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ISSN 2229 6867 IJPI’s Journal of Analytical Chemistry Visit www.ijpijournals.com A Review: Current Analytical Methods for Determination of Ketoconazole in Pharmaceutical and Biological Samples Olga Popovska 1 *, Vesna Rafajlovska 1 , Zoran Kavrakovski 2 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
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Page 1: Analytical Review Ketoconazole

ISSN 2229 – 6867

IJPI’s Journal of Analytical Chemistry

Visit

www.ijpijournals.com

A Review: Current Analytical Methods for Determination of Ketoconazole in

Pharmaceutical and Biological Samples

Olga Popovska1*, Vesna Rafajlovska

1, Zoran Kavrakovski

2

1Ss. Cyril and Methodius University in Skopje, Faculty of Technology and Metallurgy

Rudjer Boskovic 16, 1000 Skopje, REPUBLIC OF MACEDONIA 2Ss. 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

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Vol 3:13 (2013) IJPI’s Journal of Analytical Chemistry

Olga Popovska et al Page 2

1. INTRODUCTION

Ketoconazole (cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-

yl]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.

Cl

O

O

N

N

*

H

O N N*

Cl

*

HO N N

*

Cl

O

O

N

N

CH3

CH3

O

OCl

(+)-(2R,4S)

(-)-(2S,4R)

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 anti-

inflammatory 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-α-demethylase, a microsomal cytochrome P450 dependent enzyme system

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Vol 3:13 (2013) IJPI’s Journal of Analytical Chemistry

Olga Popovska et al Page 3

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 (UESA-

DLLME)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

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Olga Popovska et al Page 4

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 n-

donating 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-1,4-

benzoquinone 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,

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Olga Popovska et al Page 5

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

9-

acetylanthracene,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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Olga Popovska et al Page 6

Table 1: Conditions for HPLC analysis of ketoconazole in samples

Detector

type Sample matrix

Chromatographic

column Mobile phase Reference

UV (225 nm) Commercial and

simulated emulsion

formulations

LiChrospher® 100 RP-18

(125 mm x 4 mm, 5 µm

particle size)

Triethylamine in methanol

(1:500 v/v) and

ammonium acetate

solution in water (1:200

w/v), 75:25 v/v

20

DAD-UV

(238 nm)

Bulk drug Promosil C-18

(250 mm x 4.6 mm, 5 µm

particle size)

water-acetonitrile-buffer

(51:45:4 v/v; pH 6.8)

33

UV (225 nm)

Shampoo

LiChrospher® 100 RP-8

(150 mm x 4.6 mm, 5 µm

particle size)

Monoisopropylamine-

methanol (2:500 v/v) and

ammonium acetate-water

(1:200 w/v), 7:3 v/v

with pH adjusted to 5.5

with pH adjusted to 5.5

34

UV (231 nm)

Serum (human)

MicroPak MCH-10

(30 cm x 4 mm, 10 µm

particle size)

Methanol-phosphate

buffer

(75:25 v/v; pH 7.5)

36

No data

Lung, liver, plasma and

adrenal gland (rat)

Novapak C-18

Methanol-acetonitrile-

phosphate buffer

(35:30:35 v/v; pH 6.8)

37

LC-MS-MS

Plasma (human)

BDS

Hypersil C-18

(50 mm x 3 mm, 5 µm

particle size)

Acetonitrile-water-formic

acid (75:25:1 v/v)

38

LC-MS-MS

Plasma

(rat)

(ketoconazole,

docetaxel)

Cosmosil C-18

(150 mm x 2 mm)

Formic acid and methanol

(gradient elution)

39

DAD-UV

(224 nm)

Creams, tablets and

shampoos

(ketoconazole,

clotrimazole)

C-18

(150 mm x 4.6 mm)

Methanol-water-

diethylamine-glacial acetic

acid (80:20:0.3:0.2 v/v; pH

7)

42

UV (206 nm) Plasma (human) Inertsil ODS-80A

(150 mm x 4.6 mm, 5 µm

particle size)

Water-acetonitrile-

tetrahydrofuran-

ammonium hydroxide-

triethylamine

(45:50.2:2.5:0.1:0.1 v/v)

49

UV (225 nm)

Tablets and creams

Merck LiChrospher® 100

RP-18 (5 µm particle size)

Diisopropylamine-

methanol (1:500) and

ammonium acetate (1:200)

(8:2)

54

Electrochemical Plasma and saliva µBondapak Formic acid and 65

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Olga Popovska et al Page 7

detection (human) octadecylsilane

(3.9 mm x 300 mm)

dibutylamine in methanol

(pH 3)

UV (231 nm)

Serum, plasma,

cerebrospinal and

synovial fluid (human)

C-18

No data

67

No data Plasma and tablets µBondapak Methanol-water-glacial

acetic acid (67.5:32:0.5

v/v)

69

UV

(240 nm)

Plasma

(dog)

Hypersil BDS RP-C-18

(250 mm x 4.6 mm, 5 µm

particle size)

Methanol-water-

diethylamine (74:26:0.1

v/v)

70

Fluorimetric

Detection

(260 nm and

(375 nm)

Plasma

(human)

Metaphase

KR100-5-C-18

(250 mm x 4.6mm, 5 µm

particle size)

Disodium hydrogen

orthophosphate-

acetonitrile

(50:50 v/v; pH 6)

75

UV Shampoo

(ketoconazole at 250

nm, formaldehyde at

345 nm after

derivatisation with 2,4-

dinitrophenylhydrazine)

Interchrom Nucleosil C-8

(250 mm x 4.6 mm, 5 µm

particle size)

Acetonitrile-phosphate

buffer

(45:55 v/v; pH 4)

76

UV (225 nm) Tablets and creams Lichrosorb® RP-18

(250 mm x 4 mm, 5 µm

particle size)

Methanol-ammonium

acetate

(80:20 v/v)

77

Table 2: Conditions for HPLC analysis for simultaneous determination of azole antifungal agents in samples

Detector

type Sample matrix

Chromatographic

column Mobile phase Reference

DAD-UV

(260 nm)

Plasma

(fluconazole,

posaconazole,

voriconazole,

itraconazole and

its metabolite

hydroxyl-

itraconazole,

ketoconazole)

Gemini C-6-Phenyl

(150 mm x 4.6 mm, 5 µm

particle size)

Phosphate buffer pH 7 and

acetonitrile

(gradient elution)

40

DAD-UV

(220 nm)

Blood (human)

(ketoconazole,

econazole nitrate)

Agilent HC-C-18

(250 mm x 4.6 mm, 5 µm

particle size)

Acetonitrile-water (75:25 v/v) 41

UV (212

nm)

Residues

(cow milk)

(ketoconazole,

clotrimazole)

Zorbax XDB C-18

(250 mm x 4.6mm, 5 µm

particle size)

Acetonitrile-sodium acetate buffer

(85:15 v/v; pH 4.6)

50

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Olga Popovska et al Page 8

LC-MS-

MS

Liver and

muscles (residue,

chickens)

(voriconazole,

griseofulvin,

clotrimazole,

bifonazole,

econazole,

ketoconazole,

itraconazole,

miconazole,

terconazole,

fluconazole)

LC BEH C18

(2.1 mm x 100 mm, 1.7

µm particle size)

Water–methanol,

water–acetonitrile, water containing

0.1% formic

acid–methanol, and water containing

0.1% formic

acid–acetonitrile

(gradient elution)

66

UV (260

nm)

Pharmaceutical

formulations

(ketoconazole,

clotrimazole,

fluconazole)

Bondapak C-18

(25 cm x 4.6 mm, 10 µm

particle size)

Acetonitrile-

tris(hydroxymethyl)aminomethane

(55:45 v/v) in phosphate buffer

(pH 7)

72

UV (254

nm)

Pharmaceutical

formulations

(ketoconazole,

isoconazole,

miconazole)

C-18-BDS

(100 mm x 4.6 mm)

Acetonitrile-ammonium acetate

buffer (70:30 v/v), with pH

adjusted to 6 with phosphoric acid

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 ketoconazole-

tetraphenylborate ion pair took a role of an ion-exchanger. The electroanalytical behavior of ketoconazole in Briton-

Robinson 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 δ) relative to the benzocaine (at 1.30 δ) 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 80°C 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

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