formulation and characterizatin of transdermal

B.M. Gurupadayya, et al. / International Journal of Advances in Pharmaceutical Research

IJAPR Available Online through www.ijapronline.org

Research Paper ISSN: 2230 – 7583

DETERMINATION OF TENOFOVIR DISOPROXIL FUMARATE IN PHARMACEUTICAL FORMULATIONS BY P-CHLORANILIC ACID AND SODIUM NITROPRUSIDE AS REAGENT N.S. Disha & B.M. Gurupadayya* Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS University, Mysore – 570 015, Karnataka, India. E-mail: [email protected] Received on 07 – 07 - 2013

Revised on 10 – 08- 2013

Accepted on 29– 08 – 2013

ABSTRACT Two simple, extractions free spectrophotometric methods (Method A and B) for the quantitative estimation of Tenofovir disoproxil fumarate (TDF) in bulk drugs and pharmaceutical formulations (tablets) have been developed. The first method is based on the charge transfer complexation reaction between drug as n-electron donor and p-chloranilic acid (p-CA) as a p-acceptor to form a violet chromogen measured at 531 nm. The colored product of tenofovire shows linearity in the concentration range of 2-10 µg/ml. Second method is based on the tenofovire formation of light green colored molecular complex with sodium nitroprusside in presence of hydroxyl amine under alkaline conditions and exhibiting λmax at 401 nm and shows linearity in the concentration range of 2-10 µg/ml. Linear relationships with good correlation coefficients (0.998-0.996) were found between absorbance and the corresponding concentrations of the drug. The proposed method is validated with respect to accuracy, precision, linearity and limit of detection. The suggested procedure is successfully applied to the determination of the drug in pharmaceutical preparation, with high percentage of recovery, good accuracy and precision. The results of analysis have been validated statistically by repeatability and recovery studies. The results are found satisfactory and reproducible. The method is applied successfully for the estimation tenofovir in tablet form without the interference of excipients. Key Words: Tenofovir disoproxil fumarate (TDF) , p-chloranilic acid Sodium nitroprusside (SNP), hydroxyl amine Pharmaceutical formulations and Visible Spectrophotometry. INTRODUCTION Tenofovir disoproxil fumarate (TDF) belongs to the of antiretroviral drugs known as nucleotide analogue reverse transcriptase inhibitors (nRTIs), which blocks reverse transcriptase, an enzyme crucial to viral production in HIV-infected people. Chemically TDF is 9[(R)-2-[[bis [[(isopropoxycarbonyl) oxy] methoxy] phosphinyl] methoxy] propyl] adenine fumarate1(Figure-1). Extensive literature review reveals that several spectrophotometric2,3, HPLC4-10, HPTLC11 and LC-MS12-15 methods have been reported so far for determination of Tenofovir. We now report a sensitive and flexible visible spectrophotometric method based on the complex formation reaction of drug with sodium nitroprusside

and hydroxylamine hydrochloride and based on the charge transfer complexation reaction between drug as n-electron donor and p-chloranilic acid (pCA) as a p- acceptor . MATERIALS AND METHODS A Shimadzu UV-visible spectrophotometer model 1800 with 1 cm matched quartz cell was used for the absorbance measurements and Systonics electronic balance was used for weighing the samples. Reagents and solutions All employed chemicals were of analytical grade and All employed chemicals were of analytical grade and high high-purified water was used throughout. Purified water was used throughout. Tenofovire TDF pure sample was obtained as a gift sample

IJAPR /Sept. 2013/ Vol. 4 /Issue 09 / 2189 – 2195

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from Intas Pharmaceuticals Ltd, Matoda, Gujarat, India. Standard drug solution The pure drug (100 mg) was dissolved in 100 ml of water in a volumetric flask to form the stock solution. Aliquots of the stock solution were similarly diluted to obtain working concentrations in the range of 2 to 10 µg/ml. Sample solution: About 20 tablets of TDF were pulverized and the powder equivalent to 10mg of Tenofovir disoproxil fumarate (label claim: 300 mg) was taken in a 100ml volumetric flask and then diluted to 100ml with distilled water to get 100 µg/ml. Reagents: Sodium Nitroprusside (SNP): SD Fine Chemicals, Mumbai, 0.4%, 1.34x10 -2M, solution prepared by dissolving 400mg of sodium nitroprusside in 100ml distilled water Hydroxylamine Hydrochloride: Loba, 0.4%, 5.75 x 10-2M solution prepared by dissolving 400mg of hydroxylamine hydrochloride in 100ml of distilled water Sodium Carbonate: Loba,10%, 9.43x10-1M solution prepared by dissolving 10g of sodium carbonate in 100ml of distilled water were prepared p-Chloranilic acid (p-CA) The regent solution was freshly prepared by dissolving 486.3 g of p-chloranilic acid in 100ml of dimethyl formamide. Analytical Method Development Method -A Aliquots of standard solution TDF (0.2ml-1ml, 100 µg/ml) were transferred into a series of 10ml calibrated volumetric flask and the volume in each flask was brought to 5.0ml with distilled water. To this 0.5 ml of (1.324x10-2M) sodium nitropruside and 1ml of (5.75x10-2M) hydroxylamine hydrochloride solutions were successively added to each flask and shaken for 2 minutes. Then 1.0ml of (9.43x10 -1M) Na2CO3 solution was added and further shaken for 15 minutes. The contents were diluted to the mark with distilled water and the absorbance were measured at 401nm against a reagent blank and the absorbance spectra is shown in the figure 2 within the stability period (immediate-120 min). The amount of TDF in the sample solution was computed from its calibration graph shown in Figure 3 . Method-B Aliquots of standard solution TDF (0.2ml-1ml, 100µg/ml) were transferred into a series of 10ml calibrated volumetric flask and the volume in each flask 0.5ml of p-Chloranilic acid is added and diluted with dimethylformamide and allowed to stand for 10min and

the absorbance were measured at 533nm against a reagent blank and the absorbance spectra is shown in the Figure 4 within the stability period (immediate-120 min). The amount of TDF in the sample solution was computed from its calibration graph shown in figure 5 . Method Validation The method was validated with reference to linearity; Standard solution of TDF was accuracy, precision, and Limit of detection and Limit of quantification16. Linearity Linearity was performed by taking from stock solution flask and volume made up to the mark with water to (100 mcg/ml) aliquots of 0.2,0.4, 0.6, 0.8 & 1ml obtain the concentrations 2 to 10 µg/ml. were taken in 10ml volumetric flasks and diluted up to 10ml with water the results of both the method are shown in Table -1 Precision Precision of the methods was studied as intra-day, interday and repeatability. Intra-day study was performed by analyzing, a homogenous sample separately 6 times for interday and intraday using the same instrument employing the same analyst evaluated the same data for standard deviation relative standard deviation and coefficient of variance and the results are summarized in Table 2. The limits of detection (LOD) and quantitation (LOQ) LOD limits of detection is defined as lowest concentration of an analyte that analytical processes can be reliably differentiate from back ground levels. LOQ is defined as lowest concentration of a standard curve that can be measured with an accuracy precision. The limits of detection (LOD) and quantitation (LOQ) calculated according to ICH guidelines using the formulae: LOD = 3.3 S/m and LOQ = 10 S/m, where S is the standard deviation of sample and m is the slope of the calibration plot result shown in Table -1. Accuracy The accuracy of the proposed methods was assessed by recovery studies at three different levels i.e. 80%, 100%, 120%. The recovery studies were carried out by adding known amount of standard solution of three deferent levels. The resulting solutions were then reanalyzed by proposed methods; the results are shown in Table 3. Chemistry of colored species: Method-A In the present investigation TDF functions as a donor due to the presence of cyclic tertiary nitrogen. Sodium nitroprusside in the Tenofovir disoproxil fumarate presence of hydroxylamine and alkali exists


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B.M. Gurupadayya, et al. / International Journal of Advances in Pharmaceutical Research as aquo ferrocyanide [Fe (CN)5 H2O]3-. In a general way it may be expected that the electron transfer depends upon the extent of delocalization of the donor and acceptor metal orbitals of the intervening ligands. From this stand point, ligands such as water and ammonia, which contain single bonds, are expected to be much less effective in conducting electrons between metal ions than unsaturated ligands such as CNwhose complexes are characterized by high degree of covalency and electron delocalization. Based on the analogy, the probable sequence of reactions is presented in scheme Figure 6. Method -B In this method, the interaction of TDF with πacceptor (p-CA) at room temperature was found to yield colored charge transfer complex. In polar solvents, complete electron transferfrom Tenofovir disoproxil fumarate (D), as an electron donor, to the acceptor moiety (A) takes place resulting in the formation of intensely colored radical anions. The reaction sequence can be shown as D+A

(D-A)

D+ + A -

Complex The interaction of TDF with p-CA in dimethylformamide solvent system resulted in the formation of pink colored charge transfer complex and the absorption exhibited maximum up to 531nm Beer’s law is obeyed in the range of 2-10 µg/ml. The

reaction between p- CA and TDF was represented in Figure-7. 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 sodium nitro prusside, hydoxylamine, sodium carbonnate, and p-chloranilic acid stability of colored species and solvent for final dilution of the colored species were studied and the optimum conditions were established. The optical characteristics such as Beer’s law limit, Sandell‘s sensitivity molar absorptivity, percent relative standard deviation, were calculated and the results are summarized in Table-1. Regression characteristics like standard deviation of slope (b), standard deviation of intercept (a), shown in Table-1. As an additional demonstration of precision were performed the SD and RSD% of interday and intraday results are summarized in Table-2 accuracy, recovery , experiments were performed by adding a fixed amount of the drug to the pre analyzed formulations at three different concentration levels. These results are summarized in Table-4. The ingredients usually present in formulations of Tenofovir disoproxil fumarate did not interfere with the proposed analytical method.

Figure: 1, Chemical structure of Tenofovir disoproxil fumarate


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Method - A

Fig. 2: Showing Absorption Spectra of Absorption spectrum of the reaction product (Method A)

Fig. 3: linearity graph for method -A

Fig. 4-Showing Absorption Spectra of Absorption spectrum of the reaction product (Method B)


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Figure 5: linearity graph for method -B

Figure 6: Reaction for Method-A

p-Chloranilic acid radical anion (Colored species) Figure 7: Reaction for Method-B


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Table 1: Optical Characteristics of proposed analytical Method PARAMETERS Method-A Method -B Wavelength (nm) 401 531 Beer’s law limit(µg/ml) 2-10 2-10 Sandell’s sensitivity (µg/cm/0.001 abs. 0.0317 0.0054 unit) Molar absorptivity (Liter/mole/cm) 4595.8 8596.8 Regression equation (Y) Intercept (m) 0.0385 0.0311 Slope(c) 0.016 0.299 Correlation Coefficient (R2 ) 0.99 0.996 %RSD 0.97 1.56 SD 0.002 0.009 LOD(µg/ml) 0.415 0.9 LOQ(µg/ml) 1.25 2.9

INTRA DAY INTERDAY SD (µg/ml) RSD% SD (µg/ml) RSD% 0.0068 1.6 0.00331 0.924 0.009 1.86 0.00331 0.474 Table-2: Precision study of the proposed methods

Method A Method B

Concen. %

METHOD-A Amount recovered SD %

% RSD

Amount Recovered %

METHOD-B SD

% RSD

80 99.8

0.005

0.91

99.8

0.003

0.47

100

99.6

0.003

0.506

99.6

0.0021

0.96

120

99.9

0.005

1.23

0.0058 0.91 99.9 Table -3: Accuracy of the proposed method

CONCLUSION The reagents utilized in the proposed methods are cheap, readily available and the procedures do not involve any critical reaction conditions or tedious sample preparation. Moreover, the methods are free from interference by common additives and excipients. The wide applicability of the new procedures for routine quality control was well established by the assay of TDF in pure form and in pharmaceutical preparations.

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INTERNATIONAL CONGRESS IN PHARMACY AND HEALTH SCIENCES Pharma Science Tech Association, Foundation No: AP/PSTA/56/2012. Please visit for Details: www.icphsmembership.com Totally three types FICPHS (Fellowship in International Congress in Pharmacy and Health Sciences), MICPHS (Member in International Congress in Pharmacy and Health Sciences), AMICPHS (Associate Member in International Congress in Pharmacy And Health Sciences) Eligibility FICPHS: Ph.D in Chemistry/ Pharmacy / M.Sc / M.Pharm with 2 years experience, MICPHS: M.Sc / M.Pharm (or) B.Sc / B.Pharm with 2 years experience, AMICPHS: B.Sc (or) B.Pharmacy


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