development and validation of simultaneous equation estimation method for hamycin and ketoconazole...

Int. J. Pharm. Res. Sci., 2014, 02(1), 60-70. www.ijprsonline.com ISSN: 2348 –0882=============================================================================

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Development and Validation of Simultaneous Equation Estimation MethodFor Hamycin And Ketoconazole In Cream Formulation By U.V.

Spectrophotometric MethodSunil Kumar1*, R. K. Nanda1, Dhananjay Babar2, Rajendra Patil2

1. Department of Quality Assurance Technique, Padm. Dr. D. Y. Patil Institute of Pharmaceutical Scienceand Research, Pimpri, Pune- 411018

2. Genpharma International Pvt. Ltd., Bhosari, Pune.*Corresponding author address:Mr. Sunil Kumar,Quality Assurance Department,Uttaranchal Biotech

Ltd.,Jainagar-3, DineshpurRoad,Rudrapur(U.S.Nagar),Uttarakhand.,India.,Email:pharmacistsk@rediffmail.com---------------------------------------------------------------------------------------------------------------------------------------AbstractA simple and accurate UV method has beendeveloped for the simultaneous estimation ofHamycin and Ketoconazole cream formulationusing SHIMADZU UV-Visible 1700spectrophotometer by simultaneous equationmethod, with Acetonitrile: 0.5% w/v Ammoniumacetate (80:20v/v) as a solvent. The absorbancemaxima were found to be 381.5 nm for Hamycinand 243.5 nm for Ketoconazole. The percentagepurity of cream formulation was found to be99.08% for Hamycin and 98.22% for Ketoconazole.This method was also validated by checking theaccuracy, precision, LOQ, LOD and Ruggedness.The %RSD shows within specification limits. Thelinearity profile shows coefficient of variation 0.99for both drugs.

KEYWORDSHamycin, Ketoconazole, Acetonitrile, Ammoniumacetate, UV, SimultaneousINTRODUCTIONKetoconazole is Chemically Cis-1-acetyl-4-[4-[2-(2,4-dichlorophenyl)-2H-imidazolyl methyl)-1,3-dioxolan-4-yl] methoxy] phenyl]- piperazine is atopical as well as systemic antifungal agent.Ketoconazole is practically insoluble in water;sparingly soluble in strong acid, soluble in strong

bases. It is an imidazole derivative with molecularweight 531.44.[1,2] It inhibits cytochrome P450dependent lanosterol C14 demethylase, which isresponsible for production of ergosterol, a necessarycomponent in fungal cell wall synthesis.[3, 4]Ketoconazole is a weak base with pKa values of2.94 and 6.51.[5] Ketoconazole is an antifungaldrug approved by the US FDA in 1981. Only a fewanalytical methods for the determination of the drugin biological samples and in the presence of otherdrugs have been reported.[6,7,8]

Fig No. 1: Structure of KetoconazoleHamycin is a polyene antimycotic organiccompound. It is a heptaene antifungal compoundrather similar in chemical structure to amphotericinB except that it has anadditional aromatic group bonded to the molecule.Hamycin is obtained from a strainof Streptomyces bacteria growing in soili.e., Streptomyces pimprina. This compound is


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being produced in India by Hindustan AntibioticsLimited, located at Pimpri, Pune, Maharashtra,India. It is useful as an antifungal antibiotic drugfor topical as well as systemic mycoses. It is Yellowamorphous powder, no definite M.P., decomposeafter 1600 C. UV max (80% methanol): 383 nm(E1%1cm916). An amphoteric compd. Almostinsoluble in water, benzene, chloroform, dry loweraliphatic alcohols, ether. Solution in basic solventssuch as pyridine, and in aqueous lower alcohols. Inconc. H2SO4 gives stable blue color, no colorationwith ferric chloride or with HCl. Hamycin is a rigid,rod-shaped molecule that kills cells by disruptingthe cell membrane, causing leakage of electrolytesand small molecules. Hamycin bind to ergosterol,the major membrane lipid in fungal cells.[7,8]

Fig No. 2: Structure of HamycinMATERIALS AND METHODSA SHIMADZU 1700 Double beam UV-VISIBLEspectrophotometer with 1.0 cm matching quartzcells, All the reagents used were of analytical gradefrom Rankem, India. Reference standard ofKetoconazole was supplied as gift sample fromGenpharma International Pvt. Ltd., Pune andHamycin was supplied as gift sample fromHindustan Antibiotic Limited, Pune.

Analytical Method DevelopmentSelection of SolventSelection of solvent was based on solubility andstability of drug in solvent system as well asextraction of drug from its formulation. Hamycin

and Ketoconazole pure form and its formulation canbe freely soluble in water and organic solvents.Hence Acetonitrile: 0.5% w/v Ammonium acetate(80:20v/v) was selected as solvent for UVspectrometric determination.

Preparation of standard stock solutions

Standard stock solutions (100 µg mL-1) of Hamycinand Ketoconazole were prepared by dissolvingseparately 10 mg of drug each in 100 mLAcetonitrile: 0.5% w/v Ammonium acetate(80:20v/v). The working standard solutions of thesedrugs were obtained by dilution of the respectivestock solution with Acetonitrile: 0.5% w/vAmmonium acetate (80:20v/v).

Selection of analytical wavelengthsAppropriate dilutions were prepared for each drugfrom the standard stock solution and scanned in thespectrum mode from 400 nm to 200 nm. ForHamycin and Ketoconazole, 381.5 nm and 243.5nm were selected as λmax respectively. Fig. No.3represents the spectra of Hamycin and Fig.No.4represents the spectra of Ketoconazole.


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Fig. No. 3: Spectrum of Hamycinfor simultaneous equation method

Fig. No. 4: Spectrum of Ketoconazole forsimultaneous equation methodStability of the Drugs in the selected SolventThe stability of the drugs in the selected solvent waschecked by measuring the absorbance of the drugsolutions at the selected wavelengths. Both thedrugs were found stable in Acetonitrile: 0.5% w/vAmmonium acetate (80:20v/v). The analyte stabilitywas studied for 1 hour. Absorbance is constantduring this given time period as shown in Fig. No. 5and 6 respectively.

Fig No.5: Plot of Time Vs.Absorbance for Hamycin.

Fig No. 6: Plot of Time Vs. Absorbance forKetoconazole.Linearity profileFor each drug, appropriate aliquots of standardstock solutions were transferred to a series of 10 mlvolumetric flasks. The volume was made up to themark with Acetonitrile: 0.5% w/v Ammoniumacetate (80:20v/v) to obtain working standardsolutions of appropriate concentrations. Triplicate


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dilutions of each concentration of the drugs wereprepared separately. The absorbances of theworking standard solutions of each concentrationwere measured at the selected analyticalwavelengths in duplicate. The standard calibrationcurves of Absorbance Vs Concentration wereplotted using the mean of these six independent

observations. The concentration range over whichthe drugs obeyed Beer- Lambert’s law was found tobe between 5-30 g ml-1 for Hamycin and 10-50 gml-1 for Ketoconazole respectively. The calibrationcurves for Hamycin and Ketoconazole are given inFig. No.7 and 8 respectively.

Fig. No. 7: Standard calibration curve of Hamycin at 381.5 nm

Fig. No. 8: Standard calibration curve of Ketoconazole at 243.5 nm.

0

0.141

0.308

0.453

0.563

0.697

0.839

y = 0.027x + 0.012R² = 0.997

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 5 10 15 20 25 30 35

Abs

orba

nce

Concentration (µg/ml)

Hamycin at 381.5 nm

0

0.247

0.5490.703

1.019

1.201

y = 0.024x + 0.014R² = 0.994

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 10 20 30 40 50 60

Abs

orba

nce

Concentration (µg/ml)

Ketoconazole at 243.5 nm


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Table No.1: Linearity Profile

*Average of six determinations.Simultaneous Estimation of Hamycin andKetoconazoleDetermination of absorbtivity values of drugs atselected wavelengthsSuitable aliquots of standard stock solutions ofHamycin and Ketoconazole each were diluted withAcetonitrile: 0.5% w/v Ammonium acetate(80:20v/v) to obtain working standard solutions ofconcentrations within the Beer-Lambert’s range.The absorbance of each resulting solution wasmeasured at 381.5 nm and 243.5 nm.

The Absorbtivity values for Hamycin andKetoconazole were calculated from the followingformula:

t)Conc.(g/li

AbsorbancetyAbsorbtivi

The standard absorbtivity values of drugs atthe selected wavelengths are shown in Table No.2.Table No.2: Standard Absorptivity values ofHamycin and Ketoconazole for simultaneousequation method

Drug Absorptivity* (g/lit) at381.5 nm 243.5 nm

Hamycin 24.7 7.6

Ketoconazole 0 30

*Average of six determinationsSetting up of Simultaneous EquationsUsing the mean of standard absorbtivity values,estimation of both drugs were done by followingsimultaneous equation

A2 aY1 – A1 aY2

cx = ……………. (1)aX2 aY1 – aX1 aY2

Parameters HAMYCIN KETOCONAZOLE

Beers-Lambert’s range 5-30 g ml-1 10-50 g ml-1

Regression equation Y=0.027x + 0.012 Y=0.024x + 0.014

Slope* 0.027 0.024

Intercept* 0.012 0.014

Correlation coefficient* 0.997 0.994


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A1 aX2 – A2 aX1

cY = …………....... (2)aX2 aY1 – aX1 aY2

Analysis of the formulationsCream equivalent to 10 mg of Hamycin and 40 mgwas weighed and dissolved in 100 mL Acetonitrile:0.5% w/v Ammonium acetate (80:20v/v) with theaid of ultrasonication for 30 min. The solution wasthen filtered through Whatmann filter paper No.42.From the above solution 1 ml was taken and dilutedfurther with Acetonitrile: 0.5% w/v Ammoniumacetate (80:20v/v) up to 10ml to obtain final

concentration of 10 µg ml-1 of Hamycin and 40 µgml-1 of Ketoconazole respectively. The samplesolutions were analyzed as per the procedure formixed standards. Solutions were scanned in therange of 400 – 200 nm and their absorbances wererecorded at the selected wavelengths. Theconcentrations of each drug in sample solutionswere calculated using equations (1) and (2).

Table No.3: Analysis of formulations by Simultaneous Equation method.Sr.No.

Amount Present(µg ml-1)

Amount Found(µg ml-1)

% Amount Found

Hamycin Ketoconazole Hamycin Ketoconazole Hamycin Ketoconazole1.2.3.4.5.6.

101010101010

404040404040

9.769.839.9110.089.8310.04

39.1639.2739.1939.2539.4739.39

97.698.399.1100.8

98.3100.4

97.998.1897.9898.1398.6798.47

RESULTS AND DISCUSSIONSThe percentage content of both drugs in formulationHamycin and Ketoconazole were found to be99.08% and 98.22% respectively.Method ValidationPurpose: Validation is a process of establishingdocumented evidence, which provides a high degreeof assurance that a specific activity will consistentlyproduce a desired result or productmeeting its predetermined specifications and qualitycharacteristics.

AccuracySolutions were prepared in triplicate at levels 80%,100% and 120% of test concentration usingHamycin and Ketoconazole working Standard asper the test method and taken absorbance of eachsolution in triplicate. The recovery results showedthat the proposed method has an acceptable level ofaccuracy for Hamycin and Ketoconazole which isfrom 80% - 120% of test concentration is form 98% - 102 %. Results were shown in table 4 and 5.


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Table No. 4: Recovery Studies for formulation

Table No. 5: Statistical Validation for Recovery Studies*Average of three determinationsThe % R.S.D. was found to be less than 2%.

PrecisionRepeatabilityTo check the degree of repeatability of the method,six samples of the formulations were analyzed asper the procedure given under formulation. For eachof the developed UV-spectrophotometric methodsthe standard deviation and % Relative StandardDeviation (% R.S.D.) were calculated. The resultsof the same are given in Table No.6.

Intermediate precision (Intra-day and Inter-dayprecision)The Intra and Inter-day precision was determinedby assay of the sample solutions on the same day atdifferent time intervals and on different daysrespectively. The S.D. and % R.S.D. werecalculated and are presented in Table No.7.

Level of%Recovery

% Mean* S.D.* % R.S.D.*

HAM KETO HAM KETO HAM KETO

80%100%120%

99.098.6599.04

98.9898.4098.53

0.06650.05560.1670

0.08500.10590.18

0.37310.28180.7664

0.11920.134

0.2075

Level of%Recovery

80%100%120%

Amountpresent(mg/2gm)

Amount ofstandard added(mg)

Total amountrecovered (mg)

% Recovery*

HAM101010

KETO404040

HAM81012

KETO324048

HAM17.8219.7321.79

KETO71.2778.7286.71

HAM99.098.6599.04

KETO98.9898.4098.53


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Table No. 6: Statistical Evaluation for RepeatabilityDrugs % Mean* S.D.* % R.S.D.*

Hamycin 98.90 0.0456 0.46

Ketoconazole 98.40 0.1111 0.28

*Average of six determinations

Table No. 7: Statistical validation for Intermediate Precision

*Average of three determinationsThe % R.S.D. was found to be less than 2%.Ruggedness studyRuggedness study was performed by changing the analysts and instrument used. The S.D. and % R.S.D. werecalculated and are presented in Table No. 8.

Table No. 8: Ruggedness Study

Drug Intra-day Precision* Inter-day Precision*

%Mean

S.D. %R.S.D.

%Mean

S.D. %R.S.D.

HamycinKetoconazole

98.898.4

0.07530.1237

0.760.31

98.698.25

0.06940.1267

0.700.32


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*Average of three determinationsThe % R.S.D. was found to be less than 2%.Limit of detection (LOD) and Limit ofquantitation (LOQ)The LOD and LOQ were separately determinedbased on the standard deviation of response of the

calibration curve. The standard deviation of y-intercept and slope of the calibration curves wereused to calculate the LOD and LOQ. The results ofthe same are shown in Table No. 9.

Table No. 9: LOD and LOQ

Limit of Detection (LOD) Limit of Quantitation (LOQ)

Hamycin Ketoconazole Hamycin Ketoconazole

0.2296 0.2712 0.7407 0.875

ConclusionA simple, accurate, specific, precise, rugged andeconomical UV-spectroscopic method(Simultaneous equation method) was developed andvalidated for formulation. Linear relationships wereobtained between response and amount of drugwith high correlation coefficients (r2) in therange 5-30µg mL-1 for Hamycin (r2 =0.997) and10-50 µg mL-1 for Ketoconazole (r2 =0.994) . Theresults of validation studies of the developedmethods suggest that the developed UVspectrophotometric methods can be employedsuccessfully for the simultaneous estimationHamycin and Ketoconazole in formulation.

AcknowledgementsI express my special thanks to Dr. P. D.

Patil, Vice-Chancellor, Dr. D. Y. Patil University,Pimpri, Pune and Chairman Dr. D. Y. Patil VidyaPratishthan Society, Pimpri, Pune for providingexcellent infrastructural facility for undertaking thisresearch work.

My sincere thanks to Dr. Sohan S.Chitlange, Principal, Padm. Dr. D. Y. Patil Instituteof Pharmaceutical Sciences and Research, Pimpri,Pune for his constant support and guidance.

I owe a deep sense of gratitude andindebtedness to Mr. Sambhaji Patil, Plant Head,Genpharma International Pvt. Ltd, Pune forproviding me with all the excellent facilities forcompletion of my research work.

Parameter % Mean* S.D.* % R.S.D.*

HAM KETO HAM KETO HAM KETO

Instrument 98.8 98.42 0.0683 0.1360 0.69 0.35

Analyst 99.2 98.87 0.0420 0.3365 0.42 0.85


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Words are less to express my deep heartfeltgratitude to my guide Dr. R. K. Nanda, for hisconstant guidance, encouragement with which hehas equipped me to complete this project. I extendmy deepest sense of gratitude for his inspiration andtime he has spared despite his very busy schedule,will always be a part of immortal reminiscences andremain idol throughout my life.

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