Spectroflourimetric Method For The Estimation Of Pazufloxacin Mesylate Biology Essay

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The Pazufloxacin mesylate showed good fluorescence in presence of sulphuric acid. Hence, this reagent was selected for the development of spectrofluorimetric method for estimation of Pazufloxacin mesylate.

Pazufloxacin mesylate was readily soluble in water and the final volume made up with sulphuric acid, the solution was scanned between 350 - 600 nm for emission at an excitation wavelength 248 nm and 335 nm in presence of the reagent. The Pazufloxacin mesylate in water with the reagent shows good fluorescence intensity at 335 nm as excitation wavelength. Hence water has chosen as the solvent.

SELECTION OF EMISSION WAVELENGTH

The solution of Pazufloxacin mesylate was scanned between 350 - 600 nm for emission, by keeping the excitation wavelength as 335 nm against the blank. The Pazufloxacin mesylate showed maximum fluorescence intensity at 417 nm. Hence, 417 nm was fixed as emission wavelength (Fig:1)

Fig. 1: Emission spectrum of Pazufloxacin mesylate

4. OPTIMIZATION OF REAGENTS

4.1 Effect of Acid Strength

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Different strength of sulphuric acid ranging from 0.01 N to 0.5 N was tried. Different peaks were observed and it was found that spectrum and fluorescence intensity was good for 0.2 N strength of sulphuric acid (Fig: 2). Hence it was fixed and result of analysis is given in Table 1.

Table 1: Effect of acid strength

Concentration

Fluorescence intensity

Peak shape

0.01 N

456.032

Not good

0.05 N

573.699

Not good

0.1 N

708.22

Good

0.2 N#

897.063

Good

0.5 N

999.766

Good

# selected parameter

Fig. 2: Effect of acid strength on fluorescence intensity

5. OPTIMIZATION OF SPECTROFLUORIMETRIC VARIABLES

The excitation and emission bandwidth, response and sensitivity are the important parameters that influence the fluorescence intensity of the compounds. Bandwidth and fluorescence intensity are directly proportional to one another. It is due to the reason, as the bandwidth increases the fluorescence intensity also will increase because the intensity is depends on the amount of light fall on the sample, so it tends to increase the fluorescence intensity.

The concentration of 500 ng/mL solution of Pazufloxacin mesylate was used for the optimization of parameters.

5.1 Response time

The excitation and emission bandwidth were fixed as 5 nm and 10 nm at different responses the fluorescence intensities were measured. (Table: 2).

Table 2: Fixing the response

Time (sec)

Fluorescence intensity

0.02

443.24

0.05#

480.68

0.1

468.93

0.25

454.87

0.5

472.32

1

462.98

2

469.90

4

457.76

8

456.83

# selected response time

5.2 Excitation and Emission Bandwidth

Response was fixed as 0.05 sec and emission bandwidth as 5nm, fluorescence intensity were measured with different excitation bandwidth such as 5, 10, 20 nm. Similarly it was also done for fixing emission bandwidth (Table. 3).The excitation and emission bandwidth were fixed as 5 and 10 nm which showed good spectrum and linearity.

Table 11: Fixing the bandwidth

Mode of measurement

Bandwidth

Intensity

Excitation

5#

96.980

10

481.76

20

730.30

Emission

5

127.67

10#

479.25

20

989.08

#selected bandwidth

5.3 Sensitivity

By keeping the response, excitation and emission bandwidth as constant, fluorescence intensity were recorded by varying sensitivity as Low, medium and high (Table. 4). Sensitivity was fixed as medium hence fluorescence intensity and linearity was satisfactory.

Table 4: Fixing the sensitivity

Sensitivity

Intensity

Low

56.66

Medium#

476.47

High

900.09

#selected sensitivity

6. FIXED INSTRUMENTAL PARAMETERS

Excitation wavelength - 335 nm

Emission wavelength - 418 nm

Excitation bandwidth - 5 nm

Emission bandwidth - 10 nm

Response time - 0.05 sec

Sensitivity - medium

VALIDATION OF THE METHOD

The validation of the developed method was carried out in terms of linearity, accuracy, inter and intraday precision, stability studies and selectivity.

Linearity and Range

100 µg/mL concentration of Pazufloxacin mesylate was prepared in water. A solution of 0.2 N sulphuric acids was prepared in water.

Aliquots of standard solution were prepared by taking 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0ml from the stock solution, were added into a series of 10ml standard flask and the final volume was made up to mark with 0.2 N sulphuric acid. The fluorescence intensity of the solutions was measured with the fixed spectrofluorimetric condition (fig. 3) and the calibration graph was prepared by plotting concentration Vs fluorescence intensity of the drug (fig. 4).

Fig. 3: Overlain spectrum of standards of Pazufloxacin mesylate

Fig. 4: Calibration graph of Pazufloxacin mesylate

Concentration (ng/mL)

Fluorescence intensity

50

55.68

100

119.50

200

211.52

400

401.73

500

480.68

700

665.38

900

866.75

1000

931.71

Accuracy

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Accuracy of the developed method was determined by conducting the recovery studies. To the powdered formulation of Pazufloxacin mesylate, standard drug was added at 80%, 100% and 120% level. The concentration of the drug present in the resulting solutions was determined by the proposed method. The recovery procedure was repeated for six times and % recovery was calculated (Table. 6).

Table 6: Recovery studies for Pazufloxacin mesylate

Level

% Recovery

%RSD*

80%

100.27

1.3393

100%

100.7

1.4312

120%

100.84

1.4724

* mean of six determinations

Precision

Precision of the method was determined by the repeatability studies. Intra-day and Inter-day studies were carried out for the method by repeating the procedure six times and the %RSD was calculated. The data's are shown in Table 7 & 8.

Table 7: Intra-day Precision

Drug

Concentration (ng/ml)

Fluorescence intensity

%rsd*

Pazufloxacin mesylate

500

480.21

0.1073

479.98

479.84

480.57

481.21

480.75

* mean of six determinations

Table 8: Inter-day precision

Drug

Concentration (ng/mL)

Days

Fluorescence intensity

% RSD

Pazufloxacin mesylate

200

1

211.78

0.1789

2

210.97

3

211.09

400

1

401.89

0.1562

2

402.11

3

401.93

500

1

480.89

0.1548

2

481.57

3

482.38

ANALYSIS OF FORMULATION

Preparation of standard solution

Stock solution of Pazufloxacin mesylate (100 µg/mL) was prepared in water.

Preparation of sample solution for Pazufloxacin mesylate

Infusion contains 500 mg/100 mL of Pazufloxacin mesylate. Volume equivalent to 10 mg of Pazufloxacin mesylate was taken, transferred to a 100 mL volumetric flask and made up to volume with water (100 µg/mL).

From the above solution, 0.5ml was pipetted into 10ml standard flask, and the volume was made upto the mark with 0.2 N sulphuric acid. The fluorescence intensity of the solution was measured with the fixed instrumental conditions (Fig. 5) and the amount of drug present in the formulation was calculated by single point standardization method. The result of analysis is given in Table 9.

Fig 5: Analysis of formulation

Table 9: Analysis of formulation

Formulation

Amount (mg/infusion)

% Label Claim

± RSD*

Labeled

Found

Pazubid

500

498.48

99.22±1.0632

* Mean of three determinations

DEVELOPMENT OF IN VITRO METHOD FOR THE ESTIMATION OF PAZUFLOXACIN MESYLATE FROM PLASMA BY SPECTROFLUOROMETRY

The spectrofluorimetric method developed above was utilized for estimation of Pazufloxacin mesylate from plasma. The spectrofluorimetric conditions followed were given below,

Excitation wavelength - 335 nm

Emission wavelength - 418 nm

Excitation bandwidth - 5 nm

Emission bandwidth - 10 nm

Response time - 0.05 sec

Sensitivity - medium

Optimization of extraction procedure of Pazufloxacin mesylate from plasma

Extraction methods are employed for removal of interfering substances. Plasma consists of proteins and other substances, which may interfere with the quantification of drug. Hence the drug must be separated from interferents substance. As a primary requirement the extraction solvent must solubilize the drug to be extracted and should be immiscible with water.

Selection of organic solvent for extraction

The extraction of drug into organic solvent depends on its distribution coefficient in aqueous and organic phases. The 50 µL (10 µg/µl) of standard Pazufloxacin mesylate was spiked with 500µL plasma and extracted with various organic solvents tried here methanol, acetonitrile, diethyl ether, ethyl acetate, n-hexane and dichloromethane (fig. 6 & 7)

Diethyl ether, ethyl acetate, n-hexane and methanol were extracting the drug from plasma and interfere. But with the clear supernatant layer the interferents were much high and the fluorescence intensity of Pazufloxacin mesylate was poor. Also the emission wavelength was changed.

The acetonitrile caused protein precipitation and extract was free from interferents and the shows the fluorescence intensity was good. Hence it was chosen as the solvent for extraction.

Fig. 6: Ethyl ether Fig. 7: Acetonitrile

Fixing the volume of plasma

Different volume of plasma like 250 and 500 µL were tried. Different spectra were observed and it was found that spectrum and fluorescence intensity was good for 250 µL of plasma (Fig: 8 & 9). Hence it was fixed and result of analysis is given in Table 10.

Table. 10: fixing the volume of plasma

Volume of plasma added (µL)

Fluorescence intensity

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250

74.02

500

53.20

Fig. 8: 250 µL plasma Fig. 9: 500 µL plasma

Fixing the volume of extraction solvent

Different volume of acetonitrile such as 2.5, 3, 4 and 5 mL were tried. Different spectra were observed and it was found that spectrum and fluorescence intensity was good for 2.5 mL of acetonitrile (Fig: 10). Hence it was fixed and result of analysis is given in Table 11.

Table. 11: Fixing the volume of acetonitrile

Volume of acetonitrile (mL)

Fluorescence intensity

2.5

74.02

3

32.33

4

52.58

5

41.42

Fig. 10: 2.5 mL of acetonitrile

Fixed extraction procedure

250µL of plasma was pipetted into centrifuge tube, spiked with 50µL of Pazufloxacin mesylate (10 µg/mL) drug solutions and vortex for 3 minutes. The spiked solution was extracted with 2.5 mL of acetonitrile, vortex for 3 minutes and centrifuge at 4000 rpm for 15 minutes. Transfer the protein free supernatant into 2 ml eppendrof's tubes. The tubes were evaporated to dryness under the stream of nitrogen gas. Reconstitute the residue with 2 mL of 0.2 N sulphuric acid and measure the fluorescence intensity with the fixed instrumental condition (fig. 11).

Fig. 11: 50 µL of Pazufloxacin mesylate

VALIDATION OF THE METHOD

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 measured the fluorescence intensity. It was found that there was no interference from blank plasma, fig.15. Hence the method was said to be specific.

Fig. 12: spectrum of blank plasma

Linearity and range

Preparation of standard graph

Calibration curve for the drug was constructed using the extraction procedure developed above. A standard solution of Pazufloxacin mesylate containing 400 µg/mL was prepared.

Aliquots of standard solution prepared by taking 0.05, 0.25, 0.5, 1.25, 2.5, and 3.5 mL from the stock solution, were added to the series of standard flask and made up to mark with water.

Transfer 250 µL of plasma into a series centrifuge tubes, spiked with 50 µL of Pazufloxacin mesylate from the aliquots of standard solutions and vortex for 3 minutes. The spiked solution was extracted with 2.5 mL of acetonitrile, vortex for 3 minutes and centrifuge at 4000 rpm for 15 minutes. Transfer the protein free supernatant into 2 ml eppendrof's tubes. The tubes were evaporated to dryness under the stream of nitrogen gas. Reconstitute the residue with 2 mL of 0.2 N sulphuric acid and measure the fluorescence intensity with the fixed instrumental condition.

The calibration graphs were constructed using fluorescence intensity of standards Vs concentration of standards, the spiked samples of plasma were found to be linear in the concentration range of 0.1 to 7 µg/mL (fig.13). The linear equation and correlation coefficient was found to be Y = 19.4007 + 63.2306 * C and 0.998809 respectively (fig. 14)

Fig. 14: Overlain spectrum of standards Pazufloxacin mesylate

Fig. 13: Calibration graph of Pazufloxacin mesylate

Concentration (µg/mL)

Fluorescence intensity

0.1

35.39

0.5

50.81

1

75.08

2.5

163.44

5

342.64

7

459.85

Precision

Precision of the method was determined by the repeatability studies. Intra-day and Inter-day studies were carried out for the method by repeating the procedure six times and the %RSD was calculated. The data's are shown in Table 12 & 13.

Table 12: Intra-day Precision

Drug

Concentration (µg/mL)

Fluorescence intensity

%rsd*

Pazufloxacin mesylate

0.1 (LLOQ)

35.41

1.035

34.99

35.72

2.5 (MLOQ)

163.09

0.2975

162.49

163.45

7 (HLOQ)

458.59

0.1958

459.71

460.39

Table. 13: Inter day precision

Drug

Concentration (µg/mL)

Days

Fluorescence intensity

% RSD

Pazufloxacin mesylate

0.1 (LLOQ)

1

33.85

0.1789

2

34.67

3

32.72

2.5 (MLOQ)

1

160.37

0.1562

2

162.51

3

162.84

7 (HLOQ)

1

460.35

0.1548

2

459.45

3

458.48

Stability

The drug may undergo degradation during the sample preparation or during the analysis. Hence it is necessary to study the stability of drug in plasma and in prepared sample. The stability of the drug was studied as per ICH and USFDA guidelines such as freeze and thaw stability, table top stability, post-preparative stability and stock solution stability under room temperature and under refrigerated conditions. The samples were measured the fluorescence intensity at fixed time intervals and the fluorescence intensity was compared with that of fresh solution. A 10 % reduction in fluorescence intensity was considered as loss of stability. The drug was found to be stable for 4 hours under room temperature. The stability data's were given in the following,

Freeze and thaw stability

The stability of Pazufloxacin mesylate was studied under the conditions of freeze and thaw stability as per guidelines. The drug was found to be stable for 2 cycles. The stability data's were given in table 14.

Table. 14: Freeze and thaw stability

Cycle

Fluorescence intensity

1

148.49

2

130.91

3

114.67

Table-top stability

The table-top stability was studied as per guidelines. The stability of drug was found to be stable for 3 hours. The stability data's were given in table 15.

Table. 15: Table-top stability

Time (hrs)

Fluorescence intensity

1/2

150.12

1

150.62

1 ½

149.92

2

147.62

2 ½

138.81

3

128.57

Post preparative stability

The post preparative stability was studied as per guidelines. The stability of drug was found to be stable for 2 ½ hours. The stability data's were given in table 16.

Table. 16: Post preparative stability

Time (hrs)

Fluorescence intensity

½

150.03

1

151.23

1 ½

148.59

2

141.44

2 ½

127.36

Stock solution stability

The stability of the stock solution was studies as per guidelines. The stability of drug was found to be stable for 3 ½ hours. The stability data's were given in table 17.

Table. 17: Stock solution stability

Time (hrs)

Fluorescence intensity

1

629.12

1 ½

625.61

2

622.61

2 ½

598.54

3

550.96

3 ½

540.87

4

524.35