1. INTRODUCTION:

Oseltamivir is an ester prodrug, which is rapidly and extensively hydrolysed invivo to its active metabolite. Oseltamivir carboxylate, a potent and selective inhibitor of influenza virus neuraminidase. Oseltamivir phosphate chemically known as ethyl(3R,4R,5S)-4(acetylamino)-5-amino -3-(1-ethyl propoxy)cyclohex-1-ene-1carboxylate dihydrogen phosphate. Oseltamivir is a white to off white powder freely soluble in water and methanol and sparingly soluble in DMSO. Oseltamivir phosphate requires conversion to the corresponding carboxylate by esterases located predominantly in the liver, to be physiologically active^[1]. The chemical formula is C₁₆H₂₈N₂O₄ (free base). The molecular weight Oseltamivir is 312.4.

Literature survey revealed, few analytical methods, which include,include, FTIR methods for the quality control of Oseltamivir determination in Timiflu capsules and generic version^[2],LC-MS^[3,4,5], Colorimetric^[1]and liquid chromatographic method^[5,6] UV/Visible spectrophotometric method^[7,8,9] and stability indicating HPTLC method^[10].

Spectrophotomertic is a potent analytical technique used for the dermination of drugs, because of its high sensitivity and selectivity, low cost, and wide availability in most of laboratories. However, to our knowledge, no information related to the method development and stability studies using UV/Visible spectrophotometry in bulk form. According to the stability test guidelines issued by ICH the present study, the stress induced stability studies were carried out for Oseltamivir to establish its stability characteristics.

2. EXPERIMENTAL PROCEDURE:

2.1 MATERIALS:

2.1.1 Instrumentation:

A double beam Schimadzu UV-Visible Spectrophotometer, model (1800) with 1cm quartz cell attached with printer, UV lamp, Digital balance, Water bath.

2.1.2 Chemicals and reagents:

Oseltamivir pure drug was procured as a gift sample from industry. The Fluvir capsules containing 750mg of Oseltamivir procured from local market, analytical grade Sodium hydroxide, Hydrochloric acid, 3% and 30% Hydrogen peroxide were used.

2.1.3 Glasswares:

Volumetric flask 100ml, 10ml, pipettes 0.5ml, 1ml, 5ml, 10ml, beaker 250ml.

2.2 METHODS:

2.2.1 Selection of solvent:

Copious trials were done to find out the right solvent system for dissolving drug. The solvents like distilled water, methanol and DMSO were tried depending on solubility of Oseltamivir. Oseltamivir is soluble in distilled water and methanol, sparingly soluble in DMSO. Based on the solubility of Oseltamivir, distilled water was selected all the way through experiment.

2.2.2 Selection of detection wave length:

To determine the optimum λ_max Oseltamivir 20µg/ml of working standard solution was prepared and scanned in UV wave length range of 200-400nm utilizing distilled water as a blank. It was observed that the drug showed maximum absorbance at 216nm which was choosen as the detection wave length for estimation of Oseltamivir.

2.2.3 Preparation of stock and working standard solution:

Stock solution of Oseltamivir was prepared by accurately weigh and dissolving 10mg in 100ml distilled water to get concentration of 100µg/ml. The working standard solution of Oseltamivir was prepared by suitable dilution.

2.2.4 Preparation of calibration curve:

A calibration curve was plotted over a concentration range of 5-30µg/ml for Oseltamivir, previously measured standard solution of Oseltamivir (0.5, 1.0, 1.5, 2.0, 2.5, 3.0ml) was shifted to a series of 10ml volumetric flask and the volume was filled upto 10ml with distilled water. Calibration curve was done by plotting Oseltamivir concentration on X-axis and their respective absorbance on Y-axis. The correlation coefficient was found to be 0.999. Calibration data shown in Table (2) Figure (2) shows the overlay spectrum of Oseltamivir. Calibration curve exhibit in Figure (3).

2.3 FORCED DEGRADATION STUDIES:

To evaluate the stability indicating property of the proposed UV method, stress studies were done under ICH recommended conditions. Forced degradation of Oseltamivir was carried out by exposing the bulk sample to alkaline, acidic, oxidative, photolytic and thermal conditions. The aim was to study the ability of the developed method to measure the analyte response in presence of its degradation product.

2.3.1 Alkaline hydrolysis:

From the stock solution (100µg/ml), 2ml of drug solution was transferred to 26, 10ml volumetric flasks, 13 flasks were made up to the mark 1M sodium hydroxide and remaining flasks with 0.1M sodium hydroxide. 12 measuring flasks with 1M sodium hydroxide were heated at 50ºC and at 60ºC. 12 flasks with 0.1M sodium hydroxide were heated at 50ºC and 60ºC for 1 hour with sampling interval of every 5 minutes. Remaining 2 flasks were kept at room temperature for 1 hour. The prepared solutions were taken in cuvette and absorbance was recorded. Figure (4, 5, 6, 7, 8 and 9)

2.3.2 Acid hydrolysis:

From the stock solution (100µg/ml), 2ml of drug solution was transferred to 26, 10ml volumetric flasks, 13 flasks were made up to the mark 1M hydrochloric acid and remaining flasks with 0.1M hydrochloric acid. 12 measuring flasks with 1M hydrochloric acid were heated at 50ºC and at 60ºC. 12 flasks with 0.1M hydrochloric acid were heated at 50ºCand 60ºC for 1 hour with sampling interval of every 5 minutes. Remaining 2 flasks were kept at room temperature for 1 hour. The prepared solutions were taken in cuvette and absorbance was recorded. Figure (10, 11, 12, 13, 14 and 15)

2.3.3 Oxidative degradation:

From the stock solution (100µg/ml) 2ml of the drug solution was transferred to 4 volumetric flasks. 2 flasks were made up to the mark with 3% hydrogen peroxide and remaining with 30% hydrogen peroxide. 1 flask from 3% and 30% hydrogen peroxide were heated at 60ºC for 1 hour and the remaining 2 flasks were kept at room temperature for 1 hour and absorbance was recorded. Figure (16)

2.3.4 Thermal degradation:

2ml of stock solution was transferred into 2, 10ml volumetric flasks and made upto the mark with distilled water. The flasks were heated at 50ºC and 60ºC and absorbance was measured. Figure(17, 18)

2.3.5 Photolytic degradation:

2ml of the drug solution were transferred to 9,10ml volumetric flasks made upto the mark with distilled water. The solutions were exposed to UV light at 365nm for 9 hours with sampling interval of every 1 hour and were absorbance was measured. Figure (19)

3. RESULTS AND DISCUSSION:

3.1 Method development and validation:

Oseltamivir was soluble and stable in distilled water. So distilled water was utilized for the estimation of detection wave length and preparation of standard and working concentration. In order to check the proposed method to the pharmaceutical formulation an assay of Oseltamivir 75mg capsules was used at working concentration at 216nm was in the acceptance limit 98-102%. UV spectrophotometric method developed according to guidelines for validation of analytical procedure. The method was validated for parameters such as linearity, precision accuracy, LOD and LOQ.

Figure 1. Overlay spectrum and Calibration curve of Oseltamivir

Table 1. Summary of Optical characteristics and validation parameter

Parameters	Result
Detection wavelength	216nm
Beerʹs Law(µg/ml)	5-30µg/ml
Regression equation(y=mx+c)	0.022x + 0.024
Correlation coefficient	0.999
Slope	0.022
Intercept	0.024
Precision:
Intra-day	0.26868
Inter-day	0.1698-0.2079
Accuracy(% mean recovery):
80% level	77.9%
100% level	99.9%
120% level	120.8%
LOD and LOQ	0.3727-0.1230

3.2 Forced Degradation Studies

3.2.1 Alkali Hydrolysis of Oseltamivir using 1N NaOH and 0.1N NaOH at 50⁰C and at 60⁰C

Figure 2. UV spectra of Alkali Hydrolysis of Samples

Table 2. Results of Alkali hydrolysis

Chemicals	Temp (ºC)	Time (min)	Conc(µg/ml)	λ _max	Absorbance
0.1 N NaOH	50ºC	5	20	216.60	1.503
		10		215.80	1.529
		15		216.20	1.506
		20		216.20	1.542
		25		216.00	1.476
		30		216.00	1.504
0.1NaOH	60ºC	5	20	216.00	1.461
		10		216.40	1.408
		15		216.40	1.399
		20		216.20	1.412
		25		216.40	1.460
		30		216.60	1.503
Chemicals	Temp (ºC)	Time (min)	Conc (µg/ml)	λ _max	Absorbance
1N NaOH	50ºC	5	20	221.00	2.233
		10		220.60	2.316
		15		220.40	2.227
		20		220.00	2.328
		25		220.00	2.222
		30		220.40	2.328
1N NaOH	60ºC	5	20	220.20	2.298
		10		220.40	2.292
		15		221.00	2.225
		20		220.80	2.323
		25		220.80	2.248
		30		220.40	2.267
0.1N NaOH	Room temp	-	20	216.00	1.564
1N NaOH	Room temp	-	20	219.80	2.217

3.2.2 Acid Hydrolysis of Oseltamivirusing 1N HCl and 0.1N HCl at 50⁰C and at 60⁰C

Figure 3. UV spectra of Acid Hydrolysis of Samples

Table 3.Results for Acid hydrolysis

Chemicals	Temp ºC	Time (min)	Conc (µg/ml)	λ_max	Absorbance
0.1N HCl	50ºC	5	20	216.80	0.405
		10		217.10	0.456
		15		215.40	0.408
		20		217.40	0.473
		25		216.80	0.483
		30		217.00	0.469
0.1N HCl	60ºC	5	20	216.20	0.533
		10		216.80	0.417
		15		215.80	0.479
		20		215.20	0.403
		25		215.20	0.466
		30		216.00	0.435
Chemicals	Temp ºC	Time (min)	Conc (µg/ml)	λ_max	Absorbance
1N HCl	50ºC	5	20	212.20	0.679
		10		209.60	0.752
		15		212.80	0.741
		20		210.80	0.737
		25		212.80	0.650
		30		211.00	0.664
1N HCl	60ºC	5	20	211.80	0.583
		10		212.00	0.640
		15		214.20	0.687
		20		210.20	0.697
		25		212.00	0.716
		30		211.40	0.729
0.1N HCl	Room temp	-	20	217.80	0.452
1N HCl	Room temp	-	20	210.80	0.793

3.2.3 Oxidative Degradation of Oseltamivir

Figure 4.UV SpectrumPeroxide degradation

3.2.4 Thermal Degradation of Oseltamivir

Figure 5. UV spectra ofThermal Degradation

3.2.5 Photolytic Degradation of Oseltamivir

Figure 6. UV spectrum Photolytic degradation

Table 4. Summary of stress degradation studies

S.No	Stress condition	Observation
1	Alkali hydrolysis	Moderate degradation
2	Acid hydrolysis	Little degradation
3	Oxidative degradation	High degradation
4	Thermal degradation	Little degradation
5	Photolytic degradation	High degradation

4. CONCLUSION:

There were no UV methods have been reported (with distilled water as solvent) so for determination of Oseltamivir in bulk form. Validation parameters are found within the limits. Infact, the magnitude of degradation was in the order of photolytic > Oxidative >Alkaline > Acidic > Thermal. It was observed that all the statistical analysis results of % RSD values particularly precision, accuracy are observed below two which speaks that the method is precise and accurate. Therefore this method was simple, precise, accurate and cost effective and in actual fact feasible for routine sample analysis of Oseltamivir in bulk form.

5. ACKNOWLEDGEMENT:

The authors are thankful to industry for providing the sample of Oseltamivir. We are highly grateful to the Principal and Management ofKarpagam College of Pharmacy, Coimbatore. We obligate our teaching and non-teaching staffs from Karpagam College of Pharmacy, Coimbatore who provided insight and expertise that immensely assisted the analysis.

6. CONFLICT OF INTEREST:

It is declared none.

7. REFERENCES:

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Received on 24.09.2019 Modified on 10.12.2019

Research J. Pharm. and Tech 2020; 13(9):4323-4328.

DOI: 10.5958/0974-360X.2020.00764.7