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ArticleTitle Article Sub-Title Article CopyRight Journal Name Corresponding Author The National Academy of Sciences, India (This will be the copyright line in the final PDF) Proceedings of the National Academy of Sciences, India Section A: Physical Sciences Family Name Particle Given Name Suffix Division Organization Address Email Author Family Name Particle Given Name Suffix Division Organization Address Email Author Family Name Particle Given Name Suffix Division Organization Address Email Author Family Name Particle Given Name Suffix Division Organization Address Email Author Family Name Particle Given Name Suffix Division Foods and Drugs Laboratories, Department of Organic Chemistry C. S. P. Sasttry Foods and Drugs Laboratories, Department of Organic Chemistry Andhra University Visakhapatnam, 530003, India Psnh Ramachandra Rao Foods and Drugs Laboratories, Department of Organic Chemistry Andhra University Visakhapatnam, 530003, India Krishna Pendem Foods and Drugs Laboratories, Department of Organic Chemistry Andhra University Visakhapatnam, 530003, India Saranapu Foods and Drugs Laboratories, Department of Organic Chemistry Andhra University Visakhapatnam, 530003, India rmj.rao@rediffmail.com Naresh R. Mrutyunjayarao Three Simple Spectrometric Determination of Terbinafine Hydrochloride (TRB) in Pure State and Tablets

Organization Address Email Author Family Name Particle Given Name Suffix Division Organization Address Email Received Schedule Abstract Revised Accepted

Andhra University Visakhapatnam, 530003, India Viplava Prasad U. Foods and Drugs Laboratories, Department of Organic Chemistry Andhra University Visakhapatnam, 530003, India

19 October 2011 13 February 2012 17 February 2012

Three simple and sensitive procedures (methods A, B and C) for the assay of terbinafine hydrochloride in pure form and formulations are described. Methods A and B are based on the selective oxidation of terbinafine hydrochloride with an excess of oxidant (chloramine T) (in method A or potassium permanganate in method B) in acidic medium. The unreacted oxidant is then estimated calorimetrically by using an oxidisable dye (gallocyanine) in method A (max 540 nm) fast green FCF in method B (max 620 nm). Method C is based on the formation of coloured radical anion on treating terbinafine hydrochloride with 2,3 dichloro 5,6-dicyno 1,4 benzoquinone (max 450 nm). The variable parameters in all these methods have been optimized. The results were statistically validated. Terbenafine hydrochloride - DDQ - CAT

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Proc. Natl. Acad. Sci. Sect. A Phys. Sci. DOI 10.1007/s40010-012-0032-x

RESEARCH ARTICLE

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Three Simple Spectrometric Determination of Terbinane Hydrochloride (TRB) in Pure State and Tablets

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Received: 19 October 2011 / Revised: 13 February 2012 / Accepted: 17 February 2012 The National Academy of Sciences, India 2012

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Abstract Three simple and sensitive procedures (methods A, B and C) for the assay of terbinane hydrochloride in pure form and formulations are described. Methods A and B are based on the selective oxidation of terbinane hydrochloride with an excess of oxidant (chloramine T) (in method A or potassium permanganate in method B) in acidic medium. The unreacted oxidant is then estimated calorimetrically by using an oxidisable dye (gallocyanine) in method A (kmax 540 nm) fast green FCF in method B (kmax 620 nm). Method C is based on the formation of coloured radical anion on treating terbinane hydrochloride with 2,3 dichloro 5,6-dicyno 1,4 benzoquinone (kmax 450 nm). The variable parameters in all these methods have been optimized. The results were statistically validated.

spectrophotometric procedures involving TRB with reagents such as chloramine T (CAT) [4, 5] and gallocyanine (GC) (method A) potassium permanganate (KMnO4)[6] and fast green FCF (FGFCF) (method B) and 2,3 dichloro, 5,6-dicyno, 1,4-benzoquinone DDQ (method C) [7, 8] by exploiting its structural features (aliphatic tertiary amine double bond and triple bond). A Milton Roy spectronic 1201 and systronic 106 digital spectrophotometer were used for the spectral and absorbance measurements an Eli co LI-120 digital pH meter was used for pH measurements.

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R. Mrutyunjayarao Saranapu Naresh Krishna Pendem Psnh Ramachandra Rao C. S. P. Sasttry U. Viplava Prasad

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Reagents

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Introduction

Terbinane hydrochloride (TRB) [1-naphthalene methanamine N-[(2E) 6,6 dimethyl-2-heptene-4ynyl]-N-methyl] is a synthetic alylamine anti-fungal compound exists in the market in the form of tablets and cream. It is ofcial in USP [1], Merck Index [2], and Martindale Extra Pharmacopoeia [3]. Existing analytical methods reveal that relatively less attention was paid in developing visible

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Keywords CAT

Terbenane hydrochloride DDQ

All the reagents were of analytical grade and all the aqueous solutions were prepared in double distilled water. Freshly prepared solutions were always used. Aqueous solution of CAT (Loba; 7.10 9 10-4 M), GC (chroma; 2.969 9 10-4 M) for method A, potassium permanganate (BDH; 2.0 9 10-3 M in 1 M H2SO4), fast green FCF (chroma; 1.236 9 10-4 M in H2SO4 1 M) for method B, DDQ (Otto-kemi 4.405 9 10-3 M in chloroform) for method C were prepared.

Drug Solution A stock solution of 1 mg/ml was prepared by dissolving 50 mg of TRB in 50 ml distilled water. The solution was diluted step wise with distilled water to obtain 10 lg/ml for method A. A stock solution of 1 mg/ml was prepared by dissolving 50 mg TRB in 50 ml of dilute acetic acid. The stock solution was diluted stepwise with dilute acetic acid to

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A1 A2 A3 A4 A5

R. Mrutyunjayarao (&) S. Naresh K. Pendem P. R. Rao C. S. P. Sasttry U. Viplava Prasad Foods and Drugs Laboratories, Department of Organic Chemistry, Andhra University, Visakhapatnam 530003, India e-mail: rmj.rao@rediffmail.com

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obtain working standard solution of concentration of 10 lg/ml for method B. A stock solution of 1 mg/ml was prepared by dissolving 50 mg TRB in 50 ml of chloroform. The stock solution was diluted stepwise with chloroform to obtain working standard solution of concentration of 100 lg/ml for method C. Recommended procedures for bulk samples are given below: Method A Aliquots of the drug solution (0.53.0 ml; 10 lg/ml) were taken in a series of 25 ml calibrated tubes. Then 1.25 ml of 5.0 M HCl and 2 ml of (7.1 9 10-4 M) CAT solutions were added to each tube. The volume in each tube was made up to 15.0 ml with distilled water. After 20 min, 10 ml of GC was added and mixed thoroughly and the absorbances were measured after 10 min at 540 nm against distilled water. Blanks were prepared appropriately. The decrease in absorbance corresponding to consumed CAT, which in turn to the drug quantity was obtained by subtracting the absorbance of the blank solution from that of the test solution. The calibration graph was drawn by plotting the decrease in the absorbance of the dye (GC), against amount of drug. The amount of drug in the sample was computed from its calibration graph. Method B

well and made up to mark with dichloromethane. Reagent blank was simultaneously prepared and read at 450 nm against a reagent blank. The amount of the drug was computed from Beers law plot. For Pharmaceutical Formulations The cream or tablet powder equivalent to 100 mg of TRB HCL was treated with 4 9 20 ml portions of chloroform and the chloroform extract was transferred to 100 ml volumetric ask and made up to 100 ml with chloroform to obtain 1 mg/ml stock solution. 25.0 ml of the above stock solution was taken and chloroform portion was evaporated to dryness and residue was transferred to 25 ml volumetric ask by dissolving it in 5 ml of 0.1 N HCL and diluted to the mark with distilled water. The stock solution was diluted stepwise with distilled water to obtain working solution of 10 lg/ml for method A. Two portions of 10 ml each of chloroform stock solution was evaporated to dryness and the residues were transferred to 10 ml volumetric asks and one is dissolved in 2 ml of glacial acetic acid and diluted to 10 ml with distilled water. The stock solution was diluted stepwise with distilled water to obtained standard solution of 10 lg/ml for method B. Another residue in 10 ml ask was dissolved in methanol and made up to 10 ml with methanol. The stock solution was diluted stepwise to obtain 100 lg/ml for method C.

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Aliquots of standard drug solution (0.53.0 ml; 10 lg/ml) were taken in a series of 25 ml calibrated tubes. To these tubes 1 ml of KMnO4 solution was added and total volume in each tube was brought to 10 ml with distilled water and kept aside for 15 min at room temperature. Then 4.0 ml each of FGFCF and 1 M sodium sulphate solutions were successfully added after 10 min, the volume was made up to the mark with distilled water. The absorbants were measured at 620 nm against distilled water. A blank experiment was carried out in a similar manner omitting the drug. The decrease in absorbance corresponding to consumed permanganate and in turn the drug concentration was obtained by subtracting the decrease in the absorbance of the test solution (dye minus test) from that of the blank solution (dye minus blank). The amount of drug was deduced from its calibration graph.

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Results and Discussions

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Aliquots of standard drug solution (free base form) were taken in a series of 10 ml test tubes (0.53.0 ml; 100 lg/ml) and the chloroform in each tube was evaporated on a hot water bath to dryness. The residue was dissolved in 0.5 ml of methanol and 2.0 ml of DDQ solution was added, shaken

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Method C

The optimum conditions for the color development of methods (A, B and C) were established by varying the parameters one at time, keeping the others xed and observing the effect produced on the absorbance of the colored species. The colored solutions exhibited by kmax at 540, 620 and 450 nm, respectively for method A, B and C. The optical characteristics such as Beers law limits (lg/ ml), molar absorbtivity (mole-1 cm-1) and Sand ells sensitivity (lg/cm2/0.001 absorbance unit) for methods A, B and C and the slopes, intercepts obtained by linear least squares treatment of the results for methods A, B and C were tabulated and estimating six replicate samples of drugs with the Beers law limits tested the precision and accuracy of the methods. The percent relative standard deviation as well as the percent range of error (95 % condence limit) for methods A, B and C were also depicted in Table 1. The application of each method was veried by means of replicate estimations of commercial formulations TRB HCL (cream and tablets).The values

Proc. Natl. Acad. Sci. Sect. A Phys. Sci. Table 1 Optical and regression characteristics, precision and accuracy of the proposed methods for TRB

Parameters kmax (nm) Beers Law limits (lg m1-1) Detection limit (lg m1-1) Molar absorptivity (mol
-1

M1 (CAT/GC) 540 0.53.0 3.11 9 10-2 cm )


-2/0.01 -1

M2 (KMnO4/ FGFCF) 620 0.53.0 2.014 9 10-2 1.032 9 10 2.267 9 10


5 -3

M3 (DDQ) 450 530 9.02 9 10-2 4.951 9 103 6.622 9 10-2

1.063 9 10 4.016 9 10

5 -3

Sandells sensitivity (lg cm absorbance unit)

Intercept (a)

6.0 9 10-4 1.291 9 10 0.9999 0.3287 0.3451 0.5413


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Slope (b) Standard deviation on slope (Sb) Standard deviation in intercepts (Sa) Standard error of estimation (Se) Correlation coefcient (r) Relative standard deviation (%)a % range of error (condence limits)a
a

1.246 9 10-1 1.327 9 10-4

1.762 9 10-1 1.216 9 10-4

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1.388 9 10-3

1.2723 9 10-3

0.5999

Average of six determinations considered

0.05 level 0.01 level % Error in bulk samples

0.6298 0.9878

Average of three determinations considered

0.4016

-0.2816

Table 2 Assay of TRB in pharmaceutical formulations Formulationsa Labeled amount (mg) Amount found by proposed methods M1 CAT/GC 121.1 1.025 F = 2.01 t = 1.529 Tablets 250 F = 1.27 t = 1.0 M2 KMnO4/ FCFGF
a

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M3 DDQ

Reference method

% Recovery by proposed methodsc M1 CAT/GC M2 KMnO4/ FGFCF M3 DDQ

Tablets

125

124.25 0.588 123.31 0.867 124.36 0.72 99.28 0.820 99.40 0.468 99.44 0.69 F = 1.52 F = 1.43 t = 0.992 F = 3.10 t = 0.228

249.47 0.925 248.42 1.445 249.36 0.573 249.52 0.82 99.78 0.370 99.37 0.578 99.74 0.22 F = 2.04 t = 0.341 9.97 0.022 F = 1.95 t = 1.163 247.5 3.633 F = 1.78 t = 1.83 247.86 3.09 98.92 1.862 99.02 1.12 98.85 1.31 9.99 0.016 99.89 0.185 99.87 0.217 99.74 0.22 t = 0.853

Cream

10

Tablets

250

Theoretical values at 95 % condence limit, F = 5.05, t = 2.57


a b

Formulations from four different pharmaceutical companies

Average standard deviation on six determinations, the t- and F-test values refer to comparison of the proposed method with the reference method
c

Recovery of 10 mg added to the pre-analyzed pharmaceutical formulations (average of three determinations)

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9.98 0.018

9.98 0.021

F = 1.27 t = 1.0

F = 1.76

t = 0.162 F = 1.21

247.31 4.656 247.56 2.80 t = 0.990

F = 2.262 t = 0.596

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obtains by proposed methods and the reference methods for formulations were compare statistically with t and F tests and were found not to differ signicantly. The results are summarized in Table 2.

Selectivity of Methods Among three proposed methods for the determination of TRB HCL method A involved principle is quantitative

Regression equation (y = a ? bc)

1.512 9 10-2 2.337 9 10-5 2.666 9 10-4 4.55 9 10-4 489 9 10-2 0.9999 0.452

0.4754 0.7456 0.3311

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Acknowledgments The Authors R. M. Rao P. S. N. H. R. Rao and S. Naresh are thankful to UGC, New Delhi for the award of teacher fellowships.

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decolourisation of GC, by unreacted CAT, method B involves two steps. In the rst step, the TRB was treated with permanganate. In the second step the unreacted permanganate was determined by FGFCF. In method C TRB initially interacts with DDQ giving the semiquinone which appears to be responsible for the colour formation. The commonly used excipients and additives in tablets and cream such as talk (up to a 250 m/m excess compared with the drug), boric acid (150 fold), stearic acid (60 fold) sodium laryl sulfate (10 fold), calcium phosphate (20 fold) and calcium sulfate (20 fold) do not interfere in the proposed methods. Among the other excipient starch, manitol, sucrose, lactose and cellulose derivatives interfere in the proposed methods were eliminated.

References
1. Schatz F, Haberl H (1989) Chromotograpic determination of terbinane hydrochloride inpersence of its degradation products. Arzneim Forsch 39:527532 2. The Merck Index, 12th edn (1996) Merck & Co. Inc. New York 3. The complete drug reference (1999) Martindale extra pharmacopoeia, 32nd edn. The Pharmaceutical press, London 4. Bartos J, Pesez (1974) Sur une colorimetrie daldehydes aromatiques par lintermediaire de leurs thiosemicarbazones. Talanta 21:13041305 5. Plaizier JA, Van Damme JG, Daveni (1977) Spectrophotometric determination of hydrazides with 2,3-dichloro-1,4-napthoquinone. J Anal Chem 48:15361538 6. Evas WL (1923) J Am Chem Soc 45:171176 7. Lepley AR, Thelman JP (1966) Tetrahedron 22:101 8. Brown NMD, Foster R, Fyie CA (1967) Equilibrium constants of chargetransfer complexes determined from 19F nuclear magnetic resonance absorption measurements. J Chem Soc, pp 406410

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