Development And Validation Of NP HPTLC Method Biology Essay

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Precoated silica gel 60F254 on aluminium sheets was selected for study. Pre-washing of plate was done with methanol and then it was then activated by keeping in an oven at 115° C for 10 minutes.

Selection of Solvent

Ideal properties of a solvent employed for HPTLC are

Drug should be soluble in the solvent used.

Drug should show stability in the solvent used.

Accordingly water was selected as the solvent of sample for further studies.

Selection of wave length

Absorption spectrum of edaravone on pre-coated plate was recorded. The max of edaravone was found to be 242 nm, and hence it was selected for the study (Fig.1)

Fig.1: Absorption spectrum of Edaravone

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4. Development of optimum Mobile Phase

A solvent system that would give dense compact spots and good separation from solvent front and application position was to be selected. Initially, different solvent systems were tried and observations were as given below. (Table. 1)

Table. 1: Solvent system selection

Solvent system tried

Observation

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

Peak shape not good

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

Peak shape not good

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

Drug moved along with solvent front

Methanol : Toluene (8 :2, v/v)

Good separation with symmetric peaks

In system Methanol: Toluene (8:2, v/v) the spot was compact and well retained and hence it was chosen for further optimization.

Optimization of mobile phase (Methanol: Toluene)

Different ratios of Methanol: Toluene were tried in order to achieve an optimum separation with good peak shape. A ratio of (8:2, v/v) of methanol: Toluene was selected because it gave compact spots and well resoluted.

Optimization of chamber saturation

The above fixed mobile phase was added to one side of a twin trough chamber and different saturation times from 5 to 30 minutes were tried. It was found that a saturation time of less than 10 min caused fluctuations in Rf value and edge effects. Saturation times of 15 min and above were devoid of fluctuations and edge effects and hence a saturation time of 15 min was fixed.

Optimization of plate saturation

The above fixed mobile phase was added to one side of a twin trough chamber and the spotted TLC plate was kept in the empty side. Different plate saturation times from 5 to 30 minutes were tried. It was found that a saturation time of 15 min gave compact spot and hence fixed.

Fixed Experimental Conditions

Stationary Phase : Pre-coated silica gel 60F254 on aluminium sheets.

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

Chamber saturation time : 15 minutes

Migration distance : 85 mm

Band width : 6 mm

Slit dimension : 5 - 0.45 mm

Source of radiation : Deuterium lamp

Detection wavelength : 242 nm

Rf value : 0.67± 0.01

VALIDATION OF THE METHOD

The HPTLC method was validated in terms of ICH guidelines.

Linearity Range

Linear regression data showed a good linear relationship over a concentration range of 300to1050 ng/spot for edaravone. Calibration graphs were plotted using standard peak area Vs concentration of standard solution. The linearity was found to be in the range of 300 to 1050 ng/spot of edaravone (fig) The linear equation and correlation coefficient values for edaravone were found to be Y=-457+7.171*X and 0.99939 respectively (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 area

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 applying decreasing amounts of the drugs on the plate. The lowest concentration at which the peak is detected is called the LOD (S/N=3) which was found to be 60ng /spot (Fig. 3). The lowest concentration at which the peak is quantified is called LOQ (S/N=10) which was found to be 300ng/spot (Fig .4)

Fig. 3:LOD OF Edaravone

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Fig. 4: LOQ of Edaravone

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Accuracy

Recovery studies of the drugs were carried out for determining accuracy parameter. The percentage recovery and its % RSD were calculated (Table. 2)

Table. 2: Recovery studies for Edaravone

Level

% Recovery

%RSD*

80%

97.51

0.0783

100%

99.96

1.1875

*RSD of 6 observations

Precision

Precision of the method was demonstrated by

Intra day precision

Inter day precision

Repeatability

Repeatability of sample application

Repeatability of measurement

Intra day precision

It was found out by carrying out the analysis of the standard drug at 2 different concentrations in the linearity range of drug for 6 times on the same day. (Table. 3).

Table. 3: Intra day precision

Concentration

(ng/spot)

Peak area

%RSD*

450

2697

2686

2691

2677

2698

2665

0.5715

600

3873

3862

3876

3838

3864

3888

0.4838

*RSD of 6 observations

ii)Inter day precision

It was found out by carrying out the analysis of the standard drug at 2 different concentrations in the linearity range of drugs for 3 days and % RSD was calculated (Table. 4)

Table. 4: Inter day precision

Concentration

(ng/spot)

Days

Peak area

%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 spotting 600 ng/spot of standard drug solution 6 times on pre coated TLC plate followed by development of plate and %RSD was calculated (Table. 5 )

Table. 5: Repeatability of Sample application

Concentration

(ng/spot)

Peak area

%RSD*

600

3871

3862

3848

3865

3857

3868

0.2156

*RSD of 6 observations

b. Repeatability of measurement

Repeatability of measurement of peak area was determined by spotting 600 ng/spot of standard drug solution on pre coated TLC plate. After development of the plate the spot was scanned 6 times without changing position of the plate and %RSD was calculated (Table. 6).

Table. 6: Repeatability of Measurement

Concentration

(ng/spot)

Peak area

%RSD*

600

3873

3896

3894

3882

3886

3902

0.2717

*RSD of 6 observations

Stability Studies

The stability of edaravone solution was studied by chromatographing the standard solution at different time intervals and comparing the peak area with that of the freshly prepared solution. The solution was found to be stable up to 5 hrs. The results are shown in (Table. 7).

Table. 7: Stability of solution

Concentration

(ng/spot)

Time(hrs)

Peak area

450

0

0.5

1

1.5

2

4

6

2658

2650

2647

2628

2617

2585

2556

Robustness

In order to demonstrate the robustness of the method, the following optimized conditions were slightly varied.

± 3 min in chamber saturation time

± 0.1% change in mobile phase ratio

± 2 min in plate saturation time

± 5 mm in solvent front

The response factors for these changed chromatographic parameters were almost same as that of fixed chromatographic parameters and hence the developed method is said to be robust.

Specificity

The peak purity of edaravone was assessed by comparing their respective spectra at peak start, peak apex and peak end positions of the spot. The good correlation among spectra acquired at start (s), apex (m) and end (e) of the peaks Edaravone {correlation r(s, m) = 0.9999, r (m, e) = 0.9998} indicates the good peak purities. It can be concluded that no impurities or degradation products migrated with the peaks obtained from standard solutions of the drugs.

ANALYSIS OF FORMULATION

Preparation of standard stock solution

Standard stock solution of Edaravone (300g/mL) was prepared in water.

Preparation of sample solution

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

Developing the chromatogram

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

Peak areas of sample chromatograms were compared with that of standard chromatograms and amount of edaravone in formulation was calculated from the calibration graph. The results are shown in (Table.8).

Table. 8: Results of analysis of formulation (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)

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