Development and Statistical Validation of Spectrophotometric Method for Estimation of Esomeprazole in Tablet Dosage Form

Patil Shamkant S*¹, Dhabale Pandurang N¹ and Kuchekar Bhanudas S²

¹Govt. College of Pharmacy, Vidyanagar, Karad-415124, Maharashtra, India.

²Maharashtra Institute of Pharmacy, Pune, Maharashtra, India.

*Corresponding Author E-mail: Shamkantpatil7@rediffamil.com

ABSTRACT

Three simple, precise and economical UV methods have been developed for the estimation of Esomeprazole in bulk and pharmaceutical formulations. Esomeprazole has the absorbance maxima at 303nm (Method A), and in the first order derivative spectra, showed zero crossing at 303nm, with a sharp peak at 292nm when n=1 (Method B), Method C applied was Area Under Curve (AUC) for analysis of Esomeprazole in the wavelength range of 294-310nm. Drug followed the Beer’s Lamberts range of 5-40 µg/ml for the Method A, B C. Results of analysis were validated statistically and by recovery studies and were found to be satisfactory.

KEY WORDS: Esomeprazole, UV Spectrophotometry, Derivative Spectroscopy, Area Under Curve.

INTRODUCTION:

Esomeprazole is a proton pump inhibitor drug used for short-term treatment of erosion and ulceration of the esophagus caused by gastroesophageal reflux disease.Chemically Esomeprazole is 6-methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl]-1H-benzimidazole (Fig- 1). It is listed in Martindale-The complete drug reference¹. Literature survey reveals plane HPLCand HPLC in plasma^2,3method exists for estimation of Esomeprazole. No single UV method for Esomeprazole is reported till date using derivative spectroscopy and AUC method. Hence an attempt has been made to develop new UV method for its estimation in bulk and pharmaceutical formulations with good accuracy, simplicity, precision and economy.

MATERIALS AND METHODS:

Pure sample of Esomeprazole was obtained from Glenmark Pharmaceutical Limited, Mumbai as a gift sample. A Shimadzu UV-1700 UV/VIS Spectrophotometer was used with 1 cm matches quartz cell. Tablet of 10mg and 20mg were procured from local pharmacy.

Preparation of standard solution:

The pure drug accurately about 5mg was weighed and dissolved 100 ml methanol to give the standard stock

solution of concentration 50 µg/ml. Aliquots of standard stock solution were pipette out and suitably diluted with distilled water to get the final concentration of standard solutions.

Zero order spectroscopic method:

The solutions were scanned in the range from 400-200 nm (method A), and the peaks were observed at 218nm and 303 nm. The wavelength selected for the analysis of the drug was 303nm (figure 2). The drug followed the Beer’s- Lamberts law in the range of 5-35 µg/ml. using the calibration curve the concentration of the sample solution can be determined.

First order derivative spectroscopic method:

The first order derivative spectra at n=1 (method B), showed a sharp peak at 292.0(figure 3).the absorbance difference at n=1 (dA/dλ) is calculated by the inbuilt software of the instrument which was directly proportional to the concentration of the standard solution. The standard drug solution was diluted so as to get the final concentration in the range of 5-40 µg/ml and scanned in the first order derivative spectra. The calibration curve of dA/dλ against concentration of the drug showed linearity.

Area Under Curve Method (AUC):

The AUC (Area Under Curve) method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelength 294nm and 310nm (Figure-4). Area calculation processing item calculates the area bound by the curve and the horizontal axis. The horizontal axis is selected by entering the wavelength range over which the area has to be calculated. The wavelength range is selected on the basis of repeated observations so as to get the linearity between area under curve and concentration. Suitable dilutions of standard stock solution (50µg/ml) of the drug were prepared and scanned in the spectrum mode from the wavelength range 400-200 nm (figure 2) and the calibration curve was plotted.

Figure 1: Chemical structure of Esomeprazole

Figure 2: Zero order spectra of Esomeprazole

All the three method were checked by analyzing the samples with known concentration. As the result obtained were satisfactory, the method was applied for the pharmaceutical formulations.

Analysis of the Tablet formulation:

For the estimation of Esomeprazole in tablet formulation by three methods, 10 tablets of brand were weighed and triturate to fine powder. Tablet powder equivalent to 5mg of Esomeprazole was weighed and the dissolved and further diluted with quantity sufficient with methanol. It was kept for ultrasonication for 30 min; this was filtered through Whatman filter paper no. 41 to get the stock solution of 50 µg/ml. Various dilutions of the tablet solution were prepared and analyzed for six times and the concentration was calculated by using the calibration curve for three methods.

Validation Of the method:

All these methods were validated according to ICH guidelines^5-6 by carrying out analysis of six replicate sample of tablet (tablet 2). Recovery studies were carried out at three different levels i.e. 50%, 100%, and 150% by adding the pure drug to previously analyzed tablet powder sample. From the amount of drug found, percentage recovery was calculated (table 2).

The ANOVA test i.e. Tukey-Kramer Multiple comparison test was applied to determine whether there is significant difference between the results of analysis by three different analysis methods (table 3)

Figure 3: First order derivative spectrum of Esomeprazole with n=1

Figure 4: Wavelength range selected for AUC method of Esomeprazole

RESULTS AND DISCUSSION:

All the methods A, B, and C for the estimation of Esomeprazole in tablet dosage were found to be simple, accurate and reproducible. Beer- lambert’s law was obeyed in the concentration range of 5-35 µg/ml in all these methods. The accuracy of the method was assessed by recovery studies at three different levels i.e. 50%, 100%, 150%. The values of standard deviation were satisfactory and the recovery studies were close to

100%. The %RSD value is less than 2 indicative of accuracy of the method. The developed method was statistically compared using one way ANOVA. The P value was found to be 0.0953 and was greater than 0.05. Hence the results of the ANOVA indicate no significant difference between three methods. Hence these methods can be useful in routine analysis of Esomeprazole in bulk drug and formulations.

Table 1: Optical characteristics and Other Parameters

Parameters	Method A	Method B	Method C
λmax (nm) / wavelength range (nm)	303	2992	294-310
Beer’s-Lambert’s range (µg/ml)	535	5-35	540
Coefficient of Correlation	0.99988	0.9989	0.999032
Regression Equation Y = mx + c
a. Slope(m)	0.03963	0.029484	0.5937
b. Intercept(c)	0.006285	0.016772	0.61071
LOD (µg/ml) LOQ (µg/ml)	0.3 0.9	0.15 0.45	0.25 0.75

Where, A is zero order derivative spectrum method with n = 0. B is first order derivative method with n = 1. C is AUC method.

Table 2: Result of Analysis of Esomeprazole in Tablet and Recovery studies.

METHOD	Tablet	Label laim	% estimated	% recovery	S.D.	% COV	S.E.
A	T1	10	99.85	101.42	0.924	0.925	0.533
	T2	20	100.36	101.17	0.638	0.636	0.368
B	T1	10	99.69	98.50	1.494	1.499	0.862
	T2	20	99.97	99.60	1.304	1.305	0.753
C	T1	10	98.83	100.02	0.481	0.486	0..277
	T2	20	99.88	100.80	0.488	0.448	0.258

Where,

A is zero order derivative spectrum method with n = 0, B is first order derivative method with n = 1, C is AUC method. T1 is Nexium 10, T2 is Nexpro 20

Table 3: One Way ANOVA (Tukey -Kramer Multiple comparison test).

Comparison	Mean Difference	q value	P Value
Method A Vs Method B	2.245	5.655	ns P > 0.05
Method A Vs Method C	0.8850	2.229	ns P > 0.05
Method B Vs Method C	-1.360	3.426	ns P > 0.05

ACKNOWLEDGEMENT:

We are highly thankful to Glenmark Pharmaceutical Limited, Mumbai for providing the gift sample of the pure drug and to the Principal Govt. College Of Pharmacy, Karad for providing excellent research facilities.

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