Development And Validation Of Uv Spectroscopic Method Biology Essay

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Deferasirox is having π electrons in its structure and hence absorbs electromagnetic radiation between 200-400 nm. This character is used for its determination by UV spectroscopic method.

Solubility of drug was checked in solvents like methanol, acetonitrile, and water. Among these, deferasirox was freely soluble in methanol and gave a good spectrum with acceptable absorbance. Hence methanol was selected as solvent of choice for further studies.

2. Selection of wavelength

A solution containing deferasirox (10 µg/mL) was prepared and scanned in UV region, from which a wavelength of 248 nm was selected for further studies, Fig. 1.

Fig. 1: Selection of wavelength

VALIDATION OF THE METHOD

The developed method was validated for the parameters like linearity, accuracy, precision and stability studies.

Linearity and range

Linearity of deferasirox was found in the concentration range of 3-12 µg/mL. The absorbances of these solutions were noted at the selected wavelength of 248 nm. The calibration curves were plotted using concentration vs. absorbance. Slope, intercept and correlation coefficient values were found to be 0.0725, 0.0023 and 0.999 respectively, fig. 2. The regression equation was,

Absorbance = 0.0023 + 0.0725 Ã- concentration.

Recovery studies

In order to ensure the suitability and reliability of proposed method, recovery studies were carried out. To an equivalent quantity of formulation, a known quantity of standard Deferasirox was added at 80%, 100% and 120% level and the contents were reanalyzed by the proposed method. The % recovery and %RSD were calculated, table 1.

Recovery was calculated by using following formula

% Recovery =

Amount of drug found after the addition of the standard drug

-

Amount of drug found before the addition of the standard drug

Amount of standard drug added

Ã- 100

Precision

Precision of the method was determined by

Intraday precision

Inter day precision

i) Intraday precision

Intraday precision was studied by carrying out the analysis of the standard drug for three different concentrations in the linearity range of drug for three times on the same day and %RSD was calculated, table 2.

ii) Inter day precision

Inter day precision was studied by carrying out the analysis of the standard drug for three different concentrations in the linearity range of drug for three days over a period of one week and %RSD was calculated, table 3.

Stability studies

When the prepared solution is exposed to atmosphere, the analytes are likely to decompose. Hence it is necessary to conduct stability studies.

Stability of the analyte in the solution was studied at different time intervals and absorbances were compared with the freshly prepared solution. The solution was found to be stable for about 42 hours as reduction of absorbance was within the limits, table 4.

Table 1: Recovery Studies

Level

% Recovery

% RSD*

80%

98.87

1.657

100%

99.85

1.491

120%

100.2

0.518* RSD of six observations

Table 2: Intraday Precision

Concentration (μg/mL)

Absorbance

% RSD

4

0.2921

0.891

0.2891

0.2943

5

0.3669

1.318

0.3682

0.3592

6

0.4379

0.906

0.4398

0.4323

Table 3: Inter day Precision

Concentration (μg/mL)

Day

Absorbance

% RSD

4

1

0.2921

0.546

2

0.2993

3

0.2911

5

1

0.3669

1.09

2

0.3675

3

0.3601

6

1

0.4379

0.9551

2

0.4298

3

0.4354

Table 4: Stability of the analyte

Concentration (μg/mL)

Time (Hours)

Absorbance

4

1

0.2932

5

0.2910

10

0.2889

24

0.2657

42

0.2513

54

0.2212

Fig. 2: Calibration graph of deferasirox (3-12 µg/mL)

ANALYSIS OF FORMULATION

Preparation of standard solution

Stock solution of deferasirox (100 µg/mL), was prepared in methanol, from which different concentrations (3-12 µg/mL) were prepared and scanned in UV region. The overlain UV spectra of standards are shown in, fig. 3.

Preparation of sample solution

Ten tablets, each containing 400 mg of deferasirox were weighed and average weight was calculated. Weight equivalent to 5 mg of deferasirox was weighed, transferred to 50 mL standard flask, extracted with methanol and made up with the same solvent and filtered through Whatman filter paper. Suitable aliquots of formulation solutions were prepared, scanned in UV region and absorbances were noted at selected wavelength, fig. 4. The results of the formulation analysis are given in table 5.

Table 5: Analysis of formulation

Formulation

Amount of drug (mg/tablet)

% label claim

% RSD*

Labeled

Estimated

Asunra (Deferasirox)

400

393.7

98.44

0.427

*RSD of six observations

Fig 3: Overlain UV Spectra of deferasirox (3-12 µg/mL)

Fig 4: UV Spectrum of formulation (8 µg/mL)

DERIVATIVE SPECTROSCOPIC METHOD 17

Derivative spectra

The normal spectra of deferasirox were recorded in methanol and these were converted into first and second derivative spectra. The amplitude was determined at the selected wavelength. Good linearity was observed for the second derivative spectra at a wavelength of 248 nm and hence it was selected for the study, fig. 1.

Fig. 1: Second derivative spectrum of deferasirox

VALIDATION OF THE METHOD

The developed method was validated for the parameters like linearity, accuracy, and precision.

Linearity and range

Linearity of deferasirox was found in the concentration range of 3-12 µg/mL. Slope, intercept and correlation coefficient values were found to be 0.0002, 0 and 0.999 respectively, fig. 2. The regression equation was,

Amplitude = 0 + 0.0002 Ã- concentration.

Precision

It was done by preparing the standards six times and measuring the amplitude of the recorded second derivative spectra. The percentage RSD was calculated, table 1.

Accuracy

The recovery studies were done to confirm the accuracy of the method. To an equivalent quantity of formulation, a known quantity of standard deferasirox was added at 80%, 100% and 120% level and the contents were reanalyzed by the proposed method. The % recovery and %RSD were calculated, table 2.

Table 1: Precision studies

Concentration (µg/mL )

Amplitude

%RSD

4

0.0008

1.253

0.0007

0.0007

0.0008

0.0009

0.0010

Table 2: Recovery Studies

Level

% Recovery

% RSD*

80%

98.90

0.4653

100%

102.4

1.1598

120%

99.5

0.7689* RSD of six observations

Fig. 2: Calibration graph of deferasirox (3-12 µg/mL)

D2 amplitude

ANALYSIS OF FORMULATION

Preparation of standard solution

Stock solution of deferasirox (100 µg/mL), was prepared in methanol, from which different concentrations (3-12 µg/mL) were prepared and scanned in UV region. Normal spectra obtained were processed to get second derivative spectra. The overlain second derivative spectra of standards are shown in, fig. 3.

Preparation of sample solution

Ten tablets, each containing 400 mg of deferasirox were weighed and average weight was calculated. Weight equivalent to 5 mg of deferasirox was weighed, transferred to 50 mL standard flask, extracted with methanol and made up with the same solvent and filtered through Whatman filter paper. Suitable aliquots of formulation solutions were prepared, scanned in UV region. Normal spectra obtained were processed to get second derivative spectra. The second derivative spectrum of formulation shown in fig. 4. The results of the formulation analysis are given in table 3.

Table 3: Analysis of formulation

Formulation

Amount of drug (mg/tablet)

% label claim

% RSD*

Labeled

Estimated

Asunra (Deferasirox)

400

399.1

99.7

0.419

*RSD of six observations

Fig 3: Overlain 2nd derivative spectrum of

Deferasirox (3-12 µg/mL)

Fig. 4: 2nd derivative spectrum of formulation (8 µg/mL)

DEVELOPMENT AND VALIDATION OF

VISIBLE SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF DEFERASIROX FROM TABLET DOSAGE FORM

OPTIMIZATION OF EXPERIMENTAL CONDITIONS

Selection of solvent

Solubility of drug was checked in solvents like methanol, acetonitrile, and water. Among these, deferasirox was freely soluble in methanol. Hence methanol was selected as solvent of choice for further studies.

Selection of reagent

Since deferasirox containing two phenolic -OH group, reagents like folin ciocalteu, 2, 4-DNPH and ferric chloride were tried to produce a colour. Among these reagents, ferric chloride reagent gave a distinct coloured chromogen with the drug and hence selected for further studies.

Preparation of 1 % ferric chloride reagent

0.5 gm of ferric chloride was weighed and transferred to 50 mL standard flask. Add 0.5 mL of concentrated sulphuric acid and made up to 50 mL with water.

Selection of wavelength

A solution containing deferasirox was treated with ferric chloride reagent and scanned in visible region, from which a wavelength of 527 nm was selected for further studies, fig. 1. Blank spectrum was also recorded and it was found that there was no interference from the blank, fig. 2.

Fig. 1: Selection of wavelength

Fig. 2: Blank spectrum

Fixing the strength of ferric chloride reagent

To 0.3 mL of stock solution (1000 µg/mL), 0.5 mL of different strengths of ferric chloride reagent ranging from 0.1-2% was added, and made up to 10 mL with methanol. 1% ferric chloride reagent treated solution showed maximum absorbance and good stability; hence 1% was fixed as the ideal strength of ferric chloride reagent, table 1 and fig. 3.

Table 1: Fixing the strength of ferric chloride reagent

Strength of ferric chloride

reagent (%)

Absorbance at 527 nm

0.1

0.32985

0.5

0.33189

1

0.34066

2

0.3384

Fig. 3: Fixing the strength of ferric chloride reagent

Fixing the volume of ferric chloride reagent

To 0.3 mL of stock solution (1000 µg/mL), different volumes of 1% ferric chloride reagent ranging from 0.5-3 mL was added, and made up to 10 mL with methanol. 0.5 mL of 1% ferric chloride reagent treated solution showed maximum absorbance and good stability and hence selected for further studies, table 2 and fig. 4

Table 2: Fixing the volume of ferric chloride reagent

volume of 1% ferric chloride

reagent (mL)

Absorbance at 527 nm

0.5

0.34809

1

0.33821

2

0.32141

3

0.29767

Fig. 4: Fixing the volume of ferric chloride reagent

Fixed procedure

Aliquots of drug solution was added to the 10 mL standard flask

Add 0.5 mL of 1% ferric chloride reagent

Made up to 10 mL with methanol

Absorbance was measured at 527 nm

against blank

VALIDATION OF THE METHOD

The developed colorimetric method was validated according to ICH guidelines for the parameters like linearity, accuracy, precision and stability studies.

Linearity and range

Deferasirox was found to be linear in the concentration range of 20-90 µg/mL. The absorbances of these solutions were noted at the selected wavelength of 527 nm. The calibration curve was plotted using concentration vs. absorbance. Slope, intercept and correlation coefficient values were found to be 0.01, -0.0037 and 0.9999 respectively, fig. 5. The regression equation was,

Absorbance = (-0.0037) + 0.01 Ã- concentration.

Recovery studies

In order to ensure the suitability and reliability of proposed method, recovery studies were carried out. To an equivalent quantity of formulation, a known quantity of standard deferasirox was added at 80%, 100% and 120% level and the contents were reanalyzed by the proposed method. The % recovery and %RSD were calculated, table 3.

Recovery was calculated by using following formula

% Recovery =

Amount of drug found after the addition of the standard drug

-

Amount of drug found before the addition of the standard drug

Amount of standard drug added

Ã- 100

Precision

Precision of the method was determined by

Intraday precision

Inter day precision

i) Intraday precision

Intraday precision was studied by carrying out the analysis of the standard drug for three different concentrations in the linearity range of drug for three times on the same day and %RSD was calculated, table 4.

ii) Inter day precision

Inter day precision was studied by carrying out the analysis of the standard drug for three different concentrations in the linearity range of drug for three days over a period of one week and %RSD was calculated, table 5.

Stability studies

Stability studies of the drug solutions were carried out at room temperature and the drug was found to be stable for about 30 hours, table 6.

Table 3: Recovery Studies

Level

% Recovery

% RSD*

80%

99.71

0.7892

100%

102.45

0.6789

120%

101.60

0.5947* RSD of six observations

Table 4: Intraday Precision

Concentration (μg/mL)

Absorbance

% RSD

40

0.3992

0.301

0.3968

0.3979

50

0.4900

0.647

0.4947

0.4961

60

0.5984

0.199

0.5978

0.5961

Table 5: Inter day Precision

Concentration (μg/mL)

Day

Absorbance

% RSD

40

1

0.3992

1.2134

2

0.3948

3

0.3896

50

1

0.4900

0.6393

2

0.4921

3

0.4962

60

1

0.5984

0.7435

2

0.5921

3

0.5899

Table 6: Stability of the analyte

Concentration (μg/mL)

Time (Hours)

Absorbance

40

1

0.3990

2

0.3987

4

0.3978

6

0.3967

24

0.3940

30

0.3896

42

0.3512

48

0.3356

Fig. 5: Calibration graph of deferasirox (20-90 µg/mL)

ANALYSIS OF FORMULATION

Preparation of standard solution

Stock solution of deferasirox (1000 µg/mL), was prepared in methanol. Aliquot volumes of drug solution 0.2-0.9 mL from stock solution were added into series of 10 mL standard flasks, add 0.5 mL of 1% ferric chloride reagent and made up to 10 mL with methanol. Standard solutions were measured at 527 nm against reagent blank and the absorbances were noted. The overlain spectra of standards are given in fig. 6.

Preparation of sample solution

Ten tablets, each containing 400 mg of deferasirox were weighed and average weight was calculated. Weight equivalent to 5 mg of deferasirox was weighed, transferred to a 50 mL standard flask, extracted with methanol and made up with the same solvent, this solution was filtered through Whatman filter paper. The developed colorimetric procedure was applied to suitable aliquots of sample solution, scanned in visible region and absorbances were measured at selected wavelength 527 nm, fig. 7. The results of formulation analysis are given in table 7.

Table 7: Analysis of formulation

Formulation

Amount of drug (mg/tablet)

% label claim

% RSD*

Labeled

Estimated

Asunra (Deferasirox)

400

393.5

98.38

0.519

*RSD of six observations

Fig. 6: Overlain Spectra of deferasirox (20-90 µg/mL)

Fig. 7: Visible Spectrum of Formulation (50 µg/mL)

DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR THE DETERMINATION OF DEFERASIROX FROM TABLET DOSAGE FORM

Selection of Plate

Pre-coated silica gel 60F254 on aluminium sheet was selected for study.

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.

Solvent should be volatile.

Accordingly, methanol was selected as the solvent.

Selection of wavelength

UV spectra of deferasirox on pre-coated plate was recorded. The λmax of deferasirox was found to be 248 nm, hence selected as detection wavelength, fig. 1.

Development of optimum mobile phase

A solvent system should be selected in such a way that it would give compact spots and good separation from solvent front and application position.

Initially, different solvent systems were tried and observations were as given in table 1.

Table 1: Selection of mobile phase system

Solvent system tried

Observation

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

Peak shape not good

Methanol: ethyl acetate (7: 3, v/v)

Peak shape not good

Methanol: acetone (6: 4, v/v)

Drug moved along with solvent front

Methanol : n-butyl acetate

(3.5: 6.5, v/v)

Good separation with symmetric peaks

Among these systems, methanol: n-butyl acetate was selected because this system gave good compact spot.

Optimization of mobile phase ratio

Different ratios of methanol: n-butyl acetate like 2:8, 2.5:7.5, 3.5 :6.5, v/v etc. were tried, from which the ratio of 3.5:6.5, v/v was selected because it gave compact spots with good separation from solvent front and sample application positions and good symmetrical peaks.

Fixed Experimental Conditions

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

Mobile phase : Methanol : n-butyl acetate

(3.5: 6.5, v/v)

Chamber saturation time : 20 minutes

Migration distance : 80 mm

Band width : 6 mm

Slit dimension : 5 Ã- 0.45 mm

Source of radiation : Deuterium lamp

Detection wavelength : 248 nm

Rf value : 0.50± 0.03

Fig. 1: UV spectrum of standard deferasirox on TLC plate

VALIDATION OF THE METHOD

The validation of the developed method was carried out for various parameters like limit of detection (LOD), limit of quantification (LOQ), linearity, accuracy, precision and stability studies as per ICH guidelines.

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The LOD and LOQ of the deferasirox were determined by applying decreasing amounts of the drug in triplicate on the plate. The lowest concentration at which the peak is detected is called the 'Limit of Detection' which was found to be 20 ng/spot, fig. 2. The lowest concentration at which the peak is quantified is called 'Limit of Quantification' which was found to be 50 ng/spot, fig. 3.

Linearity and Range

Linear regression data revealed an excellent linear relationship in the concentration range of 300 to 800 ng/spot. The slope, intercept and correlation co-efficient values were found to be 9.878, 938.609 and 0.9992, fig. 4. The regression equation was,

Peak area= 938.609 + 9.878 Ã- concentration.

Accuracy

Recovery studies were done for determining accuracy parameter. It was done by mixing known quantity of standard drug with the analysed sample formulation and the contents were reanalyzed by the proposed method.

Recovery studies carried out at 80, 100 and 120% levels. The percentage recovery and its %RSD were calculated, table 2.

Precision

Precision of the method was determined by

Intra day precision

Inter day precision

Repeatability

Repeatability of measurement

Repeatability of sample application

I. Intraday precision

Intra day precision was studied by carrying out the analysis of the standard drug at three different concentrations in the linearity range of drug (500, 600, and 700 ng/spot) for three times on the same day and % RSD was calculated, table 3.

II. Inter day precision

Inter day precision was studied by carrying out the analysis of the standard drug at three different concentrations in the linearity range of drug (500, 600, and 700 ng/spot) for three days over a period of one week and % RSD was calculated, table 4.

III. Repeatability

Repeatability of sample application

Repeatability of sample application was evaluated by spotting 500 ng/spot of drug solution six times on pre-coated TLC plate. Plate was then developed, scanned and %RSD calculated, table 5.

Repeatability of measurement

Repeatability of measurement of peak area was evaluated by spotting 500 ng/spot of standard drug solutions on pre-coated TLC plate. After development of the plate, the separated spots were scanned without changing position of the plate for six times and %RSD was calculated, table 6.

Stability studies

When the developed chromatographic plate is exposed to atmosphere, the analytes are likely to decompose. Hence it is necessary to conduct stability studies.

Stability of the analyte on the plate was studied at different time intervals and peak areas were compared with the peak area of freshly scanned plate.

Stability of the developed plate was found to be 7 hours, which was indicated by the reduction in the peak areas, table 7.

Table 2: Recovery studies

Level

% Recovery

%RSD*

80%

98.97

0.7885

100%

102.26

0.5020

120%

101.08

0.6170

*RSD of six observations

Table 3: Intraday precision

Concentration (ng/spot)

Peak area

%RSD

500

5705.5

1.126

5832.4

5745.3

600

6844.8

1.108

6921.2

6998.2

700

7748.3

0.700

7845.6

7754.3

Table 4: Inter day precision

Concentration (ng/spot)

Day

Peak area

%RSD

500

1

5907.2

1.55

2

5801.3

3

5984.4

600

1

6955.1

1.22

2

6788.4

3

6891.1

700

1

7897.1

1.12

2

7812.8

3

7721.2

Table 5: Repeatability of sample application

Concentration (ng/spot)

Peak area

%RSD

500

6347.5

0.830

6368.0

6381.9

6459.8

6318.9

6317.6

Table 6: Repeatability of measurement

Concentration (ng/spot)

Peak area

%RSD

500

6328.4

0.186

6331.4

6339.4

6342.4

6360.6

6348.5

Table 7: Stability of the analytes on plate

Concentration (ng/spot)

Time (hours)

Peak area

500

0

5907.26

1

5912.5

2

5895.4

3

5879.4

4

5860.5

5

5862.4

6

5838.5

7

5267.7

8

5132.5

9

5045.7

Fig. 2: LOD of deferasirox (20 ng/spot)

Fig. 3: LOQ of deferasirox (50 ng/spot)

Fig. 4: Calibration graph of deferasirox (300-800 ng/spot)

Concentration (ng/spot)

Peak area

300

3837.6

400

4892.86

500

5907.26

600

6955.18

700

7897.14

800

8739.96

ANALYSIS OF FORMULATION

Preparation of standard solution

Stock solution of deferasirox (100 µg/mL), was prepared in methanol.

Preparation of sample solution

Ten tablets, each containing 400 mg of deferasirox were weighed and average weight was calculated. Weight equivalent to 5 mg of deferasirox was weighed, transferred to a 50 mL volumetric flask, extracted and made up to volume with methanol and filtered through a Whatman filter paper.

Recording of the chromatogram

With the fixed chromatographic conditions, 3-8 μL from standard stock solution of deferasirox (100 μg/mL) and suitable volumes from sample solution were applied on to the plate. The plate was analysed photometrically and chromatograms were recorded, fig. 5-11.

Peak areas of sample chromatograms were compared and amount of deferasirox was calculated, table 8.

Table 8: Analysis of formulation

Formulation

Amount of drug (mg/tablet)

% label claim

% RSD*

Labeled

Estimated

Asunra (Deferasirox)

400

395.4

98.85

0.721

*RSD of six observations

STANDARD CHROMATOGRAMS OF DEFERASIROX

Fig. 5: 300 ng/spot

Fig. 6: 400 ng/spot

Fig. 7: 500 ng/spot

Fig. 8: 600 ng/spot

Fig. 9: 700 ng/spot

Fig. 10: 800 ng/spot

Chromatogram of formulation

Fig. 11: 400 ng/spot

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE DETERMINATION OF DEFERASIROX FROM TABLET DOSAGE FORM

1. Selection of stationary phase

Since deferasirox is polar in nature, RP-HPLC method with C18 column was selected for the development of the method.

2. Selection of wavelength

Selectivity of HPLC method that uses UV detector depends on proper selection of wavelength. A wavelength which gives good response for the drug to be detected is to be selected. From the UV spectra 248 nm was selected as detection wavelength, fig.1.

3. Selection of mobile phase

Solvent selectivity (solvent type), solvent strength (percentage of organic solvent in the mobile phase), strength of buffer, flow rate etc. were optimized to get the chromatographic conditions, that gave the best separation, table 1.

INITIAL CONDITIONS

Stationary phase : Merk- LichroCART, C18 column

(250 mmÃ-4mm, 5µm)

Flow rate : 1 mL/minute

Operating temperature : Room temperature

Selected wavelength : 248 nm

Table 1: Selection of mobile phase

Sl. No.

Mobile phase

Observation

Retention time (min)

Fig. no.

1.

Methanol: water

(80:20, v/v)

Tailed peak

1.6

2

2.

Acetonitrile: water

(80:20, v/v)

Tailing and peak shape not good

1.68

3

3.

0.1% glacial acetic acid: methanol (20:80, v/v)

Tailed peak

5.5

4

4.

Acetonitrile: 0.1% formic acid (80: 20, v/v)

Tailed peak

3.09

5

5.

Methanol: 0.1% formic acid

(80: 20, v/v)

Good symmetrical peak

5.6

6

OPTIMIZATION OF SEPARATION CONDITIONS

1. Selection of strength of formic acid

Different ionic strengths of formic acid such as 0.05%, 0.1%, 0.5%, 0.1% etc. in the ratio 20:80, v/v with methanol were tried, fig. 7-9. Good peak characteristics was observed for strength of 0.1% and hence selected for further study, table 2.

Table 2: Selection of strength of formic acid

Strength (%)

Observation

0.05

Tailing

0.1

Good

0.5

Tailing

2. Effect of ratio of mobile phase

A mobile phase system containing methanol and 0.1% formic acid in different ratios like 75: 25, 80: 20, 85:15, 90:10, v/v etc. were tried, fig. 10-13 and the chromatograms were observed for good peak characteristics.

For a ratio of 85:15, v/v good symmetrical peak was observed.

3. Effect of flow rate

Keeping all other parameters of mobile phase system constant, the chromatograms were recorded with different flow rates like 0.9, 1.0 and 1.1mL/min, fig. 14 - 16. A flow rate of 1 mL/min gave good symmetrical peaks with good peak area response and hence selected, table 3.

Table 3: Effect of flow rate

Flow rate

(mL/ min)

Peak area

Retention time(min)

Fig.no

0.9

614904

4.639

14

1

609552

4.08

15

1.1

570704

3.767

16

FIXED CHROMATOGRAPHIC CONDITIONS

Stationary phase : Merk- LichroCART, C18 column

(250 mmÃ-4mm, 5µm)

Mobile phase : Methanol: 0.1% formic acid

Solvent ratio : 85:15, v/v

Detection wavelength : 248 nm

Flow rate : 1 mL/minute

Operating pressure : 133 kgf

Temperature : Room temperature

Fig. 1: UV spectrum of deferasirox

Fig. 2: Separation using methanol: water

(80:20, v/v)

Fig. 3: Separation using acetonitrile: water

(80:20, v/v)

Fig. 4: Separation using 0.1% glacial acetic acid: methanol

(20:80, v/v)

Fig. 5: Separation using acetonitrile: 0.1% formic acid

(80: 20, v/v)

Fig. 6: Separation using methanol: 0.1% formic acid

(80:20, v/v)

EFFECT OF IONIC STRENGTH OF FORMIC ACID ON SEPARATION

Fig. 7: Separation using methanol: 0.05% formic acid

(80:20, v/v)

Fig. 8: Separation using methanol: 0.1% formic acid

(80:20, v/v)

Fig. 9: Separation using methanol: 0.5% formic acid

(80:20, v/v)

EFFECT OF MOBILE PHASE RATIO ON SEPARATION

Fig. 10: Separation using methanol: 0.1% formic acid

(75:25, v/v)

Fig. 11: Separation using methanol: 0.1% formic acid

(80:20, v/v)

Fig. 12: Separation using methanol: 0.1% formic acid

(85:15, v/v)

Fig. 13: Separation using methanol: 0.1% formic acid

(90:10, v/v)

EFFECT OF FLOW RATE ON SEPARATION

Fig. 14: 0.9 mL/min

Fig. 15: 1 mL/min

Fig. 16: 1.1 mL/min

VALIDATION OF RP-HPLC METHOD

1. Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined by injecting progressively lower concentrations of the drug. LOD and LOQ of deferasirox were found to be 0.8 ng/mL and 50 ng/mL respectively, fig.17 & 18.

2. Linearity and range

Calibration graph was plotted using standard drug peak areas vs. concentration of standard solutions. Linear regression data revealed an excellent linear relationship in the concentration range of 100-1000 ng/mL. The slope, intercept and correlation co-efficient values were found to 69.301, -1073.211, 0.9994 respectively, fig. 19. The regression equation was,

Peak area = (-1073.211) + 69.301Ã- concentration

3. Precision

Precision of method was determined by

Intra day precision

Inter day precision

Repeatability of injection

a) Intra day precision

Intra day precision was done by carrying out analysis of standard drug solutions at three different concentrations in the linearity range (200, 400, 600 ng/mL) for three times on the same day and %RSD was calculated, table 4.

b) Inter day precision

Inter day precision was done by carrying out the analysis of standard drug solutions at three different concentrations in the linearity range (200, 400, 600 ng/mL) for three days over a period of one week and %RSD was calculated, table 5.

c) Repeatability of injection

A standard solution of drug (600 ng/mL) was injected 6 times and its %RSD was calculated, table 6.

4. Accuracy

Recovery studies were done for determining accuracy parameter. It was done by mixing known quantity of standard drug with the analysed sample formulation and the contents were reanalyzed by the proposed method.

Recovery studies carried out at 80, 100 and 120% levels. The percentage recovery and its %RSD were calculated, table 7.

5. Stability

Sample solution of deferasirox was subjected to stability studies under refrigerated and room conditions. Stabilities were studied by looking for any change in retention time, resolution, peak shape, etc. when compared to chromatogram of freshly prepared solution. The solution stored under room temperature was stable up to 16 hours and under refrigeration up to 48 hours.

6. System suitability studies

System suitability parameters like number of theoretical plates (N), peak asymmetry factor (As), resolution (Rs) etc. were studied, the results are given in table 8.

7. Robustness

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

± 5 in ratio of methanol in mobile phase

± 0.2 units in pH of buffer

The response factors for these changed chromatographic parameters were almost same as that of the fixed chromatographic parameters and hence developed method is said to be robust, table 9.

8. Specificity

Conditions of HPLC method like percentage of organic solvent in mobile phase, ionic strength, pH of buffer, flow rate etc, were changed. Although these changes were made, no additional peaks were found but there were some slight changes in retention times and peak shapes. Peak purity tests were done. The peak purity index of deferasirox was found to be 0.9999. Peak purity index values close to one proves peak purity of the drug.

Fig. 17: Limit of detection - 0.8 ng/mL

Fig. 18: Limit of quantification - 50 ng/mL

Fig. 19: Calibration graph of deferasirox

(100-1000 ng/mL)

Concentration (ng/mL)

Peak area

100

5892

200

11937

400

27922

600

39904

800

54995

1000

67746

Table 4: Intraday precision

Concentration (ng/mL)

Peak area

%RSD

200

11937

0.451

11832

11904

400

27922

0.1035

27965

27910

600

39904

0.998

39889

39210

Table 5: Inter day precision

Concentration (ng/mL)

Day

Peak area

%RSD

200

1

11937

0.378

2

11905

3

11848

400

1

27922

0.206

2

27918

3

27820

600

1

39954

1.520

2

39910

3

38890

Table 6: Repeatability of injection

Concentration (ng/mL)

Peak area

%RSD

600

38914

1.05

39928

39920

39931

39945

39992

Table 7: Recovery studies

%Recovery

% RSD*

80%

100%

120%

80%

100%

120%

100.4

104.2

99.4

0.8625

0.7655

0.5438

*RSD of six observations

Table 8: System suitability studies

Rs

N

As

Tailing factor

8.148

4298

1.5

1.3

Table 9: Robustness

Chromatographic condition

Observation

Mobile phase ratio

(methanol: 0.1% formic acid)

80: 20, v/v

Peak area

596482

85: 15, v/v

608385

90: 15, v/v

619349

pH of 0.1% formic acid

2.4

Peak shape

Good

2.6

Good

2.8

Good

ANALYSIS OF FORMULATION

Fixed chromatographic conditions were made use for the analysis of formulation.

1. Preparation of standard solution

Standard stock solution of deferasirox (100 µg/mL) was prepared in methanol. Stock solution was diluted with methanol: water (50:50, v/v) to get a series of concentrations ranging from 100-1000 ng/mL.

2. Preparation of sample solution

Ten tablets, each containing 400 mg of deferasirox were weighed and average weight was calculated. Weight equivalent to 5 mg of deferasirox was weighed, transferred to 50 mL standard flask, extracted with methanol and made up with the same solvent, this solution was filtered through Whatman filter paper and suitable aliquots of formulation solutions were prepared.

3. Recording of chromatograms

A steady baseline was recorded with the fixed chromatographic conditions, and standard drug solutions were injected and chromatograms were recorded, fig. 20-25. Retention time was found to be 4.1 minutes for deferasirox. This was followed by injection of sample solution obtained from the formulation, fig. 26.

Calibration curve was plotted using standard drug peak area vs. concentration of standard solutions. The results of formulation analysis are given in table 10.

Table 10: Analysis of formulation

Formulation

Amount of drug (mg/tablet)

% label claim

% RSD*

Labeled

Estimated

Asunra (Deferasirox)

400

388.16

97.04

0.543

*RSD of six observations

CHROMATOGRAM OF STANDARDS

Fig. 20: Deferasirox - 100 ng/mL

Fig. 21: Deferasirox - 200 ng/mL

Fig. 22: Deferasirox - 400 ng/mL

Fig. 23: Deferasirox - 600 ng/mL

Fig. 24: Deferasirox - 800 ng/mL

Fig. 25: Deferasirox - 1000 ng/mL

CHROMATOGRAM OF FORMULATION

Fig. 26: Deferasirox - 800 ng/mL

DEVELOPMENT AND VALIDATION OF BIO ANALYTICAL METHOD FOR THE DETERMINATION OF DEFERASIROX FROM HUMAN PLASMA BY HPTLC

FIXED CHROMATOGRAPHIC CONDITIONS

Stationary phase : Pre-coated silica gel 60F254 on

Aluminium sheets

Mobile phase : Methanol: n-butyl acetate

(2.5: 7.5, v/v)

Chamber Saturation time : 20 minutes

Migration Distance : 80 mm

Band width : 6 mm

Slit dimension : 5 x 0.45 mm

Source of Radiation : Deuterium lamp

Scanning Wavelength : 248 nm

Internal standard- Paracetamol 8 (µg/mL)

Rf values:

Deferasirox - 0.23

Paracetamol - 0.61

OPTIMIZATION OF EXTRACTION PROCEDURE

Selection of organic solvent for extraction

50 µL of standard drug solution and 50 µL of internal standard were added to the 250 µL of human plasma and extracted with various organic solvents listed below, table 1.

Table 1: Selection of organic solvent for extraction

Organic solvent

Observation

Fig. no.

Acetonitrile

Extraction efficiency was poor

1

Methanol

Deferasirox was not extracted

2

Ethyl acetate

Extraction efficiency was poor

3

n-Hexane

No extraction

4

Diethyl ether

Extraction efficiency was good

5

Diethyl ether with 0.1% NaOH

Deferasirox was not extracted

6

Diethyl ether with 0.1% formic acid

Extraction efficiency was better

7

Among all the organic solvents, diethyl ether showed good extraction efficiency, fig. 5. Addition of 0.1% formic acid to this extraction step improved the extraction efficiency and reduced the matrix interference, fig. 7.

Fig. 1: Extraction with acetonitrile

Fig. 2: Extraction with methanol

Fig. 3: Extraction with ethyl acetate

Fig. 4: Extraction with n-Hexane

Fig. 5: Extraction with diethyl ether

Fig. 6: Extraction with diethyl ether

(In presence of 0.1% NaOH)

Fig. 7: Extraction with diethyl ether

(In presence of 0.1% formic acid)

Fixed extraction procedure

To the clean and dry centrifuge tube, 250 µL plasma, 50 µL of aliquots of standard drug solution (40, 80, 120, 160, 200, 240, 280 µg/mL) and 50 µL of internal standard (8 µg/mL) were added. Five hundred micro litres of 0.1% formic acid was added and vortexed for 2 minutes. Three milli litres of diethyl ether was added to the above solution and vortexed for 3 minutes. This solution was centrifuged at 3000 rpm for 5 minutes. The clear supernatant solution was transferred to eppendrof's tubes and evaporated to dryness using nitrogen gas. The residue was reconstituted with 200 µL of methanol and 10 µL of this solution was applied on to the plate. The plate was analysed photometrically and chromatograms were recorded, fig. 8-14.

STANDARD CHROMATOGRAMS OF DEFERASIROX IN PLASMA

Fig. 8: Deferasirox -100 ng/spot, paracetamol -20 ng/spot

Fig. 9: Deferasirox -200 ng/spot, paracetamol -20 ng/spot

Fig. 10: Deferasirox -300 ng/spot, paracetamol -20 ng/spot

Fig. 11: Deferasirox -400 ng/spot, paracetamol -20 ng/spot

Fig. 12: Deferasirox -500 ng/spot, paracetamol -20 ng/spot

Fig. 13: Deferasirox -600 ng/spot, paracetamol -20 ng/spot

Fig. 14: Deferasirox -700 ng/spot, paracetamol -20 ng/spot

VALIDATION OF THE METHOD 18, 19

The validation of the developed method was carried out in terms of specificity, linearity, accuracy, precision, recovery and stability studies according to US-FDA guidelines.

Specificity

Specificity of the above developed extraction method was demonstrated as follows. Blank plasma samples from six volunteers were extracted using the above procedure and chromatographed. It was found that there was no interference from blank plasma, fig. 15.

Linearity and Range

Linear regression data revealed an excellent linear relationship between concentration and peak area ratio over a concentration range of 100 to 700 ng/spot. The slope, intercept and correlation co-efficient values were found to be 0.0056, -0.1981 and 0.997, fig. 16. The regression equation was,

Peak area ratio = (-0.1981) + 0.0056Ã- concentration

Lower limit of quantification (LLOQ)

The lowest concentration at which the peak is quantified is called 'Lower Limit of Quantification' which was found to be 100 ng/spot, fig. 17.

Accuracy

A minimum of five determinations per concentration was carried out to measure the accuracy of the method. At least three concentrations (high QC, middle QC, low QC) in the range is recommended. Accuracy can be measured by assessing the deviation of mean test result from the true value and it should be within 15%. The results are shown in table 2.

Precision

Precision of the method was determined by

Intraday precision

Inter day precision

I. Intraday precision

Intraday precision was studied by carrying out the analysis of the standard drug at three different concentrations in the linearity range of drug (high QC, middle QC, low QC) for five times on the same day and coefficient of variation (CV) was calculated, table 3.

II. Inter day precision

Inter day precision was studied by carrying out the analysis of the standard drug at three different concentrations in the linearity range of drug (high QC, middle QC, low QC) for five days over a period of one week and coefficient of variation (CV) was calculated, table 4.

Recovery

The extraction efficiency was explained by recovery studies. The standard drug was extracted by the proposed extraction method. The extracts were analysed with the fixed chromatographic conditions. The peak areas of developed chromatograms were compared with that of unextracted standard peak areas. The peak area responses obtained were used for determining the extraction efficiency. Recovery should be determined by using three concentration levels (high QC, middle QC, low QC).

The % extraction efficiency or % recovery was calculated by using the following formula,

The results are shown in table 5.

Stability

The drug may undergo degradation during the analysis. Hence it is necessary to study the stability of drug in prepared sample at two different concentrations (low QC, high QC). The stability of the drug was studied under room temperature and under refrigerated conditions. The samples were chromatographed at fixed time intervals and the peak area was compared with that of fresh solution. The stability of deferasirox was found to be 12 hours and 22 hours, under room temperature and refrigeration, respectively.

Fig. 15: Chromatogram of blank plasma

Fig. 16: Calibration graph of deferasirox in plasma

(100-700 ng/spot)

Fig. 17: Lower limit of quantification (LLOQ) -100 ng/spot

Table 2: Accuracy study

True concentration (ng/spot)

Mean Test result (ng/spot)

% deviation from true value

Limit for % deviation

Number of determinations

200 (Low QC)

173.5

13.25

Within 15

5

400 (Middle QC)

369.2

7.7

600 (High QC)

555.4

7.4

Table 3: Intraday precision

Concentration

(ng/spot)

Coefficient of variation (CV)

Limit

Number of determinations

200 (Low QC)

11.25 %

Within

15 %

5

400 (Middle QC)

10.56 %

600 (High QC)

11.65 %

Table 4: Inter day precision

Concentration

(ng/spot)

Coefficient of variation (CV)

Limit

Number of determinations

200 (Low QC)

12.64 %

Within 15 %

5

400 (Middle QC)

11.78 %

600 (High QC)

12.25 %

Table 5: Recovery study

Concentration

(ng/spot)

Mean % Recovery

Number of determinations

200 (Low QC)

87.12

5

400 (Middle QC)

88.09

600 (High QC)

88.62

IN VITRO INTERACTION STUDY OF DEFERASIROX WITH ALUMINIUM HYDROXIDE BY EQUILIBRIUM DIALYSIS METHOD 20, 21

Plasma protein binding properties are considered as the primary determinants of the pharmacokinetic properties of drugs. Any physiological condition that causes the alteration in the albumin binding of the drugs might lead to change in the pharmacokinetic and pharmacological properties of the drugs. Drug-drug interactions thus play a vital role in the extent of plasma protein binding and consequently the therapeutic effect of the drugs.

Deferasirox mainly used as an iron chelating agent for β-thalassemic patient. Literature survey reveals that deferasirox should not be co-administered with aluminium containing antacid, otherwise it may cause unwanted side effect like abdominal pain. So in vitro protein binding of deferasirox has been conducted by equilibrium dialysis method using UV spectroscopic method. In this study, the free fraction of drugs and the % of protein binding of deferasirox to BSA in the presence and absence of aluminium hydroxide were calculated. This study was done to evaluate the interaction of aluminium hydroxide with deferasirox at physiological pH (7.4) and temperature (37±0.5oC). BSA and HSA have structural similarity. In this study, BSA was used instead of HSA, because of its low cost and easy availability.

Preparation of standard solutions

5 mg of deferasirox was dissolved in 5 mL of methanol and the volume was made up with phosphate buffer of pH 7.4 to 50 mL. From this stock solution (100µg/mL) aliquots of standard solutions were prepared using phosphate buffer of pH 7.4.

Preparation of reagents

1) Buffer solution pH 7.4

50 mL of potassium dihydrogen phosphate solution was placed in 200ml volumetric flask. Add 39.1mL of 0.2N sodium hydroxide solution and diluted with distilled water to produce 200mL.

2) Potassium dihydrogen phosphate solution (0.2M)

27.22 gm of potassium dihydrogen phosphate was dissolved in water and diluted with distilled water to produce 1000mL.

3) Sodium hydroxide solution (0.2N)

0.8gm of sodium hydroxide was dissolved and made up to 100mL with water.

4) Bovine serum albumin (3Ã-10-4M)

0.99 gm of bovine serum albumin was dissolved in 25mL of buffer solution of pH 7.4 and diluted to volume to 50ml.

Preparation of aluminium hydroxide solution (3Ã-10-4M)

Stock solution of 1000 µg/mL was prepared using phosphate buffer of pH 7.4. From this stock solution 0.5 mL was transferred to 50 mL standard flask and final volume made up to 50 mL with phosphate buffer of pH 7.4.

Preparation of standard curve

For the spectrophotometric determination, standard calibration curve was plotted using different concentrations of the drug solutions and absorbances were noted at 248 nm. Linearity range was found between 1-10 µg/mL and the correlation coefficient value was found to be 0.9997. The regression equation was,

Absorbance = 0.0094 + 0.076 Ã- concentration

Equilibrium dialysis method

Equilibrium dialysis is one of the methods used for the determination of protein binding and this method is used to study the complexation between BSA and the drug. If binding occurs, the drug concentration in the sac containing the protein is greater at equilibrium than the concentration of drug in the vessel outside the sac. At regular intervals, samples were withdrawn and analyzed to obtain the concentration of free and complexed drug.

Study of protein binding of deferasirox

Protein binding of deferasirox was determined by equilibrium dialysis method. For this, 25 mL of 3Ã-10-4M concentrations of deferasirox was prepared in phosphate buffer pH 7.4. About 25 mL of 3 x 10-4M BSA solution was taken in glass tube attached to semi permeable membrane (Sigma dialysis sacs, 21mm diameter, and 30cm length). The dialysis membranes were previously activated by immersing it in warm water for 30 minutes. These tubes were then immersed in beakers with 25 mL of phosphate buffer containing fixed concentration of drug solution (3Ã-10-4M). Immediately at zero time, 1mL of the solution was pipetted out from the beaker and it was replaced with 1 mL of phosphate buffer of pH 7.4. Readings were taken at various time intervals by UV spectrophotometer at 248 nm till the absorbance values were constant.

INTERACTION STUDIES

Study of effect of aluminium hydroxide on in vitro protein binding of deferasirox

To study the effect aluminium hydroxide on in vitro protein binding of deferasirox, 25 mL of 3 x 10-4M BSA was taken in each of 5 cylindrical glass tubes attached to semi permeable membrane (Sigma dialysis sacs, 21mm diameter, 30cm length). The dialysis membranes were previously activated by immersing it in warm water for 30 minutes. These tubes were then immersed in beakers with 25 mL of phosphate buffer containing fixed concentration of deferasirox (3Ã-10-4M)). Aluminium hydroxide was added in increasing concentrations into five beakers containing deferasirox solution to give a final ratio (BSA: Deferasirox: Aluminium hydroxide, 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5). This system was maintained at room temperature for 8 hours. After 8 hours, 1 mL of the solution was withdrawn from the beaker, and diluted to 10 mL using potassium dihydrogen phosphate buffer and the responses of free drug was measured at a wavelength of 248 nm. The concentration of the free fraction of drug was determined from the calibration graph.

RESULTS

IN VITRO PROTEIN BINDING STUDY

Percentage protein binding and %free fraction of drugs

The concentration of the drug was determined from the calibration graph. The % protein binding and free fraction of deferasirox was calculated. Percentage protein binding of deferasirox was found to be 98.16% and the percentage free fraction of deferasirox was found to be 1.84%.

Effect of aluminium hydroxide on in vitro protein binding of deferasirox

After interaction with aluminium hydroxide, the free fraction of deferasirox increased from 1.84% to 7.27% when the ratio of aluminium hydroxide to BSA was increased from 1 to 5.

Table 1: Effect of aluminium hydroxide on

in vitro protein binding of deferasirox

Bsa:Deferasirox:

Aluminium Hydroxide

% Protein Binding

% Free Drug Concentration

1:1:1

93.29

6.71

1:1:2

93.21

6.79

1:1:3

93.08

6.92

1:1:4

92.90

7.1

1:1:5

92.73

7.27

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