A Novel Analytical approach for Simultaneous Estimation of Esomeprazole and Ondansetron by HPLC-DAD Method with Degradation Studies

 

Uttam Prasad Panigrahy¹*, Siva Prasad Panda², Biplab Kumar Dey³

^1,3Faculty of Pharmaceutical Sciences, Assam Down Town University,

Sankar Madhab Path, Gandhi Nagar, Panikhaiti, Guwahati, Assam, India.

²Institute of Pharmaceutical Research, GLA University, Mathura, UP, India.

 

ABSTRACT:

Objective: A simultaneous estimation of Esomeprazole and Ondansetron by HPLC-DAD method with degradation studies was developed. Method: It was carried out using Kromasil (150mm×4.6mm, 5µm particle size) column with mobile phase consists 0.01M KH₂PO₄ Phosphate buffer (1mL of Triethylamine added and pH was adjusted to 3.5 with Orthophosphoric acid): Acetonitrile (35:65, %v/v) with flow rate 1mL/min at 265nm. Esomeprazole and Ondansetron were eluted at 2.350 and 2.831mins respectively. Results and Discussion: Validation was performed as per ICH guidelines, which shows linearity 25-150μg/mL of Esomeprazole and 10-60μg/mL of Ondansetron with R² was 0.999. Accuracy was obtained to be 99.75-100.21% for Esomeprazole and 99.39-100.3% for Ondansetron respectively and precision which shows %RSD less than 2%. Stress studies were carried out which shows that both Esomeprazole and Ondansetron is moderately degraded in acid, base, peroxide and thermal degradation and less degraded in photolytic degradation. Conclusion: This method was applicable for routine analysis and stability of Esomeprazole and Ondansetron.

 

 

 

INTRODUCTION: 

Esomeprazole is a proton-pump inhibitor which decreases the secretion of gastric acid by particular inhibition of an enzyme H⁺/K⁺-ATPase in the gastric parietal cell. Chemically Esomeprazole is (S)-5-methoxy-2-[(4-methoxy3, 5 dimethylpyridi2yl) Methyl sulfinyl] 3H benzoimidazole was shown in Figure 1. It is also used in the treatment of GERD, gastrointestinal ulcers associated with Crohn's disease.

 

 

Figure 1: Chemical structure of Esomeprazole

 

Ondansetron is a serotonin 5-HT3 receptor antagonist and low affinity for Dopamine receptors was shown in Figure 2. Chemically Ondansetron is (RS)-9-methyl-3-[(2-methyl-1H-imidazol-1-ylmethyl]-2, 3dihydro-1H-carbazol-4(9H)-one¹. It is an antiemetic drug effective against nausea and vomiting.

 

Figure 2: Chemical structure of Ondansetron

 

Literature review revealed that there is a few analytical methods were developed either individual or combined with other drugs. Such methods are as follows, HPLC estimation for Omeprazole and Ondansetron in pharmaceutical formulation². Evaluation of drotaverine HCl and omeprazole in pharmaceutical formulation by HPLC³. HPLC method for Eluxadoline⁴. Stability indicating RP-HPLC method for determination of Ondansetron in orally disintegrating films⁵. RP-HPLC method for Sacubitril and Valsartan⁶. HPLC method for Stavudine⁷. UV-Spectrophotometer determination of Domperidone and (S)-Esomeprazole Magnesium in capsule dosage form⁸. Simultaneous Estimation of Prazosin and Polythiazide by RP-HPLC method⁹. Validation of UPLC-tandem mass spectrometry coupled with electrospray ionization method for determination of ornidazole in solid dispersion¹⁰. Determination of Tenofovir in pharmaceutical formulation using stability indicating RP-HPLC method¹¹. Stress studies are used to achieve better understanding of active pharmaceutical ingredient and drug product stability as well as to present information concerning degradation pathways and products¹². The objective of this work was to develop and validate the method with forced degradation studies of Esomeprazole and Ondansetron in bulk and pharmaceutical formulation by RP-HPLC as per ICH guidelines¹³. The basis for stability studies research is to present the information about the uniformity of a substance differs with an ecological variables such as temperature, humidity, light and permits the planned storage conditions, reanalysis phases and shelf life^14,15.

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Esomeprazole and Ondansetron API were obtained from ASB Drugs, Hyderabad, India and Maps Labs Pvt. Ltd., India respectively. Potassium dihydrogen orthophosphate, Triethylamine, orthophosphoric acid, Water and Acetonitrile (Merck, HPLC-Grade) were utilized in the study.

 

Instrumentation:

The separation done on Waters 515 HPLC system, attached with PDA 2998 Detector and Empower software; Kromasil (150mm×4.6mm, 5µm particle size) were used as stationary phase. Shimadzu 1800 double beam UV-Spectrophotometer was used for wavelength and isosbestic point determination. All amounts were weighed in an electronic balance (CA123, Contech), pH Meter (3 Star, Global) and Sonicator (UCB 70, Life care) were utilized in the study.

 

Selection of wavelength:

Both the drugs are scanned in UV-Spectrophotometer and the isosbestic point is determined by overlaying the spectra of both drugs at 265nm were shown in Figure 3.

 

 

Figure 3: Isosbestic point of Esomeprazole and Ondansetron at 265nm

 

Preparation of phosphate buffer and mobile phase:

1.36gm Potassium di-hydrogen Ortho-phosphate was exactly weighed and taken into a 1L vol. flask to it 900mL of HPLC grade water was added and then ultrasonication for degassing then final volume was made upto the mark with HPLC grade water. To above solution 1mL of Triethylamine was added and adjusted the P^H 3.5 with Orthophosphoric acid. The above phosphate buffer solution (35%) and Acetonitrile HPLC grade (65%) were mixed and degassed in an ultrasonicator about 15mins and then filtered through 0.45µ filter under vacuum filtration.

 

Standard and sample solution preparation:

Both standard and sample solution of 100µg/mL of Esomeprazole and 40µg/mL of Ondansetron were prepared from standard and sample stock solution for this study.

 

RESULTS AND DISCUSSION:

Assay Methodology:

From the above standard and sample solution, 20µL were injected inside HPLC and % assay were calculated and results was revealed in Table 1 and Figure 4 and 5.

 

Table 1: Reports of Assay studies

Serial No.	Name of drug	Labelled amount (mg)	Observed amount (mg)	Average %Assay (n=6)	SD	% RSD
1.	Esomeprazole	10	10.05	100.5	0.71	0.72
2.	Ondansetron	4	4.01	100.01	1.1	1.1

 

 

Figure 4: Chromatogram for standard Esomeprazole and Ondansetron

 

Figure 5: Chromatogram for sample Esomeprazole and Ondansetron

 

Analytical method validation:

System Suitability:

It is important for assuring the performance quality of chromatographic system. So, system suitability studies were performed and the results were revealed in Table 2.

 

Table 2: Results of System suitability studies

Parameter	Esomeprazole	Ondansetron	Acceptance Limits
Theoretical plate number	4645	5074	NLT:2000
Retention time	2.350min	2.831min	-
Tailing factor	1.27	1.22	NMT:1.5
Resolution		3.23	NLT:2
LOD (mg/mL)	1.11	1.35
LOQ (mg/mL)	3.39	4.09

 

Linearity:

A concentration range of 25-150μg/mL of Esomeprazole and 10-60μg/mL of Ondansetron was prepared and were injected inside HPLC system and the results were shown in Figure 6 and 7.

 

Figure 6: Linearity of Esomeprazole

 

Figure 7: Linearity of Ondansetron

 

Accuracy:

It expresses the closeness of agreement between the conventional true value and the value found. 50%, 100% and 150% concentration level were chosen three times for accuracy studies by standard addition method. % recovery and RSD were calculated and was shown in Table 3and 4.

 

Table 3: Recovery Studies of Esomeprazole

Amount API added (µg/mL)	Amount found (µg/mL)	% recovery	Statistical data
50	50.12	100.24	Mean	100.06
50	49.93	99.87	SD	0.183
50	50.04	100.08	%RSD	0.182
100	99.55	99.55	Mean	99.75
100	99.65	99.65	SD	0.24
100	100.02	100.02	%RSD	0.25
150	150.55	100.37	Mean	100.21
150	151.66	101.10	SD	0.98
150	148.74	99.16	%RSD	0.98

 

Table 4: Recovery Studies of Ondansetron

Amount API added (µg/mL)	Amount found (µg/mL)	% recovery	Statistical data
20	19.82	99.14	Mean	100.14
20	20.11	100.55	SD	0.87
20	20.14	100.73	%RSD	0.86
40	39.77	99.44	Mean	99.39
40	39.85	99.63	SD	0.26
40	39.64	99.11	%RSD	0.26
60	60.29	100.49	Mean	100.30
60	59.81	99.68	SD	0.55
60	60.44	100.74	%RSD	0.54

 

Precision:

It is defined as the degree of agreement between individual test outcomes acquired when the method is applied to multiple sampling of a homogenous sample.

 

(a)  System precision:

Standard solution of 100µg/mL of Esomeprazole and 40µg/mL of Ondansetron were injected six times inside HPLC and %RSD were calculated and revealed in Table 5.

 

Table 5: System Precision of Esomeprazole and Ondansetron

Injection/ concentration (100mg/mL)	Esomeprazole Peak Area	Injection/concentration (40mg/mL)	Ondansetron Peak Area
Injection-1	3931739	Injection-1	1743173
Injection-2	3909951	Injection-2	1748486
Injection-3	3904683	Injection-3	1733044
Injection-4	3931904	Injection-4	1741405
Injection-5	3913737	Injection-5	1736641
Injection-6	3931338	Injection-6	1733688
Average	3920558.7	Average	1739406
Standard Deviation	12498.04	Standard Deviation	6024.093
%RSD	0.32	%RSD	0.34

 

(b) Method precision:

Homogeneous sample solution of 100µg/mL of Esomeprazole and 40µg/mL of Ondansetron were injected six times inside HPLC and peak area, % Assay with SD and %RSD were calculated and revealed in Table 6.

 

Table 6: Method Precision of Esomeprazole and Ondansetron

Conc. (100mg/mL)	Esomeprazole Peak Area	% Assay	Conc. (40mg/ mL)	Ondansetron Peak Area	% Assay
Injection-1	3901561	99.17	Injection-1	1744164	99.80
Injection-2	3943666	100.24	Injection-2	1736679	99.37
Injection-3	3917788	99.58	Injection-3	1742848	99.73
Injection-4	3949119	100.38	Injection-4	1721810	99.52
Injection-5	3923926	99.74	Injection-5	1772380	101.42
Injection-6	3981670	101.20	Injection-6	1768463	101.19
Average	3936288	100.05	Average	1747724	100.01
SD	28212.2	0.7	SD	19328.7	1.1
%RSD	0.72	0.72	%RSD	1.1	1.1

 

 

Table 7: Intermediate Precision of Esomeprazole

Conc. (100mg/ mL)		Laboratory/Analyst-1  %Assay		Laboratory/Analyst-2   %Assay
		Day-1	Day-2	Day-1		Day-2
Injection-1		99.17	99.87	99.67		99.96
Injection-2		100.24	100.1	100.12		100.13
Injection-3		99.58	99.87	99.69		99.86
Injection-4		100.38	100.01	99.94		100.1
Injection-5		99.74	99.16	99.89		99.94
Injection-6		101.2	99.89	99.97		100.2
Average		100.05	99.81	99.88		100.03
SD		0.715	0.334	0.172		0.13
%RSD		0.715	0.335	0.173		0.13
Reproducibility
	Laboratory/ Analyst-1 % Assay		Laboratory/ Analyst-2 % Assay		Mean Reproducibility
Average	99.93		99.95		Mean Average		99.94
SD	0.52		0.15		Mean SD		0.33
%RSD	0.52		0.15		Mean %RSD		0.33

Table 8: Intermediate Precision of Ondansetron

Conc. (40mg/mL)		Laboratory/Analyst-1 %Assay			Laboratory/Analyst-2 %Assay
		Day-1	Day-2		Day-1		Day-2
Injection-1		99.80	99.81		99.68		100.01
Injection-2		99.37	99.91		99.76		99.62
Injection-3		99.73	99.72		99.81		99.79
Injection-4		99.52	99.86		99.95		99.86
Injection-5		101.42	99.29		100.02		99.61
Injection-6		101.19	100.01		100.04		99.97
Average		100.17	99.76		99.87		99.81
SD		0.89	0.25		0.14		0.17
%RSD		0.892	0.253		0.14		0.17
Reproducibility
	Laboratory/ Analyst-1 % Assay			Laboratory/ Analyst-2 % Assay		Mean Reproducibility
Average	99.96			99.84		Mean Average		99.9
SD	0.57			0.15		Mean SD		0.36
%RSD	0.57			0.15		Mean %RSD		0.36

 

Intermediate Precision:

It was also called as ruggedness and performed on different analyst and day. Standard solution of 100µg/mL of Esomeprazole and 40µg/mL of Ondansetron were injected six times inside HPLC and %RSD were calculated and revealed in Table 7 and 8.

 

Robustness:

Standard solution of 100µg/mL of Esomeprazole and 40µg/mL of Ondansetron were injected six times inside HPLC by varying flow rate at 0.9 to 1.1mL/min and organic composition of mobile phase from 40:60 to 30:70, %v/v and the robustness result were revealed in Table 9 and 10.

 

 

Table 9: Variations in flow rate

Sample Name	Change in flow rate (mL/min)	Retention Time (min) (n=6)	Average area of six injections	SD	%RSD	USP Plate count	USP Tailing
Esomeprazole	0.9	2.364	5646947	14386	0.3	4335	1.25
	1.1	2.334	5594612	21485	0.4	4284	1.23
Ondansetron	0.9	2.917	1204024	2167	0.2	5047	1.17
	1.1	2.809	1222681	1528	0.1	5020	1.24

 

Table 10: Variations in organic composition of mobile phase

Sample Name	Change in organic composition of mobile phase	Retention Time (min) (n=6)	Average area of six injections	SD	% RSD	USP Plate count	USP Tailing
Esomeprazole	40:60,%v/v	2.338	3541919	3729.6	0.1	4424	1.27
	30:70,%v/v	2.404	5634677	7064.3	0.1	4480	1.25
Ondansetron	40:60,%v/v	2.840	1244674	92.63	0.007	5003	1.22
	30:70,%v/v	3.020	1191851	10969.1	0.9	5287	1.28

 

 

Stress studies:

A stress study was performed to assess the stability and specificity of the method. A concentration of 100µg/mL Esomeprazole and 40µg/mL Ondansetron were chosen for the stress studies. 0.1 M HCl was taken for acid decomposition stress study and alkaline degradation was carried out using 0.1M NaOH and both acid and base influenced drug samples were refluxed at 80ºC for 30 mins. After cooling the solutions was neutralized and diluted with mobile phase. For Hydrogen peroxide stress testing, diluted hydrogen peroxide of 0.3 to 3% can be used at 40°C. Drug solutions was also exposed to thermal degradation and for photo stability testing was exposed to UV light for 4h UV light chamber (365nm) and analyzed. Esomeprazole is moderately degraded in acid hydrolysis (%degradation is 9.01%), in basic hydrolysis (%degradation is 8.39%), in peroxide degradation (%degradation is 7.22%), in thermal degradation (%degradation is 4.58%) and less degraded in photolytic degradation (%degradation is 1.98%). Ondansetron is moderately degraded in acid hydrolysis (% degradation is 8.07%), in basic hydrolysis (% degradation is 6.87%), in peroxide degradation (% degradation is 5.62%), in thermal degradation (% degradation is 3.77%) and less degraded in photolytic degradation (%degradation is 1.40%). The representative chromatograms for stress testing studies are presented in Figure 8. The percentage drug recovered and the drug decomposed were calculated and revealed in Table 11 and 12.

 

(A)

 

 

(B)

 

(C)

 

(D)

 

(E)

 

Figure 8: Degradation chromatograms for stress studies, (A) Acid degradation, (B) Base degradation, (C) Photolytic degradation, (D) Oxidative degradation, and (E) Thermal degradation

 

Table 11: Forced degradation of Esomeprazole

Stress conditions	Concentration (mg/mL)	Peak Area	% Drug recovered	% Drug decomposed	Purity angle	Purity threshold
Acid hydrolysis	90.54	3486612	90.99	9.01	0.123	0.302
Base hydrolysis	91.16	3510527	91.61	8.39	0.109	0.295
Photolytic degradation	97.54	3756275	98.02	1.98	0.114	0.298
Peroxide degradation	92.32	3555166	92.78	7.22	0.138	0.296
Thermal degradation	94.95	3656561	95.42	4.58	0.106	0.292

 

Table 12: Forced degradation of Ondansetron

Several mobile phase compositions were tried for the optimization of the method and a better resolution and symmetry peak of Esomeprazole and Ondansetron was achieved using 0.01M KH₂PO₄ Phosphate buffer (1ml of Triethylamine added and pH adjusted to 3.5 with Orthophosphoric acid): Acetonitrile (35:65, %v/v) mobile phase with a flow rate of 1mL/min at 265nm. Esomeprazole and Ondansetron were eluted at 2.350 and 2.831mins respectively. Validation was performed as per ICH guidelines, which shows linearity 25-150 μg/mL of Esomeprazole and 10-60μg/mL of Ondansetron with R² was 0.999. Accuracy was obtained to be 99.75-100.21% for Esomeprazole and 99.39-100.3% for Ondansetron respectively and precision which shows RSD less than 2%. The % RSD value of method precision was 0.72% for Esomeprazole and 1.1% for Ondansetron, system precision was 0.32% for Esomeprazole and 0.34% for Ondansetron and reproducibility was 0.33% for Esomeprazole and 0.36% for Ondansetron which indicates that the proposed method is precise and the LOD value of Esomeprazole was 1.11 μg/mL and Ondansetron was 1.35 μg/mL and LOQ value of Esomeprazole was 3.39 μg/mL and Ondansetron was 4.09 μg/mL. Robustness studies were successfully carried out and found to be < 2% RSD which indicates that the proposed method is robust. The results and discussion reports revealed that the developed method is unambiguous and valid for the estimation of Esomeprazole and Ondansetron by RP-HPLC.

 

CONCLUSION:

RP-HPLC method for Esomeprazole and Ondansetron in bulk and mixed dosage form were developed and validated in accordance to ICH guidelines. Esomeprazole and Ondansetron with correlation coefficient 0.999 were achieved with better linearity. Accuracy of the drug was attained in the range of 98-102% within the acceptance criteria. Precision were successfully carried out and found to be < 2% RSD which indicates that the proposed method is precise. The developed method is unambiguous and valid for estimation of Esomeprazole and Ondansetron by RP-HPLC and also provides information about stability of drugs under stress conditions.

 

ACKNOWLEDGEMENT:

The authors are thankful to Faculty of Pharmaceutical Sciences, Assam down town University, Guwahati, Assam, INDIA for providing necessary facilities for conducting this research work.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.      Tripathi KD. Essentials of medical pharmacology, Jaypee brother’s medical publishers, New Delhi, 2018; 8th edition. ISBN-13:978-8180611872

2.      Dedania Z. Dedania R. Karkhanis V. RP-HPLC Method for Simultaneous Estimation of Omeprazole and Ondansetron in Combined Dosage Forms. Asian J. Research Chem. 2009; 2(2):108-111. doi.org/10.5958/0974-4150

3.      Topagi KS. Jeswani RM. Sinha PK. Damle MC. A validated normal phase HPLC method for simultaneous determination of drotaverine hydrochloride and omeprazole in pharmaceutical formulation. Asian Journal of Pharmaceutical and Clinical Research. 2010; 3(1):20-24. https://innovareacademics.in/journal/ajpcr/Vol3Issue1/244.pdf

4.      Panigrahy UP. Reddy ASK. A novel validated RP-HPLC-DAD method for the estimation of Eluxadoline in bulk and pharmaceutical dosage form. Research J. Pharm. and Tech. 2015; 8(11):1469-1476. doi: 10.5958/0974-360X.2015.00263.2

5.      Singh PK. Development and Validation of a Stability Indicating RP-HPLC Method for Determination of Ondansetron in Orally Disintegrating Films. International Journal of Research in Pharmacy and Science. 2013; 3(1):57-66. http://www.ijrpsonline.com/pdf/0191.pdf

6.      Kadam GM. Puyad AL. Kalyankar TM. RP-HPLC Method Development and Validation for the Estimation of Sacubitril and Valsartan in Pharmaceutical Dosage Form. Research Journal of Pharmacy and Technology. 2021; 14(11):5797-2. doi: 10.52711/0974-360X.2021.01008

7.      Dayaramani RA. Patel PU. Patel NJ. Development and Validation of RP-HPLC Method for Estimation of Stavudine in Bulk and in Capsule Formulation. Research J. Pharm. and Tech. 2020; 13(1):15-21. doi: 10.5958/0974-360X.2020.00003.7

8.      Singh S. Rai J. Inamullah. Choudhary  N. Gautam H. Sharma S. Yadav AK. Agrawal VK. Chaturvedi S.  Stability indicating simultaneous equation method for determination of Domperidone and (S)-Esomeprazole Magnesium in capsule dosage form using UV-Spectrophotometer. Journal of Pharmaceutical Research International. 2013; 3(3): 435-445. doi: 10.9734/BJPR/2013/3197

9.      Panigrahy UP. Kumari KNV. Ram Mohan Reddy T. Abbulu K. RP-HPLC Method Development and Validation for Simultaneous Estimation of Prazosin and Polythiazide in Bulk and Pharmaceutical Dosage Form. Research J. Pharm. and Tech. 2020; 13(4):1779-1787. doi: 10.5958/0974-360X.2020.00321.2

10.   Agrawal GP. Maheshwari RK. Mishra P. Validation of ultra performance liquid chromatography-tandem mass spectrometry coupled with electrospray ionization method for quantitative determination of ornidazole in solid dispersion. Current Pharmaceutical Analysis. 2020; 16(5): 487-493. https://doi.org/10.2174/1573412914666181024145937

11.   Chhabra GS. Rajora A. Mishra DK. Stability indicating RP-HPLC method for the determination of Tenofovir in pharmaceutical formulation. Research Journal of Pharmacy and Technology. 2021; 14(12):6335-9. doi: 10.52711/0974-360X.2021.01095

12.   Alsante KM. Ando A. Brown R. Ensing J.  Hatajik TD. Kong W. Tsuda Y. The role of degradant profiling in active pharmaceutical ingredients and drug products. Adv Drug Del Rev. 2007; 59(1): 29–37. doi: 10.1016/j.addr.2006.10.006

13.   Shabir GA. Validation of high-performance liquid chromatography methods for pharmaceutical analysis. Understanding the differences and similarities between validation requirements of the US Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. J Chromatogr A. 2003; 987(1-2): 57-66. doi: 10.1016/s0021-9673(02)01536-4

14.   Blessy M. Patel RD. Prajapati PN. Agrawal YK. Development of forced degradation and stability indicating studies of drugs—a review. J Pharm Anal. 2014; 4(3):159–165. doi.org/10.1016/j.jpha.2013.09.003

15.   Soni NR. Stability Indicating Analytical Methods (SIAMs), Scholars Press, USA; 2018: 72. ISBN-13:978-6202305327

 

 

Accepted on 30.12.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4855-4860.