A Novel visible spectrophotometric method development for the estimation of oseltamivir phosphate in capsules based on ferric hydroxamate reaction

 

B. Kalyana Ramu2*, M. Syam Bab1, U. Viplava Prasad1

1Department of Organic Chemistry& Analysis of Foods Drugs &water Laboratories, School of Chemistry, Andhra University, Visakhapatnam-530003 Andhra Pradesh (India)

2Department of Chemistry, Maharajah’s College (Aided& Autonomous), Vizianagaram-535002 (AP) India.

 

 

ABSTRACT:

A Simple, sensitive spectrophotometric method in visible region was developed for the estimation of osetamivir phosphate in bulk and solid dosage forms. The method is based on the ferric hydroxamate reaction purple red colored species is formed with hydroxylamine-ferric per chlorate reagent in non aqueous medium which exhibits maximum absorption at 514 nm. Beer’s law obeyed in the concentration range of 20-60µg/ml. commercially available Natflu capsules were analyzed, and the results are statistically compared with those obtained by the UV reference method and validated by recovery studies.  The results obtained by the proposed method were in good agreement with the labeled amounts and found satisfactory and reproducible. The method is applied successfully for the estimation of the Oseltamivir phosphate in the presence of other ingredients that are usually present in dosage forms. The method offers the advantages of rapidity, simplicity and sensitivity and normal cost and can be easily applied to resource-poor settings without the need for expensive instrumentation and reagents.   

 

KEYWORDS: Anti viral, capsules, Hydroxylamine, Ferric per chlorate, Regression equation

 

INTRODUCTION:

Oseltamivir phosphate (OP) (Fig.1) is the best known orally active  newest addition to the group of H1N1 and H5N1 neuraminidase inhibitor and an antiviral drug that slows the spread of influenza (flu) viruses (type A and B) between cells in the body by stopping the new virus from chemically cutting ties with its host cell. The drug is considered the best treatment for the bird flu disease.  OP is an ethyl ester pro-drug that is rapidly and extensively metabolized by esterases in the gastrointestinal tract and liver to its active form, oseltamivir carboxylate(OC). OP is a white crystalline powder solid with the chemical name (3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid,ethylester, phosphate (1:1) and Its chemical formula is C16H28N2O4.H3PO4 representing molecular weight of 410.4.

 

In literature, OP can be identified by thin layer chromatography, specific optical rotation, infrared spectrophotometry and tests characteristic for ortho phosphates1, Determination, by International Pharmacopeia2, can be done by high-performance liquid chromatography1-2 or by titration with perchloric acid1.

 

Fig.1: Chemical structure of OP

 

Other analytical methods such as UV spectroscopy3-5, visible spectrophotometric6-9 colorimetric and LC10, spectrofluorimetric11, HPLC with UV detection12-19 and mass spectrometry20-23, Micellar electrokinetic chromatography24, capillary electrophoresis25 voltammetry26 and potentiometry27 have been reported for the determination of OP in biological fluids and formulations. The main purpose of the present study was to establish relatively simple, sensitive, validated and inexpensive extraction free visible spectrophotometric method for the determination of OP in pure form and in pharmaceutical preparations, since most of the previous methods involve critical reaction conditions or tedious sample preparations and less specificity. So the authors have made some attempts in this direction and succeeded in developing the method based on the reaction between the drug and hydroxylamine–ferric per chlorate28-29 reagent under specified experimental conditions. The proposed method for OP determination has many advantages over other analytical methods due to its rapidity, normal cost and environmental safety. Unlike HPLC, HPTLC procedures, the instrument is simple and is not costly. Economically, all the analytical reagents are inexpensive and available in any analytical laboratory. The method can be extended for the routine quality control analysis of pharmaceutical products containing OP.

 

MATERIALS AND METHODS:

Apparatus and chemicals: A Shimadzu UV-Visible spectrophotometer 1601 with10mm matched quartz cells was used for all spectral measurements. A Systronics digital pH meter mode-362 was used for pH measurements. All the chemicals used were of analytical grade. Neutral hydroxyl amine (prepared by mixing equal volume of 12.5% solution of hydroxyl amine hydrochloride in methanol and 12.5% solution of NaOH in the same solvent and filtered. Ferric per chlorate solution (prepared by dissolving 5.0 gram of ferric per chlorate in a mixture of 10 ml of 70% perchloric acid and 10ml of water, dilute to 100ml with ethanol while cooling) were prepared.

 

 

Preparation of Standard drug stock solution:

The standard stock solution (1mg/ml) of OP was prepared by dissolving 100mg of OP in 100 ml ethanol. This solution was further diluted stepwise with the same solvent to obtain working standard solution concentration of 200μg/ml. The prepared stock solution was stored at 4⁰C protected from light. From this stock solution, a series of standards were freshly prepared during the analysis day.

 

Fig.2: Absorption spectra of OP-HA-Ferric perchlorate system

Preparation of Sample solution:

About 20 capsules were weighed to get average capsule weight and pulverized. The powder equivalent to 100mg of OP was weighed, dispersed in 25ml of isopropyl alcohol (IPA), sonicated for 30minutes and filtered through whatman filter paper no.41. The filtrate was evaporated and the residue was used for the preparation of working sample solutions in the same way as under working standard solution.

 

Determination of wavelength maximum (λ max):

The 3.0 ml of working standard solution of OP (200µg/ml) was taken in 10 ml standard flask. To this, 0.3ml neutral hydroxylamine hydrochloride solution is added and kept in water bath at 70°C for 5 minutes. Then allowed to cool and dilute to 5ml with ferric per chlorate. Shaken for 2 minutes and kept aside for 5 minutes at room temperature and made up to the mark with ethanol. In order to investigate the wavelength maximum, the above standard stock solution was scanned in the range of 360-560nm by UV-Visible spectrophotometer. From the spectra (Fig.2), it was concluded that 514nm is the most appropriate wavelength for analyzing OP with suitable sensitivity.

 

Preparation of calibration curve: 

To aliquots of standard OP drug solution [1.0-3.0ml, 200µg/ml] in ethanol in a series of 10 ml calibrated tubes, 0.3ml neutral hydroxylamine hydrochloride solution is added and kept in water bath at 70°c for 5 minutes. Then allowed to cool and dilute to 5ml with ferric per chlorate. Shaken for 2 minutes and kept aside for 5 minutes at room temperature and made up to the mark with ethanol. The purple colored species was obtained and it was stable for 30 minutes.  The absorbance of the colored species was measured at 514 nm against the reagent blank. The amount of OP was computed from its calibration curve (Fig.3 showing Beer’s law plot).

 

RESULTS AND DISCUSSION:

 In developing this method, a systematic study of the effects of various parameters were undertaken by varying one parameter at a time and controlling all others fixed. The effect of various parameters such as time, volume and strength of hydroxyl amine, ferric per chlorate, stability of colored species and solvent for final dilution of the colored species were studied and the optimum conditions were established.

 

Fig.3: Beer’s Law plot of OP-HA-Ferric perchlorate system

 

The optical characteristics such as Beer’s  law limit, Sandell‘s sensitivity, molar absorptivity, percent relative standard deviation (calculated from the six measurements containing 3/4th of the amount of the upper Beer’s law limits ) were calculated  and the results are summarized in table-1. Regression characteristics like standard deviation of slope (Sb), standard deviation of intercept (Sa), standard error of estimation (Se) and % range of error (0.05 and 0.01 confidence limits) were calculated and are shown in Table-1.

 

Table 1: Optical characteristics, precision and accuracy of proposed method.

Parameter

Values

גmax  (nm)

514

Beer’s law limit(µg/ml)

20-60

Sandell’s sensitivity  (µg/cm2/0.001 abs. unit

0.016129032

Molar absorptivity (Litre/mole/cm)

 

Correlation Coefficient

25444.8

 

0.997

Regression equation  (Y)*

 

Intercept (a)

-0.104

Slope(b)

0.009

 %RSD

1.36

% Range of errors(95% Confidence  limits)

1.43

0.05 significance level

 2.24

0.01            significance level

 

 *Y = a +b x, where Y is the absorbance and x is the concentration of OP in µg/ml

 

 

Natflu capsules containing OP were successfully analyzed by the proposed method. The values obtained by the proposed and reference methods for formulations were compared statistically by the t-and F-test and found not to differ significantly. As an additional demonstration of accuracy, recovery experiments were performed by adding a fixed amount of the drug to the pre analyzed formulations at three different concentration levels.


Table 2: Analysis of oseltamivir phosphate in pharmaceutical formulations

Method

*Formulations

Labeled Amount (mg)

Found by Proposed Methods

Found by Reference Method  ± SD

#% Recovery by Proposed Method ± SD

**Amount found

± SD

t

F

HA-Ferric per chlorate

capsule-1

30

29.79 ± 0.14

0.28

1.40

29.80±0.16

99.31 ±  0.46

capsule-2

75

74.34  ± 0.43

0.67

4.49

74.65±0.91

99.11±  0.57

* Capsule- 1 and capsule-2: Natflu capsules of NATCO PHARMA LIMITED, Hyderabad (India)

**Average ± Standard deviation of eight determinations, the t- and f-values refer to comparison of the proposed method with reference method. (UV). Theoretical values at 95% confidence limits t =2.57 and f = 5.05.

# Recovery of 10mg added to the pre analyzed sample (average of three determinations). Reference method (reported UV method) using 0.1M NaOH (גּ max=216nm).

 

Fig.4. probable sequence reactions of the proposed method

 

 

These results are summarized in Table-2. The ingredients usually present in formulations of OP did not interfere with the proposed analytical method.

 

Chemistry of colored species:

Hydroxamic acids were discovered in 1869 by Lossen.  Feigl and his co-workers is first introduced the use of the ferric-hydroxamic acid reaction as a spot test for compounds containing carboxylic esters. Based on it this method has been developed. In the present investigation the presence of ester group of OP permits for the development of visible spectrophotometric method for its determination through the formation of hydroxamic acid-iron complex.   The nature of colored species formation may be involved initially the formation of corresponding hydroxamic acid by the alkaline hydrolysis of ester group in drug with hydroxyl amine and then followed by formation of molecular complex with ferric ion as shown in scheme (Fig.4). 

CONCLUSION:

The reagents utilized in the proposed method are normal cost, readily available and the procedure does not involve any critical reaction conditions or tedious sample preparation. The proposed colorimetric methods possesses reasonable precision, accuracy, and are simple, sensitive and can be used as alternative methods to the reported ones for the routine determination of OP depending on the need and situation. 

 

ACKMOWLEDGEMENTS:

The authors (BK Ramu & MS Bab) are thanks to the University Grants Commission, New Delhi for providing financial assistance under teacher fellow ship and also thanks to University authorities for providing facilities in this work.

 

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Received on 31.01.2013

Modified on 20.02.2013

Accepted on 25.02.2013     

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 5(2): March- April, 2013, 70-74