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ISSN 2229 6867

IJPIs Journal of Analytical Chemistry


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A Review: Current Analytical Methods for Determination of Ketoconazole in Pharmaceutical and Biological Samples
Olga Popovska1*, Vesna Rafajlovska1, Zoran Kavrakovski2
1

Ss. Cyril and Methodius University in Skopje, Faculty of Technology and Metallurgy Rudjer Boskovic 16, 1000 Skopje, REPUBLIC OF MACEDONIA 2 Ss. Cyril and Methodius University in Skopje, Faculty of Pharmacy Vodnjanska 17, 1000 Skopje, REPUBLIC OF MACEDONIA

*Corresponding Author: Olga Popovska

ABSTRACT:

Ketoconazole is an imidazole antifungal drug which is available in the different pharmaceutical dosage forms through various routes of administration, such as oral and topical. The drug is a broad-spectrum antifungal agent and it is used in the treatment of superficial or systemic fungal infections. This article reviews the current analytical methods for identification and quantitative determination of ketoconazole in samples. The most commonly used methods for the determination of ketoconazole presented in this article are chromatographic methods: high-pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC); electroanalytical methods: voltammetry and potentiometry; capillary electrophoresis (CE); spectrofluorimetric and spectrophotometric methods. Recent preferences in the analysis of ketoconazole samples prove primacy of HPLC and confirm general trends moving towards more sensitive methods, with higher resolution potential, consuming small quantities of samples and reagents and require less time of the analysis.
Keywords: Ketoconazole, Antifungal drug, UV-Vis spectroscopy, Chromatography

Vol 3:13 (2013) 1. INTRODUCTION

IJPIs Journal of Analytical Chemistry

Ketoconazole (cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4yl]methoxy]phenyl]piperazine) is a lipophilic imidazole derivative appears as white to off white crystalline powder (Figure 1). The drug is practically insoluble in water, soluble in strong bases and sparingly soluble in strong acid. Ketoconazole is a weak base with pKa values of 6.51 and 2.94, contains two nitrogen atoms in the five-membered azole ring.1,2 Figure 1: Ketoconazole enantiomer chemical structures.

N N O (+)-(2R,4S) Cl Cl * O * O H N N O CH3

Cl H (-)-(2S,4R) Cl O * O N N * O N N

CH3

Ketoconazole is a chiral drug administered as a racemic (1:1) mixture of enantiomers of the cis configuration.3,4 It is a broad-spectrum antifungal imidazole agent that has been shown to be efficient in the treatment of human systemic fungal infections, against Candida species, Cryptococcus neoformans, histoplasmosis, tinea corporis, tinea cruris, tinea manuum and tinea pedis, onychomycosis, seborrheic dermatitis, possessing antiinflammatory and some antibacterial activities with topical and systemic action.5-15 Ketoconazole undergoes very easily on chemical degradation. The stressed degradation of ketoconazole drug substance into imidazole and acetamide radical species were performed under acid, base, thermal, photo and oxidative stress conditions.16,17 The potency losing and forming harmful degradation products are the most common consequences of the degradation of the drug.2 Ketoconazole was formulated into several pharmaceutical forms through various routes of administration such as: tablets, topical creams, ointments, gels and antidandruff shampoo.2,13,18-23 The role of ketoconazole as tablet has been limited to few indications due to the variable bioavailability, liver toxicity and inhibition of steroid biosynthesis.15,24-26 Nowadays, no specific evidence of development of resistance or cross-resistance of fungi to ketoconazole used in cosmetic dandruff shampoo at concentrations up to 2 %.27 The mechanism of attacks and changes in the structure and function of the cell membrane permeability is the most important factor in the ketoconazole action of the fungi.28 The primary mechanism of action of ketoconazole as an azole derivative is the inhibition of sterol 14--demethylase, a microsomal cytochrome P450 dependent enzyme system Page 2

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which converts lanosterol into ergosterol required for fungal cell membrane synthesis.29-34 Ketoconazole prevents the converting of the lanosterol in ergosterol and disrupts the integrity of membrane-bound enzymes and fungal cell membranes. This ketoconazole action increases membrane permeability and the leakage of small ions, amino acids and proteins from the fungi,32 leading to cell death.35 The use of the ketoconazole as a drug essential in pharmaceutical formulations highlights the requirement for its determination and quantification with appropriate analytical methods. This paper gives an overview of the analytical techniques that are available and nowadays have been used for determination of ketoconazole in pharmaceutical and biological samples. 2. METHODS FOR THE DETERMINATION OF KETOCONAZOLE The analytical methods that are currently used for determination of ketoconazole in pharmaceutical samples (tablets, shampoos, creams and ointments) and biological samples (plasma, serum, urine, saliva, tissues of lung, liver, muscles) are: spectrophotometric methods, as well as chromatographic methods (thin-layer chromatography, TLC and high-pressure liquid chromatography, HPLC), capillary electrophoresis (CE), spectrofluorimetry and electroanalytical methods (differential pulse polarography, adsorptive stripping differential pulse voltammetry, rotating disk electrode voltammetry, coulometry and potentiometry). The analytical methods which are already published usually require sample preparation, including extraction and clean-up, as well as the subsequent instrumental determination of ketoconazole from the matrixes with the other azole derivatives. The developed method for determination of the ketoconazole should be selective, sensitive and reproducible, with less consumption of solvent and time for analysis, as well as they should be validated in order to prove that the developed procedure is suitable for intended analytical purpose and give accurate results. A. Sample Procedures: A number of extraction techniques such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are available for isolation of the ketoconazole from the pharmaceutical and biological matrixes. 31,36-40 Newly extraction technologies such as ultrasound-enhanced surfactant-assisted dispersive liquid-liquid microextraction (UESADLLME)41 and supercritical fluid extraction (SFE)42 were introduced, also. The application of the liquid-liquid extraction technique was tested in the ketoconazole isolation from tablets or creams using chloroform43-45; dichloromethane for creams and tablets46; tetrachloromethane for creams47 and toluene for tablets.48 The frequently used organic solvents in LLE for the analyses of ketoconazole in human and rat plasma are: acetonitrile and 1-chlorobutane in mixture ratio of 1:4,49 diethyl ether38,41; chloroform38,41 and ethyl acetate.39 In the extraction step of analyzing ketoconazole in samples, buffers were used, also. The citrate buffer (pH 2) and phosphate buffer (pH 3) were used in the procedure for the ketoconazole determination in tablets and creams, 43,44 while for the plasma analyses phosphate buffer (pH 2) or dihydrogen phosphate-sodium hydroxide buffer solution (pH 6).40,41 The solid-phase extraction of ketoconazole from pharmaceutical samples and endogenous substances was performed using SDS-coated Al2O3 for tablet, cream and shampoo samples,31 Sep-Pak C18 cartridges for serum36,50 and Bond Elute Plexa cartridge for plasma samples,40 followed by elution with methanol36,40 or with ethanol.50 B. Spectrophotometric Methods: The quantitative determination of ketoconazole with UV-Vis spectrophotometric methods was based on forming coloured complexes43,46,51-53 or stabilizing diprotonated form of ketoconazole in HCl54,55 followed by measuring of the absorbance maximum. The ketoconazole in pharmaceutical samples was quantified by the method based on the formation of ion-pair complexes of the ketoconazole with reagents like bromocresol green, bromocresol purple, bromophenol blue and bromophenol red in acidic medium.56 Olga Popovska et al Page 3

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An analytical procedure for the determination of ketoconazole in bulk drugs and tablets was developed by the forming of pink complex from the reaction between ketoconazole and iron(III) chloride in the presence of potassium thiocyanate absorbed at 510 nm.51 Two procedures for spectrophotometric determination of three piperazine derivatives: ketoconazole, piribedil and prazosin hydrochloride as a result of charge-transfer and ion-pair complexation reactions was described in the literature.52 The reaction of the drugs with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in acetonitrile was studied. The maximum absorbance of formed orange-red complex was measured at 460 nm. A stable yellow ion-pair complex which absorbs at 410 nm was constituted in the interaction of the drugs in chloroform with bromophenol blue in acetonitrile. The two methods were applied for determination of the piperazine derivatives contents in tablets. A highly colored product was produced in the reaction of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and ketoconazole. The spectrophotometric method was developed for determination of the six drugs with different ndonating groups: clotrimazole, clozapine, ketoconazole, oxamniquine and pimozide in form of referent material and pharmaceutical dosage forms.53 The formation of a charge-transfer complex between antifungal drugs from the pharmaceutical dosage forms: clotrimazole, econazole, ketoconazole and miconazole as n-donor and iodine as acceptor were described in the literature.57 A procedure with picric acid for determination of ketoconazole in tablets and creams, followed by reading the absorbance at 410 nm was introduced in the literature.45 A spectrophotometric method for the determination of clotrimazole, econazole nitrate, ketoconazole, miconazole and tolnaftate based on the reaction between antifungal agents and 2,3-dichloro-5,6-dicyano-1,4benzoquinone in methanol or with p-chloranilic acid in acetonitrile by subsequently measured the absorbance at 460 nm and 520 nm, respectively was introduced in the literature.47 Blue-colored stable complexes were formed when ketoconazole as a ligand reacted quantitatively with copper(II) and cobalt(II). The values of the absorbance of the Cu(II) or Co(II) complexes were measured at 720 nm and 612 nm, respectively.46 The spectrophotometric method based on the coupled redox-complexation reactions which proceed in the ketoconazole-iron(III) and 1,10-phenanthroline systems was described for determination of ketoconazole in samples of tablets, creams and shampoos.58 The reactions of a triiodide ion and alizarin red S with clotrimazole and ketoconazole were studied with aim to analyze the studied drugs in pure forms and formulated in commercial preparations. Highly stable products were formed with the reaction of protonated forms of ketoconazole and clotrimazole with triiodide ion. The absorption maximum was achieved at 425 nm.44 A spectrophotometric method for determination of three pharmaceutical piperazine derivatives: ketoconazole, piribedil and trimetazidine hydrochloride was introduced in the literature.59 The yellow orange complexes were obtained in the reaction between iron(III) chloride and the pharmaceutical piperazine derivatives. A kinetic-spectrophotometric method for determination of the ketoconazole in tablets, creams and shampoo samples was presented.31 The manganate ion formed by the alkaline oxidation of ketoconazole with potassium permanganate in micellar SDS medium was measured at 610 nm. Zero-crossing and the first derivative ultraviolet spectrophotometric method with methanol as background solvent for quantitative determination of ketoconazole in commercial and simulated emulsion formulations were used in the literature.20,21 A method for quantitative determination of seven azole antifungal agents was introduced in the literature.60 Analysis was performed directly by using zero-order (fluconazole), first derivative (bifonazole, clotrimazole, Olga Popovska et al Page 4

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econazole, itraconazole, miconazole) and second derivative (ketoconazole) UV spectrometry. The antifungals were dissolved in methanol. The UV absorption spectra in range 200-400 nm were recorded for solutions of the standards and the analysed samples. The absorbance of the red complex formed in the reaction between ketoconazole from the pharmaceutical preparations and iron(III) ions in an acid medium was measured at 495 nm.61 The procedure for determination of ketoconazole, clotrimazole and fluconazole in their pure forms and pharmaceutical preparations in form of ion-pair yellow complex obtained with bromothymol blue and picric acid was reported.43 The absorbances were recorded at 410 nm and 415 nm for ketoconazole, 410 nm and 413 nm for clotrimazole and at 373 nm and 415 nm for fluconazole using picric acid and bromothymol blue, respectively. A method for determination of the ketoconazole in tablets based on amplification reactions was described in the literature.48 In the first stage, ketoconazole was oxidized with periodate in KC2+ and iodate ions. The iodate was treated with iodide to release iodine, after masking the excess periodate with molybdate. The absorbance was measured at 535 nm. C. Chromatographic Methods: The high-pressure liquid chromatography (HPLC) and the thin-layer chromatography (TLC) are widely used chromatographic methods in the analysis of imidazole derivatives including ketoconazole. I. Thin-layer chromatography The quantification of azole derivatives in pharmaceutical creams and ointments using thin-layer chromatography (TLC) densitometric methods was reported.62 The method was used for separation, qualitative and quantitative analysis of pure ketoconazole or in mixture with the clotrimazole and miconazole. A precoated silica gel and solvent system consist of n-hexane-chloroform-methanol-diethylamine (50:40:10:1 v/v) were applied in the analysis. A high-pressure thin-layer chromatographic method for determination of ketoconazole in shampoos and creams was reported in the literature.63 The samples were separated on a silica gel plate and developed in mobile phase consist of ethanol-acetone-1 M H2SO4 in ratio of 80:10:10 v/v. II. High-pressure liquid chromatography HPLC was commonly used technique in developing and validating assay methods for determination of the ketoconazole in biological samples and pharmaceutical formulations where it was introduced as a pure drug or together with others azole derivatives. The detection modes extensively applied in HPLC analysis of ketoconazole were: UV (ultra-violet) and DAD (diode array detector); spectrofluorimetric and detector with electrochemical detection. 64,65 On the other hand, mass spectrometry (MS) coupled with HPLC or with liquid chromatography (LC) as tandem methods were more often used in the direct analysis of ketoconazole and azole substances in the procedure for their identification.66 The new trends in mass spectrometry involved multiple steps in tandem MS-MS with intention to increase the selectivity and sensitivity of the determination resulting in far better limits of detection than those achieved by using LC-MS.38,66 The reversed-phase column and isocratic elution with mobile phase consist of two solvents or a mixture of solvents with buffers were reported in the literature as the most appropriate conditions for quantification of ketoconazole in pharmaceutical and biological samples (Table 1). The internal standards used in HPLC methods for quantification of the ketoconazole were phenothiazine,67 clotrimazole,37,49 terconazole,20,68 benzafibrate,69 R51012,38 9acetylanthracene,70 paclitaxel39 and linezolid.40 In the literature simultaneous determination of ketoconazole and other azole derivatives in pharmaceutical formulations, as well as in biological samples was reported, also.40,41,66,71-74 The separation methods were based on adjusting parameters such as mobile phase, pH, polarity and flow speed (Table 2).

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IJPIs Journal of Analytical Chemistry Table 1: Conditions for HPLC analysis of ketoconazole in samples

Detector type UV (225 nm)

DAD-UV (238 nm) UV (225 nm)

UV (231 nm)

No data

LC-MS-MS

Chromatographic Mobile phase column Commercial and LiChrospher 100 RP-18 Triethylamine in methanol simulated emulsion (125 mm x 4 mm, 5 m (1:500 v/v) and formulations particle size) ammonium acetate solution in water (1:200 w/v), 75:25 v/v Bulk drug Promosil C-18 water-acetonitrile-buffer (250 mm x 4.6 mm, 5 m (51:45:4 v/v; pH 6.8) Shampoo LiChrospher Monoisopropylamineparticle size) 100 RP-8 (150 mm x 4.6 mm, 5 m methanol (2:500 v/v) and particle size) ammonium acetate-water (1:200 w/v), 7:3 v/v with pH adjusted to 5.5 with pH adjusted to 5.5 Serum (human) MicroPak MCH-10 Methanol-phosphate (30 cm x 4 mm, 10 m buffer particle size) (75:25 v/v; pH 7.5) Lung, liver, plasma and Novapak C-18 Methanol-acetonitrileadrenal gland (rat) phosphate buffer (35:30:35 v/v; pH 6.8) Plasma (human) BDS Acetonitrile-water-formic Hypersil C-18 acid (75:25:1 v/v) (50 mm x 3 mm, 5 m particle size) Sample matrix Plasma (rat) (ketoconazole, docetaxel) Creams, tablets shampoos (ketoconazole, clotrimazole) Plasma (human) Cosmosil C-18 (150 mm x 2 mm) Formic acid and methanol (gradient elution)

Reference
20

33

34

36

37

38

LC-MS-MS

39

DAD-UV (224 nm)

UV (206 nm)

UV (225 nm)

Electrochemical

Methanol-waterdiethylamine-glacial acetic acid (80:20:0.3:0.2 v/v; pH 7) Inertsil ODS-80A Water-acetonitrile(150 mm x 4.6 mm, 5 m tetrahydrofuranparticle size) ammonium hydroxidetriethylamine (45:50.2:2.5:0.1:0.1 v/v) Tablets and creams Merck LiChrospher 100 DiisopropylamineRP-18 (5 m particle size) methanol (1:500) and ammonium acetate (1:200) (8:2) Plasma and saliva Bondapak Formic acid and

and C-18 (150 mm x 4.6 mm)

42

49

54

65

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Vol 3:13 (2013) detection UV (231 nm) (human) octadecylsilane (3.9 mm x 300 mm) Serum, plasma, C-18 cerebrospinal and synovial fluid (human) Plasma and tablets Bondapak

IJPIs Journal of Analytical Chemistry dibutylamine in methanol (pH 3) No data

67

No data

Methanol-water-glacial acetic acid (67.5:32:0.5 v/v) UV Plasma Hypersil BDS RP-C-18 Methanol-water(240 nm) (dog) (250 mm x 4.6 mm, 5 m diethylamine (74:26:0.1 particle size) v/v) Fluorimetric Plasma Metaphase Disodium hydrogen Detection (human) KR100-5-C-18 orthophosphate(260 nm and (250 mm x 4.6mm, 5 m acetonitrile (375 nm) particle size) (50:50 v/v; pH 6) UV Shampoo Interchrom Nucleosil C-8 Acetonitrile-phosphate (ketoconazole at 250 (250 mm x 4.6 mm, 5 m buffer nm, formaldehyde at particle size) (45:55 v/v; pH 4) 345 nm after derivatisation with 2,4dinitrophenylhydrazine) UV (225 nm) Tablets and creams Lichrosorb RP-18 Methanol-ammonium (250 mm x 4 mm, 5 m acetate particle size) (80:20 v/v)

69

70

75

76

77

Table 2: Conditions for HPLC analysis for simultaneous determination of azole antifungal agents in samples Detector type DAD-UV (260 nm) Sample matrix Chromatographic Mobile phase column Gemini C-6-Phenyl Phosphate buffer pH (150 mm x 4.6 mm, 5 m acetonitrile particle size) (gradient elution) Reference 7 and
40

Plasma (fluconazole, posaconazole, voriconazole, itraconazole and its metabolite hydroxylitraconazole, ketoconazole) DAD-UV Blood (human) (220 nm) (ketoconazole, econazole nitrate) UV (212 Residues nm) (cow milk) (ketoconazole, clotrimazole)

Agilent HC-C-18 Acetonitrile-water (75:25 v/v) (250 mm x 4.6 mm, 5 m particle size) Zorbax XDB C-18 Acetonitrile-sodium acetate buffer (250 mm x 4.6mm, 5 m (85:15 v/v; pH 4.6) particle size)

41

50

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66

Liver and LC BEH C18 Watermethanol, muscles (residue, (2.1 mm x 100 mm, 1.7 wateracetonitrile, water containing chickens) m particle size) 0.1% formic (voriconazole, acidmethanol, and water containing griseofulvin, 0.1% formic clotrimazole, acidacetonitrile bifonazole, (gradient elution) econazole, ketoconazole, itraconazole, miconazole, terconazole, fluconazole)

UV (260 Pharmaceutical nm) formulations (ketoconazole, clotrimazole, fluconazole) UV (254 Pharmaceutical nm) formulations (ketoconazole, isoconazole, miconazole)

Bondapak C-18 (25 cm x 4.6 mm, 10 m particle size)

Acetonitriletris(hydroxymethyl)aminomethane (55:45 v/v) in phosphate buffer (pH 7) Acetonitrile-ammonium acetate buffer (70:30 v/v), with pH adjusted to 6 with phosphoric acid

72

C-18-BDS (100 mm x 4.6 mm)

74

D. Alternative Methods I. Capillary zone electrophoresis A method of capillary zone electrophoresis for simultaneous determinations of a mixture of antifungal drugs: ketoconazole, clotrimazole and econazole in pharmaceutical forms were adapted. 78 The separation was achieved using a fused-silica capillary column with acetic acid-Tris buffer at pH 5 and UV detection at 196 nm. A capillary zone electrophoresis for determination of ketoconazole in tablets and creams with phosphate buffer adjusted with phosphoric acid at pH 2.3 and UV detection at 225 nm was introduced in the literature.77 II. Electroanalytical methods A non-aqueous potenciometric titration where the equivalence point of ketoconazole was potentiographically located using D1 differential potentiograph was introduced.79 The oxidation of ketoconazole by rotating disk electrode voltammetry, cyclic voltammetry, and controlled potential coulometry in chloroform or acetonitrile at platinum, gold and glassy carbon electrodes using tetrabutylammonium perchlorate as supporting electrolyte was introduced in the literature.80,81 The method was used for quantification of ketoconazole in tablets and cream. The oxidation of ketoconazole on a bare carbon electrode voltammetrically was investigated.82 The results obtained by determination of ketoconazole in human urine and formulations indicated that the process was irreversible and controlled by an adsorption-extraction process that allowed accumulation of the drug at the electrode surface. A simple potenciometric method using liquid-plasticized PVC membrane sensor for determination of the ketoconazole in pharmaceutical preparations and biological matrixes was described in the literature.83 A water-insoluble ketoconazoletetraphenylborate ion pair took a role of an ion-exchanger. The electroanalytical behavior of ketoconazole in BritonRobinson buffer in a gel formulation and spiked urine samples was studied.84 III. Proton nuclear magnetic resonance

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1H-NMR was used for determination of ketoconazole in pure form and in tablets involving integration of the methyl protons-signal of ketoconazole (at 2.07 ) relative to the benzocaine (at 1.30 ) used as an internal standard.79 IV. Spectrofluorimetric methods Spectrofluorimetric methods used as an analytical technique are based on the measuring of the native fluorescence of ketoconazole. A synchronous spectrofluorimetric determination of famotidine, fluconazole and ketoconazole in bulk powder and pharmaceutical formulations was reported in the literature. 85 The fluorescence intensity was recorded at 364 nm with excitation at 239 nm for ketoconazole, 384 nm with excitation at 284 nm for famotidine and 285 nm with excitation at 255 nm for fluconazole. The quantitative determination of antifungal drugs: clotrimazole, econazole nitrate, ketoconazole, miconazole and tolnaftate in pharmaceutical formulations using spectrofluorimetric method was introduced in the literature.47 The spectrofluorimetric method measured native fluorescence of ketoconazole at 375 nm with excitation at 288 nm or the induced fluorescence after alkaline hydrolysis of tolnaftate with NaOH solution at 420 nm with excitation at 344 nm. V. Thermogravimetric method A thermogravimetric method for quantification of the ketoconazole in raw materials and tablets was studied in the literature.86 The method included changes in physical and chemical properties of analytes as a function of increasing temperature. The samples were analyzed by dynamic thermogravimetry in nitrogen and nitrogen-synthetic air mixture at heating rates of 10, 20, 40, 60 and 80C min 1. The concentrations of ketoconazole were obtained from the vapour pressure curves. 3. CONCLUSION Presented systematic review covers the current analytical methods for the determination of ketoconazole in pharmaceutical and biological samples. The limitation of the reported methods requires developing new optimized method which would be suitable for intended analytical purpose for analyzing the content of ketoconazole in pharmaceutical, as well as in biological samples. The new trends and advances for quantification of ketoconazole are based on using high-pressure liquid chromatography which is widely available and flexible method with the ability of coupling with mass spectrometry. The HPLC method could be automated; there are different column fillings; different solvents with different polarity as mobile phases and different detection modes. The faster time, high sensitivity, specificity and better separation efficiency enable HPLC to be used frequently for the simultaneous qualitative and quantitative determination of azole derivatives in the comparison with the other methods. The development of a new established method should reduce existing analytical problems including many steps for the preparation of the sample, as well as improving the resolution which can give accurate results for the concentration of all ketoconazole samples and could be used for routine analysis. 4. REFERENCES (1) Beggs WH (1991) Protonation of ketoconazole in relation to fungistatic activity. Mycopathologia 116(1): 3-4. (2) Skiba M, Skiba-Lahiani M, Marchais H, Duclos R, and Arnaud P (2000) Stability assessment of ketoconazole in aqueous formulations. Int J Pharm 198(1): 1-6. (3) Hamdy DA and Brocks DR (2008) A stereospecific high-performance liquid chromatographic assay for the determination of ketoconazole enantiomers in rat plasma. Biomed Chromatogr 22(5): 542-547. (4) Hamdy DA and Brocks DR (2010) High performance liquid chromatographic assay for the simultaneous determination of midazolam and ketoconazole in plasma. J Pharm Biomed Anal 53(3): 617-622. (5) Ginsburg CM, McCracken GH-Jr, and Olsen K (1983) Pharmacology of ketoconazole suspension in infants and children. Antimicrob Agents Chemother 23(5): 787-789.

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(6) Perez H (2004) Ketoconazole as an adjunct to finasteride in the treatment of androgenetic alopecia in men. Med Hypotheses 62(1): 112-115. (7) Wang L and Tang X (2008) A novel ketoconazole bioadhesive effervescent tablet for vaginal delivery: design, in vitro and in vivo evaluation. Int J Pharm 350(1-2): 181-187. (8) Chander Shekar B, Shireesh Kiran R, and Nagendra Babu B (2010) Preparation and evaluation of gastro retentive floating tablets of ketoconazole. IJPRD 2(9): 174-184. (9) Kaur IP and Kakkar S (2010) Topical delivery of antifungal agents. Expert Opin Drug Deliv 7(11): 13031327. (10) Patel GM and Patel AP (2010) A novel effervescent bioadhesive vaginal tablet of ketoconazole: formulation and in-vitro evaluation. Int J PharmTech Res 2(1): 656-667. (11) Agrawal TK and Murti Y (2011) Synthesis and bioevaluation of ketoconazole thiosemicarbazone analogues. IJPSR 2(12): 3156-3160. (12) Patel MH, Patel AM, Patel SM, Ninama GL, Patel KR, and Lavingia BC (2011) Antifungal susceptibility testing to determine MIC of amphotericine B, fluconazole and ketoconazole against ocular fungal infection. NJCM 2(2): 302-305. (13) Patel MR, Patel RB, Parikh JR, Solanki AB, and Patel BG (2011a) Investigating effect of microemulsion components: in vitro permeation of ketoconazole. Pharm Dev Technol 16(3): 250-258. (14) Rao Purushotham K, Venkateshwarlu P, Shashikala P, Sagare P, Saran SV, Ravindranath A, Ashok Kumar C, Anand C, and Jayanthi C (2012) Ketoconazole derma sticks for topical applications. Int J Res Chem Environ 2(1): 255-259. (15) Xin C, Li-hong W, Jing Y, Yang Y, Yue Y, Qi-fang W, and San-ming L (2012) Ketoconazole ion-exchange fiber complex: a novel method to reduce the individual difference of bioavailability in oral administration caused by gastric anacidity. Pharm Dev Technol doi: 10.3109/dmd.10837450.2012.696266. (16) Morsy MA, Sultan SM, and Dafalla H (2009) Electron paramagnetic resonance method for the quantitative assay of ketoconazole in pharmaceutical preparations. Anal Chem 81(16): 6991-6995. (17) Mhaske RA and Sahasrabudhe S (2011) Identification of major degradation products of ketoconazole. Sci Pharm 79(4): 817-836. (18) Staub I, Schapoval EES, and Bergold AM (2005) Microbiological assay of ketoconazole in shampoo. Int J Pharm 292(1-2): 195-199. (19) Faergemann J, Ausma J, and Borgers M (2006) In vitro activity of R126638 and ketoconazole against Malassezia species. Acta Derm Venereol 86(4): 312-315. (20) Kedor-Hackmann ERM, Santoro MIRM, Singh AK, and Peraro AC (2006) First-derivative ultraviolet spectrophotometric and high performance liquid chromatographic determination of ketoconazole in pharmaceutical emulsions. Braz J Pharm Sci 42(1): 91-98. (21) Shivanand P, Devmurari V, Manish G, and Pandey D (2009) Formulation, optimization and in-vitro evaluation of ketoconazole cream. Der Pharmacia Lettre 1(2): 18-24. (22) Jain A, Gautam SP, Gupta Y, Khambete H, and Jain S (2010) Development and characterization of ketoconazole emulgel for topical drug delivery. Der Pharmacia Sinica 1(3): 221-231. (23) Mitu MA, Lupuliasa D, Dinu-Prvu CE, Rdulescu F, Miron DS, and Vlaia L (2011) Ketoconazole in topical pharmaceutical formulations. The influence of the receptor media on the in vitro diffusion kinetics. Farmacia 59(3): 358-366. (24) Bajad S, Johri RK, Singh K, Singh J, and Bedi KL (2002) Simple high-performance liquid chromatography method for the simultaneous determination of ketoconazole and piperine in rat plasma and hepatocyte culture. J Chromatogr A 949(1-2): 43-47.

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