Vardenafil is a PDE5 inhibitor used to treat erectile dysfunction. This document discusses several studies that developed and validated analytical methods for quantifying vardenafil concentration. One study used thin layer chromatography with densitometric detection to simultaneously estimate vardenafil and dapoxetine in pharmaceutical formulations. Another used HPLC with amperometric detection employing a boron-doped diamond electrode to determine vardenafil and related metabolites in plasma. A third developed a HPLC-chemiluminescence method to quantify trace levels of vardenafil in dietary supplements.
2. WHAT IS VARDINAFIL
Vardenafil is a PDE5 inhibitor used for treating ED .that is
sold under the trade names Levitra (Bayer AG, GSK, and SP),
Staxyn in India, and Vivanza in Italy
3. PHYSICOCHEMICAL PROPERTIES
Computed properties
Molecular Weight 488.607 g/mol
XLogP3 2.5
Hydrogen Bond Donor Count 1
Hydrogen Bond Acceptor Count 7
Rotatable Bond Count 8
Exact Mass 488.221 g/mol
Monoisotopic Mass 488.221 g/mol
Topological Polar Surface Area 118 A^2
Heavy Atom Count 34
Formal Charge 0
Complexity 854
Covalently-Bonded Unit Count 1
4. PHYSICOCHEMICAL PROPERTIES
Experimental Properties
• Melting Point
• The melting point is the temperature at which a substance changes state
from solid to liquid at atmospheric pressure. When considered as the
temperature of the reverse change, from liquid to solid, it is referred to as
the freezing point.
• 192 °C
• Solubility
• The solubility of a substance is the amount of that substance that will
dissolve in a given amount of solvent. The default solvent is water, if not
indicated.
• In water, 3.5 mg/L at 25 deg C /Estimated/US EPA; Estimation Programs
Interface (EPI). ver. 3.11. U.S. EPA version for Windows. Washington, DC:
U.S. EPA (2003). Available from, as of Dec 15, 2004:
http://www.epa.gov/oppt/exposure/pubs/episuitedl.htm
• Water Solubility:
• 0.11 mg/mL (HCl salt)
5. PHYSICOCHEMICAL PROPERTIES
• Vapor Pressure
• Vapor pressure is the pressure of a vapor in thermodynamic
equilibrium with its condensed phases in a closed system.
• 3.4X10-19 mm Hg at 25 deg C /Estimated/
• LogP
• Octanol/Water Partition Coefficient, used as a measure of
molecular lipophilicity
• log Kow = 2.79 /Estimated/
• Dissociation Constants
• A specific type of equilibrium constant that measures the
propensity of a larger object to separate (dissociate)
reversibly into smaller components, as when a complex falls
apart into its component molecules, or when a salt splits up
into its component ions.
• pKa1= 4.72; pKa2= 6.21 (tertiary amine) /Estimated/
7. CHRONOLOGICAL LISTING
OF THE ARTICLES
1. Simultaneous estimation and validation of vardenafil and
dapoxetine hydrochloride in pharmaceutical formulation by thin
layer chromatographic densitometric method(2012)
2. High-performance liquid chromatographic method with
amperometric detectionemploying boron-doped diamond electrode
for the determination of sildenafil,vardenafil and their main
metabolites in plasma (2011)
3. A High-Performance iquidChromatography:Chemiluminescence
Method for Potential Determination of Vardenafil in Dietary
Supplement (2010)
4. Development of a method for the determination of vardenafil in
human plasma by high performance liquid chromatography
with UV detection (2009)
8. CHRONOLOGICAL LISTING
OF THE ARTICLES
5. Liquid chromatography/tandem mass spectrometry
method for the simultaneous determination of vardenafil and
its major metabolite, N-desethylvardenafil, in human plasma:
Application to a pharmacokinetic study (2009)
6. Multi-response optimization of a capillary electrophoretic
method for determination of vardenafil in the bulk drug and
in a tablet formulation (2007)
7. Development and validation of a high-performance liquid
chromatographic method using fluorescence detection for
the determination of vardenafil in small volumes of rat
plasma and bile (2007)
9.
10. SIMULTANEOUS ESTIMATION AND VALIDATION OF
VARDENAFIL AND DAPOXETINE HYDROCHLORIDE
IN PHARMACEUTICAL FORMULATION BY THIN
LAYER CHROMATOGRAPHIC DENSITOMETRIC
METHOD(2012)
11. ADVANTAGES
1. (1) Simultaneous estimation and validation of vardenafil and
dapoxetine hydrochloride in pharmaceutical formulation by
thin layer chromatographic densitometric method(2012)
• Low operation cost
• High sample throughput
• No need for a lot of samples preparation
• Several samples can be run simultaneously using a small
quantity of mobile phase unlike HPLC
• The technique is precise , accurate and specific
• The statistical analysis proved that it can test vardenafil
accurately and specifically despite the presence of
excipients without interference's
12. SIMULTANEOUS ESTIMATION AND VALIDATION OF
VARDENAFIL AND DAPOXETINE HYDROCHLORIDE IN
PHARMACEUTICAL FORMULATION BY THIN LAYER
CHROMATOGRAPHIC DENSITOMETRIC METHOD
13. SIMULTANEOUS ESTIMATION AND VALIDATION OF
VARDENAFIL AND DAPOXETINE HYDROCHLORIDE IN
PHARMACEUTICAL FORMULATION BY THIN LAYER
CHROMATOGRAPHIC DENSITOMETRIC METHOD
14. SIMULTANEOUS ESTIMATION AND VALIDATION OF
VARDENAFIL AND DAPOXETINE HYDROCHLORIDE IN
PHARMACEUTICAL FORMULATION BY THIN LAYER
CHROMATOGRAPHIC DENSITOMETRIC METHOD
15.
16. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC
METHOD WITH AMPEROMETRIC
DETECTIONEMPLOYING BORON-DOPED DIAMOND
ELECTRODE FOR THE DETERMINATION OF
SILDENAfiL,VARDENAfiL AND THEIR MAIN
METABOLITES IN PLASMA (2011)
17. ADVANTAGES
1. BDD electrodes surpass conventional electrode materials in
low capacitance (resulting in lower inherent noise), higher
chemical stability, an absence of surface oxide formation,
chemical fouling and other undesirable side-reactions
occurring during electrode reaction within their potential
operational range
2. The present method requires only a small volume of blood
plasma; such assays can be beneficial if just limited
amounts of biological material are available, as in the case
of neonatal PAH patients treated with this compounds
3. simple, sensitive, isocratic HPLC method for the one-run
determination of sildenafil and vardenafil as well as their
main active metabolites, N-desmethyl sildenafil and N-
desethyl-vardenafil, in human plasma, based on their
electrochemical oxidation
18. REAGENTS AND
CHROMATOGRAPHIC SYSTEM
Reagents :
• Standards of the actives
• Mobile phase :sodium dihydrogen or disodium hydrogen phosphate,
phosphoric acid (TraceSelect purity)and sodium perchlorate
monohydrate (HPLC grade) obtained fromFluka (Fluka AG, Buchs,
Switzerland), and gradient grade acetonitrile,methanol, tert-butyl methyl
ether and ethyl acetate (LabScan,) were used
19. REAGENTS AND CHROMATOGRAPHIC
SYSTEM
Chromatographic system :
Parte used
pump ESA isocratic pump (Model
582), (ESA Inc., Chelmsford, MA, USA) with a pulse damper
injector Rheodyne manual injector (Rheodyne, Cotati, CA, USA) equipped with a
2.5µL loop
detector
analytical cell potential
Electrochemical detection was performed using an ESA coulometric detector
Coulochem III, an amperometric cell (Model5040) equipped with a boron-
doped diamond electrode, and combined with a guard cell (Model 5020)
prior to the injector, (all ESAInc., Chelmsford, MA, USA).
maintained at +1520 mV(vs. Pd/H2). The set-up detector gain ranged from
100 nA Vto 5 A V-1 . The guard cell potential was set to +1000 mV (vs. d/H)
syringe Glass 25µL syringe (Hamilton, Reno, NV, USA).
column Ascentis®C18 3 m,100 mm× 2.1 mm I.D. (Supelco, Bellefonte, PA, USA).
Phase reversed
mobile phase Sodium dihydrogen phosphate and sodium perchlorate were dissolved in a
common aqueous solution to yield final concentrations of 20 mmol L-1for
phosphate and 40 mmol L-1
for perchlorate, respectively. The prepared aqueous portion was mixed with
pure acetonitrile in the ratio 70/30 (vaq/v) pH 3.5 of the resulting
mobile phase was adjusted with concentrated phosphoric acid. The
mobile phase was vacuum-filtered through a 0.2 m porous filter
(Supelco, Bellefonte, PA, USA) and degassed by helium sparging
before use. The flow rate was 0.2 mL minorg-1.
20. CHROMATOGRAPHIC ANALYSIS
Several HPLC columns were for the tested HPLC separation:
• Agilent Zorbax Eclipse XDB-C8 4.6 mm× 150 mm (5µm),
Phenomenex Gemini C18 3.0 x 150 mm (5 µm)
• Ascentis C18 3µm, 100 mm× 2.1 mm I.D.
For the purpose of this study, the latter column provided the best
peak symmetry and chromatographic performance; its smallest
internal diameter not only reduces the consumption of the mobile
phase but also the amount of sample required.
21. CHROMATOGRAPHIC ANALYSIS
Effect of buffer composition and concentration:
Several buffers were tested to determine their influence on :
1. Chromatographic selectivity,
2. Peak symmetry
3. Resolution
Phosphate buffer was used as a general component of the mobile phase to ensure a
sufficient buffering capacity
It was observed that adding perchlorate to the background buffer significantly
improved:
o peak shape
o Symmetry
o altered selectivity, and increased retention of the studied compounds
22. CHROMATOGRAPHIC ANALYSIS
We suppose that the presence of perchlorate in the mobile
phase :
promotes ion pair formation with the studied analytes due
to the relatively hydrophobic nature of the perchlorate ion.
Perchlorate anions desolvate more easily than does strongly hydrated
phosphate. Such anions (also described as “chaotropic”) tend to break
the hydrogen bonding structure of the mobile phase. In addition,the
dynamic coating of the stationary phase might also beinvolved. The
formed dynamically coated (quasi-stationary) phase has reduced
hydrophobicity compared to the original stationary phase. It has been
also shown that
even at pH ~ 3 and high quality silica, there are still some silanophilic
interactions with the ionizedsilanol groups present Both these effects
contribute to increased retention and improved peak shape (Fig. 2). The
phenomenon has been thoroughly studied and discussed
23.
24. CHROMATOGRAPHIC ANALYSIS
Effect of pH
As the compounds of interest have ionizable groups (two pK values are
reported changes in chromatographic behavior within the pH range are to be
expected.
The mobile phase of pH 3.5, 5.0 and 6.5 was used to study the influence
of pH on chromatographic separation.
The observed electrode response was comparable for the pH
values examined. Although the chromatographic resolution
achieved at pH 6.5 is the highest of the mobile phases tested,
it is not desirable to extend the analysis time from both
economical as well as analytical point of view. On the other
hand, the separation at pH 3.5 offered sufficient retention as
well as resolution for these of studied compounds and was
therefore chosen for this study.
25.
26. CHROMATOGRAPHIC ANALYSIS
Effect of organic modifier content :
• Acetonitrile was preferred over methanol as an organic modifier why?
because of lower mobile phase viscosity resulting in narrower peaks.
At 35% (v/v) of acetonitrile, loss of resolution and co-elution
of DSL and IS occurs.
decreasing the acetonitrile content to 25% (v/v) dramatically
increases analysis time while resolution between VR and IS
deteriorates At the same time, overall peak broadening
makes the analysis less capable of detecting trace analyte
concentrations
Thus mobile phase with an acetonitrile content of 30%,
offering the best separation of compounds of interest, was
used for next experiments.
27.
28. LINEARITY & SELECTIVITY
The calibration curves of SL, DSL, VR and DVR in human plasma were
found to be linear in the range of 10–400 ng mL The data also indicate
good linearity of this method for the intra- and inter day assays the
achieved LOD and LOQ values also confirm that the present method can
be used for monitoring the studied drugs in plasma.
29.
30. LINEARITY & SELECTIVITY
Application to human plasma
To test the applicability of the method for routine use, samples of
human plasma were subjected to analysis. A typical chromatogram
of blank plasma is shown in Fig. 7. As can be seen, no interfering
endogenous peaks occur within the area of the expected elution
of analytes. Blank plasma spiked by standards of SL, VR, DSL, DVR,
and processed by the procedure described in Section 2, was analyzed
using the above-described HPLC-ECD method. Fig. 8 shows
the complete chromatographic separation of all the analytes at the
physiological concentration level achieved within 12 min.
33. ADVANTAGES
1. Chemiluminescence (CL) detection is a highly sensitive
analytical technique.
2. sensitive for a large CL signal-to-noise ratio (S/N) and wide
linear working range
3. The combination of HPLC high resolution, CL sensitive
detection and FI manipulation would be one of the plausible
techniques for a sensitive, precise, and facile determination of
trace levels of compounds in the complex mixture of
substances
4. The proposed method exhibited the advantages of automated
operation, high analytical throughput, simple instrumentation,
wide linear range, and reagent-saving and was applied to oral
liquid, wine, and capsule samples analysis
34. REAGENTS AND
CHROMATOGRAPHIC SYSTEM
Reagents and Chemicals:
• Ethanol and methanol were of HPLC grade. All the other reagents
were of analytical reagent grade unless specified otherwise, and
deionized and doubly distilled water was used throughout
• Vardenafil hydrochloridetrihydrate (purity, 99.6%)
• Luminol
• The mobile phase consisting of ethanol and an aqueous solution
containing 1.0 × 10-2 mol/L H3po4 and 2.0 ×10-4 mol/L Na2EDTA
(25 : 75, v/v%) was prepared. Mobile phase was filtered through
0.45 µm Millipore membrane of polytetrafluoroethylene (PTFE)
and further degassed in an ultrasonic bath before use.
36. CHROMATOGRAPHIC ANALYSIS
• CL Reaction Selection:
• For vardenafil CL determination we considered several known CL reactions
including :
1. Alkaline luminol-H2O2
2. Alkaline luminol-K3Fe(CN)6
3. Alkaline luminol –KI04
4. Acidic KMnO4
5. Acidic KMnO4-HCHO
6. Acidic KMnO4-NA2SO4
NaClO-NaOH. To prevent the possible interference from metal ions, 2.0 × 10-4 mol/L Na2EDTA
was used in all luminol concerned reactions
Experimental results indicated that alkaline luminol-K3Fe(CN)6 reaction contributed the
strongest CL response to 1.0 × 10-5 mol/L vardenafil presence. Therefore, alkaline
luminol-K3Fe(CN)6 CL reaction was selected which reached amaximum within 1.5 s.
Thus, the studied CL reaction was a fast reaction
37. CHROMATOGRAPHIC ANALYSIS
• For HPLC-CL analysis, the mobile phase should be, not only suitable for
vardenafil separation in complex matrix but also compatible with CL
detection.
• Tested buffers :
1. Acetonitrile-acidic aqueous buffer [1, 4, 11, 19]
2. methanol-acidic aqueous buffer [8, 20]
• acetonitrile brought about very high CL background. This high
background went against the sensitive vardenafil detection
• methanol and ethanol were used, relatively low CL backgrounds and
smooth baselines could be obtained. Thus, methanol and ethanol were
selected.
38.
39. The effect of organic phase volume percentage (v/v%, organic phase
volume/organic + aqueous phase volume) on The S/N value and column
pressure (P) :
the higher the v/v%, the shorter the t, and 30% ethanol contributed the
biggest S/N value. However, 30% ethanol also caused relatively high P.
Thereupon, as a compromise among the bigger S/N value, shorter tr and
suitable P, 25% ethanol was chosen in subsequent experiments.
Experiment indicated that H3PO4 could effectively suppress the peak-
tailing, and a relatively narrow and symmetric chromatographic peak
could be achieved
40.
41. CHROMATOGRAPHIC ANALYSIS
The effect of flow rate on SN, tr , p:
The effect of mobile phase flow rate upon CL detection was
investigated. It was found that the higher the mobile phase
flow rate, the shorter the tend the higher the P.Lastly, aflow
rate of 0.8 mL/min was chosen as a compromise between the
biggest S/N value and relatively shorter tR
.
42. CHROMATOGRAPHIC ANALYSIS
The effect of NaOH :
The effect of NaOH concentration on the CL detection was examined in the
range of 0.2~1.3 mol/L. Experiments showed that the S/N value increased
with the NaOH concentration increasing, and a leveling off could be found
when the NaOH concentration was higher than 1.0 mol/L. Sequent
experiments were performed using 1.0 mol/L NaOH .
The effect of luminol concentration on CL detection :
It was found that the S/N value increased with luminol concentration
increasing
The effect of K3Fe(CN)6 concentration on CL detection :
It was found that the S/N value increased with K3Fe(CN)6 concentration
43.
44. DEVELOPMENT OF A METHOD FOR THE DETERMINATION
OF VARDENAFIL IN HUMAN PLASMA BY HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY
WITH UV DETECTION (2009)
45. ADVANTAGES
1. The method is rapid and sensitive enough for the routine
analysis
2. Efficient for the separation and quantitative determination of the
investigated substance in human plasma. There was no
interference in the samples examined, which confirms the good
selectivity of the method.
3. The method is rapid and sensitive enough for the routine
analysis
4. The liquid- liquid extraction procedure used for the sample
preparation eliminates endogenous interference which is
frequently present in a biological sample.
5. rapid, sensitive, specific and robust, and can be used for
the quantification of vardenafil in human plasma specimens
46. REAGENTS AND
CHROMATOGRAPHIC SYSTEM
Parte used
pump model 515 pump
injector Rheodyne manual injector (Rheodyne, Cotati, CA, USA) equipped with a
2.5µL loop
detector model 996 diode array detector (Waters, Milford, MA, USA)
column KR 100 C18 (5 μm particle size) column
(Eka Chemicals AB, Bohus, Sweden), protected by a 20 × 4.6 mm
i.d. (40 μm particle size) disposable Pelliguard
precolumn(Supelco,Bellefonte, PA, USA)
Phase reversed
mobile phase acetonitrile and potassium dihydrogen phosphate (70 mM; pH 4.5
with phosphoric acid) (30:70, v/v).
49. CHROMATOGRAPHIC ANALYSIS
The method employing orthophosphoric acid instead of
trifluoroacetic Acid is advantageous because of the consequent
deterioration of the stationary phase and the column lifetime
reduction The liquid-liquid extraction procedure used for the sample
preparation Eliminates endogenous interference,which is frequently
Present in a biological sample Finally the HPLC method presented
here is rapid, sensitive, specific and robust, and can be used for
the quantification of vardenafil in human plasma specimens treated
with this drug.
50. Development and validation of a high-performance liquid
chromatographic method using fluorescence detection for
the determination of vardenafil in small volumes of rat
plasma and bile
51. ADVANTAGES
1. This method requires only 50µl of plasma and 10 µl of
bile, making it suitable for studying the pharmacokinetics
of vardenafil in small animals .
2. provides wider linear range for monitoring concentration
of vardenafil.
52. REAGENTS AND
CHROMATOGRAPHIC SYSTEM
Parte used
pump Model L-7100 pump
Sampler L-7200 autosampler
detector L-7485 fluorescence detector, an L-7455 diode array detector
and a Hitachi D-7000 Chromatography Data Station
column Hypersil-100 C18 (5 m,25 cm× 4.6 mm I.D., Astmoor, Runcorn,
UK) column. A Hypersil guard column (H5 ODS, 10 mm× 3.2 mm)
was used The temperature of the columns was maintained at
35 C.
Bandwidths for excitation and emission 15 nm
mobile phase acetonitrile and 50 mM ammonium acetate aqueous solution (pH
6.8) (40/60,v/v). Flow rate 1mlmin
56. MULTI-RESPONSE OPTIMIZATION OF A CAPILLARY
ELECTROPHORETIC METHOD FOR DETERMINATION OF
VARDENAFIL IN THE BULK DRUG AND IN A TABLET
FORMULATION
57. ADVANTAGES
1. Has many advantages over HPLC, including speed and greater
separation efficiency (an order of magnitude more Theoretical plates)
2. Unlike HPLC, reduction in sample pretreatment is possible in CE, in
which uncharged molecules remain at the start of the capillary
3. Consumption of reagents and sample volumes, rather than capillary
columns, is, moreover, relatively low in CE
4. relatively inexpensive
5. Simple versatile technique
58. REAGENTS AND
CHROMATOGRAPHIC SYSTEM
Parte used
Software and instrument A Beckman (Fullerton, USA) P/ACE MDQ CE system coupled
with a diode-array detector (DAD) was used throughout the
experiments
Separations fused-silica capillary tubing 31.2 cm long (21.2cm to the detector) × 50 µm ,
housed in a cartridge, with a 100 µm ×800 µm detection window.
Buffers Acetate–acetic acid, borate–boric acid, and phosphate–phosphoric
acid buffers were prepared
samples Levitra tablets , Viagra tablets
60. CHROMATOGRAPHIC ANALYSIS
In general, it can be concluded that pH had the largest effect on
sensitivity and repeatability for both PA and tm whereas EC had the
largest effect on speed. Interaction of EC and PA had a slight effect
on all CE responses except the repeatability of tm .
.