A Review On Different HPLC Analysis Method Of Voriconazole
Verma Vikrant1* Singh Umesh Kumar1
1
Department Of Pharmacy Kharvel Subharti College of Pharmacy Swami vivekanand Subharti
University Meerut
*
Corresponding author [email protected]
Keywords-Voriconazole, Detector, Antifungal
Abstract:
This review article was prepared with an aim to review different methods developed for
Voriconazole drug by HPLC analytical method and strategies developed and optimized for
efficient method development.There is a contionous research in this field and different
researchers are exploiting different mobile phase combinations for their research work.
Voriconazole is generally used as an antifungal drug under the brand name Vfend. The use of the
Voriconazole as a drug essential in pharmaceutical formulations highlights the requirement for
its determination and quantification with appropriate analytical methods
INTRODUCTION
Literature survey revealed that method development by HPLC has been taken in plasma samples,
solid, liquid and semisolid dosage formulations for estimation of antifungal drug voriconazole.
Till now few methods have been developed for estimation of antifungal drugs voriconazole
Thus the aim of this article is to is to discuss various high performance liquid chromatographic
methods developed by various researcher for determination of these antifungal drugs with
different mobile phase combinations .
M. Vamsi Krishna et al. developed an RP-HPLC method for evaluation of in vitro permeability
of voriconazole in the presence of enhancers through rat skin In this method an isocratic RPHPLC–UV method for the analysis of voriconazole in skin diffusate samples has been developed
and validated. In this method experimental design was employed to optimize the method. The
method was validated as per ICH guidelines. Linearity was observed over the concentration
range of 2–100 lg mL_1 (r2= 0.999). Limits of detection and quantification were 0.6 and 2 lg
mL_1, respectively. Intra-day and inter-day precision (% RSD) was within the ICH limits (62%).
The method was successfully used to analyze skin diffusate samples, and the effectiveness of
different permeation enhancers was compared with respect to flux and permeability coefficient.
The main purpose of this research was to develop and validate an isocratic RP-HPLC method for
the quantitative analysis of VCZ in skin diffusate samples as well as to evaluate the enhancing
effects of three penetration enhancers, i.e., DMSO, 1% SLS, and eucalyptus oil on the
percutaneous absorption of VCZ through rat skin in vitro.1-6
B. Sridhar et al validated RP-HPLC method for the Estimation of Voriconazole in Bulk and
tablet dosage form .In this a simple, selective, linear, precise and accurate RP-HPLC method was
developed and validated for rapid assay of Voriconazole in bulk and tablet dosage form. Isocratic
elution was done at a flow rate of 1 mL/min on a Inertsil C8 column (250 x 4.6 mm; 5μ) at
ambient temperature. The mobile phase consisted of Buffer (0.01M sodium dihydrogen
orthophosphate, pH was adjusted to 5.0):Acetonitrile (50:50 v/v) the UV detection wavelength
was 254 nm and 20μl of sample was injected. The retention time for Voriconazole was found to
be 6.905 min. The % recovery was within the range between 99.73% and 99.85%. The
percentage RSD for precision and accuracy of the method was found to be less than 2%. The
method was validated as per the ICH guidelines. The method was successfully applied for
routine analysis of Voriconazole in bulk samples and its formulations.
In this method important parameters found are given in the Table 1 and Table 2 and the
chromatogram obtained is given in Fig.1
S. No
1
2
3
4
5
Parameters
λ max (nm)
Beer’s law limit (μg/ml)
Correlation coefficient
Regression equation
Limit of detection (μg/ml)
Voriconazole
1.11
25-75
0.9996
Y = 24.875X+1.65
0.65
6
Limit of quantification (μg/ml)
1.98
Table 1: Statistical analysis of calibration curves in the HPLC determination of
Voriconazole (n=6) Parameters Values
S. No
Parameters
1
Tailing factor
2
Asymmetric factor
3
Theoretical plates
4
Capacity factor
5
HETP
Table 2: System suitability and study of Voriconazole
Voriconazole
1.11
1.20
5622
1.50
0.0366
Fig.1 Typical chromatogram obtained from the analysis of voriconazole standard solution712
Saidulu Goli et al. developed and validated voriconazole for Injection by RP-HPLC Method
In this a new simple, precise, accurate and selective RP-HPLC method has been developed and
validated for voriconazole in parental dosage form. The method was carried out on an Intersil
ODS-C18 (150X4.6X5μ) column with a mobile phase consisting of mixed phosphate buffer,
ACN and Methanol (65:30:5) and flow rate of 2.0 mL min-1. Detection was carried out at 257
nm. The retention time for VCZ was found to be 6.413 min. The VCZ % recovery was within the
range between 99.55-99.63%. The percentage RSD for precision and accuracy of the method was
found to be less than 2%. The method was validated as per ICH guidelines. The developed
method was validated for precision, accuracy, sensitivity and robustness. The developed method
can be used for routine analysis of titled drug in formulation.
In this method important parameters found are given in the table 3 and table 4 and the
chromatogram obtained is given in Fig.2 and Fig.313-18
S.No
1
2
3
4
5
Table 3 Accuracy data
Conc.(ppm)
80%
90%
100%
110%
120%
Standard concentration
7.94
8.93
9.92
10.92
11.91
S.No
1
2
3
Parameters
%R.S.D
%Tailing factor
%No of theoretical plates
Table 4 System suitability parameters
Fig.2 Chromatoghaphic conditions
Voriconazole
0.12
1.04
8.022
Fig.3 Linearity calibration graph
F. Péhourcq et al. developed direct injection HPLC micro method for the determination of
voriconazole in plasma using an internal surface reversed-phase column.The method was found
to be easy to perform and requires 10 µL of a plasma sample. The chromatographic run time was
less than 9 min using a mobile phase of 17:83 v/v acetonitrile–potassium dihydrogen phosphate
buffer, 100 mm, pH 6.0 and UV detection at 255 nm. The flow rate was 1 mL/min. A linear
response was observed over the concentration range 0.5–10 µg/mL A good accuracy (bias
≤7.5%) was achieved for all quality controls, with intra-day and inter-day variation coefficients
inferior to 6.7%. The lower limit of quantitation was 0.2 µg/mL, without interference of
endogenous components. In this method stability of voriconazole in plasma stored at different
temperatures was checked. Finally, the possibility of direct injection of plasma samples into the
column permits a reduction in reagent consumption and in analytical steps, and hence in
analytical error.The chromatogram obtained during the study is given in figure 4.19-24
Figure 4. Chromatogram obtained from the study
Yuru Li et al. developed and validated a stability-indicating HPLC Method for Determination
of Voriconazole and its Related Substances.An isocratic reversed-phase high performance liquid
chromatographic (RP-HPLC) method has been developed and validated for the determination of
voriconazole and its related substances. The drug substance was subjected to stress conditions of
UV light, water hydrolysis, acid, base, oxidation, and deoxidization to observe the degradation
products. The successful separation of voriconazole from its synthetic impurities and degradation
products formed under stress conditions was achieved using an Agilent Zorbax SB-C18 (250mm
× 4.6 mm i.d., 5 μm) column maintained at 25°C with a mobile phase of a mixture of ammonium
phosphate dibasic buffer (pH adjusted to 6.0 using diluted orthophosphoric acid; 50 mM)–
acetonitrile (52:48, v/v). The mobile phase flow rate was 1.0 mL/min, and the detection
wavelength was 250 nm. The stress sample solutions were assayed against the qualified
reference standard of voriconazole and the mass balance in each case was close to 99.7%,
confirming its stability-indication capacity. The developed HPLC method was validated with
respect to linearity, accuracy, precision, specificity, and robustness. The developed HPLC
method to determine the related substances and assay determination of voriconazole can be used
to evaluate the quality of regular production samples. It can be also used to test the stability
samples of voriconazole.25-28Important parameters found in the study are given in table 5 and
table 6.
Table 5. Recovery Results of Voriconazole Sample
Added(μg) (n = 3)*
25.3
50.6
7.5
Stress condition
Acid
hydrolysis(0.5N
HCl)
Recovered(μg
% Recovery
25
98.8
50.8
100.4
74.8
99.5
* n = number of determinations
Table 6 Summary of Forced Degradation Results
Time
% Assay of
% Mass
active substance
balance(%
assay +
impurities)
48 h
87.9
99.7
Base
hydrolysis(0.5N
NaOH)
48 h
0
99.8
Oxidation
(3% H2O2)
48 h
91.9
99.6
% RSD
0.9
0.6
0.7
Remarks
Degraded to impA and imp-D
Degraded to impA, imp-D, and
other unknown
degradants
Degraded to impD and other
unknown
Deoxidization
(3.0% NaHSO3)
Water hydrolysis
at 60°C
UV (254 nm)
10 days
99.6
99.6
48 h
77.5
99.7
48 days
99.6
99.6
degradants
No degradation
products formed
Degraded to impA and imp-D
No degradation
products formed
.
Gennethel J. Pennick et al.
developed and validated
a High-Performance Liquid
Chromatography Assay for Voriconazole.In this a analytical method for the determination of
voriconazole (UK-109,496; Pfizer) in plasma was developed and validated. The method utilizes
solid-phase extraction technology and high-performance liquid chromatography.The lower limit
of quantitation is 0.2 _g/ml, and the range of linearity tested was 0.2 to 10 _g/ml.
Important parameters found in the study are given in figure 5. 29-30
Figure 5 Representative chromatograms of VRC and the internal standard
Rafael Linden et al. developed Ultra-Performance Liquid Chromatographic Method for
Measurement of Voriconazole in Human Plasma and Oral Fluid.In this a simple, sensitive and
selective ultra-performance liquid chromatography method for the determination of voriconazole
in plasma and oral fluid was developed and validated. After a liquid-liquid extraction with
methyl-tert-butyl ether, the analyte and internal standard were separated on a Hypersil Gold C18
column (2.1 × 100 mm, p.d. 1.9 μm), eluted with a mobile phase composed of thietylammonium
phosphate buffer and acetonitrile (70:30, v/v). Total run time was found to be 4 min, total
mobile phase consumption of 2.2 mL. Detection was performed with a photodiode array detector
with quantitation at 256 nm. Voriconazole concentrations in oral fluid were on average 57.5% (±
5.3) of those measured in paired plasma samples.
Important parameters found in the study are given in figure 6.31-32
Figure 6
Michael Vogeser et al. quantified voriconazole in plasma by liquid chromatography-tandem
mass spectrometry.A convenient liquid chromatography-tandem mass spectrometry method for
the quantification of the triazole antifungal agent voriconazole in plasma samples is described.
Fenbuconazole is used as an internal standard. After protein precipitation, automated solid-phase
extraction is applied. Electrospray ionization in the positive mode is used. The analytical run
time is 4 min. The response was linear from 78 to 5000 mg/L. The total coefficient of variation
(ns16) was 12.6% for a low-concentration pool (143 mg/L), 4.7% for a medium-concentration
pool (419 mg/L), and 5.0% for a high-concentration pool (4304 mg/L). The method is proposed
for future investigations that should be performed to test the hypothesis that therapeutic drug
monitoring of voriconazole is clinically useful. Important parameters found in the study are given
in figure 7.
Figure 7 Representative chromatogram of voriconazole (350)126.6) and fenbuconazole
(337)125) used as the internal standard (concentration of voriconazole, 143 mg/L; the Yaxis represents the relative intensity of the signal with respect to the base peak of the
chromatogram).33-37
Peter H Tang et al. quantified Antifungal Drug Voriconazole in Serum and Plasma by HPLCUV.In this a simple, sensitive and rapid high-performance liquid chromatographic (HPLC)
method for the determination of voriconazole in human serum or plasma was developed.
Voriconazole and internal standard clonazepam were extracted from plasma or serum with
methanol and analyzed on a Microsorb-MV C18 column with ultraviolet (UV) detection set at
wavelengths of 256 and 310 nm, respectively. The calibration curve was linear through the range
of 0.1-10 mg/L using a 0.1 mL sample volume. The within-run and between-run precisions were
all less than 6%. Accuracies ranged from 97 to 106%. Absolute recovery was 96.4 ± 1.3% for
voriconazole. The method has been applied to monitor voriconazole use in order to ascertain
clinical efficacy and minimize toxic effects.38-44
Reehana Shaik et al. developed an RP-HPLC method for quantitative estimation of voriconazole
for injection in pharmaceutical dosage forms In this a simple RP-HPLC method for the
determination of voriconazole in pharmaceutical dosage forms. Numerous HPLC conditions
were tested for determination of voriconazole. The best result was achieved by using inertsil ods2, c-18 (150×4.6mm) 5μm column and a mobile phase consisting of OPA: acetonitrile:
methanol(65:30:5 v/v/v), a flow rate of 2.0 ml/min with ultraviolet detection at 257nm. The
retention time of the drug was 6.438 min. The method produced liner responses in the
concentration range of 7 to 12 ppm of voriconazole. The method was found to be applicable for
determination of the drug in injection. Important parameters found in the study are given in
figure 8 and table7. 45-48
Figure 8 A model chromatogram of Voriconazole
S.No
Mobile phase
composition
Type of
column
Flow
rate
1
OPA buffer:
acetonitraile:
methanol(65:30:5)
OPA buffer:
acetonitraile:
methanol(65:30:5)
OPA buffer:
acetonitraile:
methanol(65:30:5)
OPA buffer:
acetonitraile:
methanol(65:30:5)
OPA buffer:
acetonitraile:
methanol(65:30:5)
kromosil
2.0ml
Column
Voriconazole Tailing
temperature retention
factor
time
ambient
6.472
1.54
Inertsil
1.6ml
ambient
7.904
1.05
Inertsil
1.8ml
ambient
7.112
1.04
Inertsil
1.9ml
ambient
6.734
1.09
Inertsil
2.0ml
40oc
6.438
1.07
2
3
4
5
Table 7: Method development conditions
Mrinalini C. Damle et al. determined Voriconazole in Human Plasma by High Performance
Liquid Chromatography with Ultraviolet Detection.In this a simple, selective, and sensitive high
performance liquid chromatography method for the determination of voriconazole in human
plasma was developed. Fluconazole was used as an internal standard. The method utilizes liquidliquid extraction with n-hexane:ethyl acetate (3:1 v/v) as the sample preparation technique. The
samples were then analyzed on Neosphere C18 column with UV detection at 254 nm. The
calibration curve was linear through the range of 0.2-12 μg/ml. The lower limit of quantification
was found to be 0.2 μg/ml. % R.S.D. was less than 3% for intra- and inter-day precision. The
mean recovery was found to be 93.09% for voriconazole. The method showed acceptable values
for accuracy, precision, recovery, sensitivity and stability. The method is well suited for routine
analysis of voriconazole in human plasma and can further be extended for pharmacokinetic
studies.49-58
kabeer a. shaikh et al. validated Stability-Indicating Liquid Chromatographic Method for
determination of degradation Impurities and Diastereomers in Voriconazole Tablets.In this a
reversed-phase gradient liquid chromatographic method has been developed for the quantitative
determination of Voriconazole, along with its degradation and diastereomeric impurities in tablet
dosage form. Chromatographic separation has been achieved on an Inertsil ODS 3V, 150 x 4.6
mm, 5 μm column. The mobile phase consisting of solvent A 0.05 molar (M) potassium
dihydrogen phosphate (pH 2.5 buffer) and solvent B (mixture of acetonitrile and methanol in the
ratio 90:10 (v/v)), was delivered at a flow rate of 1.2 mL min−1 with the detection wavelength at
256 nm. Resolution of Voriconazole and all five potential impurities was achieved at greater than
2.0 for all pairs of compounds. The drug was subjected to stress conditions such as oxidative,
acid and base hydrolysis, and thermal and photolytic degradation. Voriconazole was found to
degrade significantly under base hydrolysis stress conditions compared to acid hydrolysis stress
conditions. The degradation products were well-resolved from the main peak and its impurities,
thus proving the stability-indicating power of the method. The stressed samples were assayed
against a reference standard and the mass balance was found to be close to 99.0%. The
developed method was validated as per ICH guidelines with respect to specificity, linearity, limit
of detection, limit of quantification, accuracy, precision, and robustness.58-60
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CONCLUSION
It can be concluded from the entire review that HPLC is a versatile, reproducible
chromatographic technique for the estimation of drug products. It has wide applications in
different fields in term of quantitative and qualitative estimation of active molecules.
HPLC method development has taken place of voriconazole drug in different drug formulations
and there is huge potential for new methods to be developed .Research is a continous process and
several regulatory agencies require the submission of analytical data and hence method
development gives an advantage for the anaysis of drugs.Voriconazole is an antifungal drug.In
this article different HPLC methods of voriconazole are presented with different parameters
found after the research work ,the parameters analysed are accuracy,precision,LOD,LOQ,system
suitability ,robustness parameters,if we compare these methods we came to the conclusion that
these is huge poential of developing a HPLC method for the drug voriconazole in order to
increase accuracy, system suitability and precision by overcoming the loopholes of the present
methods and lower limits of LOD and LOQ can be achieved by chossing suitable mobile phase
combinations and altering the flow rate and column parameters.