ANALYSIS OF ANTIFUNGAL AGENTS BY RP-HPLC: A - wjpps

13 Pages·2014·0.43 MB·English

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Talaviya et al. World Journal of Pharmacy and Pharmaceutical Sciences
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ANALYSIS OF ANTIFUNGAL AGENTS BY RP-HPLC: A REVIEW

*1Smita Talaviya and 2Falguni Majmudar

1Department of Pharmaceutical Chemistry, K. B. Raval College of Pharmacy, Gandhinagar,
Gujarat, India.
2Department of Pharmacology, Smt. N.H.L. Municipal Medical College, Ahmedabad,
Gujarat, India.

Article Received on ABSTRACT
01 Oct 2014,
Analytical method development and its validation is an important

Revised on 25 Oct 2014,
aspect in drug discovery process. Development of analytical method
Accepted on 18 Nov 2014
producing accurate and precise data is necessary to ensure the quality
and safety of the drug. In today’s scenario, the most common
*Correspondence for Author
Smita Talaviya analytical method employed for estimation of drugs is Reverse Phase
Department of Pharmaceutical High Pressure Liquid Chromatography (RP-HPLC) because of its high
Chemistry, K. B. Raval College
sensitivity and speed. Many types of analytical methods are available
of Pharmacy, Gandhinagar,
for estimation of antifungal agents including RP-HPLC. This review
Gujarat, India.
article briefly discusses analytical methods available for the estimation

of currently available antifungal agents specifically focusing on RP-HPLC.

KEYWORDS: Antifungal agents, Analytical methods, RP-HPLC, analysis of antifungals.

INTRODUCTION
Fungal infections are caused by microscopic organisms that can invade the epithelial tissue.
The fungal kingdom includes yeasts, molds, rusts and mushrooms. Fungi, like animals, are
heterotrophic, i.e. they obtain nutrients from the environment and not from the endogenous
sources. Some of these fungi are pathogenic and can produce mild to severe fungal
infections. An antifungal agent is a drug that selectively eliminates fungal pathogens from a
host with minimal toxicity to the host.[1] They can be categorized in to several categories
according to different pharmacophores and different mechanisms. Polyene antifungal drugs
like amphotericin B, nystatin interacts with sterols in the cell membrane to form channels
through which small molecules leak from the inside of the fungal cell to the outside. Azoles
like fluconazole, itraconazole, ketoconazole, clotrimazole, voriconazole, posaconazole etc.
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inhibit cytochrome P450-dependent enzymes (particularly C14-demethylase) involved in the
biosynthesis of ergosterol, which is required for fungal cell membrane structure and function.
Allylamines like naftifine, terbinafine inhibit ergosterol biosynthesis at the level of squalene
epoxidase. Antimetabolites like 5-Fluorocytosine act as an inhibitor of both DNA and RNA
synthesis via the intracytoplasmic conversion of 5-fluorocytosine to 5-fluorouracil.
Echinocandins like anidulafungin, caspofungin and micafungin are used for systemic fungal
infections in immunocompromised patients, they inhibit the synthesis of glucan in the cell
wall via the enzyme 1,3-β glucan synthase[2,3].

Analytical method Development and validation for newly introduced pharmaceuticals is of
importance, as drug or drug combination may not be official in pharmacopoeia and so
analytical method for quantification is not available. To check and ensure the quality
standards of drug molecules and their formulation various analytical methods are employed.
Most of the drugs in single or multi component dosage forms can be analyzed by HPLC
method because of the associated advantages like speed, greater sensitivity, improved
resolution, specificity, accuracy, precision, reusable columns and ease of automation in this
method. [4,5,6] This review article briefly discusses the RP-HPLC methods available for the
estimation of antifungal agents in bulk and in various formulations concentrating mainly on
the mobile phase, stationary phase and detector type.

Table 1 Chromatographic conditions for RP-HPLC methods reported for the estimation
of polyene antibiotics.
Name of Chromatographic Type of
Sample matrix Mobile phase
antifungal agent Column detector
Nystatin[7] Bulk drug and Symmetry C18 Methanol:water:dimeth UV
pharmaceutical (4.6 x 250 mm, 5 ylformamide (55:30:15 detection at
preparation μm) v/v/v) 305 nm
Nystatin[8] Ointment Inerstil ODS-3 Methanol:water (75:25 UV
column (250 x 4.6 v/v) detection at
mm, 5 μm) 305 nm
Nystatin[9] Nystatin, Inertsil ODS‐3v Acetonitrile: 25 mM DAD
Miconazole, (250 mm × 4.6 KH PO buffer, acquisition
2 4
Hydrocortisone mm, 5 μm) pH=2.8) (50:50 v/v) wavelength
acetate and from 200 to
Neomycin in 400 nm
pharmaceutical
preparations
Nystatin[10] Nystatin and muBondapark C18 Methanol: water (65:35 UV
triamcinolone in (150mm × 4.6m, 5 v/v) detection at
cream μm) 254 nm
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Nystatin[11] Liposomal muBondapak C18 10 mM sodium UV
Nystatin in (150mm × 4.6m, 5 phosphate, 1 mM detection at
plasma and tissue μm) EDTA, 30% methanol 305 nm
and 30% acetonitrile
adjusted to pH 6
Nystatin[12] Miconazole and Phenomenex Luna Methanol:acetonitrile:5 UV
nystatin C18 column (250 0 mM ammonium detection at
× 4.6 mm, 5 µm) acetate buffer (pH 230 nm
adjusted to 6 using
glacial acetic acid)
(60:30:10 v/v/v)
Nystatin[13] Nystatin LichrCARTŖ C18 Methanol- Diode-array
hydrocortisone, (250 × 4.6 mm, 5 NaH PO /Na HPO detector
2 4 2 4
and µm) gradient elution
oxytetracycline
Amphotericin Bulk and dosage Spheri-5, CYANO 10 mM sodium acetate UV
B[14] form column (30 × 4.6 buffer:acetonitrile detection at
mm, 5 μm ) (72:28 v/v with pH-4 ) 408 nm
Amphotericin Liposomal C18 μBondapac Acetonitrile:0.02M UV
B[15] amphotericin B column (300 × 4.6 ethylenediamine tetra- detection at
mm, 5 μm) acetic acid disodium 407 nm
Salt at pH 5 (45:55 v/v)
Amphotericin Amphotericin B, LiChrosorb-RP-8 Acetonitrile:methanol:0 UV
B[16] liposomal column .010 M NaH PO detection at
2 4
amphotericin B, buffer (41:10:49 v/v) 405 nm
and amphotericin
B colloidal
dispersion in
plasma
Amphotericin Amphotericin B C-18 column Acetonitrile:20 mM UV
B[17] in human plasma disodium edetate at pH detection at
5 (45:55 v/v) 407 nm
Amphotericin Bulk and dosage Luna C18 column Acetonitrile:Tetrahydro UV
B[18] form (250 x 4.6 mm; furan:o-phosphoric detection at
5μ) acid, pH=6 adjusted 287 nm
with TEA (60:30:10
v/v/v)

Table 2 Chromatographic conditions for RP-HPLC methods reported for the estimation
of azoles.
Name of Sample matrix Chromatographic Mobile phase Type of
antifungal agent Column detector
Fluconazole[19] Human urine LichroCART® Methanol:water UV detection
C18 (150 mm x (70:30 v/v) at 254 nm.
4.6 mm, 5 μm)
Fluconazole[20] Fluconazole and Purospher STAR Water:methanol UV detection
Related RP-18 (150 x 4.6 (60:40 v/v) at 260 nm
Compounds in mm, 5 μm)
Capsule
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Fluconazole[21] Bulk and solid SunFire C18 (250 Methanol:water UV detection
pharmaceutical × 4.5 mm, 5 μm) (70:30 v/v) at 211 nm
dosage form
Fluconazole[22] Capsules, C-18 Phenomenix Water:acetonitrile UV detection
uncoated and (150 × 4.6 mm, 5 (65:35 v/v) at 260 nm
dispersible μm)
Tablets
Fluconazole[23] Biological Skin Lichrospher RP 18 Methanol:0.025 UV detection
Matrices column (250 × 4 mol/L phosphate at 260 nm
mm, 5 μm) buffer pH 7.0
(45:55 v/v)
Clotrimazole[24] Lozenges Gracemart C18 Methanol:0.1% UV detection
(250 × 4.6 mm, TEA in water pH at 215 nm
5μm) 3 adjusted by
Ortho-phosphoric
acid (75:25 v/v)
Clotrimazole[25] Beclomethason Reversed phase Acetate buffer- UV detection
e, Clotrimazole, C18 column (250 acetonitrile with at 254 nm
Chloramphenic x 4.6 mm, 5.0 μm) different gradient
ol, lidocaine in program
Formulations
Clotrimazole[26] Clotrimazole C-18 Column (150 Methanol:water:di DAD-UV
and x 4.6 mm, 5 μm) ethylamine:glacial detection at
ketoconazole in acetic acid, pH 7 224 nm
Creams, tablets (80:20:0.3:0.2
and shampoos v/v)
Ketoconazole[27] Plasma Inertsil ODS-80A Water:acetonitrile UV detection
(150 mm x 4.6 :tetrahydrofuran:a at 206 nm
mm, 5 μm) mmonium
hydroxide:triethyl
amine(45:50.2:2.5
:0.1:0.1 v/v)
Ketoconazole[28] Tablets and Merck Diisopropylamine UV detection
creams LiChrospher® 100 :methanol (1:500) at 225 nm
RP-18 (5 μm) and ammonium
acetate (1:200)
(8:2)
Ketoconazole[29] Commercial LiChrospher® 100 Triethylamine in UV detection
and simulated RP-18 (125 mm x methanol(1:500 at 225 nm
emulsion 4 mm, 5 μm) v/v):ammonium
formulations acetate in water
(1:200w/v) (75:25
v/v)
Ketoconazole[30] Serum (human) MicroPak MCH- Methanol:phosph UV detection
10 (30 cm x 4 mm, ate buffer, pH 7.5 at 231 nm
10 μm) (75:25 v/v)
Ketoconazole[31] Tablets and Lichrosorb® RP- Methanol:ammoni UV detection
creams 18 (250 mm x 4 um acetate (80:20 at 225 nm
mm, 5 μm) v/v)
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Ketoconazole[32] Shampoo Interchrom Acetonitrile:phos UV detection
Nucleosil C-8 phate buffer, pH 4 at 250 nm
(250 x 4.6 mm, 5 (45:55 v/v)
μm)
Econazole[33] Cream and RP-18 column Methanol, UV detection
lotion aqueous
ammonium
carbonate,
tetrahydrofurane
Voriconazole[34] Bulk and Tablet Inertsil C8 column 0.01M sodium UV
Dosage Form (250 x 4.6 mm, dihydrogen detection at
5μm) orthophosphate, 254 nm
pH 5:Acetonitrile
(50:50 v/v)
Voriconazole[35] Voriconazole Intersil ODS-C18 Phosphate buffer: UV detection
for injection (150 X 4.6 mm, acetonitrile:Metha at 257 nm
5μm) nol (65:30:5
v/v/v)
Voriconazole[36] Voriconazole Inertsil ODS-2, C- OPA:Acetonitrile: UV detection
for injection 18 (150 × 4.6 mm, Methanol at 257nm
5μm) (65:30:5 v/v/v)
Voriconazole[37] Voriconazole Zorbax SB-C18 Ammonium UV detection
and its related (250mm × 4.6 phosphate dibasic at 250 nm
substances mm, 5 μm) buffer, pH=6 by
orthophosphoric
acid:acetonitrile
(52:48 v/v)
Voriconazole[38] Degradation Inertsil ODS 3V 0.05 M potassium UV detection
impurities and (150 × 4.6 mm, 5 dihydrogen at 256 nm
Diastereomers μm) phosphate, pH
in tablets 2.5:mixture of
acetonitrile and
methanol (90:10
v/v)
Voriconazole[39] Voriconazole Inertsil ODS-2, C- Ortho phosphoric UV detection
For Injection 18 (150 × 4.6 mm, acid:acetonitrile: at 257nm
5μm) methanol (65:30:5
v/v/v)
Voriconazole[40] Pure and RP-C-18 Hypersil Water:acetonitrile UV detection
Pharmaceutical BDS column (250 :methanol at 256 nm.
Dosage Forms × 4.6 mm, 5 μm) (50:25:25 v/v/v)
Voriconazole[41] Stability Hypersil C18 (250 Acetonitrile:water Photodiode
indicating × 4.6 mm , 5 μm) (40:60 v/v) array
method detector
Voriconazole[42] Bulk and Hypersil C18 (250 Water:acetonitr UV detection
formulation × 4.6 mm, 5 μm) ile (35:65 v/v) at 256 nm
Itraconazole[43] Capsule Inertsil C-18, (250 Tetrabutyl UV detection
× 4.6 mm, 5 μm) ammonium at 225nm
hydrogen sulphate
buffer:Acetonitril
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e (40:60 v/v)
Itraconazole[44] Itraconazole Zorbax Eclipse Acetonitrile:water UV detection
and its XDB-C18 (4.6 × , pH 2.5 adjusted at 256 nm
impurities 150 mm, 5 μm) with o-phosphoric
acid (50:50 V/V)
Itraconazole[45] Capsule dosage ThermoHypersil Tetra Butyl UV detection
form BDS C18 (150mm Ammonium at 225nm
x 4.6 mm, 5μm) Hydrogen
Sulphate:acetonitr
ile (65:35 v/v)
Itraconazole[46] Itraconazole Shimadzu C-18 Methanol:water Fluorometric
and hydroxyl column (3.9 mm × (75:25 v/v) detection at
itraconazole in 150 mm, 5μm) 250 and
plasma 380nm
Itraconazole[47] Capsule Dionex C-18 (250 Potassium UV detection
x 4.6 mm, 5 μm) dihydrogen at 306 nm
phosphate
buffer:Methanol
(60:40 v/v)
Itraconazole[48] Itraconazole Acclaim RSLC Ammonium UV detection
and Related 120, C18 (2.1 × acetate at 225 nm
Substances 100 mm, 2.2 μm) buffer:CH CN
3
gradient technique
Posaconazole[49] Human serum Sunfire C18 (4.6 x Acetonitrile:water UV detection
150 mm, 5 μm) (60:40 v/v) at 262 nm
Posaconazole[50] Posaconazole Sunfire C-18 (250 Acetonitrile:water UV detection
and its related mm x 4.6 mm, (90:10 v/v) at 210 nm
substances 5μm)
Posaconazole[51] Bulk Assay C column Methanol:water UV detection
8
(75:25 v/v) at 260 nm
Bifonazole[52] Cream Hypersil ODS. 0.08 mol/L UV detection
triethylamine at 254 nm.
phosphate, pH 7
:acetonitrile:meth
anol (20:10:70
v/v/v)
Sertaconazol[53] Cream Spherisorb CN Acetonitrile:0.01 UV detection
column (10 M sodium at 260 nm
microns) phosphate (37:63
v/v)

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Table 3 Chromatographic conditions for RP-HPLC methods reported for the estimation
of allylamines.
Name of Sample matrix Chromatographic Mobile phase used Type of
antifungal agent Column detector
Terbinafine[54] Terbinafine Intersil L1ODS Methanol:acetonitrile PDA
Hydrochloride in (4.6 mm × 15cm 5 (60:40 v/v) with detection at
Semi Solids Dosage μg) (0.15% triethylamine 224 nm
Form and 0.15%
phosphoric acid )
PH=7.68
Terbinafine[55] Terbinafine RP C18 (250 mm Methanol:water UV
Hydrochloride in × 4.6mm 5μ) (80:20 v/v) detection at
pharmaceutical solid 282 nm
dosage form
Terbinafine[56] Pharmaceutical Vertical® RP-C Methanol:water (95:5 UV
18
hydroalcoholic (250 mm × 4.6 v/v) detection at
solutions and tablets mm, 5 μm) 254 nm
Terbinafine[57] Terbinafine C18 Enable (250 × Methanol:Water UV
Hydrochloride and 4.6 mm, 5 μm) (95:5 v/v) detection at
Mometasone furoate 248 nm
in combined dosage
form
Terbinafine[58] Terbinafine Neosphere C18 Methanol:0.5% UV
Hydrochloride in (250 x 4.6 mm, triethanolamine (v/v) detection at
Bulk and in Tablet 5μm) 250 nm
dosage form
Butenafine[59] Butenafine Inertsil C18 (250 × Methanol and water UV
Hydrochloride and 4.6 mm, 5μm) in gradient technique detection at
Betamethasone 254 nm
Dipropionate in a
Cream Formulation

Table 4 Chromatographic conditions for RP-HPLC methods reported for the estimation
of echinocandins.
Name of Sample matrix Chromatographic Mobile phase used Type of
antifungal agent Column detector
Anidulafungin[60] Human Plasma and Zorbax SB-C8 0.005 M ammonium UV
Saline column (4.6 × 250 phosphate buffer: detection
mm, 5 μm) methanol (55:45 v/v) at 310 nm.
Anidulafungin[61] Bulk sample and YMC ODS Pack AQ Acetonitrile:water:0.1 UV
parenteral dosage C18 (150 x 4.6 mm, 3 % v/v trifluoroacetic detection
form μm) acid (48:52:1 v/v/v) at 300nm.
Micafungin[62] Micafungin Sodium Agilent Zorbax SB Buffer consisting DAD at
in drug substances (250 × 4.6 mm, 5 μm) sodium dihydrogen 210 nm
phosphate and sodium
perchlorate PH
2.9:acetonitrile (62:38
v/v)
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CONCLUSION
Presented review covers the analytical methods for the determination of antifungal agents in
various pharmaceutical and biological samples alone or in combination with other drugs with
help of RP-HPLC. For quantitative estimation of antifungal agents, RP-HPLC method is the
most common among others. All the reported methods are sensitive, precise and accurate;
consisting mainly RP C18 column as stationary phase and variety of polar solvents (like
methanol, water, acetonitrile, buffers) in different ratios as mobile phase. For development of
analytical methods, for newly developed or for upcoming novel antifungal agents, this can be
taken for consideration.

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