Spectrophotometric Determination and Validation of Zidovudine Concentration in Bulk and Dosage form

 

Ghanshyam B. Jadhav1*, Amar G.  Zalte1, Ravindranath B. Saudagar1 and Ashok P. Pingale2
1KCT’s R. G. Sapkal College of Pharmacy, Anjaneri, Tal-Trimbakeshwar, Dist- Nashik, Maharashtra, India.

2Dept. of Pharmaceutical Chemistry, M.V.P's College of Pharmacy, Gangapur Road, Nashik, Maharashtra, India.

*Corresponding Author E-mail: aaryajadhav@rediffmail.com

 

ABSTRACT:

A simple, accurate, precise, sensitive and a highly selective ultra violet spectrophotometric method has been  developed for the simultaneous estimation of  in bulk and solid dosage form. The estimation of Zidovudine was carried out at 266 nm. The developed method was validated for linearity, range, precision, recovery studies and interference study for mixture. All these parameters showed the adaptability of the method for the quality control analysis of the drug in bulk and in marketed formulations.

 

KEYWORDS: Zidovudine, UV- method, Spectrometric method, Determination

 

 

INTRODUCTION:

Zidovudine (INN) or azidothymidine  (AZT)  is a synthetic drug with pyrimidine nucleoside analogue active against HIV-1, AIDS and pre- AIDS.  It has a molecular formula of C10H13N5O4 and a molecular weight of 267.24 g/mol. Zidovudine is a white to beige, odorless, crystalline solid and it is soluble in ethanol (95%),  sparingly soluble in water. Chemically Zidovudine  is1- (3-azide-2, 3-di deoxy-β-D-ribofuranosyl)-5-methyl Pyrimidin-2, 4 (1H, 3H) – dione. Zidovudine also has been referred to as 3′azido-3′-deoxythymidine. It has the structural formula shown as,

 

The drug is officially listed in United States of Pharmacopeia. Various analytical methods that have been reported for the estimation of Zidovudine in biological fluids or pharmaceutical formulations include UV-Visible Spectrophotometry, High Performance Liquid Chromatography and HPTLC. Thus efforts is to develop a simple, accurate, precise and economic UV spectrophotometric method for the estimation of Zidovudine in bulk and pharmaceutical dosage forms.

 

MATERIALS AND METHODS:

Instrument: Absorbance was measured, and the Spectra was recorded over the wavelength of 200-400 nm using a double beamUV- Spectrophotometer Jasco V-630.

 

Drugs: Zidovudine was obtained as a gift sample from Matrix Laboratories Ltd, Hyderabad. Tablet Retrovir by GSK Ltd.

 

Reagents:  Methanol, Distilled water and other reagents were of analytical grade.

 

Preparation of Stock solutions

Standard Zidovudine 10 mg was weighed and dissolved in5 mL of methanol in a 100 mL volumetric flask. The flask was shaken and volume was made up to the mark with distilled Waterto give a solution containing 100 µg / mL (stock solution A).

 

Selection of analytical concentration ranges

From the standard stock solution A of Zidovudine, appropriate aliquots were pipetted out into 10  ml volumetric flasks and dilutions were made with distilled water to obtain working standard solutions of concentrations from 2 to 100 μg / mL.Absorbance for these solutions were measured at 266 nm and the spectra. For the standard solution analytical concentration range were found to be 8 -28 µg / mL and those values were reported in Table no. 2.

 

Calibration curve for the Zidovudine (8 – 28 µg / ml)

Appropriate volume of aliquots from standard Zidovudine stock solution A were transferred to different volumetric flasks of 10 mL capacity. The volume was adjusted to the mark with distilled water to obtain concentrations of   8, 12, 16, 20, 24 and 28 µg / mL. Absorbance spectra of each solution against distilled water as blank were measured at 266 nm and the graphs of absorbance against concentration were plotted and shown in Figure 1. The regression equation and coefficient of determination was determined.

 

Sample preparation for determination of Zidovudine from dosage form

Ten tablets of a brand were weighed and finely powdered. The powder equivalent to 10mg of Zidovudine was accurately  weighed and transferred to  volumetric  flask of 100  mL capacity containing 5 mL of the methanol and sonicated for 5 min. The flask was shaken and volume was made up to the mark with distilled water  to give a solution of 100 µg / mL (stocksolution B ). The above solution carefully filtered through Whatmann filter paper(No. 41) and used for the estimation of Zidovudine. To examine the absence of either positive or negative interference of excipients used in formulation, recovery studies were carried out.

 

Validation Method

Accuracy

Accurately weighed formulation sample equivalent to 10 mg of sample were mixed with 10 mg of Zidovudine pure drug. From above equivalent 20 mg mixture 10mg equivalent weight of sample were dissolved in 5 ml of methanol and further volume make up with distilled water. Solutions were analysed.

 

Precision

The parameter was validated by assaying number of aliquots samples of zidovudineand its validity was estimated using parameters such as Standard deviation and Relative Standard deviation.

 

Recovery Studies

Accurately weighed formulation sample equivalent to 10 mg of sample were mixed with 10 mg of Zidovudine pure drug. From above equivalent 20 mg mixture 10mg equivalent weight of sample were dissolved in 5 ml of methanol and further volume make up with distilled water. Different concentrations like 8, 12, 16, 20, 24 and 28 µg / mL were taken and absorbance was recorded.

 

RESULT AND DISCUSSION:

Determination of wavelength and calibration graph

The λmax of Zidovudine was found to be 266 nm in methanol and distilled water. The absorbance was measured at 266 nm against methanol and distilled water. The calibration curve was prepared by plotting absorbance versus concentration of drug.

 

Determination of Molar Absorptivity

Absorptivity constant is the ratio of the absorbance of the sample of the product of the thickness of the medium and concentration of the sample. Increase or decrease in absorbance depends upon Increase or decrease in concentration which always remain constant. The absorbance of different concentrations was determined at 266 nm and molar absorptivity calculated using following formula,

 

a= A/ bc

Where,

a= Absorptivity

A= Absorbance

b= Path length

c= Concentration

 

Effect on Absorbance with Time (Stability)

The stability of sample was checked by taking absorbance at regular interval of time. Absorbance remains stable for 240 min. than the absorbance decreased with time.

 

 

 

 

Table no. 1: Optical Characteristics of Zidovudine

Parameters      

Results

Absorption maximum                                                                 

 266 nm

Beer’s law limit (μg / ml)

8-28 (μg / ml)

Correlation coefficient (r2)

0.9979

Molar absorptivity (mol-1 cm -1)

Regression equation (y = mx + c)

Slope (m)

Intercept (c)

4.4 X 103

y = 0.0301x + 0.0099

0.0301x

0.0099

 

 

 

 

 

 

Table no. 2: Results of calibration curve at 266 nm for Zidovudine by UV spectroscopy

Sr. no

Concentration (µg / ml)

Absorbance (nm)

1

8

0.2468

2

12

0.3820

3

16

0.4777

4

20

0.6256

5

24

0.7280

6

28

0.8533

 

 

Figure no.1 Linearity plot or calibration curve for Zidovudine at 266 nm by UV spectroscopy

 

Table no. 3: Accuracy results of Zidovudine at 266 nm

Amount of

Sample

(µg / ml)

Amount of Drug Added

(µg / ml)

Amount recovered

(µg / ml)

% Recovery ± SD

10

8

              17.74

            98.55

20

 10

              29.36

            97.86

 30

12

              31.66

            98.93

 

 

 

 

Table no. 4: Precision results of Zidovudine at 266 nm

Conc.                                                 Inter-day                                          % RSD                                              Intra-day                                          %RSD

 μg / ml                                              Absorbance**                                                                                             Absorbance**

8                                                          0.2468± 0.4043                               0.0066                                               0.2446 ± 0.3667                              0.0152

12                                                       0.3820± 0.3146                               0.0513                                               0.3819 ± 0.3700                              0.0173

16                                                       0.4777± 0.3333                               0.0291                                               0.4745 ± 0.3633                              0.0152

20                                                       0.6256± 0.3156                               0.0049                                               0.6252 ± 0.3000                              0.0171

24                                                       0.7280± 0.6033                               0.0152                                               0.7276 ± 0.3149                              0.0042

28                                   0.8533± 0.3533                               0.0057                                               0.8532 ± 0.3996                              0.0066

 

 

Table no. 5: Ruggedness results of Zidovudine at 266 nm

Analyst 

Label Claim

 mg                                                                                                                

Amount found      mg

% Recovery ± SD

Analyst I

300

299.43

99.81 ± 0.11

Analyst II

300

298.91

99.63 ± 0.18

 

 

 

 

 

 

CONCLUSION:

From the results, it can be concluded that the proposed method for the estimation of Zidovudine is simple, convenient, accurate, sensitive and reproducible. It can be successfully used for routine analysis of the Zidovudine in bulk and pharmaceutical dosage forms.

 

REFERENCES:

1.       USP 28, NF 23, The United States Pharmacopoeial Convention Inc, 1985, 3489

2.       AshenafiDunge, Nishi Sharda, Baljinder Singh and Saranjit Singh.2005. Validated specific High Performance Liquid Chromatography method for determination of Zidovudine during stability studies. Journal of Pharmaceutica Bio Anal, 37(5), 1109-1114.

3.       International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human use. 1996.Validation of Analytical procedures: Methodology. ICHQ2B, Geneva, (CPMP/ICH/281/95).

4.       I. C. H. 1996. Harmonized Tripartite guideline. Recommended for adaptation at step-4 of the ICH process, By ICH steering committee.

5.       Sharma B. K. Instrumental Method of Chemical Analysis, 18th edition, Krishna Prakashan Media Pvt. Ltd., Merrut, 1999,  39-139.

6.       Vogel’s Textbook of Quantitative Chemical Analysis, 5th edition, ELBS Longman, London, 1997, 661-672.

7.       N. Hari krishanan,2008. Simultaneous estimation of Lamivudine, Zidovudine and Nevirapine by R. P. HPLC in pure and pharmaceutical dosage form, Asian Journal of Chemistry, 20 (4), 2551-2556.

8.       GeethaRamachandran, A. K. Hemanthkumar, V. Kumaraswami and Soumya Swaminathan,2006. A simple and rapid Chromatographic method for simultaneous determination of Zidovudine and Nevirapine in plasma. Journal of Chromatography B. 843 (2), 339-344.

9.       NeerajKaul, 2004. Stability indicating HPTLC determination of Zidovudine as the bulk drug and in pharmaceutical dosage form. Journal of Planar Chromatography-Modern TLC, 17 (1), 264-274.

 

 

 

Received on 15.03.2014       Accepted on 26.04.2014     

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Asian J. Pharm. Ana. 4(2): April-June 2014; Page 51-53