Validation of Esomeprazole Magnesium Tri-hydrate in Pharmaceutical dosage form by RP-HPLC Method

 

Rajan V. Rele*, Prathamesh P. Tiwatane

Central Research Laboratory, D. G. Ruparel College, Matunga, Mumbai 400016.

 

ABSTRACT:

A sensitive and robust novel reverse phase liquid chromatographic method was developed for assay of esomeprazole magnesium trihydrate in its dosage form. For chromatographic study BDS Hypersil C18 (150mm x 4.6mm, 5 µm) was used in on isocratic system at ambient temperature. The selected mobile phase made up of buffer and acetonitrile in proportion 75:25 % (v/v). The flow rate of 1 ml/min. was used in study. The effluent study was done at wavelength 280 nm. The retention time was observed at 3.8 min. The linearity was observed in concentration range as 20 to 60 µg/ml. The correlation coefficient is less than 1. The ICH guidelines were used for system suitability, linearity, accuracy and precision studies. The accuracy and precision were found to be well within the acceptable limit. The method was successfully applied for assay of Esomeprazole magnesium trihydrate in tablets with good recoveries.

 

 

 

INTRODUCTION:

The research article is useful for validation and application of RP-HPLC method for the assay of Esomeprazole magnesium trihydrate in bulk drug and dosage form.

 

Esomeprazole magnesium trihydrate 1 (ESO) is bis(5-methoxy-2-[(S)-[(4-methoxy-3,5- dimethyl - 2 -pyridinyl)methyl ]sulfinyl ] - 1-H - enzimidazole - 1 -yl) magnesium trihydrate. Such compound prevents secretion of gastric acid. It is S-isomer of omeprazole. It is a first single optical isomer proton pump inhibitor. It gives better acid control than other inhibitors of racemic proton pump. It produces a favourable pharmacokinetic than omeprazole.

 

 

 

According to the literature review several assay methods has been developed for drug, like spectroscopy methods^1-6. HPLC^7-11. The proposed aim of the study was to develop simple, accurate, specific and precise RP-HPLC method for the estimation of Esomeprazole magnesium trihydrate in the bulk and pharmaceutical formulation.

 

Structure of esomeprazole magnesium tri-hydrate

 

MATERIAL AND METHODS:

Instrumentation:

The Merck-Hitachi La-Chrome, isocratic pump with UV detector equipped with EZ Chrom Elite software for quantification of recorded chromatograms.

 

Materials and reagents:

A esomeprazole magnesium tri-hydrate, reference standard with certificate of analysis was obtained as gift sample. HPLC grade acetonitrile and AR grade of tri-ethyl amine and phosphoric acid were used from Merck and the Millipore water system was used HPLC grade water.

 

Preparation of mobile phase:

The buffer solution and acetonitrile in ratio as 75:25 % (v/v) as mobile phase. They were sonicated for degassed.\

 

Preparation of buffer solution:

A 1 ml of AR grade tri-ethyl amine was dissolved in 1000 ml of HPLC grade water and pH of solution was adjusted to 7.3 with the help of dilute phosphoric acid.

 

Conditions of Chromatographic separation:

The column was used as BDS hypersil C18 (150mm x 4.6mm, 5 µm) and flow rate was 1. 0 ml/min. The UV detector was adjusted at 280 nm and injection volume was 15 μl for each injection.

 

Standard solution:

Weigh about 4 mg of standard esomeprazole magnesium tri-hydrate accurately in 10 ml of standard flask. Add about 5 ml of diluent (buffer: acetonitrile (75:25 % v/v)) It was sonicated for 10 minutes for complete dissolved the esomeprazole magnesium tri-hydrate. The solution was further diluted to mark to give concentration as 400 μg/ ml.

 

Sample preparation:

It is equivalent of 4. 0 mg of esomeprazole magnesium tri-hydrate. It was transferred into 100 ml volumetric flasks and sonicated with dissolved with 25 ml mobile phase and filtered through Whatman filter paper. Further dilutions were made based on the required concentrations. A 15 µl was injected for analysis.

 

Method Development:

A UV spectrum of the drug is given in fig 1 and chromatogram of the drug assayed is depicted in fig. 2.

 

Fig no. 1. UV spectrum of esomeprazole magnesium tri-hydrate

 

Figure.2: chromatogram of standard esomeprazole magnesium trihydrate

Validation of Method:

The method was validated for assay of esomeprazole magnesium tri-hydrate using given parameters.

System performance parameters of developed HPLC method were determined by injecting standard solutions. Parameters such retention time, area, % area and asymmetry were shown in Table-1.It indicated good performance of the system.

 

Table – 1 : System performance parameters for esomeprazole magnesium tri-hydrate. (n = 6).

 

System suitability:

A standard solution of esomeprazole magnesium tri-hydrate was prepared. The working standard was injected in six replicate in HPLC. The system suitability parameters were calculated from standard chromatograms. the % RSD was calculated from six replicates for peak areas and retention time. The data is given in table no.2

 

Table 2: Values of System precision

 

Linearity:

The linearity study was determined for Esomeprazole magnesium trihydrate at five concentrations level 50%, 80%, 100%, 120% and 150% in ranging from 20 to 60 µg/ml. The linearity curve was plotted by plotting area against concentration of the drugs. The regression equation was given as y = 77562x - 2906. The correlation coefficient (r²) was 0.9999 and concentration range indicated above. The linearity graph is given in fig no.3

 

Figure 3: Linearity curve

 

The results are given in the table 3.

 

Table 3: parameters of linearity study

Parameter	Esomeprazole magnesium tri-hydrate
Range of linearity	20-60 μg /ml
Slope	77562
Intercept	-2906
Coefficient of correlation	0.9999

 

Accuracy:

The accuracy study of the method was carried by recovery experiments in the concentration range as 80%, 100% and 120 % in 3 replicates with test concentration. The % recovery studies and RSD were calculated and presented in Table 4.

 

Method Precession:

The method precision was established by carrying out the analysis of esomeprazole magnesium tri-hydrate. The assay was carried out of the drug using analytical method in six replicates. The value of relative standard deviation lies well with the limits (0.10 %). The results of the same are tabulated in the table 5.

 

Table 4 : Accuracy - %Recovery

Step	No.	Amount in mg	Recorded area	Amount added in μg /ml	Amount recovered in μg /ml	% recovery	Recovery
80%	1	4.21	2604760	33.6	33.61	100.03	100.08
	2	4.2	2608189	33.6	33.66	100.16
	3	4.22	2604673	33.6	33.61	100.03
100%	1	4.19	3254577	42	42.00	99.99	100.06
	2	4.17	3256854	42	42.03	100.06
	3	4.25	3258838	42	42.05	100.12
150%	1	4.21	3910720	50.4	50.46	100.12	100.25
	2	4.19	3915831	50.4	50.53	100.26
	3	4.21	3920500	50.4	50.59	100.37
					Mean recovery		100.13

* Average of triplicate analysis.

 

 

 

 

Table 5 : Precision – method precision.

Replicate no	Sample in mg	Recovered Peak Area	% Assay
1	4.21	3255776	99.09
2	4.22	3257345	99.37
3	4.18	3251534	99.42
4	4.15	3251990	98.72
5	4.23	3247948	100.50
6	4.21	3250346	98.92
	Mean Assay		99.34
	SD		0.628
	RSD		0.633

 

Stability of solution:

The solution stability study experiment was carried out of the solutions by keeping the solutions at room temperature for 24 hours. The results indicated no significant change in the assay results of the same solutions. It confirmed the stability of the drug in the solvents used for the analysis.

 

Robustness:

Robustness study of the method was performed by making slight changes in the chromatographic conditions as per given below.

 

 

The observed chromatograms does no showed marked changes hence the proposed method was robust.

 

Application of Method to dosage:

The validated high performance liquid chromatographic method was applied for determination of Esomeprazole magnesium trihydrate its formulation. A portion equivalent to 4 mg of esomeprazole magnesium trihydrate was weighed accurately. It was dissolved in 10 ml of diluents and sonicated for 15 minutes and filtered through whatmann filter paper. It gave the concentration 400 µg/ml.

 

A 15 µl of this solution was injected under specified conditions. The analyte peaks were identified by comparison with respective standard and chromatogram was recorded. (Fig. no.4).

 

 

 

The assay results expressed as mg are shown in Table-3. It indicated the amount of each drug in the product meet the requirement.

 

RESULTS AND DISCUSSION:

In the new method, use of 0.1 % tri-ethyl amine pH 7.3 and acetonitrile (75:25 % (v/v) gave peak with good resolution. The data given below of suitability of method.

 

Parameters	Range/ values
Retention time	3.84
Linearity range	20-60 μg/ml
Regression equation	y = 77562x - 2906
Coefficient of co-relation	0.9998
The relative standard deviation	0.663 (limit %RSD < 2.0%)
Mean recovery	99.34

The results of robustness with change in flow rate, wavelength and mobile composition indicated that there was no effect on the drug study hence method is found to be robust. No interfering peaks were found in run time of 7 mins in the chromatogram of the formulation indicted that excipents used in the formulation did not interfere the estimation of drug.

 

The proposed method was found to be satisfactory as low values of standard deviation 0.628 and percent relative standard deviation 0.633.( Table no.5). It proves reproducibility, repeatability and accuracy of Method. The accuracy and reproducibility of the proposed method was done by standard addition method (Table no.4). The non- interference from the excipients was observed by percent recovery obtained. The methods reported in literature have greater retention time. Hence more time will be required for validation of drug and its formulation as well it requires more amount of chemicals.

 

This can be successfully used for validation of drug as well as for determination stability of drug in various conditions as per ICH guidelines.

 

CONCLUSION:

Thus the proposed RP-HPLC method is used for assay of esomeprazole magnesium trihydrate from active pharmaceutical ingredient and marketed formulation i.e. ointment due to its simplicity and non interference of other peaks and relatively short retention time as 3.84 mins as compared to methods suggested in literature. It is more precise, accurate, linear, robust, simple and rapid method. Hence the proposed RP-HPLC method is strongly recommended for the quality control of the raw material, active pharmaceutical ingredient and pharmaceutical formulation per ICH guidelines.

 

ACKNOELEDGEMENT:

Authors express sincere thanks to the Principal, Dr. T.M. Desai, D. G. Ruparel college for providing laboratory facilities.

 

REFERENCES:

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Received on 04.07.2018         Modified on 16.08.2018

Accepted on 07.09.2018         © AJRC All right reserved