A Novel RP-HPLC Method for Determination of Esomeprazole in Pharmaceutical Dosage Forms in Human Plasma:

A Pilot Stress Degradation Study

 

Shanthi Diraviyam Shanmugakumar*, Anjali, Nasreen Begum, Boga Uday

Department of Pharmaceutical Analysis, Jyothishmathi Institute of Pharmaceutical Sciences,

Thimmapur, Karimnagar, Telangana. India.

 

ABSTRACT:

Esomeprazole, an proton pump inhibitor used for the remedy of acid reflux disorders including healing and maintenance of erosive esophagitis, gastroesophageal reflux disease and peptic ulcer disease. It is utilized in a prolonged treatment of pathological hypersecretory conditions such as Zollinger –Ellison syndrome. In this study, a novel RP-HPLC method was developed for the determination of esomeprazole both in API and tablets (Esmoz (40mg)), Phenomenex Luna, 5µm C18 100 A^◦ LC column (250X4.6mm) was used at 40^◦C. Isocratic elution was utilized with acetonitrile: water (96:4v/v) mixture. The flow rate was 1.0mL min^-1 and UV detection was at 270nm. The RT value was observed at 3.713 minutes respectively. The developed method was validated according to ICH guidelines and found to be linear within the range of 100-500µg/mL. In similar lines, linearity was achieved by spiking esomeprazole in human plasma within the range of 20-80µg/mL. The developed method was accurate, precise, robust, and rapid. Hence it can be applied for both stability studies and routine quality control analyses.

 

 

 

 

INTRODUCTION:

Proton pump inhibitors (PPI) are utilized to decrease gastric acid secretion through inhibition of H+/K+ ATP ase in gastric parietal cells by inhibiting the functioning of this enzyme. Esomeprazole (ESM) an oral dimethyl pyridine methyl benzimidazole derivative that has molecular mass 345.41g mol-1 that is almost sparingly soluble in water and produces high plasma protein binding (93-95%) in humans with serum albumin as the main building component¹. An abundance of literature was present in the estimation of esomeprazole related to gastroesophageal diseases. In the literature, there was an HPLC-UV method was adopted for the determination of esomeprazole in bulk and pharmaceutical dosage forms. However, there is no simple and appropriate method reported for the quantification of esomeprazole in pharmaceutical dosage forms in human plasma. In the pharmaceutical industry, where a huge number of quality control samples and HPLC method is a precise and unique technique for the analyses of a wide variety of samples in a simplified mobile phase². In this analytical perspective, it was focused to develop an accurate, precise, robust, rapid, and selective RP-HPLC method for the determination of esomeprazole in tablet dosage forms. The stability of esomeprazole was evaluated and also forced degradation procedure was applied under stress conditions like high temperature, acidic –alkali conditions, and irradiation with UV-light. The developed method was applied to exhibit linearity studies in the esomeprazole spiked in human plasma. The developed method was completely validated according to ICH guidelines³. Hence this method could be concluded as a proposed method for the quality control process of esomeprazole in the pharmaceutical industry.

 

MATERIALS AND METHODS:

Chemicals and reagents:

ESM (Esomeprazole) working standard was supplied from Gland pharma, Hyderabad, India. Acetonitrile (ACN) and water (HPLC grade) were procured from spectrochem, Mumbai. The tested pharmaceutical formulations (ESMO, 40mg) approved in India were procured from Sun pharma, Sikkim, INDIA. (Figure 1)

 

Figure 1 Esomeprazole

 

Apparatus and chromatographic conditions:

HPLC investigation was performed on a Shimadzu HPLC system (LC 2010 AD). Separations were achieved on a Phenomenex Luna, 5µm, C18 100A◦ LC column (250X4.6mm). The column temperature was at 40◦ Centigrade and the flow rate at 1.0mL/min^-1 while utilizing isocratic elution with ACN: Water (96:4 volume/volume) mixture. Injection volume was 20µL and UV detection was achieved at 270nm. Peak identity was confirmed by retention time.

 

Preparation of standard solution:

Two diluting solvents were utilized to make the sample and standard solution. Diluent-A was a integration of ammonium acetate buffer (pH 9.0 adjusted by ammonia solution, 0.04 M ammonium acetate solution) and methanol in the ratio of 60: 40 v/v also diluent-B was a integration of ammonium acetate buffer (0.025 M ammonium acetate solution pH 7.4 adjusted by ammonia solution) and methanol in the ratio of 60:40 v/v. The standard stock solution of ESM -esomeprazole (100µg mL^-1) was prepared in ACN: water (96:4 v/v) mixture. The working stock solutions (100.0, 200.0, 300.0, 400.0 and 500µg mL^-1) were prepared by diluting the stock solution in the mobile phase solution. The stock solution was kept at 10^◦C where it is stable for one month. Standard solutions were daily prepared by diluting the stock solution with a mobile phase solution.

 

Preparation of sample solution:

Twenty tablets were weighed to obtain the average weight and finely grounded into a powder. An amount of powder equivalent to 10mg of esomeprazole (ESM) was transferred to a 100mL volumetric flask (100µg/mL) and added 70mL of diluents (ACN: Water (96:4 v/v) and sonicated for 20 minutes. The volume was made up with diluents to obtain a solution containing 100µg/mL-1 esomeprazole (ESM). The resultant solution was filtered using a 0.45µm membrane filter and a 20µL solution was injected into the chromatographic system.

 

Forced degradation:

Within this phase, forced degradation studies have been undertaken to degrade the sample [e.g., drug product or API (Active Pharmaceutical Ingredient)] intentionally. These studies were utilized to assess an analytical method's capacity to quantify an active ingredient and its degradation products without interference. Drug Product or sample (spiked placebo) and drug substance were subjected to acid (1 N HCl), base (1 N NaOH), oxidizing agent (10% Hydrogen Peroxide), decreasing agent (10% Na bisulfite solution) and water for 24 h to make 10–30% degradation of the active ingredient. The degraded samples were subsequently examined using the proposed system to determine whether there are interferences with the energetic or related chemical.

 

Plasma sample preparations:

Accurately weigh 10mg of esomeprazole in a 100mL volumetric flask. 70mL of the diluents was added and the mixture was sonicated for 10 minutes. Make up the volume with diluents. The working stock solutions (20, 30, 40, 50, 60, 70 and 80µg mL^-1) were achieved by diluting the stock solution with a mobile phase solution. The liquid-liquid extraction method was performed by adding working stock solutions in 500µL plasma and vortexed for 20 secs. Add 5mL of dichloromethane was added and the mixtures were vortexed for 3 minutes. Afterward, 20µL was injected into the HPLC system.

 

Method Validation:

The stability-indicating method was validated for assay and related substances, observing the recommendations of the ICH guidelines for industry Q2 (R1)³. The following parameters were evaluated: selectivity, the limit of detection (LOD) and limit of quantification (LOQ), linearity, precision, accuracy, robustness, and stability of solutions.

 

Specificity:

The LC model specificity has been assessed to verify that there was no interference from the degradation products, impurities or excipients present in the pharmaceutical formulation⁶. The specificity was analyzed by injecting the stressed and unstressed standard solutions, excipients and pharmaceutical preparations of ESP and NPX Mg.

 

System suitability test:

After obtaining the optimum conditions for esomeprazole analyses. The following parameters were evaluated: Selectivity, the limit of detection (LOD) and limit of quantification (LOQ), linearity, precision, accuracy, robustness, and stability of solutions.

 

LOD and LOQ:

Limits of detection and quantitation were estimated at a signal to noise ratio of 3:1 and 10:1 respectively, according to ICH, 2005³.

 

Linearity:

Linearity data was evaluated in ranges from 100-500µg/mL. The solutions were prepared in triplicate mode. The resultant data were statistically analyzed to prove that they met the assumptions for linear regression. The average peak areas were plotted against concentrations. The linearity of the proposed method was evaluated by using a calibration curve to calculate the coefficient of correlation, slope and intercept values.

 

Precision:

For evaluating the impurities, the intraday precision was determined by carrying in triplicate mode by spiking esomeprazole (50µg/mL) of nominal sample preparation. The precision of the assay method was assessed in terms of intraday and inter-day precision by the quantification of esomeprazole with independent replicates prepared at 100% of the working concentration. Results were reported in terms of relative standard deviation (RSD) among responses using the formula [% RSD = (standard deviation/mean) × 100%].

 

Accuracy:

The accuracy of an analytical model expresses the nearness between the value found and expected value. To assess the accuracy, the known amounts of esomeprazole were spiked with placebo in three-level concentrations: 80%, 100% and 120% respectively. Each solution was prepared in triplicate and the recovery percentage of esomeprazole was calculated. standard solution were carried out to determine the accuracy of the proposed method.

 

Robustness:

Robustness evaluate the ability of an analytical method to remain unaffected by small even deliberate variations in the parameters of the model. The reliability of the analytical model indication will be provided by robustness during normal usage. The robustness of the developed method was evaluated by analyzing samples prepared in the assaying test. Samples were assayed under nominal conditions and by varying the following analytical parameters: Flow rate change (0.5mL, 1.0mL and 1.5mL/min) and wavelength change (269, 270, 271 nm) respectively.

 

Assay:

Twenty tablets were weighed and the content was finely pulverized. The powder was weighed equivalent to 50mg into a 100mL volumetric flask. Make up the volume to 100mL with the diluent. Filter the solution using 4.1 Whatman filter paper. Further, pipette and dilute 5mL of the resulting solution to 100mL of the volumetric flask to obtain the final volume of 50µg/mL. The quantification was performed in triplicate under the chromatographic conditions.

 

RESULTS AND DISCUSSION:

The chromatographic condition was optimized with different ratios of the mobile phase and the best results were obtained with acetonitrile and water (96:4) and with Luna Phenomenex C18 (250x4.6mm,5µ). The column temperature was analyzed in the range of 25◦ centigrade to 50^◦ centigrade. A uniform sharp peak was achieved with the detection wavelength at 270nm. The resolution was achieved at an RT 3.713. The chromatogram which was obtained in the optimized condition is shown in Figure 2, Table 1.

 

Table 1: Optimized chromatographic conditions

Column	C-18 Luna Phenomenex (250 x 4.6 mm, 5 μ).
Elution mode	Isocratic
Mobile phase (Ratio)	Acetonitrile; Water (96:4)
Flow rate	1ml/min
Detection wavelength	270nm
Injection volume	20µl
Run time	10 mins
Column temperature	36°C
sample temperature	25°C
Remarks	Uniform sharp peak

 

Figure 2: Optimized Chromatogram of esomeprazole

 

Forced degradation ⁵

Table 2 shows a summary of the results obtained in the forced degradation study for API. In acidic hydrolysis, 0.05M hydrochloric acid was used for 2hrs. Esomeprazole API showed around 4.8% degradation (Figure 3). In alkaline hydrolysis, esomeprazole API was exposed to 0.1M sodium hydroxide for 2 hrs at 80◦ centigrade. API showed degradation of 6.8%. In thermal degradation, esomeprazole was subjected to a water bath for 24 hrs at 80◦ C (Figure. 4). Two impurities A and B were found. API showed degradation of 5.1%. When esomeprazole was subjected to oxidation mechanism with 3% hydrogen peroxide at room temperature. The API showed a degradation of 11.3% (Figure 5). One impurity was found to be C. Esomperazole API was more susceptible to photolysis when exposed to UV light for 24 hrs. After the exposure, there was a formation of impurities D.

 

In the perusal of literature. It revealed that the chasteness angle was less than the threshold angle and there was no purity flag for esomeprazole⁵. In this study, the stress conditions employed resulted in esomeprazole degradation under all conditions and inadequate extension (4.8 to 11.3%). It is difficult to observe the degradation obtained, once the pharmaceutical forms used are different, and API from different manufacturers can lead to distinct degradation profiles.

 

 

Table 2: Forced degradation studies of esomeprazole

S. No	Stress conditions	Temperature and Time	Area of the esomeprazole for assay	% assay of the active substance	Area of the esomeprazole on degradation	% Degradation of the active substance
1	Acid 0.05M -HCl	Room temp	978974	98	20358790	4.8
2	Alkali 0.1M -sodium hydroxide	Room temp	978974	98	14633888	6.7
3	Oxide (3%H2O2)	Room temp	978974	98	8694402	11.3
4	Thermal	80◦ C for 24hrs	978974	98	50227	5.1
5	Photo degradation	24hrs	978974	98	373115	38.1

 

 

Figure 3 Acid degradation of esomeprazole

 

Figure 4 Thermal degradation of esomeprazole

 

Figure 5: Oxidative degradation of esomeprazole

 

Plasma sample preparations:

Recovery studies of esomeprazole were achieved at the concentration range of 80,100 and 120 % by spiking the esomeprazole with human plasma. The percentage recovery was calculated as 72.5 %, 77.8% and 79.2% respectively. The amount of drug bound with plasma was calculated as 2.0,2.3 and 2.5 respectively. The corresponding percentage of relative standard deviation value found to be 51.23, 0 and 65.08 respectively.

 

Linearity studies were carried out as per the procedure postulated by Madhavi and Prameela rani⁴. The concentration range is 20-80µg/mL and calibration was partially linear with the correlation co-efficient R² =0.8501. The data were shown in Table 3 and Table 4 and Figure .6.

Table 3: Recovery studies of esomeprazole in human plasma

S. No	Accuracy	Drug spiked in plasma (mg)	Percentage recovery	Amount recovered (mg)	Amount of drug bound with plasma (mg)	SD	% RSD
1	80	8	72.5	6	2	39.19821425	51.23949575
2	100	10	77.8	7.7	2.3	0	0
3	120	12	79.2	9.5	2.5	49.78835205	65.08281314
			229.5
			76.5

 

Table 4: Linearity studies of esomeprazole in human plasma

Analyte	Concentration µg/mL	Peak area*	Sum of peak area	Average	Regression equation
Esomeprazole	20	961109	2279025	1139513	Y=784218X R2 =0.8501
	20	1319716
	40	1082391	2200749	1100375
	40	1118358
	60	2677091	5360143	2680072
	60	2683052
	80	3208184	6073030	3036515
	80	2864846

 

 

Figure 6–Linearity studies of the esomeprazole

 

Figure 7: Calibration curve of esomeprazole

 

Method validation:

Selectivity:

The selectivity of the method was investigated by injecting the stress samples. No peaks were eluting in the same retention time as esomeprazole. The peaks were well separated and the resolution was not less than 1.5

 

LOD and LOQ:

Employing the signal to noise ratio method, the detection and quantification limits were 0.284µg/mL and 0.4393µg/mL respectively.

Linearity:

The data obtained in the calibration studies when subjected to linear regression analysis showed a linear relationship between peak areas and concentrations in the range of 100-500µg/mL. The equation of the calibration curve obtained was Y=305.4X+6.05X with the correlation coefficient of the linear study were found to be R² =0.9786. The data were shown in Table 5 and Figure 7.

 

Table 5: Linearity studies of esomeprazole

Analyte	Concentration µg/mL	Peak area*	Sum of peak area	Average	Regression equation
Esomeprazole	100	3523042	6931429 Y=20098X-41035 R2=0.9996	3465715	Y=3E+06X R2 =0.9786
	100	3408387
	200	5730734	11418975	5709488
	200	5688241
	300	7302238	14601763	7300882
	300	7299525
	400	10745155	21644247	10822124
	400	10899092
	500	13181431	26373074	13186537
	500	13191643

Acceptance criteria: R²= 0.999

 

Table 6: Intraday and Inter-day Precision studies of esomeprazole

Concentration	Injection number	Intraday Precision	Inter day precision
		Area	Area
Esmoperazole (100µg/mL)	1	786372	829950
	2	754061	796111
	3	764162	792569
	4	758273	793474
	5	768178	781285
	6	786473	791285
Mean		769586.5	797445.6667
Standard deviation		12697.456	15258.82
%RSD		1.64	1.91

Acceptance criteria: < 2%

 

 

 

 

 

Precision:

Injection repeatability values (% RSD) of esomeprazole was found to be 0.314 and 0 for intraday and inter-day precision. The data were shown in Table 6.

 

Accuracy:

Accuracy was assessed using placebo solutions spiked with known amounts of esomeprazole in three levels. The recovery percentages were between 95.03,100 and 99.96 respectively. Moreover, the RSD values were 0.94,0.76 and 0.79 respectively. The data were represented in Table –7.

 

Figure 8 shows that recovery and RSD achieving greater performance compared to existing model in the medical fields. The recovery for both drugs was found to be >99% which showed the accuracy of this model. Intra- and inter-day precision anlysis of the new proposed model were less than the maximum permissible limit [% RSD (Relative Standard Deviation) ≤2.0 according to FDA.

 

Figure 8: Recovery and RSD Analysis

 

Table 7: Recovery (Accuracy) studies of esomeprazole

Amount (%) of drug added to the analyte	Wt has taken (mg)	Sample added (mg)	Peak area*	Sample recovery	Recovery
					% Recovery	%RSD
80	25	25	1035048	0.956	95.03	94.23
	25	24	1033734	0.956
	25	24	1016594	0.939
100	50	49	1004991	0.665	100	76.54
	50	49	1762913	1.168
	50	49	1759406	1.165
120	75	74	1495777	0.710	99.96	79.47
	75	74	2401471	1.140
	75	74	2421954	1.149

Acceptance criteria: 97 -103%

 

Table 8: Assay of esomeprazole

Tablet Formulation	Label Claim per Tablet (mg)	% Drug found ± SD (n=6)	RSD (%)
Esomeprazole – 40mg	Esmo-40	98.4 %	0.123

Acceptance criteria: R>0.999

 

Figure 9: RP-HPLC overall performance comparison with existing model.

 

Assay:

The developed method precisely detected the concentration of esomeprazole in a tablet formulation as indicated by the peak area of 826414. The percentage labeled claim for esomeprazole was found to be 98.4% and relative RSD was found to be 0.123. The data has been expressed in Table – 8.

 

Figure 9 shows that RP-HPLC achieving greatest performance of 91.88%, 92.71%, 93.11%, 93.83% and 94.08% respectively for N=20, 40, 60, 80 and 100 Drugs. Where as Existing model HPLC and ION-PAIR-HPLC achieving 93.93% and 90.56% respectively. Concluding this result clearly that RP-HPLC achieving the greater performance in all aspects for estimation of naproxen (NPX) and esomeprazole (ESP) in pharmaceutical preparations.

 

CONCLUSION:

In the present investigation, an attempt was made to develop a simple, accurate, selective, and sensitive RP-HPLC method of esomeprazole. The developed method was validated for selectivity, accuracy, linearity, precision (Inter–day and Intraday), robustness, and ruggedness following ICH guidelines. The observation from stress degradation studies including the separation of the degradation product and quantification of esomeprazole after exposure to stress conditions shows the method is stability-indicating and capable of determining esomeprazole in presence of its degradation products which indicates the selectivity of the method. In human plasma studies, the esomeprazole recovery was achieved at 94.25%, 77.8%, and 79.2% respectively. In similar lines, linearity was achieved in the concentration range of 20-80µg/mL with the correlation co-efficient R² = 0.8501. Hence in this method, a simple mobile phase without the aid of any buffer solution or ion-pairing agents and short run-time is advantageous and makes this method suitable for routine analyses of a large number of samples per day.

 

CONFLICT OF INTEREST:

No conflicts of interest are declared.

 

ACKNOWLEDGEMENTS:

The authors expressed their gratitude to Dr. Venkatesan R and D, Vice President, Gland Pharma, Hyderabad, Telangana, India for providing the drug sample. Further, the authors thank Jyothishmathi Management Shri J. Sagar Rao, Chairman and Shri. J. Sumith Sai, Secretary and Correspondent for their valuable support and encouragement during the sessions of the work.

 

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Received on 11.09.2020            Revised on 01.10.2020                                                                                                        

Accepted on 16.10.2020     ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2021; 11(1):9-16.