vardinafil methods of analysis

VARDINAFIL

PHARMACIST LUBNA MOHAMMAD

1ST

YEAR PHARMACEUTICAL

SCIENCES STUDENT

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

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

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)

https://pubchem.ncbi.nlm.nih.gov/compound/water

http://www.epa.gov/oppt/exposure/pubs/episuitedl.htm


• 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/


Spectral Properties

GC-MS:

NIST Number 335369

Library Main library

Total Peaks 43

m/z Top Peak 113

m/z 2nd Highest 70

m/z 3rd Highest 56

NIST Number 335369

Library Main library

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)

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)

SIMULTANEOUS ESTIMATION AND VALIDATION OF

VARDENAFIL AND DAPOXETINE HYDROCHLORIDE

IN PHARMACEUTICAL FORMULATION BY THIN

LAYER CHROMATOGRAPHIC DENSITOMETRIC

METHOD(2012)

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

SIMULTANEOUS ESTIMATION AND VALIDATION OF

VARDENAFIL AND DAPOXETINE HYDROCHLORIDE IN

PHARMACEUTICAL FORMULATION BY THIN LAYER

CHROMATOGRAPHIC DENSITOMETRIC METHOD

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)

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

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

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.

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.


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


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


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.


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.

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.

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.

A HIGH-PERFORMANCE LIQUID

CHROMATOGRAPHY:CHEMILUMINESCENCE METHOD FOR

POTENTIAL DETERMINATION OF VARDENAfiL IN DIETARY

SUPPLEMENT (2010)

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

REAGENTS AND


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.

REAGENTS AND



• 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


• 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.

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


The effect of flow rate on S\N, 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

.


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

DEVELOPMENT OF A METHOD FOR THE DETERMINATION

OF VARDENAFIL IN HUMAN PLASMA BY HIGH

PERFORMANCE LIQUID CHROMATOGRAPHY

WITH UV DETECTION (2009)

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

REAGENTS AND


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).


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.

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

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.

REAGENTS AND


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 1ml\min

CHROMATOGRAPHIC

ANALYSIS

MULTI-RESPONSE OPTIMIZATION OF A CAPILLARY

ELECTROPHORETIC METHOD FOR DETERMINATION OF

VARDENAFIL IN THE BULK DRUG AND IN A TABLET

FORMULATION

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

REAGENTS AND


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


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 .

.

vardinafil methods of analysis

Health & Medicine