Int J Curr Pharm Res, Vol 12, Issue 2, 111-114Original Article



D. S. T. S. Mandal’s College of Pharmacy, Solapur 413004 Maharashtra, India

Received: 25 Nov 2019, Revised and Accepted: 22 Jan 2020


Objective: A new, simple, economical, precise, sensitive, linear, accurate, rapid UV spectrophotometric method has been developed for the estimation of Oseltamivir Phosphate in pure form and pharmaceutical formulation.

Methods: This UV method was developed using Methanol as a solvent. In the present method, the wavelength selected for analysis was 218 nm. UV-Visible double beam spectrophotometer (Systronic 2201) was used to carry out spectral analysis. The ICH guidelines were used to validate the method.

Results: The method was validated for linearity, range, accuracy, precision, robustness, LOD and LOQ. Linearity was found in the range of 10-50µg/ml. Accuracy was performed by using a recovery study. The amount of drug recovered was found to be in the range of 99.01-100.1%. The % RSD value was found to be less than 2.

Conclusion: The developed UV spectrophotometric method was found to be simple, economic, sensitive, easy, accurate, linear, specific and highly sensitive and can be used for routine estimation of Oseltamivir Phosphate.

Keywords: Oseltamivir Phosphate, Methanol, UV-Visible spectrophotometric method, Method validation


UV-Visible spectrophotometry is one of the most frequently employed techniques in pharmaceutical analysis. By using UV-Visible spectrophotometry, the amount of ultraviolet or visible radiation absorbed by an analyte in a solution is determined [1].

Fig. 1: Chemical structure of oseltamivir phosphate [2]

Oseltamivir Phosphateis also is known as Tamiflu. Its molecular formula is C16H28N2O4. IUPAC name of Oseltamivir Phosphate is ethyl (3R, 4R, 5S)-5-amino-4-acetamido-3-(pentan-3-yloxy) cyclohex-1-ene-1-carboxylate (fig. 1). Oseltamivir is administered orally, it is an antiviral drug for the management of influenza A and B infections in children>1 y and adults of all ages [3]. Oseltamivir phosphate is an ethyl ester prodrug requiring ester hydrolysis for conversion to the active form, oseltamivir carboxylate. Oseltamivir carboxylate is an inhibitor of influenza virus neuraminidase affecting the release of viral particles [4]. The concentrations of oseltamivir carboxylate required for inhibition of influenza virus in cell culture were highly variable depending on the assay method used and the virus tested. Standard dose of oseltamivir in adults is 75 mg, while children have unit doses that are selected on the basis of body weight. Oral capsule (35, 40 and 75 mg) and suspension formulations are now readily available [5]. Antiviral drugs are a class of medication used specifically for treating viral infections rather than bacterial ones. Most of the antiviral drugs are available for treating various types of viral diseases such as, HIV, influenza A and B, herpes viruses, hepatitis B and C viruses [6].



UV/Visible double beam spectrophotometer Systronic 2201. Standard cuvettes having 10 mm of path length are used for analysis. Ultra Sonicator (micro clean-103) was used to sonicate the formulation sample. Drug sample was weighed by using an electronic analytical balance (Shimadzu AY220).

Chemicals and reagents

Active pharmaceutical ingredient of Oseltamivir Phosphate is gifted as a sample from Zydus Cadila Healthcare Ltd, the pharmaceutical company, Vadodara, Gujarat. Marketed formulation of Oseltamivir Phosphate was procured from a local pharmacy.

Experimental work

Method development

Preparation of standard stock solution of oseltamivir phosphate

Accurately weighed 10 mg of drug was transferred to 10 ml volumetric flask and dissolved in methanol, this was considered as a stock solution. From stock solution 0.1 ml, 0.2 ml,0.3 ml, 0.4 ml, 0.5 ml, were taken and was make up the volume to 10 ml with methanol to get respective concentrations of (10,20,30,40 and 50) μg/ml. Prepared samples were analyzed by using an ultraviolet double beam spectrophotometer at λmax 218 nm.

Assay of oseltamivir phosphate capsules

Weigh 20 Capsule of Oseltamivir Phosphate equivalent to10 mg of Oseltamivir Phosphate was weighed, transferred into 10 ml volumetric flask and dissolved in methanol. This solution was sonicated for 10 min and the final volume was made up to the mark with water. 1 ml of solution was transferred into 10 ml volumetric flask and diluted up to 10 ml with methanol. The absorbance of this solution was measured at 218 nm.


Method validation

The method was validated for several parameters like Linearity, Accuracy, Precision, Robustness, Limit of Detection (LOD), Limit of Quantification (LOQ) and Specificity of Oseltamivir Phosphate capsule [7-10].

Linearity and range

The linear relation between absorbance and concentration of drug was evaluated using three replicates over concentration rangein10-50µg/ml by making the replicates (table 1 and fig. 2).

Table 1: Results of linearity

S. No. Concentration(µg/ml) Absorbance
1 10 0.04
2 20 0.093
3 30 0.142
4 40 0.192
5 50 0.252

Fig. 2: Calibration curve for oseltamivir phosphate

The wavelength for linearity was scanned at 218 nm. By taking five different concentrations for linearity the regression coefficient was found to be 0.9987 i.e. in the limit of standard. Hence the linearity parameter was found to be validated.


The percentage recovery results for Oseltamivir Phosphate were varied from 99.01% to 100.1% at three different concentration levels that is (30, 40, 50µg/ml), and the results were shown in table 2. Based on the % recovery data, it was concluded that the developed method is capable for the estimation of Oseltamivir Phosphate drug substance and is adequate for routine analysis.


Method precision was determined by analyzing the test solution of six determinations, and the observed values of % RSD were shown in table 3 and 4. The % RSD for Oseltamivir Phosphate in the test solution for six formulations was not more than 2%. The solution was analyzed in 6 replicates for intra-day precision and in two successive days for inter-day precision.

Results confirmed that the precision of the method was found to be accepted. Precision results were given in table 3 and table 4 for intra and inter-day precision respectively.


The robustness of the proposed method was performed by preparing the standard solutions and test solutions of Oseltamivir Phosphateat 100% level were analyzed by a change in wavelength for absorbance readings. The wavelength selected was±2 nm to the λmax, i.e., 218 and 234 nm for Oseltamivir Phosphate drug for standard and sample solutions. Robustness was carried out on two different instruments and also carried out by using two different analysts (table 5).

Table 2: Results of accuracy

Name of drug Recovery levels Concentration (µg/ml) Amount recovered % Recovery with SD
Oseltamivir 80 % 30 30.002 100.02±0.80
100 % 40 40.001 100.01±0.26
120% 50 50.004 100.02±0.5

Table 3: Results for Intra-day precision

S. No. Concentration (µg/ml) Absorbance
1 30 0.142
2 30 0.143
3 30 0.141
4 30 0.142
5 30 0.143
6 30 0.144
SD 0.001049
%RSD 0.736006%

Table 4: Results for Inter-day precision

S. No. Concentration (µg/ml) Absorbance (Day1) Absorbance (Day2)
1 30 0.142 0.144
2 30 0.143 0.143
3 30 0.141 0.145
4 30 0.142 0.142
5 30 0.143 0.143
6 30 0.144 0.144
SD 0.001049 0.001049
%RSD 0.736006% 0.730877%

For Intra-day and the inter-day precision relative standard deviation is in the limit.

Table 5: Results for robustness

Wavelength 218 nm 234 nm
Concentration 25µg/ml 25µg/ml
Absorbance 0.111 0.113
0.112 0.112
0.114 0.111
0.112 0.112
0.111 0.111
0.115 0.114
SD 0.001643 0.001169
% RSD 1.460593 1.042239

By change in concentration and wavelengths i.e. 218 nm and 234 nm % RSD is less than 2% i.e. within the range. So the parameter was validated.

Table 6: Results for ruggedness

Concentration Analyst 1 Analyst 2
20(µg/ml) 0.093 0.092
0.092 0.091
0.094 0.095
0.091 0.093
0.095 0.094
0.093 0.092
SD 0.001414 0.001472
%RSD 1.52066% 1.585594%

By change in analyst and laboratory, there is no effect on absorbance with the same conditions (table 6). Hence, the parameter was validated.


The degree of reproducibility of test results of the same sample within different laboratories and different analysts under the same condition with the same concentration.

Limit of detection (LOD)

The limit of detection (LOD) is the lowest concentration of an analyte in a sample that can be detected but not necessarily to be determined quantitatively under specified experimental conditions. The limit of detection, as calculated statistically for Oseltamivir Phosphate was found to be 2.08 µg/ml. The low value of Limit of detection suggests that a very low concentration of drugs can be detected.

Limit of quantitation (LOQ)

The limit of quantification (LOQ) is the lowest concentration of an analyte in a sample that can be quantitatively determined within an acceptable level of accuracy and precision under the stated operational conditions of the method. The limit of quantification was found to be 6.93µg/ml. Hence, a very low concentration of drug can be quantified satisfactorily.


The developed UV spectrophotometric method was found to be simple, economic, easy, accurate, precise, linear, specific, and highly sensitive and can be used for routine estimation of Oseltamivir Phosphate.


The authors are thankful to the Principal Dr. R. Y. Patil sir, DSTS Mandal’s College of Pharmacy Solapur, for providing the facilities and continuously support for carried out this Research Programme. The authors are also thankful to Prof. Shrishail M. Ghurghure for providing consistent support and timely guidance.




All the authors have contributed equally.


Declare none


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