard sample of LVT (i.e., might be expected to be present) and expressing that analytes peak did not interfere from excipients.

Linearity:

Method A:

Levetiracetam (10mg) was separately weighed and dissolved in 10ml of methanol to get the stock solution of concentrations 1000µg/mL. It was further diluted appropriately with methanol to get 100µg/mL working standard solution. From working standard solution, in the range of 4-32µL were spotted on the TLC plate to obtain the final concentration range of 400–3200ng/spot for Levetiracetam. The plate was developed using the mobile phase Toluene: Methanol: Ethyl acetate: (4:2:4 mL v/v/v). The peak area was plotted against the corresponding concentrations to obtain the calibration graph (Figure3).

Method B

Aliquot 0.8, 1.2, 1.6, 2, 2.4 and 2.8mL were pipetted out from working standard mixture solution into a series of 10 mL volumetric flasks. The volume was made up to the mark with methanol to get 80-280µg/mL concentration of LVT. Each sample was scanned three times and absorbance was measured. Calibration curve was plotted and regression line equation was calculated (Figure 4).

Precision:

The precision of the method was demonstrated by intra-day and inter-day studies. In the intraday studies, 3 different concentrations (1600, 2000, 2400ng/spot, 200, 240 and 280µg/mL) and their six replicates were analysed for three times in a day for HPTLC and First Derivative spectrophotometric method respectively and percentage RSD was calculated. In the inter day variation studies, above mentioned 3 different concentrations (1600, 2000, 2400ng/spot and 200, 240 and 280µg/mL) and their six replicates were analysed on 3 consecutive days for Method A and Method B respectively and percentage RSD were calculated²⁷.

Recovery Study (Accuracy):

For accuracy study data from nine determinations over three concentrations at 50%, 100%, and 150% of expected sample concentration covering the specified range was calculated using regression equation of the calibration curve²⁸ and expressed as recovery values (For Method A as well as Method B).

Limit of detection (LOD) and limit of quantification (LOQ):

The limit of detection (LOD) and the limit of quantification (LOQ) of the drug determines sensitivity of methods and were derived by calculating the signal-to-noise ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the following equations as per International Conference on Harmonization (ICH) guidelines. [19] LOD = 3.3 × σ/S LOQ = 10 × σ/S Where σ= the standard deviation of the response and S = Slope of calibration curve²⁹.

Robustness:

The robustness of the method was determined to check the reliability of analysis with variation in method parameters such as solvent front and saturation time for Method A and change in solvent and change in wavelength for Method B.

Ruggedness:

Change in analyst as well as performing analysis in different days were the parameters to study ruggedness of the methods.

Specificity:

Magnesium stearate as an excipient was selected for specificity study of both analytical methods.

Analysis of Tablet dosage form:

Weight of twenty tablets (Levera)were measured accurately and powdered. An equivalent to 10mg of LVT powder was weighed and transferred to a 10mL calibrated volumetric flask containing methanol and was sonicated for 5 min. The solution was filtered through whatmann filter paper no.41. Further volume was made to 10mL with the same solvent. From the sample stock solution volume accurately 1.2 and 1.6µL was spotted on to the HPTLC plates to get concentration of 1200 and 1600ng/spot of LVT. Development of plate was done up to a distance of 70 mm, using the mobile phase Toluene: Methanol: Ethyl acetate: (4:2:4mL v/v/v) in normal conditions of temperature and humidity. The peak areas of the spots were scanned at 218nm. The amount of drug present in the tablet was calculated using the regression equation³⁰.

Method B:

Weight of twenty tablets (Levera)were measured accurately and powdered. An equivalent to 10mg LVT powder was weighed and transferred to a 10mL calibrated volumetric flask containing methanol and was sonicated for 5min. The solution was filtered through whatmann filter paper no. 41. Further volume was made to 10mL with the same solvent. From the sample stock solution volume accurately 2 and 2.5mL was transferred to 10 ml volumetric flask and volume was made up to mark with methanol to get concentration of 200 and 250 µg/mL of LVT. The amount of drug present in the tablet was calculated using the regression line equation³¹.

Comparison of the proposed methods using t test:

The assay results for LVT in their tablet dosage forms obtained using both analytical methods and then paired t test was applied for comparison.

RESULT:

Optimization of chromatographic and spectrophotometric conditions:

Various solvents were tried as mobile phases to achieve good separation of LVT. Mobile phase consisting toluene: methanol: Ethyl acetate (4:2:4v/v/v) was optimized as provide acceptable Rf values and resolution. The Rf value for LVT 0.47±0.02 was obtained on 10 × 10cm HPTLC silica gel 60F254 aluminium-backed plates detected at wavelength 218nm. Plate activation was performed at 60⁰C for 5 minutes and then developed in presaturated chamber at room temperature (20minutes) to get well defined spots. A typical densitogram of drug shown in (Figure2)

Figure 2: HPTLC densitogram of LVT 2800 ng/spot (R_F 0.47±0.02)

The standard solution of LVT was scanned from 200-400nm to get normal order spectrum. It was then processed to obtain first derivative spectra. The first derivative absorbances was recorded at 215nm and overlain spectra at 215nm (Figure 3)

Figure 3: Overlain first order derivative spectra of LVT (80-280µg/mL)

Validation of analytical methods:

Linearity:

Linear correlation was observed when graph was plotted between peak areas Vs concentrations of LVT in range of 400-3200ng/spot for Method A and between absorbance Vs concentrations of LVT in range of 80–280µg/mL for Method B. Calibration graphs of both methods were shown as (Figure 4 and Figure 5)

Figure 4: Calibration curve for LVT (400-3200 ng/spot- Method A)

Figure 5: Calibration curve for LVT (80–280 µg/mL -Method B)

The linearity data for both analytical methods was summarized in (Table 1).

Table 1: Linearity data and summary of validation parameters for the proposed Method A and Method B

Parameters	Method A	Method B
Concentration range	400-3200 (ng/spot)	80-280 µg/mL
Slope	0.679	0.001
Intercept	54.62	0.012
Correlation coefficient	0.998	0.998
LOD a	0.091 ng/spot	5.77 µg/mL
LOQ b	0.278 ng/spot	16.90 µg/mL

a-LOD = Limit of detection. b-LOQ = Limit of quantification.

Precision:

Intra and Inter day precision studies for both methods determined to assure the repeatability of the test results. The %R.S.D. was calculated (Table 2).

Accuracy:

Standard addition method was used for recovery experiments³². The mean recoveries obtained for LVT by Method A and Method B was shown in (Table 3).

Table 3: Accuracy study results

% Level	Method A			Method B
	Amount of sample+ Amount of std. spiked (ng/spot)	Mean Drug recovered (n=3)	% Mean Recovery± SD(n=9)	Amount of sample+ Amount of std. Spiked (µg/mL)	Mean Drug recovered (n=3)	% Mean Recovery± SD (n=9)
50%	1000+500	1514.92	100.7±1.20	100+50	152	101.4±0.48
	1000+500	1529.8		100+50	154
	1000+500	1492.5		100+50	153
100%	1000+1000	1991.0	99.25±0.58	100+100	203	101.5±0.50
	1000+1000	1992.5		100+100	205
	1000+1000	1971.6		100+100	202
150%	1000+1500	2488.0	100.70±1.04	100+150	252	100.8±0.40
	1000+1500	2529.8		100+150	251
	1000+1500	2537.8		100+150	253

Table 4: Robustness data

Method A			Method B
Change in saturation Time-30min			Change in wavelength
Conc. (ng/spot)	Mean Peak Area ±SD (n=3)	% RSD	Conc.(µg/mL)	Mean Absorbance at 213 nm±SD (n=3)	Mean Absorbance at 217nm±SD (n=3)	% RSD
1600	1146±0.21	0.63	200	0.0322±0.00057	0.327±0.00115	0.179
Change in migration distance-80mm			Change in solvent
Conc. (ng/spot)	Mean Peak Area ±SD(n=3)	% RSD	Conc.(µg/mL)	Mean Absorbance in 0.1NHCL±SD(n=3)	Mean Absorbance in water±SD(n=3)	%RSD
1600	975±13.11	0.0077	200	0.326±0.000577	0.328±0.00101	0.177

Table 5: Ruggedness data

Method A					Method B
(Change in analyst)					(Change in analyst)
Conc. (ng/spot)	Mean Peak Area ±SD (n=3)		% RSD		Conc. (µg/mL)	Mean Absorbance ±SD (n=3)		%RSD
	A 1	A 2	A1	A2		A1	A 2	A 1	A2
1200	973.66±15.27	937±18.52	1.56	1.97	200	0.350±0.00070	0.336± 0.00070	0.211	0.221
Change in day					Change in day
Conc. (ng/spot)	Mean Peak Area ±SD (n=3)		%RSD		Conc. (µg/mL)	Mean Absorbance ±SD (n=3)		%RSD
	D1	D 2	D 1	D 2		D 1	D 2	D 1	D2
1600	1045.33±6.65	1075.66±15.53	0.63	1.10	200	0.343±0.000141	0.388±0.00070	0.412	0.180

A1: Analyst 1, A2: Analyst 2 D1: -Day 1, D2: Day 2

Specificity:

The excipient magnesium stearate was not showing any interaction with drug (Table 6).

Table 6: Specificity data

%Level	Std. conc. (ng/spot)	Magnesium stearate conc. Spiked ((ng/spot)	Mean Peak area±SD
Method A
Std	1200	-	856±2.11
50	1200	600	862±1.56
100	1200	1200	852±1.89
150	1200	1800	860±2.11
Method B
% Level	Std. conc. (µg/mL)	Magnesium stearate conc. Spiked (µg/mL)	Mean absorbance ± SD
Std	100	-	0.140±0.31
50	100	50	0.142±0.29
100	100	100	0.141±0.189
150	100	150	0.140±0.45

Analysis of Tablet dosage form:

Content of LVT in marketed tablet dosage form was analysed by both analytical methods (Method A and B). The results obtained were in agreement with the corresponding labelled amounts (Table 7).

Table 7: Assay results for the tablet dosage form (Levera)

Tablet	Method A	Method B
Levera	Mean % Label claim ±SD	Mean % Label claim ±SD
Levera	99.66±0.98	101.23±0.46

Comparison of the proposed methods using t test

The tabulated t value (2.571) is more than calculated t value 2.05 for LVT at 95 % confidence interval (Table 8).

Table 8: Comparative data of Method A and Method A

Parameters	Method A	Method B
Assay results ± S.D^a	99.66±0.98	101.23±0.46
N^b	6	6
t-value (2.571)^c	2.05

^aS.D = Standard deviation ^bN = number of determinations c= corresponding to t-tabulated value at 95% confidence interval

DISCUSSION:

Analytical method development is the process of demonstrating whether an analytical method is acceptable for use in the literature. Through literature survey it was cleared that for estimation of LVT first derivative spectrophotometric method as well as HPTLC methods has not been tried so in the present work, we selected these two analytical methods.

In case of HPTLC method a number of experimental parameters, such as activation time, saturation time, mobile phase composition, scan modes, detection wavelength were optimized during method development in order to provide accurate and reproducible results for the determination of LVT. Finally, method was optimized with mobile phase Toluene: ethyl acetate: methanol (4:4:2 v/v/v) with maximum resolution and Rf value 0.47. First order derivative spectrophotometric method was developed using methanol as solvent and the derivative amplitude of solutions was measured at 215nm for LVT. Paired t test was used to compare the assay results of tablet dosage forms by both analytical methods. The calculated t value 2.05 for LVT which was less than the tabulated t-value (2.571) at 95% confidence interval. Statistical comparison of the results obtained by proposed HPTLC method with the results obtained by proposed first derivative UV spectrophotometric method shows good agreement and indicates no significant difference in the content of LVT by the proposed RP-HPTLC and first derivative spectrophotometric method³³.

Further, the developed method was subjected to validation. Validation was executed per the ICH Q2R1 guidelines for the parameters specificity, linearity, system suitability, LOD, LOQ, precision, accuracy, and robustness. All the parameters were within limits.

CONCLUSION:

The results obtained by both methods in current project are proved that HPTLC and first order derivative spectrophotometric methods are highly reproducible and reliable and can be used for the routine analysis of the LVT in tablet dosage form.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

Authors are very thankful to Dr. R.S. Bhambar, Principal, MGV’s pharmacy college Panchavati, Nasik -03 and management for providing facilities to carry out mentioned research work. Authors are also thankful to INTAS Pharmaceuticals for providing gift sample of drug.

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Received on 07.09.2022 Modified on 30.12.2022

Research J. Pharm. and Tech 2024; 17(3):979-985.

DOI: 10.52711/0974-360X.2024.00151

Intra- day Precision	Conc. (ng/spot)	Mean Peak Area ± SD(n=3) (Method A)	% RSD	Conc. (µg/ml)	Mean absorbance± SD(n=3) (Method B)	% RSD
	1600	1124.33±18.57	1.65	200	0.332±0.0085	0.025
	2000	1362.3±20.14	1.47	240	0.381±0.0075	0.019
	2400	1757.9±24.00	1.37	280	0.467±0.001	0.021
Intra- day Precision	1600	1049.50±14.95	1.42	200	0.308±0.023	0.074
	2000	1303.18±18.51	1.42	240	0.349±0.032	0.091
	2400	1658.68±23.20	1.39	280	0.433±0.033	0.077

Figure 2: HPTLC densitogram of LVT 2800 ng/spot (RF 0.47±0.02)

Figure 3: Overlain first order derivative spectra of LVT (80-280µg/mL)

A1: Analyst 1, A2: Analyst 2 D1: -Day 1, D2: Day 2

Specificity:

Figure 2: HPTLC densitogram of LVT 2800 ng/spot (R_F 0.47±0.02)