Development of UV Spectrophotometry and RP-HPLC Methods for the Estimation of Levosulpiride in Bulk and in Tablet Formulation

 

S.P. Silambarasan¹, K. Anandakumar², R. Venkatalakshmi³* and C. Sasikala⁴

¹Axis Clinicals Ltd, Hyderabad, Andhrapradesh, India.

²Dept. of Pharmaceutical Analysis, Adhiparasakthi College of Pharmacy, Melmaruvathur – 603 319, Tamilnadu.

³Department of Pharmaceutics, Shri Vishnu College of Pharmacy, Bhimavaram, Andhrapradesh, India.

⁴Department of Pharmacology, Shri Vishnu College of Pharmacy, Bhimavaram, Andhrapradesh, India.

*Corresponding Author E-mail: rvenkatalakshmi@yahoo.co.in

ABSTRACT:

Two new simple, sensitive, rapid, accurate and precise methods, namely UV spectrophotometric and RP-HPLC methods were developed for the estimation of Levosulpiride in bulk and in tablet formulation. In UV spectrophotometric method, Levosulpiride exhibited maximum absorbance at 291.5 nm with apparent molar absorptivity of 2.6031 x 10³ L mol^-1 cm^-1 in 0.1M HCl. Beer’s law was obeyed in the concentration range of 10 - 50 mg/ml. In RP-HPLC method, the elution was done using a mobile phase consisting of methanol and 25 mM phosphate buffer pH 3.5 (pH adjusted with phosphoric acid, 15:85 v/v) on Shimadzu HPLC C₁₈ (4.6 x 150 mm) column at a flow rate of 0.8 ml/min with UV detection at 293 nm. An external standard calibration method was employed for quantization. The elution time was 5.08 minutes.  Beer’s law was found to be obeyed in the concentration range of 4 – 24 mg/ml.  The results of proposed methods were validated statistically and by recovery studies. The % RSD values for recovery studies were found to be less than 2% for the both methods. Hence the proposed methods were successfully used to determine the drug content in bulk and in tablet formulation.

 

 

 

INTRODUCTION:

Levosulpiride (LVS)¹ is chemically, (S)-(-)-5-aminosulfonyl-N [(1-ethylpyrrolidinyl) methyl] 2- methoxybenzamide, which is selective dopamine receptor (DA2) blocker in the CTZ with antiemetic effect. It is used for the treatment of depression and somatoform disorders². It is not official in IP, BP and USP. The literature survey revealed that three chromatographic methods have been reported for estimation of LVS in body fluids (Blood, plasma and urine)^3-5. There are no reports on UV and RP- HPLC methods for the estimation of LVS in bulk and in formulation. The present study was aimed to develop simple, rapid, precise, accurate and specific UV spectrophotometric and RP-HPLC methods for determination of LVS in bulk and in tablet formulation.

 

MATERIAL AND METHODS:

All the chemicals and solvents used were of HPLC and analytical grade and are procured from Qualigens and Loba Chemicals India Pvt. Ltd., Mumbai. Reference standard of LVS was obtained as a gift sample from Suriyen Pharmaceuticals Ltd., Chennai. One marketed brand LVS tablet (Lesuride) was procured from local pharmacy. For UV method, a standard stock solution of LVS (500 mg/ml) was prepared by dissolving 25 mg of the drug in 50 ml of 0.1M HCl in a volumetric flask. For HPLC method, standard solution of LVS (1000 mg/ml) was prepared by dissolving 50 mg of the drug in 50 ml of methanol in a volumetric flask.  Working standard solution (40 mg/ml) was obtained from stock solution by dilution with the mobile phase.

 

UV SPECTROPHOTOMETRIC METHOD:

In UV spectrophotometric method, Shimadzu 1700 UV-Visible spectrophotometer with 1 cm matched quartz cells was used for estimation.  Linearity of the method was investigated by serially diluting the stock solution to give a concentration range of 10 – 50 mg/ml. Absorbance was measured at 291.5 nm against 0.1M HCl as a blank.  Calibration graph was constructed by plotting absorbance against concentration. The spectrum is shown in fig.1.

 

Figure 1: UV spectrum of Levosulpiride in 0.1M HCl (10 µg/ml)

 

RP-HPLC Method:

In RP-HPLC method, Shimadzu HPLC equipped with Rheodyne injector with injection volume 20 ml, LC-10 ATVP solvent deliver module, SPD–10 A_VP UV–Vis detector, Winchrom software and Luna C₁₈ column having 150 mm length and 4.6 mm internal diameter was used.  Mobile phase was prepared by mixing methanol and 25 mM potassium di hydrogen phosphate (pH was adjusted to 3.5 with ortho phosphoric acid), in proportion of 15:85% v/v, respectively. The mobile phase was filtered through   0.4 micron membrane filter paper and degassed by ultrasonication for 10 minutes. Aliquots of standard solution (40 mg/ml) of LVS were suitably diluted with mobile phase to give final concentration of 4, 8, 12, 16, 20 and 24 mg/ml and injecting 20 ml with Hamilton syringe. Calibration graph was constructed by plotting peak area against concentration.

 

ASSAY OF MARKETED LESURIDE TABLET:

Assay of the marketed formulation Lesuride tablet containing 25 mg of LVS (Sun Pharmaceuticals Ltd., Mumbai) was performed. Twenty tablets were weighed and powdered. An amount of powder equivalent to 25 mg of LVS was dissolved in 0.1M HCl to obtain 500 mg/ml concentration, ultrasonicated and filtered through whatmann filter paper No. 41. An aliquot corresponding to 30 mg/ml was analyzed by UV spectrophotometric method described above.  An amount of powder equivalent to   50 mg of LVS was dissolved in methanol to obtain 1000 mg/ml concentration, ultrasonicated and filtered through whatmann filter paper No. 41. The solution was subjected to analyze by RP-HPLC method as described earlier after suitable dilution (10 mg/ml). Amount of LVS was determined by UV spectrophotometric and RP-HPLC method by referring to the respective calibration curve. The optical characteristics of UV spectrophotometric method are shown in Table 1.

 

TABLE 1: OPTICAL CHARACTERISTICS OF UV SPECTROPHOTOMETRIC METHOD

Parameters	Value
lmax (nm)	291.5
Beer’s law limit (mg/ml)	10 - 50
Molar absorptivity (L mol–1 cm–1)	2.6031×103
Correlation coefficient (r)*	0.9999
Regression equation (y = mx+c)	Y= 0.0068x + 0.0024
Slope (m)	0.0068
Intercept (c)	0.0024
LOD (mg/ml)	0.2339
LOQ (mg/ml)	0.7091

*Mean of six observations.

 

RESULT AND DISCUSSION:

To optimize the RP-HPLC parameters, several mobile phase ratios, different ionic strength of phosphate buffer and different flow rates were tried. Better parameters were obtained with mobile phase consisting of methanol: 25 mM phosphate buffer pH 3.5 (15:85% v/v). Quantification was achieved with UV detection at 293 nm based on external standard calibration. An optimized chromatogram for LVS is shown in fig. 2. Parameters of chromatogram are shown in Table 2.

 

Figure 2: Optimized RP-HPLC Chromatogram of Levosulpiride

 

TABLE 2: VALIDATION AND SYSTEM SUITABILITY PARAMETERS

Parameters	RP-HPLC Method
Retention time (min)	5.08
Linearity range (mg/ml)	4-24
Correlation coefficient (r)*	0.9997
Regression equation (y = mx+c)	Y= 103976.17x + 9390.69
Slope (m)	103976.17
Intercept (c)	9390.69
Tailing factor	1.25
Asymmetric factor	1.24
Capacity factor	2.10
HETP	0.0237
Theoretical plates	6323
Limit of Detection (mg/ml)	0.01229
Limit of Quantification (mg/ml)	0.0373

* Mean of  three observations.

 

The regression data showed a good linear relationship over a concentration range of 10 - 50 µg/ml for UV Spectrophotometric method and 4 - 24 mg/ml for RP-HPLC method. Correlation co-efficient of UV and RP-HPLC method was found to be 0.9999 and 0.9997, respectively. The limit of detection and limit of quantification for UV spectrophotometry was found to be 0.2339 and 0.7091 µg/ml, respectively, and for RP-HPLC method 0.01229 and 0.0373 µg/ml, respectively. As per the USP⁶, system suitability parameters of LVS for RP-HPLC were carried out. The results are summarized in Table 2. The results of the analysis of tablet formulation are well agreed with the label claim (Table 3). The methods were validated as per ICH guidelines⁷. The study was made to test ruggedness of the method through an intraday, inter day, different instruments and different analyst for analysis of sample.

 

TABLE 3: ASSAY OF LEVOSULPIRIDE IN TABLET FORMULATION

Method	S. No	Formulation	Amount found (mg)	Percentage obtained	Average (%)	SD	%RSD	SE
UV method	1		24.57	98.28
	2	Lesuride (25 mg)	24.79	99.16
	3		24.72	98.87	98.72	1.4186	1.4370	0.0394
	4		24.45	97.81
	5		24.23	97.04
	6		25.29	101.17
RP-HPLC method	1		24.88	99.52
	2		24.95	99.79
	3		25.40	101.61	99.79	0.8075	0.8077	0.0224
	4		24.90	99.62
	5		24.89	99.57
	6		24.93	99.72

(SD= Standard Deviation; % RSD= Percentage Relative Standard Deviation;    SE= Standard Error).

 

TABLE 4: PERCENTAGE RECOVERY STUDY

Formulation	Method	Amount added  µg/ml	Amount recovered µg/ml	% Recovery	SD	% RSD	SE
Lesuride (25 mg)	UV method
		6	6.03	100.48	0.2581	0.2565	0.0071
		12	12.27	100.90
		18	18.20	100.43
	RP-HPLC  method	2	2.011	101.11	0.8782	0.8700	0.0244
		4	4.044	101.65
		6	5.969	100.03
		8	8.066	101.38
		10	10.125	101.81
		12	11.898	99.69

(SD= Standard Deviation; % RSD= Percentage Relative Standard Deviation;    SE= Standard Error).

 

 

Results obtained confirmed ruggedness of the method. To study the accuracy, reproducibility and the precision of the proposed methods, recovery experiments were carried out. Recovery studies are carried out by adding a known quantity of pure drug to the fixed amount of pre analyze formulation, the proposed methods were followed.  From the amount of drug found, percentage recovery was calculated.  The results of recovery studies are presented in Table 4. The % RSD values were found to be less than 2% for both the methods. Hence the accuracy of the method was confirmed. The results indicate that there is no interference due to excipients used in formulation.

 

CONCLUSION:

The developed methods were found to be simple, accurate, precise and repeatable.  The statistical data proved that methods are reproducible and selective for the analysis of LVS in bulk and in tablet formulation. This indicates that the method can be effectively used for routine analysis of LVS in bulk and in tablet formulation.

 

ACKNOWLEDGEMENT:

The authors thankful to Arulthiru Amma, President, Thirumathi Amma, Vice-President, Adhiparasakthi Educational Institutions, for providing necessary facilities to carry out this work and also thankful to Suriyen Pharmaceuticals, Chennai for providing gift sample of LVS.

 

REFERENCES:

1.       www. google.co.in.

2.       Mucci A, Nolfe G, Maj M. Levosulpiride: A review of its clinical use in psychiatry. Pharmacol Res. 1995; 31: 95-101.

3.       In Bok Paek, et.al. Hydrophilic interaction liquid chromatography–tandem mass spectrometry for the determination of Levosulpiride in human plasma. J Chromatogr B. 2004; 809: 345-350.

4.       Su-Eon Jin, et.al. Development of HPLC method for the determination of Levosulpiride in human plasma. J  Pharm Biomed Anal. 2004; 35: 929-936.

5.       Hea-Young Cho, Yong-Bok Lee. Improvement and validation of a liquid chromatographic method for the determination of Levosulpiride in human serum and urine. J Chromatogr B. 2003; 796: 243-251.

6.       The United States Pharmacopoeia, XXIII, National Formulary, 22^nd Revision, United States of Pharmacopoeia Convention, Inc., Rockville, MD., USA, 1995, 1983.

7.       Code Q2B, Validation of Analytical Procedure: Methodology. ICH Harmonized Tripartite Guidelines, Geneva, Switzerland, 6^th November 1996, 1-8.

 

 

Received on 26.11.2009        Modified on 15.02.2010

Accepted on 19.03.2010        © AJRC All right reserved