Visible Spectrophotometric Estimation of Tenofovir Disoproxil Fumarate in Pharmaceutical Formulations
P. Janaki Pathi1*, P. Raveendra Reddy2 and N. Appala Raju3
1Analytical Department, Vishnu Chemicals Limited, Jeedimetla, Hyderabad. 1,2Department of Chemistry, Sri Krishnadevaraya University, Anantapur-515 003 3Department of Pharmaceutical Chemistry, Sultan-Ul-Uloom College of Pharmacy Mount Pleasant,Road # 3, Banjara Hills, Hyderabad-500 034
.*Corresponding Author E-mail: pjp02002@rediffmail.com; pjp02002@yahoo.com
ABSTRACT:
Three simple, accurate, rapid and sensitive methods (A, B and C) have been developed for the estimation of Tenofovir Disoproxil Fumarate in its pharmaceutical dosage form. The Method A is based on the formation of golden yellow colored chromogen, due to ion-association of Tenofovir with Metanil Yellow dye in Chloroform, which exhibits λ max at 425 nm. Method B is based on the formation of reddish brown colored chromogen due to ion-association of Tenofovir with Solochrome Black-T dye in Chloroform, which exhibits λ max at 438 nm. The Method C is based on the formation of blood red colored chromogen with Ferric Chloride and 2,2-Bipyridyl which shows absorption maximum at 523 nm. The absorbance-concentration plot is linear over the range of 10-60 µg/mL for Method A, 10-60 µg/mL for Method B and 20-90 µg/mL for Method C. Results of analysis for all the methods were validated statistically and by recovery studies. The proposed methods are economical and sensitive for the estimation of Tenofovir Disoproxil Fumarate in bulk drug and in its formulations.
KEYWORDS: UV-Visible Spectrophotometry, Tenofovir Disoproxil Fumarate, Metanil Yellow, Solochrome black-T, 2, 2-Bipyridyl
INTRODUCTION:
Tenofovir1is a acyclic phosphonate nucleotide analogue, chemically it is ({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid. It is official in Indian pharmacopoeia. Its molecular weight is 287.21 with molecular formula C9H14N5O4P. Tenofovir is a nucleotide reverse transcriptase inhibitor used in combination with other antiretrovirals for the treatment HIV infection2,3,4. Survey of literature reveals that the drug is determined by using HPLC5-10 in biological fluids. However there is only one study has been done to estimate the tenofovir disoproxil Fumarate in pharmaceutical dosage forms by visible spectrophotometric methods11. The present investigation aims to develop simple, sensitive, accurate, rapid and cost effective spectrophotometric methods A, B, and C for the estimation of Tenofovir Disoproxil Fumarate in its tablet formulations.
Fig-1 Structure of Tenofovir
EXPERIMENTAL:
Instrument: Elico Ultraviolet-Visible double beam spectrophotometer SL-164 with 1 cm matched quartz cells was used for all spectral measurements.
Reagents:
All the chemicals used were of analytical reagent grade.
1. 2,2-Bipyridyl (0.1M)-(15.6% w/v) AR Grade: 780 mg 2,2-Bipyridyl in 25 mL of in distilled water.
2. Ferric chloride hexahydrate (0.03 M) AR Grade: 405 mg of Ferric chloride hexahydrate is dissolved in 50 mL of 0.1N Hydrochloric acid.
3. Metanil yellow (0.2%w/v): Prepared by dissolving 200 mg of the dye in 100 mL of distilled water.
4. Solochrome black-T (0.2%w/v): Prepared by dissolving 200mg of dye in 100 mL of water.
5. Acid phthalate buffer (pH 2.4): Accurately weighed quantity of 4.0846 gms of potassium hydrogen phthalate was dissolved in 100 mL distilled water (0.2M Potassiumhydrogen phthalate). To 25mL of this solution 21.1mL of 0.2M hydrochloric acid was added and diluted to 100mL with distilled water to obtain pH 2.4.
6. Chloroform AR Grade
PROCEDURE: Standard stock solution was prepared by dissolving 10 mg of Tenofovir Disoproxil Fumarate in distilled water. The volume was made up to 10 mL with distilled water to get a concentration of 1000 µg/mL. This was further diluted to get the working standard solution of 100µg/mL for all three methods A, B, and C.
ASSAY PROCEDURE:
Method A: Aliquots of standard solutions containing 1 to 6 mL (100µg/mL) were transferred into series of 250mL separating funnels. 1mL of dye solution (0.2% w/v), 2mL of buffer solution were added and the drug dye complex with 10mL chloroform. The contents were centrifuged thoroughly for 5 min and allowed to stand, so as to separate the aqueous and clear chloroform layer. The yellow colored chloroform layer was separated out and absorbance was measured at 425 nm against reagent blank. Calibration curve was plotted from absorbance values so obtained. Similarly the absorbance of the sample solution was measured, and the amount of Tenofovir Disoproxil Fumarate was determined by referring to the calibration curve.
Method B: Aliquots of standard solutions of Tenofovir Disoproxil Fumarate containing 1 to 6mL (100µg/mL) were transferred into series of 250mL separating funnels. 1mL of dye solution (0.2% w/v), 2mL of buffer solution were added and the drug dye complex with 10 mL chloroform. The contents were shaken thoroughly for 5 minutes and allowed to stand, so as to separate the aqueous and chloroform layer. The absorbance of the reddish brown colored species was measured at 438 nm against reagent blank. The colored species is stable for 45 min. The amount of Tenofovir Disoproxil Fumarate present in the sample solution was computed from its calibration curve. Calibration curve was prepared from absorbance values so obtained. Similarly the absorbance of the sample solution was measured, and the amount of Tenofovir Disoproxil Fumarate was determined by referring to the calibration curve.
Method C: Aliquots of standard drug solution of Tenofovir Disoproxil Fumarate containing 2-9mL (100µg/mL) were taken and transferred into series of graduated test tubes. To each test tube 2 mL of Ferric chloride (0.03M) and 2 mL of 2’2-bipyridyl (2%) were added. The test tubes were allowed to stand in water bath at 700 c for 20 minutes. The test tubes were then cooled to room temperature and the solutions were made up to 10 mL with distilled water. The absorbance of the red colored chromogen was measured at 523 nm against reagent blank and a calibration curve was constructed. The absorbance of the sample solution was measured, and the amount of Tenofovir Disoproxil Fumarate was determined by referring to the calibration curve.
Assay for Pharmaceutical Dosage forms: The above specified methods were extended for the determination of Tenofovir Disoproxil Fumarate from Tablet formulations. Twenty tablets of Tenofovir (Tenof 300mg, Hetero Drugs) were accurately weighed and powdered. Tablet powder equivalent to 100 mg of Tenofovir Disoproxil Fumarate was dissolved in 50 mL of distilled water, sonicated for 15 mins, filtered and washed with distilled water. The filtrate and washings were combined and the final volume was made to 100 mL with distilled water for all the three Methods. The solution was suitably diluted and analyzed as given under the assay procedure for bulk samples.
The analysis procedure was repeated three times with Tablet formulations and the results of analysis are shown in Table II.
Recovery Studies:
To ensure the accuracy and reproducibility of the results obtained, adding known amounts of pure drug to the previously analyzed formulated samples and these samples were reanalyzed by the proposed method and also performed recovery experiments. The percentage recoveries thus obtained were given in Table II.
RESULTS AND DISCUSSIONS: In the present study, the Method A and B involves ion association complex between active pharmaceutical ingredient and dye in the prescence of Acid phthalate buffer system. Tenofovir DF being a base, it forms an ion-association complex with an acidic dye (Metanil yellow-Method A or Solochrome black-T Method B) which is extractable into chloroform from the aqueous phase. The protonated nitrogen (positive charge) of the drug molecule in acid medium is expected to attract the oppositely charged part (negative charge) of the dye and behave as a single unit being held together by electrostatic attraction. Based on the analogy, the structures of ion-association complexes are shown in Fig-2 and Fig-3 for method-A and method-B respectively.
Fig-2: Probable Mechanism involved in Method-A
Fig-3: Probable Mechanism involved in Method-B
The Method C is based on the reduction of Ferric chloride to ferrous (Fe2+) form by the Tenofovir Disoproxil Fumarate, which forms complex with 2,2- Bipyridyl to yield red colored chromogen, having absorbance maximum at 523 nm. The linearity was found to be in the concentration of 20-90 µg/mL. The colored chromogen was stable for 3 hrs. Based on the analogy, the most probable reaction mechanism involved in the formation of chromogen is shown in Fig-4.
Fig-4: Probable mechanism involved in Method-C
Table: I - OPTICAL CHARACTERISTICS AND PRECISION DATA:
|
Parameters |
Method A |
Method B |
Method C |
|
l max (nm) |
425 |
438 |
523 |
|
Beer’s law limits |
10-60 |
10-60 |
20-90 |
|
Molar absorptivity (l/mol.cm) |
1.535x103 |
1.722 x 103 |
1.246 x 104 |
|
Sand ell’s sensitivity (micrograms/cm2/0.001 absorbance unit) |
0.414 |
0.369 |
0.051 |
|
Regression Equation* (Y) Slope (m) Intercept (c) |
0.00243 0.00033 |
0.0026 -0.0027 |
0.0204 -0.018 |
|
Correlation Coefficient(r) |
0.9999 |
0.9999 |
0.9999 |
|
Precision (%Relative Standard Deviation) |
0.284 |
0.343 |
0.0812 |
|
Standard error of estimate |
0.0174 |
0.0265 |
0.0054 |
*Y=mx+c, where X is the concentration in micrograms/ml and Y is absorbance unit.
Table II: ASSAY OF TENOFOVIR IN TABLET FORMULATIONS.
|
Tablet formulation |
Labeled Amount(mg) |
Amount Obtained (mg)* By proposed method |
** % Recovery by the Proposed method |
||||
|
Method A |
Method B |
Method C |
Method A |
Method B |
Method C |
||
|
1 |
300 |
299.5 |
301.5 |
299.8 |
99.5 |
99.9 |
99.7 |
|
2 |
300 |
298.5 |
302.3 |
300.2 |
99.4 |
101.2 |
99.3 |
|
3 |
300 |
301.3 |
299.8 |
301.6 |
99.9 |
100.1 |
99.5 |
*Average of three determinations. ** After spiking the sample.
The optical characteristics such as absorption maxima, Beer’s law limits, molar absorptivity and Sand ell’s sensitivity are presented in Table I.
The regression analysis using the method of least squares was made for slope (m), intercept (b) and correlation obtained from different concentrations and the results are summarized in Table I.
The reproducibility and precision of the methods are very good as shown by the standard values of %RSD. The Mean percentage recovery value of 99.6% for Method A, 100.4% for Method B and 99.5% for Method C, indicates non-interferences from the formulation excipients. All the validated parameters are summarized in Table II.
In conclusion, the proposed methods are simple, sensitive, accurate and economical for the routine analysis of Tenofovir in bulk and in its formulations.
ACKNOWLEDGEMENT:
The authors acknowledge M/s Hetero Drugs Ltd, Hyderabad for the supply of Tenofovir Disoproxil Fumerate as gift sample and to the Vishnu Chemicals Limited, Jeedimetla, Hyderabad for providing the necessary facilities to carry out the research work.
REFERENCES:
1. S. Budhavari, The Merck Index, 14th edition, 2006; 1573.
2. C. Sean Sweetman, Martindale-The Complete Drug Reference, 2005; 34: pp. 655.
3. Grim S.A., Romandh F., Ann Pharmacother 2003; 37: 849-59.
4. Gallant J.E., Deresinkski.S., Clin Infect Dis 2003;37: 944-50.
5. Sparidans, Rolf W.; Crommentuyn, Kristel M. L.; Schellens, Jan H. M.; Beijnen, Jos H. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences, 2003; 791(1-2): 227-233.
6. Jullien, Vincent; Treluyer, Jean-Marc; Pons, Gerard; Rey, Elisabeth. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences, 2003: 785(2): 377-381.
7. Sentenac, S.; Fernandez, C.; Thuillier, A.; Lechat, P.; Aymard, G. Journal of Chromatography, B: AnalyticalTechnologies in the Biomedical and Life Sciences, 2003; 793(2): 317-324.
8. Takahashi, Masaaki; Kudaka, Yuichi; Okumura, Naoya; Hirano, Atsushi; Banno, Kazuhide; Kaneda, Tsuguhiro. Biological andPharmaceutical Bulletin, Pharmaceutical Society of Japan, 2007; 30(9): 1784-1786.
9. King, Tracy; Bushman, Lane; Kiser, Jennifer; Anderson, Peter L.; Ray, Michelle; Delahunty, Thomas; Fletcher, Courtney V. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences, 2006; 843(2): 147-156.
10. Gehrig, Anne-Kathrin; Mikus, Gerd; Haefeli, Walter E.; Burhenne, Juergen. Rapid Communications in Mass Spectrometry, 2007; 21(16): 2704-2716.
11. N.Appala Raju, Dr .J. venkateswara Rao, K.Vanitha Prakash, Dr. K. Mukkanti andK.Srinivasu; Biosci.Biotech.Research Asia, 2008; 5(1): 439.
Received on 16.09.2010 Modified on 06.10.2010
Accepted on 24.10.2010 © RJPT All right reserved
Research J. Pharm. and Tech. 4(3): March 2011; Page 446-449