1. INTRODUCTION:

Esomeprazole (ESO) (Figure1) bis(5-methoxy-2-[(S)-[(4-methoxy-3,5-dimethyl-2-pyridinyl) methyl]sulfinyl]-1-H-benzimidazole-1-yl) is a compound that inhibits gastric acid secretion. Esomeprazole is cost effective in the treatment of gastric oesophageal reflux diseases. Esomeprazole is the S-isomer of omeprazole, the first single optical isomer proton pump inhibitor, generally provides better acid control than racemic counterpart and has a favourable pharmacokinetic profile relative to omeprazole[1-3]. White to slightly coloured crystalline powder. It is slightly soluble in water[4].

Received on 01.06.2015 Modified on 20.06.2015

Research J. Pharm. and Tech. 8(7): July, 2015; Page 820-824

DOI: 10.5958/0974-360X.2015.00133.X

Naproxen (NAP) (Figure 2) (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid. Naproxen is a non-steroidal anti-inflammatory drug (NSAID)commonly used for the reduction of moderate to severe pain, fever, inflammation and stiffness. It works by inhibiting both the COX-1 and COX-2 enzymes. Like other NSAIDs, Naproxen is capable of producing disturbances in the gastrointestinal tract[5-6]. Naproxen is an odourless, white to off-white crystalline substance. It is lipid soluble practically insoluble in water [7].

Literature survey of Esomeprazole and Naproxen revealed few methods based on UV Spectrophotometry [8-9] and Chromatography [10-11] have been reported for determination of both drugs in single and combined dosage forms. The present work describes the development and validation as per ICH guidelines[12] of reverse phase high performance liquid chromatographic (RP-HPLC) method, which can quantify these components simultaneously.

Fig.1: Chemical structure of Esomeprazole

Fig.2: Chemical structure of Naproxen

2. EXPERIMENTAL

Materials and Methods:

Reagents required

Acetonitrile : HPLC grade, Merck

Water : HPLC grade, Merck

Potassium dihydrogen phosphate: AR grade, Merck

Dipotassium hydrogen phosphate: AR grade, Merck

Drugs used

The gift samples of Esomeprazole and Naproxen were kindly provided by AstraZeneca Pharmaceutical Ltd and the marketed formulations containing Naproxen (500 mg) and Esomeprazole (20 mg) were procured from local pharmacy (trade name: VIMOVO).

Instrumentation and Chromatographic Conditions

The developed method HPLC system with UV detector data were acquired and processed by Empower software. The separation was carried out at ambient temperature by using a Agilent CN (4.6 x 250mm, 5µm) .The mobile phase consisting of Acetonitrile :K₂HPO₄: KH₂PO₄ (60:20:20v/v). The flow rate was 2 ml/min. The injection volume was 0.02 µL and detection at a wavelength of 257nm.

Preparation of Mobile phase

Mix the 200 ml of KH₂PO₄ (20%) and 200ml of K₂HPO₄(20%) and 600 ml of acetonitrile (60%). Filter through 0.45 µ filter under vacuum filtration.

The mobile phase liquid is also used for making working dilution of drugs.

Preparation of stock solutions

Stock Solution A of Naproxen and Esomeprazole (20µg/ml and 5mg/ml respectively)

The stock solution of naproxen and esomeprazole were prepared by weighing accurately 2 mg of naproxen and 500 mg of esomeprazole pure drug and transferred to a 100ml volumetric flask and dissolved in the mobile phase and made up to the mark with mobile phase. Stock solution A was prepared.

Stock Solution B of Naproxen and Esomeprazole (2µg/ml and 500µg/ml respectively)

From the above Stock Solution A 10 ml was taken and transferred to a 100ml volumetric flask and made up to the mark with mobile phase. Stock solution B was prepared.

Working Stock Solution of Naproxen and Esomeprazole

From the above Stock Solution B 10 ml was taken and transferred to a 100ml volumetric flask and made up to the mark with mobile phase.

Linearity of pure standard solution:

The linearity of the samples of Naproxen and Esomeprazole was prepared by suitably diluting working solution and found to be linear response of drug over a range of 150-350 µg/ml concentration. for the Naproxen and 6-14µg/ml for Esomeprazole respectively. The three such linearity’s of Naproxen and Esomeprazole were taken for correlation co-efficient and standard deviation calculation.

Table 1 : Area Of Different Concentration Of Naproxen and Esomeprazole Obeying Beer’s Law

S. No	Naproxen (µg/mL)	Area (mV.s)	Esomeprazole (µg/mL)	Area (mV.s)
1	150	508.413	6	149.046
2	200	717.59	8	199.190
3	250	893.46	10	253.149
4	300	1104.37	12	306.856
5	350	1262.83	14	358.255

Fig 3 : Graph showing Linearity of Naproxen

Fig 4 : Graph showing Linearity of Esomeprazole

Fig.5: Chromatogram showing peaks of Esomeprazole and Naproxen

Preparation of Sample Solution

VIMOVO of strength 500mg of Naproxen and 20mg of Esomeprazole. respectively. Average weight of twenty tab were taken and crushed to make powder, weighed powder containing 500 mg Esomeprazole was transferred to 100ml of volumetric flask and volume was made up to the mark with diluent (Acetonitrile: K₂HPO₄ : KH₂PO₄)(60:20:20) and filtered through whatmann filter paper in to another 100ml volumetric flask and make up to mark with same diluent which gives the solution of 5 mg/ml concentration of Esomeprazole and 20 µg/ml of Naproxen, further dilutions of the solution made to obtain the concentration of 500 µg/ml concentration of Esomeprazole and 2 µg/ml of Naproxen with the same diluent and again filter it with milli pore filter through syringe filter and used for further analysis. The same procedure as mentioned for the pure drug was followed for the formulation. The concentrations of both Esomeprazole and Naproxen were determined by measuring peak area at 257 nm.

Assay for Marketed formulation

The Assay performed by the marketed formulation of Esomeprazole and Naproxen (VIMOVO). The prepared standard and sample solutions were injected into HPLC and peak areas were recorded. The amount of drug present and percentage purity was calculated by comparing the peak areas of the standards with that of samples.

Table 2 : Assay of marketed formulation

Drug

Labeled amount

( mg )

Amount found

mg/tab

% Recovery

%RSD

(n=5)

NAPROXEN

500 mg

499.22

98.84

0.0146

ESOMEPRAZOLE

20 mg

19.77

98.85

0.0662

Fig. 6: Chromatogram showing peaks of Esomeprazole and Naproxen of formulation

VALIDATION OF HPLC METHOD

The HPLC method was validated in accordance with ICH guidelines.

Precision

System Precision for Naproxen and Esomeprazole: The system precision was evaluated by measuring the peak responses of Naproxen and Esomeprazole for five replicate injections of standard solution, prepared as the proposed method. The results shown in the table-3 indicate that the precision of the system is with in the limit. (Acceptance criteria: % rsd nmt 2.0%)

Method Precision for Naproxen and Esomeprazole was determined by preparing a sample solution of single batch Naproxen and Esomeprazole Tablet five times and analyzing as per the proposed method. The results shown in Table-3 indicate that the proposed method is precise. (Acceptance criteria: % RSD NMT 2.0%

Accuracy

To check the accuracy of the developed method and to study the interference of formulation excipients, recovery study was carried out by using standard addition method by adding 100% concentration to a fixed amount of the pre analysed sample and the amount of drug were analyzed by the proposed method. Results from the recovery studies are given in table 4and5.

Limit of detection (LOD) and Limit of quantification (LOQ)

Limit of detection and limit of quantification were estimated from signal to noise ratio. LOD is the lowest concentration resulting in a peak area of three times the baseline noise and the equation is LOD = 3.3 x ASD/S. LOQ is the lowest concentration that provide signal to noise ratio more than 10 and the equation is LOQ = 10 x ASD/S, where ‘ASD’ is the average standard deviation and ‘S’ is the slope of the line.

Robustness

Robustness was performed by deliberately changing the chromatographic conditions. The important parameter to be studied was the resolution factor between two peaks. Robustness of the method was carried out by deliberately made small variation in the flow rate, pH of mobile phase, organic phase ratio and column oven temperature by using 100 µg mL^-1 of Naproxen and 4 µg mL^-1 solution of Esomeprazole, respectively.

Linearity

The linearity of the method was determined by comparing the known concentration Vs response, a series of calibration standards 150, 200, 250, 300, 350 µg/ml of Naproxen and 6, 8, 10, 12, 14 µg/ml of Esomeprazole were prepared. The solutions were injected into the chromatographic system and peak area of each peak at each concentration was noted. The calibration curve was plotted using peak area versus concentration of the standard solution.

Degradation Studies:

Acid degradation

About 100 mg of Naproxen and 80 mg of Esomeprazole was transferred to a 100 ml volumetric flask and dissolved in minimum quantity of diluent, 5 ml of 0.1 N HCL was added and the volume made up to the mark with diluent, the solution was heated at 60-70^oc for 1 hour. Cool the solution at room temperature further dilutions of the solution made to obtain the concentration of 50 µg/ml and 20 µg/ml concentration of Naproxen and Esomeprazole respectively with the same diluent and used for further analysis. The same procedure as mentioned for the pure drug was followed for the formulation.

Base Degradation

About 100 mg of Naproxen and 80 mg of Esomeprazole was transferred to a 100 ml volumetric flask and dissolved in minimum quantity of diluent, 5 ml of 0.1 N NaoH was added and the volume made up to the mark with diluent, the solution was heated at 60-70^oc for 1 hour. Cool the solution at room temperature further dilutions of the solution made to obtain the concentration of 50 µg/ml and 20 µg/ml concentration of Naproxen and Esomeprazole respectively with the same diluent and used for further analysis. The same procedure as mentioned for the pure drug was followed for the formulation.

Oxidative Degradation

About 100 mg of Naproxen and 80 mg of Esomeprazole was transferred to a 100 ml volumetric flask and dissolved in minimum quantity of diluent, 5 ml of 1% H₂O₂ was added and the volume made up to the mark with diluent, the solution was heated at 60-70^oc for 1 hour. Cool the solution at room temperature further dilutions of the solution made to obtain the concentration of 50 µg/ml and 20 µg/ml concentration of Naproxen and Esomeprazole respectively with the same diluent and used for further analysis. The same procedure as mentioned for the pure drug was followed for the formulation.

Thermal Degradation

About 100 mg of Naproxen and 80 mg of Esomeprazole was placed in a china dish. The dish was covered by aluminium foil and kept in hot air oven at 60-70ºC for 1 hour and transferred to 100 ml volumetric flask and dissolved in minimum quantity of diluent and the volume made up to the mark with diluent, the solution was heated at 60-70^oc for 1 hour. Cool the solution at room temperature further dilutions of the solution made to obtain the concentration of 50 µg/ml and 20 µg/ml concentration of Naproxen and Esomeprazole respectively with the same diluent and used for further analysis. The same procedure as mentioned for the pure drug was followed for the formulation. Results of degradation data was given in table no-7.

Table: 3 Precision Data

Drug

Concentration

( µg/ml )

System Precision

% Recovery (% RSD, n=5)

Method Precision

% Recovery

(%RSD, n=5)

Naproxen

500

98.44 (0.0146)

99.42 (0.0204)

Esomeprazole

98.16 (0.0662)

98.93 (0.0141)

Table 6: Optical Characteristics of the Proposed Method for Naproxen and Esomeprazole

Parameter	Naproxen	Esomeprazole
λmax (nm) selected	257	257
Beer’s law limits (µg/ml)	150-350	6-14
Correlation coefficient (r)	0.999	0.998
Relative standard deviation (%)	0.0146	0.0662
% Error at 99%(0.01 level)	1.249	0.321
% Error at 95%(0.05 level)	0.844	0.217
Limit of detection (µg/ml)	0.0550	0.0090
Limit of Quantification (µg/ml)	0.1813	0.0296

Table 7: Degradation Data

Drug	Degradation	Area (mV.s)	% Recovered	% Degraded
Naproxen	Acid	114.905	89.73	10.27
	Base	116.667	99.69	0.31
	Oxidative	107.328	88.12	11.88
	Thermal	97.928	78.12	21.88
Esomeprazole	Acid	599.116	92.81	7.19
	Base	611.585	101.47	-
	Oxidative	568.840	93.64	6.36
	Thermal	530.780	85.21	14.79