INTRODUCTION:

Lansoprazole (LNS), 2-{(3-methly-4-(2,2,2-trifluoroethoxy)-2-pyridyl) methyl} sulfinyl benzimidazole as shown in Figure 1, a benzimidazole derivative is a proton pump inhibitor with antisecretory and antiulcer activities¹. It is successfully used for the treatment of those resistant to treatment with histamine H₂ receptor antagonists, hence effective in treating various peptic diseases such as duodenal ulcer, gastric ulcer, reflux oesophagitis, Zollinger-Ellision syndrome and other hyper acidic-related conditions². This drug is official in the United States Pharmacopoeia³ and the British Pharmacopoeia⁴. USP and BP describe a high performance liquid chromatographic method; in addition BP recommends potentiometric titration too for LNS estimation. Different analytical methods have been reported in the literature for the determination of LNS in either individual or in pharmaceuticals combinations and include many techniques as high pressure liquid chromatography^5-9, high pressure thin layer chromatography¹⁰, visible spectrophotometry¹¹, flow-injection analysis with UV-detection¹² and UV-spectrophotometry^13-14.

According to our best of knowledge, there is no validated stability indicating method covering degradation behavior of LNS along with its determination in bulk drug and capsule dosage form appeared in the literature.

Figure 1: Lansoprazole

MATERIALS AND METHOD:

Instrumentation:

Spectrophotometric analysis was carried out on a JASCO V-630 UV- Visible spectrophotometer using a 1 cm quartz cell. The instrument settings were zero order and band width of 1.0 nm in the range of 200–400 nm.

Sample:

Pharmaceutical grade LNS was obtained from Macleods Pharmaceuitcals Ltd, Mumbai, India. Commercial formulation of brand name Lanzol-30 (Cipla Ltd, Sikkim, India) was purchased from local commercial sources.

Reagents and Chemicals

All reagents and chemicals used were of analytical reagent grade whereas acetonitrile (ACN) was of HPLC grade (Finar Chemicals Ltd., Ahmedabad, India). Double-distilled water was used to prepare solutions wherever required. Aqueous solutions of hydrochloric acid (Loba Chemie Pvt Ltd., Mumbai, India), sodium hydroxide (Merck, Mumbai, India) and Hydrogen Peroxide (Loba Chemie Pvt. Ltd., Mumbai, India; 30% w/v) were used to prepare required volumetric reagents.

Selections of solvent:

In the present study, different solvents were investigated to develop a suitable UV spectrophotometric method for the analysis of LNS in bulk and formulation. For selection of diluent, the criteria employed were the solubility of the drug, the easiness of the sample preparation, specificity of the method. ACN:Water (70:30 v/v) was used as the diluent because of the total solubilization of the drug in this diluent. Both the components of the diluent were selected as the cut off wavelengths of both of them is below 200 nm hence it will not interfere in the absorption of wavelength in the UV range.

Forced degradation studies:

For the degradation studies, LNS at a concentration of 20 µg/ml was used. Forced degradation was carried out to analyze the degradation behavior and also explaining the stability-indicating property, specificity of proposed method. The degradation was done under strong as well as moderate conditions. The initial absorbance of the drug, at zero time was considered as 100% concentration and degradation was correlated with this concentration [Figure 2].

Acid and base induced degradation:

Acid decomposition studies were performed by exposing 1 ml of drug solution (200 µg/ml) to 2 ml of 1M hydrochloric acid at 80°C in different 10 ml calibrated flask dedicating for each hour respectively up to 8 hours. Similar procedure was employed for alkali degradation studies, where in LNS was exposed to alkali conditions by replacing 1M hydrochloric acid with 1M sodium hydroxide. For analysis of degraded samples, flask at respective time interval was cooled at room temperature and diluted to 10 ml with the solvent to get 20 µg/ml concentrations. Absorbance was measured against acid and alkali blank for acid and alkali hydrolysis respectively.

Hydrogen peroxide induced degradation:

To study the effect of oxygen, 1 ml of the drug solution (200 µg/ml) was exposed to 2 ml of 3% hydrogen peroxide at 80°C in different 10 ml calibrated flask dedicating for each hour up to 8 hours. The resultant solutions at regular time interval were cooled to room temperature and diluted to obtain 20 µg/ml concentrations. Absorbance was measured against blank prepared by diluting 2 ml of 3% hydrogen peroxide to 10 ml with solvent.

Thermal and Photochemical Degradation:

Standard LNS sample was taken in two different petriplate. One petriplate was kept in an oven at 105°C for 5 hours satisfying dry heat stress conditions whereas the other was exposed to UV light in a UV chamber at 12000 lux hour for 5 days. The same samples were used to prepare the solution of concentration 20 µg/ml. The absorbance of the working solution was measured at λ_max (284nm).

Effect of placebo blank interference:

To assess the usefulness of the method for estimation of LNS pharmaceutical dosage forms, the effects of diluents, excipients and additives which often accompany LNS in its dosage forms (talc, starch, acacia, methyl cellulose, sodium citrate, magnesium stearate and sodium alginate) were studied. The results indicated that there is no placebo interference, indicating a high selectivity for determining the LNS in its dosage forms.

Procedure for capsules:

The granules of ten capsules (Lanzol-30, 30 mg LNS per capsule) were crushed, powdered, weighed out and the average weight of one capsule was determined. An accurate weight equivalent to 20 mg LNS was transferred into a 100 ml volumetric flask containing 30 ml of ACN:Water (70:30v/v) and the flask was kept for ultrasonication for 15 min, then it was diluted up to the mark with the same diluent and the solution was filtered through Whatman filter paper No. 42. From the above solution 1 ml was pipetted out into a 10 ml volumetric flask and the volume was made up to the mark with the solvent. The final concentration of LNS was brought to 20 μg/ml and used for the analysis.

RESULTS AND DISCUSSION:

Spectral characteristics:

The zero order absorption spectrum of 20 μg/ml LNS solution was recorded between 200-400 nm and showed an absorption maximum at 284 nm, and at this wavelength the solvent had insignificant absorbance. Therefore, 284 nm was used as analytical wavelength (λ_max). Figure 2 represents the absorption spectra of LNS (20 µg/ml) against the diluent blank. The solutions which were partially degraded by various stress conditions shows decrease in absorbance and hence was considered as a direct measure of extent of degradation.

Degradation Profile:

For acid hydrolysis, drug solution was exposed to 0.1 M, 0.5 M and 1 M hydrochloric acid (HCl) at 80°C for up to 8 hour but 1 M concentration for degradation study was found suitable. It was observed that LNS remains surprisingly stable in 0.1 M and 0.5 M HCl challenging its weak basic nature. On the otherside LNS when exposed to 1 M HCl at 80°C, 21.66% of LNS was found degraded after 4hour. The drug remains nearly stable after undergoing partial degradation from 4^th till 8^th hour in acidic stress condition (Figure 8). The degradation after 8 hour was found to be 24.66%(Figure 3). The decomposition route is proposed on the basis that under acidic conditions, the imidazole nitrogen is protonated because of the high basicity of benzimidazole nitrogen in LNS (lack of electron withdrawing group in benzimidazole ring¹⁵. Then the lone pair of electrons on the imidazole nitrogen undergoes nucleophilic attack at the electron deficient carbon (2^nd carbon) of the imidazole ring. This results in the formation of sulfinamide derivative of LNS.

For alkali stress condition, drug solution was exposed to 0.1 M, 0.5 M and 1 M sodium hydroxide (NaOH) at 80°C for up to 8 hour, but 1 M concentration for degradation study was found suitable. The degradation was much slower in alkali conditions as 11.14% drug was degraded in 1 M NaOH at 80°C after 2 hour. The drug remains stable after undergoing partial degradation from 2^nd till 8^th hour in alkali stress condition (Figure 8). The degradation after 8 hours was found to be 12.66%(Figure 4).

For oxidative hydrolytic study, drug solution was subjected to 1%, 3% and 6% concentration of hydrogen peroxide (H₂O₂), but at 3% concentration suitable degradation resulted. The drug was found to be unstable in presence of oxygen. The reaction in 6% H₂O₂ at 80°C was so fast that around 37% of the drug was found degraded in 1 hour. 13% degradation was observed when exposed to 3% H₂O₂ at 80°C after 3 hours and increasing sharply with due course of time. (Figure 8). The degradation after at 8 hour was found 74.12%(Figure 5). During H₂O₂ oxidation, it is proposed that may be the sulphoxide gets converted to sulphonyl group giving sulfone.

LNS was found thermally and photo-chemically stable as the UV spectra of stressed LNS samples were similar to that of the standard LNS sample (Figure 6&7), having nearby same absorbance indicating no degradation under these conditions.

METHOD VALIDATION¹⁶

Linearity:

A stock standard solution containing 20 mg LNS in 100mL of diluent was prepared to give the final concentration 200 µg/ml. Linearity was studied by analyzing eleven concentrations of the drug prepared in the solvent in the range of 2-40 μg/ml in triplicate and fitting the data into best fitted curve. Suitable aliquots were prepared ranging from 0.1 to 2.0 ml by transferring accurately from the LNS stock standard solution and diluted to 10 ml with the solvent to give concentration in the range of 2 to 40 μg/ml. The absorbance of the resulting solution was measured at 284 nm against ACN:Water (70:30v/v) blank. Calibration curve was prepared by plotting the absorbance versus concentration of drug.(Figure 9) Correlation coefficient, intercept and slope for the calibration data are summarized in Table 1. The limit of detection and limit of quantification are calculated as per the current ICH guidelines are compiled in the same table; speak of the excellent sensitivity of the proposed method.

Figure 9: Linearity and Calibration curve of LNS at 284 nm.

Table No. 1: Optical Characteristics.

Parameters	Results
Wavelength (nm)	284 nm.
Beer-Lambart’s Law (µg/ml).	2-40 µg/ml.
LOD (µg/ml)	0.13 µg/ml
LOQ (µg/ml)	0.38 µg/ml
Regression Equation	Y= mX+c
Slope (m)	0.046
Intercept (c)	-0.009
Correlation coefficient (r²)	0.998

Accuracy:

Accuracy of the proposed method was tested by performing recovery study by standard addition method. The standard LNS sample was added to pre-analysed marketed dosage form (20 µg/ml) at the level of 80, 100 and 120%. The percentage of the recovery, lower values of standard deviation and relative standard deviation indicates the accuracy of the method (Table No. 2).

Table No. 2: Recovery of the drug from synthetic mixture.

Name of Formulation	Level of Recovery.	Percent recovery* ± S.D.	% R.S.D.
Lansol-30	80%	99.40±1.0392	1.0455
	100%	105.70±1.0392	0.9832
	120%	100.20±0.3464	0.3457

* Average of three determinations.

Precision:

Precision was verified by repeatability and intermediate precision studies. Repeatability was established by analyzing three different concentrations of LNS marketed dosage form in triplicate on the same day whereas intermediate precision was checked by repeating the studies on 3 consecutive days. Precision of the method was expressed in terms of percentage relative standard deviation (%RSD). The intra-day and inter-day assay of LNS by the proposed method was found to be suitable with very low values of standard deviation. This justifies the reproducibility and repeatability of the proposed method (Table No. 3).

Table No. 3: Evaluation of intra-day and inter-day precision.

Lanzol-30 (µg/ml)	Intra-day assay precision(n=3)		Inter-day assay precision(n=3)
Lanzol-30 (µg/ml)	Percent Found* ± S.D.	% R.S.D.	Percent Found* ± S.D.	% R.S.D.
10	101.30± 0.5196	0.5129	100.83± 0.9712	0.9632
20	98.10± 0.7506	0.7651	100.20± 0.3464	0.3457
30	99.40± 0.5749	0.5784	101.90± 0.5196	0.5001

* Average of three determinations.

Application to capsules:

In order to evaluate the analytical applicability of the proposed method to the quantification of LNS in commercial capsules, assay was performed for Lansol-30 capsules by the proposed method. The results (Table No. 5) showed that there is a good agreement between the results obtained by the proposed method and the label claim of the marketed preparation.

Table No. 4: Results of analysis of capsules.

Name of Formulation

(Brand Name)

Label Claim

(mg/capsule)

% Label Claim Found* ± S.D.

% R.S.D.

Lansol-30

102.55±0.8092

0.7891

* Average of five determinations.

CONCLUSION:

The present study reports a simple method for quantification of LNS in capsules as well as stress testing study. Comparing the results of various stress conditions, LNS proves itself to be stable from thermal and photolytic exposure. The conditions maintained for acidic, basic and oxidative stress conditions were quite harsh to withstand so LNS API shows degradation but still justifies its stability. Moreover, Stress testing is an important aspect of the drug development process. The previously reported HPLC and other methods for degradation studies are complex and cumbersome and are crucially depend on judicious control of pH of the medium besides requiring expensive and sophisticated instruments. In contrast, the present method requires simple and inexpensive instrument producing relatively more and accurate results, and thus can be used for the routine determination of LNS in its available dosage forms.

ACKNOWLEDGEMENTS:

The authors wish to acknowledge, Macleods Pharmaceuticals Ltd, Mumbai, India, for providing the gift sample of lansoprazole. The authors also thank the authorities of the Bharati Vidyapeeth’s College of Pharmacy, Kolhapur, for permission and facilities. The corresponding author also wishes to thank All India Council of Technical Education (AICTE), for the award of PG Research Fellowship.

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Received on 08.05.2012 Modified on 27.05.2012

Research J. Pharm. and Tech. 5(6): June 2012; Page 822-828