MATERIALS AND METHODS:

Drugs and Chemicals:

ESO was received as gift sample from Metrochem API Pvt. Ltd., Hyderabad and DOM was received as gift sample from Sidmech Laboratories India Pvt. Ltd., Dehradun. Methanol was used of analytical grade. All other reagents used were of analytical grade for the forced degradation studies. The pharmaceutical dosage form used in this study was Esofag-D labelled to contain 40mg of ESO and 30mg of DOM per capsule were purchased from local market.

Apparatus:

A UV-Visible spectrophotometer, model UV-3200 (Labindia) with 1cm quartz cells. A electronic balance (Roy electronics- LCBCN5) was used for weighing the samples. Hot Air Oven (Coslab CLE-101) was used for the thermal degradation study. A Sonicator (Labfit) was also used.

Standard stock solution:

Standard stock solution (1000µg/ ml) of ESO and DOM were prepared by dissolving accurately about 50 mg of each drug separately in methanol in 50 ml volumetric flask. The working solution was in the range of 1-6µg/ ml for ESO and 5-30µg/ ml for DOM were prepared by further dilution for calibration curves (Figure 3).

METHOD DEVELOPMENT:

Absorption ratio method:

Selection of λ₁and λ₂ for ESO and DOM:

The isobestic point (where both the drugs show equal absorbance) was obtained from the overlain spectra of ESO and DOM. The overlain spectra showing isobestic point at 291 nm. Thus 291 nm was selected as λ₁and λ₂was selected at 299 nm which was λ_max for ESO (Figure 2).

Fig. 2: Isoabsorptive point of esomeprazole and domperidone.

Fig. 3: Linearity curve of esomeprazole and domperidone.

Preparation of standards for test for linearity:

A calibration curve was plotted over a concentration range of 1-6 µg/ml for ESO and 5-30 µg/ml for DOM. Accurately measured standard stock solution of ESO (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 ml) and standard stock solutions of DOM (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ml) were transferred to a separate series of 10 ml of volumetric flasks and diluted to the mark with methanol. The different concentration of solutions were scanned over the range of 200 nm to 400 nm and absorbance at 291 nm and 299 nm for ESO and DOM were

measured respectively in the quantitative mode of the instrument and the absorptivity was calculated.

From the absorbance value the absorptivity value at respective wavelength i.e. λ₁ (291nm) and λ₂(299 nm) was calculated by dividing absorbance and respective concentration. Similarly for ESO the absorbance value was recorded at 291 and 299 nm and the absorptivity was calculated. The concentration of ESO and DOM were calculated by using the following equations.

C_x = (Q_M- Q_Y) A₁/ (Q_X- Q_Y) a_x1

Where, A₁and A₂are the absorbance of the mixture at 291 nm and 299 nm respectively; a_x1and a_y1are absorptivities of ESO and DOM respectively at 291 nm; a_x2and a_y2are absorptivities of ESO and DOM respectively at 299 nm.

Q_M= A₂/ A₁

Q_{X =}a_x2/a_x1and

Q_Y= a_y2/ a_y1

Analysis of commercial formulation:

Twenty capsules were accurately weighed and its contents crushed to fine powder. Powder equivalent to 40 mg of ESO and 30 mg of DOM was weighed and dissolved in methanol, sonicate for 10 minute and filtered through Whatman’s filter paper no. 41. After rejecting first few ml, different concentrations of capsule sample were prepared by serial dilution technique and analyzed at 291 and 299 nm wavelength (Table 2).

METHOD VALIDATION (Table 1):

Recovery studies:

To study the accuracy of the proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120% of the test concentration as per ICH guidelines). A known amount of drug was added to pre analyzed capsule powder and percentage recoveries were calculated. The result of recovery studies was satisfactory.

Linearity and range:

The six point calibration curve that were constructed were linear over the concentration range between 1-6 µg/ ml for ESO and 5-30 µg/ ml for DOM respectively. Each concentration was repeated for 3 times.

Precision:

For evaluation of intraday precision repeatability of the result was evaluated for the concentration of 1 µg/ ml for ESO and 15 µg/ ml for DOM by 3 replicate determination at interval of 1 hours and for evaluation of interday precision repeatability of the result was evaluated for the concentration of 1 µg/ ml for ESO and 15 µg/ ml for DOM by 3 replicate determination at interval of 1 hours for 3 days.

Limit of detection:

Limit of detection for ESO was found to be 0.116 and for DOM was found to be 0.657.

Limit of quantification:

Limit of quantification for ESO and DOM was found to de 0.386 and 2.18 respectively.

Robustness:

Robustness of proposed method was performed by changing the UV analyst and remaining condition was keeping constant.

STABILITY INDICATINGASSAY METHOD (Table 3)

Acid degradation:

In the 1µg/ml solution of ESO and 15µg/ml solution of DOM 10 ml 1N HCl were added and kept at room temperature for 24 hours (Figure 4).

Base degradation:

In the 1µg/ml solution of ESO and 15µg/ml solution of DOM 10 ml 1N NaOH were added and kept at room temperature for 24 hours (Figure 5).

Table1. Validation parameters for UV-Spectroscopic methods

Sr. No.	Validation Parameter	Mean±SD
Sr. No.	Validation Parameter	Esomeprazole magnesium	Domperidone
1.	Linearity range	1-6µg/ ml	5-30 µg/ ml
2.	Correlation coefficient	0.998	0.999
3.	Slope	0.215	0.0292
4.	Intercept	0.0014	0.0149
5.	Accuracy
6.	Interday Precision
	(1^st day)	104±0.000772	172± 0.1212
	(2^nd day)	136± 0.01928	156± 0.00442
	(3^rd day)	152± 0.003478	153±0.003813
	Intraday precision
	(1^st hrs)	104± 0.000115	116±0.000287
	(2^nd hrs)	104± 0.000125	196±0.000386
	(3^rd hrs)	104± 0.000126	203±0.000562
7.	Recovery
	80%	95±0.000327	43±0.000712
	100%	67±0.00106	53±0.00245
	120%	72±0.00332	56±0.000356
8.	LOD (mg/ ml)	0.116mg/ ml	0.657mg/ ml
9.	LOQ (mg/ ml)	0.386mg/ ml	2.18mg/ ml
10.	Robustness	120±0.00156	107±0.00272

Table.2 Analysis of commercial formulation in capsule dosage form

Formulation

Drug

Label claim (mg)

% Label claim

(Mean± SD)

Capsule

Esomeprazole magnesium

Domperidone

40 mg

30 mg

92± 0.01845

98± 0.000573

Table.3 Forced Degradation Studies

Condition	Absorbances (λ)		Mean ± SD^*		Result (% degradation)
Condition	ESO	DOM	ESO	DOM	ESO	DOM
Acid degradation			-	-	-	-
Alkaline degradation
Thermal degradation
Photolytic degradation

Fig. 4: Acidic degradation of esomeprazole and domperidone.

Fig. 5: Basic degradation of esomeprazole and domperidone.

Thermal degradation:

About 50mg of drug substance kept at 60ºC for 8 hours. Then the solution was prepared to achieve 1µg/ml for ESO and 15µg/ml for DOM respectively (Figure 6).

Fig. 6: Thermal degradation of esomeprazole and domperidone.

RESULTS AND DISCUSSION:

In a quantitative assay of two components in admixture by absorbance ratio method, absorbances are measured at any two wavelengths, one being the iso absorptive point of two components and other being the wavelength of maximum absorption of one of the two components. Thus, two selected wavelengths for this method were 291 nm (iso-absorptive point) and 299 nm λ_max of ESO. The absorbencies and absorptivity at these wavelengths were determined and substituted in equation 1 to obtain the concentration of both the drugs. Percent label claim in given tablet formulation were 100.05 for ESO and 100.10 for DOM were found to be satisfactory. The method was validated as per ICH norms.

CONCLUSION:

All these factors lead to the conclusion that the stability indicating absorption ratio method development is accurate, precise, simple, sensitive and rapid and can be applied successfully for the estimation of ESO and DOM in bulk and in pharmaceutical formulations without interference. The relative standard deviation (RSD) for all parameters was found to be less than one, which indicate that the validity of method are also within the limit so the proposed method can be used for routine quantative simultaneous estimation of both the drug.

ACKNOWLEDGEMENT:

The authors are thankful to Metrochem API Pvt. Ltd., Hyderabad and Sidmak Laboratories India Pvt. Ltd., Dehradun for providing gift sample of ESO and DOM and also thankful to the project guide and management, Invertis Institute of Pharmacy, Invertis University, Bareilly to providing the equipment and facility for entire duration of research work.

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Received on 15.04.2013 Modified on 25.05.2013

Asian J. Research Chem. 6(8): August 2013; Page 735-739