INTRODUCTION:

Levosulpiride (LVS); 2-methoxy-N-((1-propylpyrrolidin-2-yl) methyl)-5-sulphamoyl benzamide (Fig. 1) is a widely prescribed anti-psychotic drug. It is a selective dopamine D2 antagonist with antidepressant activities^1,2. Several methods have been reported for the determination of LVS in pharmaceutical preparations and biological fluids. These methods include: spectrophotometry, HPLC and spectrofluorimetry^3-9.

Pantoprazole (PAN); 5- (difluoromethoxy) - [[(3,4- dimethoxy-2-pyridiynyl) methyl] sulphinyl] - 1H benzimidazole (Fig. 2) is a gastric proton pump inhibitor^1,2. The methods reported for quantitative determination of pantoprazole in bulk or pharmaceutical formulations include colorimetry, spectroscopy, HPLC and HPTLC^10-16.

No spectrophotometric method was found in literature for the simultaneous estimation of levosulpiride and pantoprazole. The present paper describes a simple, rapid, accurate and precise first order derivative spectrophotometric method for the simultaneous estimation of LVS and PAN in their combined capsule dosage form.

Fig. 1. Chemical structure of levosulpiride

Fig. 2. Chemical structure of pantoprazole

MATERIALS AND METHODS:

Apparatus

Instrument used was UV-Visible double beam spectrophotometer, SHIMADZU (model UV-1800) with a pair of 1 cm matched quartz cells. Calibrated glass wares were used to prepare the solutions.

Reagents and chemicals

All reagents and solvents were of Analytical Reagent grade.

Both LVS and PAN were kindly gifted by Sunpharma Research Centre, Baroda. Marketed formulation, Pantocid-L capsules containing 75 mg LVS and 40 mg PAN, were procured from local pharmacy.

Preparation of standard solutions

Accurately weighed LVS (25 mg) and PAN (25 mg) were transferred to separate 25 ml volumetric flasks, dissolved in and diluted to the mark with 0.1N NaOH to obtain standard solutions having concentration 1000 ug/ml.

Selection of analytical wavelengths

Standard solutions of LVS (25, 50, 75, 100 and 125 ug/ml) and PAN (5, 10, 15, 20 and 25 ug/ml) were scanned in the range of 200 to 400 nm. Zero crossing points (ZCP) of both drugs were selected by converting both the spectra into first order derivative. ZCP of LVS and PAN were found to be 291.0 nm and 252.5 nm, respectively (Fig. 3).

Fig. 3 First order derivative overlain spectra of LVS 75 ug/ml) and PAN (15 ug/ml)

Stability of standard solutions

Stability of standard solutions, LVS (25, 50, 75, 100 and 125 ug/ml) and PAN (5, 10, 15, 20 and 25 ug/ml) was checked by measuring their absorbance at 0, 2, 4, 6, 8 and 10 hrs of preparation at room temperature
(25^o C).

Validation of Method¹⁷

The method was validated as per ICH guidelines Q2(R1).

RESULTS AND DISCUSSION:

Zero-order absorption spectra of levosulpiride and pantoprazole sodium showed overlapping peaks that interfere with their simultaneous determination in formulation. First derivative spectroscopy, based on a mathematical transformation of the zero-order spectra into the derivative spectra, allows a fast, accurate and precise resolution of a multicomponent mixture.

Stability of standard solutions

The standard solutions were found to be stable for the period of 10 hrs after preparation at room temperature (25^o C).

VALIDATION OF THE PROPOSED METHOD²⁰

Linearity

The calibration curves were plotted over a concentration range of 25-125 ug/ml for LVS and 5-25 ug/ml for PAN. The absorbance of solutions was measured at 252.5nm (ZCP of PAN) and at 291.0nm (ZCP of LVS) for estimation of levosulpiride and pantoprazole, respectively (Fig. 4 and 5).

Fig. 4. Calibration curve of LVS at 252.5 nm (ZCP of PAN)

Fig. 5. Calibration curve of PAN at 291.0 nm (ZCP of LVS)

Method precision (repeatability)

The precision of the method was checked by repeated scanning and measurement of the absorbance of six different solutions of LVS (75 ug/ml) and PAN (15 ug/ml) without changing the parameters of the proposed method.

Intermediate precision (reproducibility)

The intraday and inter day precision of the proposed method was determined by estimating the corresponding responses 3 times on the same day and on 3 different days over a period of one week for 3 different concentrations of standard mixture solutions containing LVS (50, 75, 100 ug/ml) and PAN (10, 15, 20 ug/ml).

Accuracy

The accuracy of the method was determined by standard addition method. Known amount of standard solutions of LVS and PAN were added to pre-quantified sample solutions of combined capsule dosage form. The amounts of LVS and PAN were estimated by applying obtained values to the respective regression line equations and percent recovery of each drug was determined. Results of the recovery data are given in Table 1.

Limit of detection and Limit of quantification

The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were obtained using the following equations designated by (ICH) guidelines.

LOD = 3.3 × s/S

LOQ = 10 × s/S

Where,

s = the standard deviation of the response and

S = mean slope of 5 calibration curves.

Results of the validation parameters are presented in Table 2.

Table 1: Recovery data of proposed method

Drug	Amount taken from sample (ug/ml)	Amount of Standard added (ug/ml)	% Recovery ± SD (n=3)
LVS	30	24	101.53 ± 0.87
	30	30	101.22 ± 0.51
	30	36	98.89 ± 0.48
PAN	10	08	98.33 ± 1.67
	10	10	102.22 ± 1.02
	10	12	99.44 ± 1.67

Table 2: Summary of Validation Parameters

Parameters	LVS	PAN
Linearity Range (μg/ml)	25-125	5-25
Correlation coefficient	0.9992	0.9972
Precision (% RSD) Repeatability (n=6) Intraday (n=3) Interday (n=3)	0.72 0.42-1.20 0.53-1.87	0.84 0.59-1.42 0.64-1.91
Accuracy (% recovery)	98.89 -101.53%	98.33 - 102.22 %
LOD (μg/ml)	0.41	0.34
LOQ (μg/ml)	1.25	1.03

Assay of marketed formulation by proposed method

Twenty capsules were individually weighed, emptied and their contents were mixed. The powder equivalent to 75 mg LVS or 40 mg PAN was accurately weighed and transferred to 100 ml volumetric flask. To this, about 50 ml of 0.1N NaOH was added and sonicated for 15 min. The volume was made up to the mark with 0.1N NaOH and mixed well. The solution was filtered through watman filter paper and 1 ml filtrate was diluted to 10 ml with 0.1N NaOH to give a solution containing 75 μg/ml LVS and 40 μg/ml PAN. From this solution, 4 ml was further diluted to 10 ml with 0.1N NaOH to give a solution containing 30 μg/ml LVS and 16 μg/ml PAN which was used for the estimation of LVS and PAN. The results obtained for LVS and PAN were compared with the corresponding labelled amounts (Table 3).

Table 3: Assay of formulation by proposed method

Formulation	Drug	Label claim (mg)	Amount found (mg) ± SD (n=3)	% Label claim ± SD (n=3)
Pantocid-L Capsules	LVS	75	74.20 ± 0.60	98.93 ± 0.80
Pantocid-L Capsules	PAN	40	39.60 ± 0.53	99.00 ± 1.32

CONCLUSION:

The present study describes a simple, precise and accurate first derivative UV-Vis Spectroscopy method for the simultaneous estimation of levosulpiride and pantoprazole. The method was successfully validated and applied to the estimation of the two drugs in their combined capsule dosage form.

ACKNOWLEDGEMENT:

The authors are thankful to Sunpharma Research Center, Baroda and Principal Maliba Pharmacy College for providing all the resources for the experiment.

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