Development And Validation Of NP HPTLC Method Biology Essay

Precoated silica gel 60F254 on aluminum sheets was selected for survey. Pre-washing of home base was done with methyl alcohol and so it was so activated by maintaining in an oven at 115 & A ; deg ; C for 10 proceedingss.

Choice of Solvent

Ideal belongingss of a solvent employed for HPTLC are

Drug should be soluble in the dissolver used.

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Drug should demo stableness in the dissolver used.

Consequently H2O was selected as the dissolver of sample for farther surveies.

Choice of moving ridge length

Absorption spectrum of edaravone on pre-coated home base was recorded. The i?¬max of edaravone was found to be 242 nm, and hence it was selected for the survey ( Fig.1 )

Fig.1: Absorption spectrum of Edaravone

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4. Development of optimal Mobile Phase

A dissolver system that would give dense compact musca volitanss and good separation from solvent forepart and application place was to be selected. Initially, different solvent systems were tested and observations were as given below. ( Table. 1 )

Table. 1: Solvent system choice

Solvent system tried

Observation

Methanol: Chloroform ( 7:3, v/v )

Peak form non good

Methanol: THF ( 7:3, v/v )

Peak form non good

Methanol: n-hexane ( 7:3, v/v )

Drug moved along with solvent forepart

Methanol: Toluene ( 8:2, v/v )

Good separation with symmetric extremums

In system Methanol: Toluene ( 8:2, v/v ) the topographic point was compact and good retained and hence it was chosen for farther optimisation.

Optimization of nomadic stage ( Methanol: Toluene )

Different ratios of Methanol: Toluene were tried in order to accomplish an optimal separation with good extremum form. A ratio of ( 8:2, v/v ) of methyl alcohol: Toluene was selected because it gave compact musca volitanss and good resoluted.

Optimization of chamber impregnation

The above fixed nomadic stage was added to one side of a duplicate trough chamber and different impregnation times from 5 to 30 proceedingss were tried. It was found that a impregnation clip of less than 10 min caused fluctuations in Rf value and border effects. Saturation times of 15 min and above were devoid of fluctuations and border effects and therefore a impregnation clip of 15 min was fixed.

Optimization of home base impregnation

The above fixed nomadic stage was added to one side of a duplicate trough chamber and the patched TLC home base was kept in the empty side. Different home base impregnation times from 5 to 30 proceedingss were tried. It was found that a impregnation clip of 15 min gave compact topographic point and hence fixed.

Fixed Experimental Conditions

Stationary Phase: Pre-coated silicon oxide gel 60F254 on aluminum sheets.

Mobile stage: Methanol: Toluene ( 8:2, v/v )

Chamber impregnation clip: 15 proceedingss

Migration distance: 85 millimeter

Band breadth: 6 millimeter

Slit dimension: 5 – 0.45 millimeter

Beginning of radiation: Deuterium lamp

Detection wavelength: 242 nanometer

Rf value: 0.67± 0.01

Validation OF THE METHOD

The HPTLC method was validated in footings of ICH guidelines.

Linearity Range

Linear arrested development informations showed a good linear relationship over a concentration scope of 300to1050 ng/spot for edaravone. Calibration graphs were plotted utilizing standard peak country Vs concentration of standard solution. The one-dimensionality was found to be in the scope of 300 to 1050 ng/spot of edaravone ( fig ) The additive equation and correlativity coefficient values for edaravone were found to be Y=-457+7.171*X and 0.99939 severally ( Fig. 2 ; Table.2 )

Fig. 2: Linear graph of Edaravone ( 300-1050ng/spot )

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Table. 2: Calibration Data of Edaravone

CONCENTRATION ( ng/spot )

Peak country

300

1670.9

450

2703.1

600

3879.3

750

5020.2

900

6072.84

1050

6950.3

Detection Limit ( LOD ) and Quantification Limit ( LOQ )

The LOD and LOQ of the drug were determined by using diminishing sums of the drugs on the home base. The lowest concentration at which the extremum is detected is called the LOD ( S/N=3 ) which was found to be 60ng /spot ( Fig. 3 ) . The lowest concentration at which the extremum is quantified is called LOQ ( S/N=10 ) which was found to be 300ng/spot ( Fig.4 )

Fig. 3: LOD OF Edaravone

F: pen drivesaravannanRahul Baghla – P’Analyisdavid projectxfin.edLOD.bmp

Fig. 4: LOQ of Edaravone

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Accuracy

Recovery surveies of the drugs were carried out for finding truth parametric quantity. The per centum recovery and its % RSD were calculated ( Table. 2 )

Table. 2: Recovery surveies for Edaravone

Degree

% Recovery

% RSD*

80 %

97.51

0.0783

100 %

99.96

1.1875

*RSD of 6 observations

Preciseness

Preciseness of the method was demonstrated by

Intra twenty-four hours preciseness

Inter twenty-four hours preciseness

Repeatability

Repeatability of sample application

Repeatability of measuring

Intra twenty-four hours preciseness

It was found out by transporting out the analysis of the standard drug at 2 different concentrations in the one-dimensionality scope of drug for 6 times on the same twenty-four hours. ( Table. 3 ) .

Table. 3: Intra twenty-four hours preciseness

Concentration

( ng/spot )

Peak country

% RSD*

450

2697

2686

2691

2677

2698

2665

0.5715

600

3873

3862

3876

3838

3864

3888

0.4838

*RSD of 6 observations

two ) Inter twenty-four hours preciseness

It was found out by transporting out the analysis of the standard drug at 2 different concentrations in the one-dimensionality scope of drugs for 3 yearss and % RSD was calculated ( Table. 4 )

Table. 4: Inter twenty-four hours preciseness

Concentration

( ng/spot )

Dayss

Peak country

% RSD*

450

1

2

3

2689

2677

2658

0.3443

600

1

2

3

3874

3892

3879

0.4301

*RSD of 3 observations

Repeatability

Repeatability of sample application

It was assessed by descrying 600 ng/spot of standard drug solution 6 times on pre coated TLC home base followed by development of home base and % RSD was calculated ( Table. 5 )

Table. 5: Repeatability of Sample application

Concentration

( ng/spot )

Peak country

% RSD*

600

3871

3862

3848

3865

3857

3868

0.2156

*RSD of 6 observations

b. Repeatability of measuring

Repeatability of measuring of peak country was determined by descrying 600 ng/spot of standard drug solution on pre coated TLC home base. After development of the home base the topographic point was scanned 6 times without altering place of the home base and % RSD was calculated ( Table. 6 ) .

Table. 6: Repeatability of Measurement

Concentration

( ng/spot )

Peak country

% RSD*

600

3873

3896

3894

3882

3886

3902

0.2717

*RSD of 6 observations

Stability Surveies

The stableness of edaravone solution was studied by chromatographing the standard solution at different clip intervals and comparing the peak country with that of the freshly prepared solution. The solution was found to be stable up to 5 hour. The consequences are shown in ( Table. 7 ) .

Table. 7: Stability of solution

Concentration

( ng/spot )

Time ( hour )

Peak country

450

0

0.5

1

1.5

2

4

6

2658

2650

2647

2628

2617

2585

2556

Robustness

In order to show the hardiness of the method, the following optimized conditions were somewhat varied.

± 3 min in chamber impregnation clip

± 0.1 % alteration in nomadic stage ratio

± 2 min in home base impregnation clip

± 5 millimeters in solvent forepart

The response factors for these changed chromatographic parametric quantities were about same as that of fixed chromatographic parametric quantities and therefore the developed method is said to be robust.

Specificity

The peak pureness of edaravone was assessed by comparing their several spectra at peak start, peak vertex and peak terminal places of the topographic point. The good correlativity among spectra acquired at start ( s ) , apex ( m ) and terminal ( vitamin E ) of the extremums Edaravone { correlativity R ( s, m ) = 0.9999, R ( m, vitamin E ) = 0.9998 } indicates the good extremum purenesss. It can be concluded that no drosss or debasement merchandises migrated with the extremums obtained from standard solutions of the drugs.

Analysis OF FORMULATION

Preparation of standard stock solution

Standard stock solution of Edaravone ( 300i?­g/mL ) was prepared in H2O.

Preparation of sample solution

A measure of H2O for injection contain 1.5mg/mL equivalent to 3 milligram of edaravone was transferred to a volumetric flask ( 10 milliliter ) and made up to volume with H2O.

Developing the chromatogram

With the fixed chromatographic conditions 1-3.5?L i.e. 300-1050ng/spot from standard stock solution and suited volumes from sample solution was spotted on a 20-10 pre coated TLC home base.The home base was analysed photometrically and chromatograms were recorded ( Fig. 5-11 )

Peak countries of sample chromatograms were compared with that of standard chromatograms and sum of edaravone in preparation was calculated from the standardization graph. The consequences are shown in ( Table.8 ) .

Table. 8: Consequences of analysis of preparation ( EDAVON )

Amount of drug mg/mL

% Label claim

% RSD*

Labeled

Estimated

99.33

1.1562

1.50

1.49

*RSD of 6 observations

Fig.5: Chromatogram of standard 1 ( Edaravone 300ng/spot )

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Fig. 6: Chromatogram of standard 2 ( Edaravone 450 ng/spot )

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Fig. 7: Chromatogram of standard 3 ( Edaravone 600 ng/spot )

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Fig. 8: Chromatogram of standard 4 ( Edaravone 750 ng/spot )

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Fig. 9: Chromatogram of standard 5 ( Edaravone 900ng/spot )

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Fig. 10: Chromatogram of standard 6 ( Edaravone 1050ng/spot )

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Fig. 11: Chromatogram of Edaravone injection ( Equivalent to 600 ng/spot )