Development and Validation of High Performance Liquid Chromatography Method for Levosulpiride and its Intermediate in Synthetic Mixture

Monika A. Rana*, Dr. Hasumati A. Raj

Department of Quality Assurance, Shree Dhanvantary Pharmacy College, Kim Surat, Gujarat India

*Corresponding Author E-mail: monika92rana@gmail.com

ABSTRACT:

A simple, accurate and precise chromatography method was developed for Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in synthetic mixture using RP-HPLC Method. In this chromatography method, both drug show its peak at 232 nm. Both the drugs show linearity in a concentration range of 10-50 μg/ml at their respective λmax. Optimize Mobile Phase was Water : Methanol : Acetonitrile (70 : 15 : 15). The relative standard deviation for accuracy, precision studies were found to be within the acceptance range (<2%). The limit of determination was 0.14μg/ml and 0.19μg/ml for Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester, respectively. The limit of quantification was 0.42μg/ml and 0.59μg/ml for Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester, respectively. Recovery of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester were found to be 100.16 % and 99.15 % respectively confirming the accuracy of the proposed method. The proposed method is recommended for routine analysis since they are rapid, simple, accurate and also sensitive and specific by no heating and no organic solvent extraction.

KEY WORDS: Levosulpiride, 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester, RP-HPLC method.

1. INTRODUCTION:

Levosulpiride is the levo enantiomer of sulpiride. It is a substituted benzamide which is meant to be used for several indications: depression, psychosis, somatoform disorders, emesis and dyspepsia.[1] It is physically present as a white crystalline powder. The Levo enantiomer shows better/similar pharmacological actions and lower incidence of toxic effects than both Dextro as well as the racemic forms of the drug. Levosulpride is an atypical antipsychotic agent that blocks the presynaptic dopaminergic D2 receptors.1 Like its parent compound, Levosulpiride shows antagonism at D3 and D2 receptors present presynaptically as well as postsynaptically in the rat striatum or nucleus accumbens2. The preferential binding of the presynaptic dopamine receptors decreases the synthesis and release of dopamine at low doses whereas it causes postsynaptic D2 receptor antagonism at higher dose.

This receptor profile of the drug along with its limbic selectivity explains its effectiveness in the management of both positive and negative symptoms of schizophrenia. The parent drug is given in a dose of 400-1800 mg orally daily although a much lower dose is effective for producing antidepressant effect (about 50-300 mg).The plasma t1/2 of the drug is about 6-8 hours. The drug is chiefly excreted through the renal route.[2] IUPAC name of Levosulpiride is (S)-5-Aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2- methoxybenzamide.[3] Structure of Levosulpiridein (Figure 1)

Figure 1: Structure of Levosulpiride

Levosulpiride is white or almost white, crystalline powder.[4] Solubility is given in soluble in water and freely soluble in Methanol, 0.1 N NaOH and 0.1 N HCl.

2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester is a white crystalline powder. Its melting point is 175˚C -177˚C. [5] Solubility is given in soluble in water and methanol and freely soluble in 0.1 N NaOH and 0.1 N HCl.

The review of literature regarding quantitative analysis of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester revealed that no attempt was made to develop analytical methods for Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester. Derivative spectrometric methods and chromatographic methods have been reported for the estimation of the Levosulpiride drugs. But no Analytical Method have been reported for 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester.The focus of the present study was to develop and validate a rapid, stable, specific, and economic spectroscopic method for the estimation of Levosulpirideand 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Synthetic mixture.[6] Structure of 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in (Figure 2)

Figure 2: Structure of 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester

2. MATERIALS AND METHODOLOGY:

Ø Levosulpiride and Its Intermidiate 2-Methoxy 5-Sulfamoylbenzoyl Benzoic Acid Methyl Ester was received as gift sample from Prayosha Health Care Pvt. Ltd.

Ø Company: Shimadzu

Ø Model No: SPD 10A-LC

Ø Software: WINCHROME SOFTWER

Ø Operation: semi Automatic

Ø An Semi micro analytical balance (Sartorius CD2250, Germany) was used for weighing purpose.

Ø HPLC water was obtained using arium^® 611VF (Sartorius).

Ø Magnetic stirrer (Remi) was used for mixing purpose.

Ø pH titer was used for pH measurement.

Ø Sonication of solutions were done using Ultrasonic cleaner (D 120/1H, Trans-O-Sonic).

Ø Nylon membrane filters (0.22 µm, 47 mm D) and Nylon syringe filters (0.22 µm, 25 mm D) were used for filtration purpose.

Ø All volumetric glassware used were calibrated.

Ø Methanol HPLC Grade (Rankem),

Ø HPLC Grade Water

Ø Acetonitrile Gradient Grade (Finar)

2.1. Materials and reagents

2.1.1 Preparation of stock solution of Levo

Accurately weighed quantity of Levosulpiride 10 mg was transferred to 100 ml volumetric flask, Add 25 ml of Mobile Phase [Water, Methanol, Acetonitrile (70, 15, 15) pH 3.5], sonicate it for 15min and dilute it up to the mark with Mobile Phase to give a stock solution having strength of 100μg/ml.

2.1.2 Preparation of stock solution of MSB

Accurately weighed quantity of 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester 10mg was transferred to 100 ml volumetric flask, Add 25 ml of Mobile Phase [Water, Methanol, Acetonitrile (70,15 ,15) pH 3.5], sonicate it for 15min and dilute it up to the mark with Mobile Phase to give a stock solution having strength of 100μg/ml.

2.1.3 Preparation of standard mixture solution

From the stock solution of LEVO take 1 ml and from stock solution of MSB take 1 ml and transferred in to 10ml volumetric flask and diluted up to mark with Mobile Phase to give a solution having strength of LEVO was 10μg/ml and MSB was 10 μg/ml.

2.1.4 Preparation of test solution

From the stock solution of LEVO take 1 ml and from stock solution of MSB take 1 ml and transferred in to 10ml volumetric flask and diluted up to mark with Mobile Phase to give a solution having strength of LEVO was 10 μg/ml and MSB was 10 μg/ml.

2.2. Calibration curves for Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester

Pipette out 1.0, 2.0, 3.0, 4.0 and 5.0 ml of the stock solution of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester (100μg/ml) into a series of 10ml volumetric flasks and the volume was adjusted to mark with Mobile Phase. The data of peak area versus concentration were treated by linear least square regression analysis.

2. RP- HPLC Method:

Selection of Wavelength: Both drug show its peak at 232 nm.

Figure 3 : Overlain zero order spectra of LEVO and MSB

3. Development of Mobile Phase:

Various mobile phases with different ratio of different solvents and pH were used are shown in Table 1. The mixture of Water, Methanol and Acetonitrile in ratio of (70:15:15, v/v) provided optimum polarity for proper migration, separation and resolution of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl Benzoic Acid Methyl Ester peaks. Under these conditions, the eluted peaks were well defined, resolved and free from tailing. Due to the non-polar nature of the stationary phase more polar component Levosulpiride will be eluted first because of its more affinity towards the polar mobile phase and less polar component 2-methoxy 5-sulfamoylbenzoyl Benzoic Acid Methyl Esterwill be eluted later due to its more affinity towards non-polar stationary phase. Figure 4.1-4.18 show chromatograms for various mobile phases tried.

_{Table 1 Optimization of Mobile Phase}

Trial	Mobile Phase		Ratio V/V	Remark
1.	Water : Methanol pH 7		50 : 50	Both Drug Show its Peak At same time very poor resolution
2.		Water : Methanol pH 6	70 : 30	Shifting of peak but poor resolution
3.	Water : Methanol pH 3.5		70 : 30	Good resolution but sharp peak was not obtain
4.	Water : Methanol pH 3.5		60 : 40	Good resolution but splitting of Levosulpiride
5.	Water : Methanol : Acetonitrile pH 3.5		60 : 20 : 20	Good resolution but tailing of both drug peak
6.	Water : Methanol : Acetonitrile pH 3.5		80 : 10 : 10	Good resolution but tailing of both drug peak
7.	Water : methanol : Acetonitrile pH 4.5		70 : 15 : 15	Good resolution and sharp peak was observed but tailing of both drug peak
8.	Water : methanol : Acetonitrile pH 3.5		70 : 15 : 15	Good resolution, sharp peak and no tailing in both drug peak

STANDARD CHROMATOGRAM OF BLANK

Figure shows chromatogram of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl Benzoic Acid Methyl Ester

TRIAL-1

TRIAL-2

TRIAL-3

TRIA

TRIAL-5

TRIAL-6

TRIAL-7

TRIAL-8

_{igure 4.18: Chromatogram using Mobile phase} = Water : methanol : Acetonitrile (70 : 15 : 15) pH 3.5

4. RESULT AND DISCUSSION

Validation Parameters [7]

5.1. Linearity and Range

Calibration curve were constructed by analysis of working standard solutions of Levosulpiride and2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester with different concentrations in the range between 10-50µg/ml and 10-50µg/ml concentration was injected and measured at 232 nm.

Table 2: Linearity Results obtained by RP-HPLC methods to laboratory prepared mixtures of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Mobile Phase

LEVO (µg/ml)	AREA*± SD	MSB ((µg/ml))	AREA*± SD
10	255854±618	10	260299±1303
20	530563±615	20	519831±721
30	826238±1921	30	830329±6293
40	1147866±1438	40	1124813±6230
50	1418845±6481	50	1387504±12618

Correlation coefficient (r2) for calibration curve of LEVO and MSB at 232 nm was found to be 0.9993and 0.9931, respectively.

The regression line equation for LEVO and MSB are as following,

y = 29433x – 47112 for LEV at 232 nm___________ (1) (figure 5)

y = 28594x – 33262 for MSB at 232 nm__________ (2) (figure 6)

5.2. Precision

5.2.1. Intraday precision

Mixed solutions of LEVO and MSB containing 10, 20 and 30 μg/ml and 10, 20 and 30 μg/ml respectively series were analyzed three times on the same day using developed RP-HPLC method and %RSD was calculated. The % RSD was found to be 0.34-0.69 % for LEVO and 0.35-0.69 % for MSB. These %RSD value was found to be less than ±2.0 indicated that the method is precise. (Table 3)

5.2.2. Interday precision

Mixed solutions of LEVO and MSB containing 10, 20 and 30 μg/ml and 10, 20 and 30 μg/ml respectively series were analyzed three times on the different day using developed RP-HPLC method and %RSD was calculated. The % RSD was found to be 0.69-0.90 % for LEVO and 0.47-0.81 % for MSB. These %RSD value was found to be less than ±2.0 indicated that the method is precise. (Table 4

Table 3: Intraday precision results obtained by applying RP-HPLC methods to laboratory prepared mixtures of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Mobile Phase.

Conc. (μg/ml)		Area* (LEVO) Avg. ± SD(231.50 nm)	% RSD	Area* (MSB) Avg.± SD(240.60nm)	% RSD
LEVO	MSB
10	10	255666±192	0.65	260196±102	0.35
20	20	530364±203	0.69	519631±198	0.69
30	30	826341±101	0.34	830231±113	0.39

Table 4: Interday precision results obtained by applying RP-HPLC methods to laboratory prepared mixtures of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Mobile Phase

Conc. (μg/ml)			Area* (LEVO) Avg. ± SD(232 nm)		% RSD		Area* (MSB) Avg.± SD(232nm)		% RSD
LEVO	MSB
10	10		255830±233		0.79		260650±137		0.47
20		20		530700±266		0.90		519812±234		0.81
30	30		826759±205		0.69		830490±209		0.73

5.3. Accuracy

The developed RP-HPLC method was checked for the accuracy. It was determined by calculating the recovery of LEVO and MSB from formulation solution by standard addition method in the combined mixture solution. The spiking was done at three levels 80 %, 100 % and 120 %. % recovery for LEVO and MSB by this method was found in the range of 99.80-100.47 % and 99.36-101.70%, respectively (Table 5 and 6)

The value of %RSD within the limit indicated that the method is accurate and percentage recovery shows that there is no interference from the excipients.

5.4. Limit of detection and quantitation

The LOD for LEVO and MSB was conformed to be 0.14μg/ml and 0.19μg/ml, respectively. The LOQ for LEVO and MSB was conformed to be 0.42μg/ml and0.59μg/ml, respectively. The obtained LOD and LOQ results are presented in Table 7.

5.5. Robustness and Ruggedness

The obtained Ruggedness and Robustness results are presented in table 6.24. The % RSD was found to be for 0.30 – 0.77 % LEVO and 0.31 - 0.85 % for MSB. These %RSD value was found to be less than ± 2.0 indicated that the method is robust and rugged.

No significant changes in the spectrums were observed, proving that the developed method is rugged and robust.(Table 8)

5.6. Application of the proposed method for analysis of LEVO and MSB in formulation

A zero order spectrum of the test solution was recorded and Measure the absorbance at 232 nm for estimation of LEVO and MSB. (Table 9, 10 )

Table 5: Recovery data results obtained by applying RP-HPLC methods of Levosulpiride in Mobile phase.

Conc. of LEVO from formulation (µg/ml)	Amount of Std. LEVO added (µg/ml)	Total amount of LEVO (µg/ml)	Total amount of LEVO found (µg/ml)* Mean± SD	% Recovery (n=3)	% RSD LEVO
20	8	28	27.94±5184	99.80	0.63
20	10	30	30.03±8466	100.10	0.95
20	12	32	32.15±2152	100.47	0.22

Table 6: Recovery data results obtained by applying Q Absorbance ratio methods of 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Mobile Phase.

Conc. of MSB from formulation (µg/ml)	Amount of Std. MSB added (µg/ml)	Total amount of MSB (µg/ml)	Total amount of MSB found (µg/ml)* Mean± SD	% Recovery (n=3)	% RSD MSB
00	8	8	8.13±1323	101.70	0.56
00	10	10	9.93±1830	99.36	0.64
00	12	12	12.17±2536	101.46	0.72

Table 7: Limit of detection and limit of quantitation data results obtained by applying Q Absorbance ratio methods to laboratory prepared mixtures of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Mobile Phase.

Conc (µg/ml)		Levosulpiride				MSB
LEVO	MSB	Avg±SD (n=3)		%RSD	Slop	Avg±SD(n=3)		%RSD	Slop
10	10	256245±1265		0.49	29433	261051±1713		0.65	28594
LOD (µg/ml)		0.14				0.19
LOQ (µg/ml)			0.42				0.59

Table 8: Robustness data results obtained by applying RP-HPLC methods to laboratory prepared mixtures of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Mobile Phase.

1. change in pH (3.5± 0.2)

Sr. No.	pH	Conc. (µg/ml)		Peak area ± SD ( n=3 )		%RSD
		LEVO	MSB	LEVO	MSB	LEVO	MSB
1	3.3	30	30	837745±2570	846432±3015	0.30	0.35
2	3.7	30	30	814956±3166	825780±4254	0.38	0.51

2. Change in Flow rate (1.0 ± 0.2 ml/min)

Sr. No.	Flow rate (ml/min)	Conc. (µg/ml)		Peak area ± SD ( n=3 )		%RSD
		LEVO	MSB	LEVO	MSB	LEVO	MSB
1	0.8	30	30	835897±2653	847411±2692	0.31	0.31
2	1.2	30	30	815245±4811	825692±2882	0.59	0.34

3. Change in Mobile Phase composition (±2)

Sr. No.		Mobile phase (Water : Methanol : Acetonitrile)	Conc. (µg/ml)		Peak area ± SD ( n=3 )		%RSD
			LEVO	MSB	LEVO	MSB	LEVO	MSB
1	68 : 16 : 16		30	30	826145±2479	835344±2963	0.30	0.35
2	69 : 14 : 17		30	30	825567±2577	836411±3130	0.31	0.37

4. Change in Drug Ratio

Sr. No.	Ratio (MSB : LEVO)	Conc. (µg/ml)		Peak area ± SD ( n=3 )		%RSD
		MSB	LEVO	MSB	LEVO	MSB	LEVO
1	1 : 1	10	10	305372±1219	275747±2012	0.39	0.72
2	1 : 2	15	30	447774±1530	776278±3347	0.34	0.43
3	2 : 1	30	15	835291±2570	466602±3867	0.30	0.82
4	1 : 4	10	40	346113±2670	1056448±4159	0.77	0.39
5	4 : 1	40	10	1234910±3722	317605±2723	0.30	0.85

Table 9: Assay of formulation results obtained by applying RP-HPLC methods to laboratory prepared mixtures of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Mobile Phase.

Drugs	Result of Synthetic Mixture analysis (n=3)
	Amount of drug	Amount of drug found		Area	% Assay± S.D.	% R. S.D.	Limit
LEVO	30µg/ml	30.05µg/ml	884490		100.16±2626	0.29	98.5 -101 %
MSB	30µg/ml	29.74µg/ml	850612		99.15±4486	0.52	-

Table 10: Assay of formulation results obtained by applying RP-HPLC methods to available of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Mobile phase.

Drugs	Result of Formulation Analysis (n=3)
	Amount of Drug	Amount of Drug Found	Area	% Assay± S.D.	%R.S.D.	Limit
LEVO	30 µg/ml	30.18 µg/ml	888541	100.62±6351	0.71	98.5 -101 %

Table 11: Summary results obtained by applying RP-HPLC methods to laboratory prepared mixtures of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Mobile Phase.

Sr No.	Parameters	LEVO	MSB
1.	Concentration Range (µg/ml)	10-50	10-50
2.	Regression Equation	y = 29433x - 47112	y = 28594x + 33262
3.	Correlation Coefficient (r^2)	0.9993	0.9991
4.	Accuracy (%Recovery)	100.12	100.84
5.	Intraday Precision (%RSD)	0.34-0.69	0.35–0.69
6.	Interday Precision (%RSD)	0.69-0.90	0.47-0.81
7.	Robustness	0.30-0.77	0.31-0.85
8.	LOD (µg/ml)	0.14	0.19
9.	LOQ (µg/ml)	0.42	0.59
10.	%Assay	100.62%	-

6. CONCLUSION:

A new, RP-HPLC method has been developed for estimation of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in synthetic mixture. The method was validated by employment of ICH (18) guidelines. The validation data is indicative of good precision and accuracy, and prove the reliability of the method.

7. ACKNOWLEDGEMENT:

We are highly thankful to Prayosha Health Care Pvt. Limited, for providing us the gift sample of the pure drug and to our Principal, Dr M.N. Noolvi and HOD Dr. Hasumati A. Raj, Shree Dhanvantary Pharmacy College for providing excellent research facilities.

8. REFERENCE:

1. Depomed, Inc. Gastric retentive pharmaceutical compositions for immediate and extended release of levosulpiride. United State patents US 20110052700 A1, 2011.

2. Gupta S, Rai Gobind, Halder S and Sharma K, "Levosulpiride: A Review." Drug Rev.2007, 10, 14-146.

3. Drug Profile available from, www.prahoshahealthcare.com

4. British Pharmacopeia; the Indian Pharmacopeia Commission, Ghaziabad, Govt. of India; Ministry of Health and Family Welfare, 1998, Volume Ӏ, pp 1252-1253.

5. www.chemicalbook.com

6. Monika A. Rana, Hasumati A. Raj.”A Review On Analytical Methods For Determination of Levosulpiride In Pharmaceutical Dosage Forms And Biological Sample.”Pharma Tutor, 2014.

7. International Conference on Harmonization, Harmonized Tripartite Guideline, Validation of Analytical Procedures Text and Methodology, ICH Q2(R₁), 2005.

Received on 27.03.2015 Accepted on 25.04.2015

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech. 2015; Vol. 5: Issue 2, Pg 97-106

DOI: 10.5958/2231-5713.2015.00015.X