1. INTRODUCTION:

Esomeprazole (ESO) belongs to gastrointestinal drugs category to suppress the gastric acidity and treatment of peptic ulcer by inhibiting the proton pump. Chemically it is bis (5-methoxy-2-((S)-((4-methoxy-3, 5-dimethyl-2-pyridinyl) methyl) sulfinyl)-1H-benzimidazole-1-yl) magnesium trihydrate¹. The chemical structures of drugs are shown in Figure 1. Esomeprazole ( S-isomer of omeprazole), the first single optical isomer proton pump inhibiter, generally provides better acid control than current racemic proton pump inhibiters and has a favorable pharmacokinetic profile relative to omeprazole. Esomeprazole blocks the enzyme in the wall of the stomach that produces acid. By blocking the enzyme, the production of acid is decreased, and this allows the stomach and esophagus to heal^2-6. The literature survey reveals few HPLC^7-15,HPTLC^16,17 and spectrophotometric^18-22 methods for estimation of from its formulations. The present paper describes simple, rapid and precise HPTLC method for the determination of esomeprazole in pharmaceutical dosage form.

2. MATERIALS AND METHODS:

2.1 Materials

An analytically pure sample of Esomeprazole magnesium trihydrate was a gift from Torrent Pharmaceuticals, Ahemadabad. All chemicals, including methanol, ethyl acetate and ammonia were of analytical reagent grade (S.D. Fine Chemicals, Mumbai, India) and were used without further purification.

2.2 Standard solutions:

An accurately weighed 10 mg of esomeprazole magnesium trihydrate was transferred to100.0 mL volumetric flask. It was dissolved in 25 mL of methanol and the volume was made up to the mark using same solvent to obtain concentration of 100µg/mL. The above stock solution ranging from 1- 6 μL was applied on TLC plates by microlitre syringe with the help of automatic sample applicator. The plates were developed in twin trough glass chambers. After development, the plates were immediately dried and densitometrically scanned at 301 nm. Peak areas were recorded for each concentration of drug and curve of concentration against peak area was plotted. The standard curve was found to be linear between concentration range of 100- 500 ng/spot.

2.3 Sample preparation:

Twenty tablets were weighed and powdered and an amount of powder equivalent to 20 mg esomeprazole magnesium trihydrate was transferred to a 100 mL volumetric flask. The powder was extracted with methanol for 10 min, by shaking mechanically, and the extract was then diluted to volume with the same solvent and filtered. A sample solution (1 µL, containing 200 ng) was applied on TLC plates followed by development and scanning as described in chromatographic conditions. The analysis was repeated in triplicate.

Figure 1. The chemical Structure of esomeprazole magnesium trihydrate.

2.4 Chromatographic conditions:

Chromatography was performed on 10cm x 10cm aluminium backed silica

gel 60 F₂₅₄TLC plates (E.Merck, Darmstad, Germany) stored in a desiccator (prewashed with Methanol). Spotting was done on the TLC plates by means of Hamilton micro syringe (Switzerland), mounted on a Linomat V applicator (Camag, Muttenz Switzerland). Samples were applied as 6 mm wide bands at a spraying rate of 15s µL^-1; and the distance between the bands was 11.6 mm. Ascending development of the plates, migration distance 70mm, (distance to the lower edge was =10mm) was performed at 25±2°C with ethyl acetate- ammonia, 8: 0.8 (v/v), as mobile phase in a Camag chamber previously saturated with mobile phase for 10 minutes. The average development time was 15 minutes. After development the plates were dried at 50°C in an oven for 5 minutes. Densitometric scanning was then performed with a Camag TLC scanner III equipped with Wincats Software Version 1.3.0 at λ _max= 301 nm using Deuterium light source; the slit dimensions were 6.00 Х 0.45 mm.

3. RESULT AND DISCUSSION:

Validation of the Method:

The method was validated in accordance with ICH guidelines^23-27.

3.1 Linearity

Amounts of standard solutions equivalent to 100, 200, 300, 400, and 500 ng/spot esomeprazole magnesium trihydrate were applied to a prewashed TLC plate. The plate was developed, dried, and scanned as described above. A calibration plot was constructed by plotting peak area against amount of esomeprazole magnesium trihydrate (ng/spot). The linearity of the response to esomeprazole magnesium trihydrate was assessed in the concentration range 100 – 500 ng/spot; the slope, intercept, and correlation coefficient were also determined. Over the concentration range studied, the correlation coefficient for the calibration plot was r = 0.9992 and the slope was 10.61 ± 0.06 (n = 6). The linearity data is given in table 1.

Table 1. Linearity data

Parameters	Esomeprazole magnesium trihydrate
Beer’s law limit (ng/spot)	100-500
Correlation Coefficient	0.9992
Intercept	1251.8
Slope	10.616

3.2 Analysis of the marketed formulation

A single spot at R_f 0.60 was observed in the chromatogram of the drug samples extracted from tablets. There were no interferences from the excipients commonly present in the tablets. The results are shown in Table 2.

Table 2. Estimation of esomeprazole magnesium trihydrate in tablets.

	Esomeprazole magnesium trihydrate
Labeled Claim (mg)	20
Amount found (%) ±SD (n = 5)	99.95 ± 0.21
% RSD	1.47

3.3 Sensitivity:

The sensitivity of measurement of esomeprazole magnesium trihydrate using the proposed method was estimated in terms of the limit of quantitation (LOQ) and the lowest concentration detected under the chromatographic conditions as the limit of detection (LOD). The LOQ and LOD were calculated using equations LOD = 3 × N/B and LOQ = 10 × N/B; where, ‘N’ is the standard deviation of the peak areas of the drug (n = 3), taken as a measure of noise, and B is the slope of the corresponding calibration plot. The LOQ and LOD were found to be 1.24 ng and 0.37 ng respectively.

3.4 Accuracy:

The accuracy of the method was determined by analysis of standard additions at three different levels, i.e. multiple-level recovery studies. A stock solution of a tablet formulation containing 200 ng per µL esomeprazole magnesium trihydrate was prepared. Amounts of standard drug solutions equivalent to 80%, 100%, and 120% of the concentration in the tablet solution were added and recovery (%) was determined. Values were found to be within the limits given in Table 3.

Figure 2. Typical HPTLC Chromatogram of esomeprazole magnesium trihydrate Standard Drug Solution. , measured at 301 nm, mobile phase Ethyl acetate- ammonia, 8 + 0.8 (v/v).

Table 3. Results of recovery studies

Drug	Level of recovery (%)	Excess drug added [ng]	Drug recovered mean±SD [ng]	%Recovery ( n = 3)	% RSD
ESO	80	160	162.26±0.67	101.41	0.41
	100	200	201.49±1.14	100.75	0.57
	120	240	241.52±1.36	100.63	0.56

Table 4. Results of precision studies.

		Intra-day precision		Inter-day precision
Drug	Amount applied (ng/spot)	Amount found (ng/spot) mean±SD	%RSD (n = 3)	Amount found (ng/spot) mean±SD	%RSD (n = 3)
ESO	200	198.47±0.33	0.16	198.95±1.18	0.59
	300	301.11±1.28	0.43	299.68±1.49	0.49
	400	399.09±1.95	0.49	398.33±0.24	0.06

3.5 Precision:

Precision was measured by analysis of sample solutions containing esomeprazole magnesium trihydrate at concentrations covering the entire calibration range. The precision of the method in terms of intra-day variation (% R.S.D.) was determined by analysis of esomeprazole magnesium trihydrate standard solutions in the range 100 – 500 ng/spot, three times on the same day. Inter-day precision (%R.S.D.) was assessed by, analysis of the same solutions on three different days over a period of one week. The results of the precision studies are shown in Table 4.

3.6 Repeatability:

The repeatability of sample application was assessed by spotting drug solution (2 µL) seven times on a TLC plate, then developing the plate and recording the peak heights and areas of the spots. The % R.S.D. for peak-height and peak-area values of esomeprazole magnesium trihydrate was found to be 1.43 and 1.68 respectively.

Figure 3. Typical Absorption Spectrum obtained from standard esomeprazole magnesium trihydrate solution.

3.7 Specificity:

The mobile phase designed for the method resolved the drug very efficiently shown in Figure 2. The R_fof esomeprazole magnesium trihydrate was 0.6. A typical absorption spectrum of esomeprazole magnesium trihydrate is shown in Figure 3. The wavelength 301 nm was selected for detection because it is resulted in better detection sensitivity for the drug. The spot for esomeprazole magnesium trihydrate from the tablet formulation was identified by comparing its R_fvalue and its absorbance reflectance spectrum with those of standard esomeprazole magnesium trihydrate. The peak purity of esomeprazole magnesium trihydrate was tested by comparison of spectra acquired at the peak-start (S), peak-apex (A), and peak-end (E) positions of the spot. The correlation between these spectra were indicative of the purity of esomeprazole magnesium trihydrate peak (correlation r(S, M) = 0.999, r (M, E) = 0.998), shown in Figure 4.

Figure 4. Peak purity spectras of esomeprazole magnesium trihydrate extracted from esomeprazole magnesium trihydrate tablet, scanned at the peak-start, peak-apex, and peak-end positions of the spot (correlation > 0.99).

3.8 Ruggedness and Robustness:

Ruggedness is a measure of the reproducibility of a test results under normal, expected operating conditions from instrument to instrument and from analyst to analyst. The results of ruggedness testing are reported in Table 5. Robustness is a measure of the capacity of a method to remain unaffected by small but deliberate variations in the method conditions, and is an indication of the reliability of the method. Typical results from robustness studies are as shown in Table 6.

Table 5. Results of ruggedness studies.

Amount found

mean±SD (ng/spot)

Amount found (%)

%RSD

(n = 6)

Analyst I

199.89±3.57

99.94

1.78

Analyst II

199.70±3.27

99.85

1.63

Table 6. Results of robustness studies.

Development distance (cm)	ESO (%) (20 mg)
7.0	97.97
7.5	98.96
8.0	98.34

3.9 System Suitability:

According to USP 28, method 621, system-suitability tests are an integral part of chromatographic analysis and it is used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis. To ascertain the effectiveness of the method developed in this study, system-suitability tests were performed on freshly prepared standard stock solutions interspersed with replicate sample solutions of esomeprazole magnesium trihydrate.

3.10 Stability studies:

To test the stability of drugs on the TLC plates, freshly prepared solutions of the analyte were applied to the plate and the plates were developed after intervals of 3 h, 24 h, and 48 h (36). No decomposition of the drug was observed during chromatogram development. No decrease in the concentration of drug on the plate was observed within 3 h. The decrease in the amount of esomeprazole magnesium trihydrate was observed 24 h after development. The results of the stability studies are shown in Table 7.

Table 7. Results of stability studies

Time (h)	Drug loss ± SD (%)
3	No loss
12	2.12 ± 0.27
24	5.13 ± 0.24

REFERENCES:

1. Neil J.O. (Ed.) The Merck Index, 13th edn, Merck, White House Station, NJ, USA, 2001, p. 6913

2. Martindale – The Complete Drug Reference, 33rd edn, Pharmaceutical Press,London, 2002, p.1225

3. Riley T. N. and Deruiter J., Block J. H., Beale J.M.; ‘Wilson and Gisvold’s Textbook of Organic Medicinal and Pharmaceutical Chemistry’, Lippincott Williams and Wilkins, 2004, p 724.

4. Tripathi K.D., Essentials of medical pharmacology, 5thedn., Jaypee Brothers, Medical Publisher Ltd, New Delhi, 2003, p. 588-593.

5. Kale-Pradhan P. B., Landry H. K., and Sypula. W. T., Ann. Pharmacology 2002, p. 655-663.

6. Scott L.J., Dunn C.J., Mallarkey G.and Shape M., Drugs, 2002, pp. 1503-1538.

7. Hultman I., Stenhoff H. and Liljeblad M.; Determination of esomeprazole and its two main metabolites in human, rat and dog plasma by liquid chromatography with tandem mass spectrometry. J. Chrom. B, 2007; 848(2); 317.

8. Setty R., Subramanian G., Ranjith Kumar A., Pandey S., Udupa N.; Estimation of esomeprazole in human plasma by reverse phase high performance liquid chromatography, Indian Drugs, 2005; 42(3); 158.

9. Onal A. and Oztunc A. J., Development and Validation of High Performance Liquid Chromatographic Method for the Determination of Esomeprazole in Tablets, J. Food and Drug Anal., 20061; 4(1); 12.

10. Roosewelt C., Magesh, A. R., Ravisundar, A. , Pandiyan, P. S., Muthuprasanna, P. Gunasekaran V., Estimation of Esomeprazole by RP-HPLC in Pure and Pharmaceutical Dosage Form, Asian J. Chem., 2007; 19(5); 3959-3962

11. Patel B. H. , Suhagia B. N. , Patel M. M. and Patel J. R. , Determination of Pantoprazole, Rabeprazole, Esomeprazole, Domperidone and Itopride in Pharmaceutical Products by Reversed Phase Liquid Chromatography Using Single Mobile Phase Chromatographia 2007; 65(11-12);743-748 .

12. Roosewelt C., Magesh A. R., Ravisudar A., Shanmuga Pandian P., Muthuprasanna P.and Gunasekaran V. , Simultaneous Method Development and Validation of Esomeprazole and Domperidone in Pure and Pharmaceutical Dosage Forms by RR-HPLC, Asian J. Chem., 2007; 19(7) 46 PP: 5313 - 5318

13. Roosewelt C., Magesh A.R., Ravisundar A., Shanmuga Pandian P., Muthuprasanna P. and Gunasekaran V., Estimation of Esomeprazole by RP-HPLC in Pure and Pharmaceutical Dosage Form, Asian J. Chem., 2007; 19(5); 3959 - 3962

14. Gawande V. V. and Puranik M. P., RP-HPLC Method for Simultaneous Estimation of Domperidone in Combination with Esomeprazole Magnesium in Solid Dosage Form, Asian J. Chem., 2009; 21(8); 6459 - 6462

15. Tang X.H.,. Hou J.P, Liu A., Xie Y.M., Hu X. and Song X., HPLC Determination of Esomeprazole Magnesium in Tablets, Asian J. Chem., 2010; 22(5); 3711 - 3718

16. Dalindre H.N., Thorve R.R., Bugdane P.M., Vekariya N.R., Londhe S.G., Validated HPTLC method for simultaneous estimation of esomeprazole and domperidone in tablets, ACAIJ, 2008; 7(6), 350-353,.

17. C. Roosewelt, A. R. Magesh, A.G. Sheeja Rekha, P. Shanmuga Pandian and V. Gunasekaran, Simultaneous Estimation and Validation of Esomeprazole and Domperidone by HPTLC in Pure and Pharmaceutical Dosage Forms, Asian J. Chem.,2007; 19(4); 2955 - 2960

18. P. M. Raj, R. V. Rao, P. B. Mukherjee, V. S. Sarvanan, N.Gopal, T. M. Kalyankar, T. Shivakumar; UV-Spectrophotometric Determination of Esomeprazole in Tablet Dosage Forms, Asian J. Chem., 2007; 19(4); 3250 .

19. Patil S. S, Dhabale P. and Kuchekar B., Development and Statistical Validation of Spectrophotometric Method for Estimation of Esomeprazole in Tablet Dosage Form, Asian J. Research Chem. 2009; 2(2): 154-156

20. Prabu S. L, Shirwaikar A, Shirwaikar A., Kumar C. D., Joseph A, Kumar R, Simultaneous estimation of esomeprazole and domperidone by UV spectrophotometric method, Ind. J. Pharm. Sci., 2008; 70(1); 128-131.

21. Nafisur R., Zehra B. and Syed N., Hejaz A., Spectrophotometric Determination of Esomeprazole Magnesium in Commercial Tablets Using 5-Sulfosalicylic Acid and N-Bromosuccinimide, J. Chinese Chem. Soc., 2008; 55; 557-566

22. Pillai S. and Singhvi I., Spectrophotometric Methods for Simultaneous Estimation of Esomeprazole Magnesium and Domperidone from Pharmaceutical Dosage Form, Asian J. Chem., 2007; 19(6); 4785 – 4789.

23. Sethi P.D., HPTLC: Quantitative Analysis of Pharmaceutical formulations, CBS Publication, New Delhi, 1996, pp. 162-165.

24. ICH guidelines Q2A, Validation of Analytical Procedures – Methodology, 1994

25. Reviewer Guidance, Validation of Chromatographic Methods, 1994

26. ICH Guidelines Q2B, Validation of Analytical Procedures – Methodology, 1996

27. United States Pharmacopeia – The National Formulary (USP28/NF 23), United States Pharmacopeial Convention, Rockville, MD, 2004, p. 2622-24

Received on 06.05.2010 Modified on 19.05.2010

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1185-1188