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Development And Validation Of Spectroflourimetric Method Biology Essay

The pazufloxacin mesylate showed good fluorescence in presence of sulphuric acid. Hence, the solutions of pazufloxacin mesylate were prepared in sulphuric acid.

Selection of excitation and emission wavelengths

Solution of pazufloxacin mesylate were prepared in sulphuric acid and scanned using spectrofluorimeter to fix the excitation and emission wavelengths. The drug showed two excitation wavelengths at 248 nm and 335 nm. The emission spectrum was recorded at these excitation wavelengths. It was found that at an excitation wavelength of 335 nm, the spectral pattern (emission wavelength – 418 nm) and response was good. Hence, an excitation wavelength of 335 nm and emission wavelength of 418 nm were selected for further studies, (fig. 1-4).

Fig. 1: Excitation spectrum of pazufloxacin mesylate at emission wavelength 417 nm

Fig. 2: Excitation spectrum of pazufloxacin mesylate at emission wavelength 418 nm

Fig. 3: Emission spectrum of pazufloxacin mesylate at excitation wavelength 248 nm

Fig. 4: Emission spectrum of pazufloxacin mesylate at excitation wavelength 335 nm

OPTIMIZATION OF REAGENTS

Effect of Acid Strength

Different strengths of sulphuric acid ranging from 0.01 N to 0.5 N were tried and it was found that a strength of 0.2 N gave good response and hence selected, (fig. 5 & table 1).

Table 1: Effect of acid strength

Strength of acid (N)

Fluorescence intensity

0.01

456.032

0.05

573.699

0.1

708.22

0.2

897.063

0.3

996.519

0.5

999.766

Fig. 5: Effect of acid strength on fluorescence intensity

OPTIMIZATION OF INSTRUMENTAL PARAMETERS

Excitation bandwidth, emission bandwidths and response are the important instrumental parameters that influence the response.

Response time

At the excitation and emission bandwidths of 5 nm and 10 nm the fluorescence intensity of pazufloxacin mesylate (0.5 µg/mL) solution was determined at different response. It was found that a response of 0.05 sec gave good response and hence selected, 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

Excitation and emission bandwidths

Keeping the response fixed as 0.05 sec, the excitation and emission bandwidths were changed to get the best response. Excitation bandwidth of 5 nm and emission bandwidth of 10 nm gave good results and hence selected for further studies, table 3.

Table 3: 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

FIXED PARAMETERS

Excitation wavelength - 335 nm

Emission wavelength - 418 nm

Excitation bandwidth - 5 nm

Emission bandwidth - 10 nm

Response time - 0.05 sec

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

Suitable aliquots of drug solutions were transferred in 10 mL standard flask and diluted with 0.2 N sulphuric acid to get standard solutions of concentration ranging from 0.1 – 1 µg/mL. The fluorescence intensities of these solutions were measured with the fixed spectrofluorimetric parameters and a calibration graph was prepared by plotting concentration Vs fluorescence intensity of the drug, (table 4 & fig. 6). The slope, intercept and correlation coefficient were found to be 922.469, 22.6898 and 0.9995, respectively.

Table 4: Calibration data

Concentration (µg/mL)

Fluorescence intensity

0.1

55.68

0.2

211.52

0.4

401.73

0.7

665.38

1

931.75

Fig. 6: Calibration graph of pazufloxacin mesylate (0.1-1µg/mL)

Accuracy

Accuracy of the developed method was determined by performing the recovery studies. To the formulation of pazufloxacin mesylate, standard drug was added at 80%, 100% and 120% levels. The concentrations of the drug present in the resulting solutions were determined by the proposed method. The recovery procedure was repeated six times and % recovery was calculated, table 5.

Table 5: Recovery studies results

Level

% Recovery

%RSD*

80%

98.27

1.3393

100%

100.74

1.4312

120%

102.84

1.4724

* RSD of six determinations

Precision

Precision of the method was determined by repeatability studies. Intra and Inter-day studies were carried out by repeating the procedure six times and the %RSD was calculated, table 6 & 7.

Table 6: Intra-day Precision

Concentration (ng/ml)

Fluorescence intensity

%rsd

500

480.21

0.1073

479.98

479.84

480.57

481.21

480.75

Table 7: Inter-day precision

Concentration (ng/mL)

Days

Fluorescence intensity

% RSD

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. Suitable aliquots of drug solutions were transferred in 10 mL standard flask and diluted with 0.2 N sulphuric acid to get standard solutions of concentration ranging from 0.1 – 1 µg/mL. The fluorescence intensities of these solutions were measured with the fixed spectrofluorimetric parameters.

Preparation of sample solution

Volume equivalent to 10 mg of pazufloxacin mesylate was taken from the infusion of strength 500 mg/mL and 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 intensities of these solutions were measured, (fig. 8). The amount of drug present in the formulation was calculated, (fig. 9). The result of formulation analysis is given in table 8.

Table 8: Analysis of formulation

Formulation

Amount (mg/infusion)

% Label Claim

± RSD*

Labeled

Found

Pazubid

500

498.48

99.22±1.0632

* RSD of three determinations

Fig. 8: Overlain spectrum of standards of Pazufloxacin mesylate

Fig. 9: Analysis of formulation

DEVELOPMENT OF BIOANALYTICAL METHOD FOR THE DETERMINATION OF PAZUFLOXACIN MESYLATE IN HUMAN PLASMA BY SPECTROFLUORIMETRY

The spectrofluorimetric method developed was applied for the bioanalysis of pazufloxacin mesylate in plasma 24, 25.

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 interfering substance.

Selection of organic solvent for extraction

The extraction of drug into organic solvent depends on its distribution coefficient in aqueous and organic phases. A volume of 500 µL plasma was spiked with 50 µL of pazufloxacin mesylate solution (10 µg/mL) and extracted with various organic solvents like methanol, acetonitrile, diethyl ether, ethyl acetate, n-hexane, dichloromethane etc., (fig. 10 - 13).

Diethyl ether, ethyl acetate, n-hexane and methanol extracted the drug from plasma. But in the extracts the interferents were high and fluorescence intensity of pazufloxacin mesylate was poor. The emission wavelength of pazufloxacin mesylate also changed in these solvents.

Acetonitrile was selected as the solvent for sample preparation because with acetonitrile, the extraction efficiency of the analyte was good.

Fig. 10: Ethyl ether Fig. 11: Acetonitrile

Fig. 12: Methanol Fig. 13: Ethyl acetate

Fixing the volume of plasma

To different volumes of plasma like 250 and 500 µL, the drug solutions were spiked, processed and response noted. It was found that a plasma volume of 250 µL was apt and hence fixed for further studies, table 9.

Table 9: Fixing the volume of plasma

Volume of plasma added (µL)

Fluorescence intensity

250

74.02

500

53.20

Fixing the volume of extraction solvent

Different volumes of acetonitrile such as 2, 2.5, 3, 4 and 5 mL were tried. The response was good for 2.5 mL of acetonitrile and hence it was fixed table 10.

Table 10: Fixing the volume of acetonitrile

Volume of acetonitrile (mL)

Fluorescence intensity

2

63.17

2.5

74.02

3

32.33

4

52.58

5

41.42

Fixed extraction procedure

250µL of plasma was transferred into a centrifuge tube, spiked with 50 µL of pazufloxacin mesylate (10 µg/mL) solution and vortexed for 3 minutes. This solution was deproteinated with 2.5 mL of acetonitrile, vortex for 3 minutes and centrifuge at 4000 rpm for 15 minutes. The clear supernatant liquid was transferred into a 2 ml eppendrof’s tube and evaporated to dryness under a stream of nitrogen gas. The residue was reconstituted with 2 mL of 0.2 N sulphuric acid and the fluorescence intensity was measured at excitation and emission wavelengths of 335 nm and 418 nm.

VALIDATION OF THE METHOD 26

Specificity

Specificity of the developed method was carried out as follows. Blank plasma samples from six volunteers were processed and analysed using the above procedure and fluorescence spectra were measured. It was found that there was no interference from blank plasma, (fig. 13).

Fig. 13: Spectrum of blank plasma

Linearity and range

Preparation of standard graph

Calibration curve for the drug was constructed using the extraction procedure developed. A standard solution of pazufloxacin mesylate containing 400 µg/mL was prepared. Aliquots of standard solutions were by transferring 0.05, 0.25, 0.5, 1.25, 2.5, and 3.5 mL from the stock solution to series of standard flasks and made up to mark with water.

Transferred 250 µL of plasma into a series centrifuge tubes, spiked with 50 µL of pazufloxacin mesylate from the aliquots of standard solutions and processed according to the developed extraction procedure.

The calibration graphs were constructed using fluorescence intensity of standards Vs concentration of standards and the linearity was found to be in the range of concentration range of 0.1 to 7 µg/mL, (fig. 14). The calibration data is shown in table 11.The slope, intercept and correlation coefficient values were found to be 63.2306, 19.4007 and 0.9988, respectively.

Table 11: calibration data

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

Fig. 14: Calibration graph of pazufloxacin mesylate

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 0.1 µg/mL, (fig. 15).

Fig. 15: lower limit of quantification

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, table 12 & 13.

Table 12: Intra-day Precision

Concentration (µg/mL)

Coefficient of variation (CV)

Limit

Number of determinations

0.5 (Low QC)

12.46 %

Within 15 %

5

2.5 (Middle QC)

11.89 %

5 (High QC)

10.65 %

Table 13: Inter day precision

Concentration (µg/mL)

Coefficient of variation (CV)

Limit

Number of determinations

0.5 (Low QC)

13.76 %

Within 15 %

5

2.5 (Middle QC)

12.91 %

5 (High QC)

13.53 %

Recovery

The extraction efficiency was determination by recovery studies. The extracts were analyzed with the fixed spectrofluorimetric conditions. The fluorescence intensities of spiked samples were compared with that of fluorescence intensity of standard sample. Recovery was determined at 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 14.

Table 14: Recovery study

Concentration (µg/mL)

Mean % recovery

Number of determination

0.5 (Low QC)

88.12 %

5

2.5 (Middle QC)

86.91 %

5 (High QC)

87.53 %

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 USFDA guidelines in terms of freeze and thaw stability, table top stability, post-preparative stability and stock solution stability under room temperature and under refrigerated conditions. The fluorescence intensities of samples were measured at fixed time intervals and the fluorescence intensity was compared with that of freshly prepared sample. The results of stability studies data is given in table 15 – 16.

Table 15: Freeze and thaw stability

Freeze & thaw

cycle

Fluorescence intensity

1

148.49

2

130.91

3

114.67

Table 16: 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

Table 17: Post preparative stability

Time (hrs)

Fluorescence intensity

½

150.03

1

151.23

1 ½

148.59

2

141.44

2 ½

127.36

Table 18: 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

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