Development And Validation Of RP HPLC Method Biology Essay

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The chemical nature of the sample provides valuable information for LC separation. The nature of the sample i.e. ionic, non-ionic or neutral, its molecular weight and solubility plays a major role in the selection of the method. Since the drug, pazufloxacin mesylate is polar in nature, reverse phase chromatography was selected for separation.

Selection of wavelength

Good analytical results will be obtained only by careful selection of wavelength used for detection. This choice requires knowledge of absorption spectra of the sample. An ultraviolet spectrum of pazufloxacin mesylate was recorded and it was found the drug showed maximum absorbance at λmax of 241 nm and 330 nm, (fig. 1). A wavelength of 241 nm was selected as it gave good response.

Fig. 1: Ultraviolet spectrum of pazufloxacin mesylate

Optimization of chromatographic conditions

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 chromatographic conditions for the separation

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Column: Merck - LichroCART, C18 column (250 mm X 4.0 mm, 5 µm)

Detection wavelength: 241 nm

Flow rate: 1 mL/min

Operating temperature: Room temperature

Mobile phase: 0.1 % orthophosphoric acid: methanol

For developing LC method, different mobile phases with different ratios were tried, table 1 & fig. 2-5.

Table 1: Selection of mobile phase

S.No

Mobile phases

Chromatogram

(Fig. 2 - 5)

1

Water: methanol

(20: 80, v/v)

2

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

3

0.1% Acetic acid: methanol

(20: 80, v/v)

4

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

Optimization of separation conditions

Ionic strength (v/v)

Different ionic strengths like 0.05%, 0.1%, 0.2% and 0.5% of orthophosphoric acid were tried and chromatograms were observed. Among these strengths, 0.1% orthophosphoric acid: methanol showed symmetrical peak with good separation, (fig. 6-8). Hence 0.1% was selected as the strength of formic acid for separation, table 2.

Table 2: Effect of ionic strength

S.No

Ionic strength of

orthophosphoric acid

Chromatogram

(Fig. 6 - 8)

1

0.05%

2

0.1%

3

0.5%

Mobile phase ratio

A mobile phase of 0.1% orthophosphoric acid: methanol in different ratios 15:85, 25:75, 10:90 and 20:80, v/v were tried and the chromatograms were recorded, (fig. 9-12). With the ratio 20:80, v/v, pazufloxacin mesylate eluted at 5.6 min with good peak characteristics. Hence 0.1% orthophosphoric acid: methanol in the ratio of 20:80, v/v was selected as the ideal ratio for successive steps, table 3.

Table 3: Effect of mobile phase ratio

S.No

1% orthophosphoric acid : methanol

Chromatogram

(Fig. 9 - 12)

1

10:90, v/v

2

15:85, v/v

3

20:80, v/v

4

25:75, v/v

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. 13). A flow rate of 0.9 mL/min gave good symmetrical peaks with good peak area response and hence selected, table 4.

Table 4: Effect of flow rate

Flow rate (mL/ min)

Peak area

Retention time(min)

0.9

1908527

5.6

1

1752891

4.8

1.1

1576828

4.0

Fig. 13: A typical chromatogram showing pazufloxacin mesylate at flow rate 0.9mL/min

Fixed chromatographic parameters

Stationary phase : LichroCART, C18 column (250 mm X

4.0 mm, 5 µm)

Mobile phase : 0.1% orthophosphoric acid: methanol

Ratio : 20:80, v/v

Flow rate : 0.9 mL/min

Operating temperature : Room temperature

Detection wavelength : 241nm

VALIDATION OF THE METHOD

Specificity

No additional peaks were found with the alteration in the experimental conditions such as ionic strength, flow rate, ratio etc., which revealed that the developed method was specific for the drugs. Peak purity tests were also done. The peak purity index of pazufloxacin mesylate was found to be 0.999. Peak purity index values close to one proves peak purity of the drug.

Linearity

Adequate dilutions were prepared from the standard stock solution to get a concentration ranging from for 100 - 1000 ng/mL of pazufloxacin mesylate using methanol. Peak areas of these solutions were measured at 241 nm. The calibration curve was plotted using the mean peak area vs. concentration of standard solutions, (fig. 14). From the graph it was found that pazufloxacin mesylate showed the linearity range between 100 - 1000 ng/ml. The calibration data is shown in table 5. The slope, intercept and correlation co-efficient values were found to 179204.71, 6944.68 and 0.999, respectively.

Table 5: Calibration data

Concentration (ng/mL)

Peak area

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100

25480

200

41485

300

62637

500

96512

700

132952

1000

185410

D:\Book\New\Image\Hplc\lnearity1.png

Fig. 14: Calibration graph of pazufloxacin mesylate

(100-1000 ng/mL)

Precision

Repeatability

The working solutions of different concentrations in the range of 100 & 200 ng/mL were injected 3 times and chromatograms were observed for the peak areas and % RSD was calculated, table 6.

Table 6: Repeatability data

Concentration (ng/mL)

% RSD*

100

0.2897

200

0.5789

*RSD of six determinations

Intra-day precision

Intra-day precision was determined by injecting a concentration of standard solutions (100, 300 and 500 ng/mL) for three times on the same day and the response for each injection was measured. Peak areas were noted and %RSD was calculated, table 7.

Table 7: Intra-day precision data

Concentration (ng/mL)

Peak Area(1)

Peak Area(2)

Peak Area(3)

% RSD

100

25480

25672

25581

0.3754

300

62457

62316

62628

0.2501

500

97681

97462

97645

0.1203

Inter-day precision

Inter-day precision was determined by injecting a concentration of the standard solutions (100, 300 and 500 ng/mL) for three days and %RSD was calculated, table 8.

Table 8: Inter-day precision data

Concentration (ng/mL)

Peak Area(1)

Peak Area(2)

Peak Area(3)

% RSD

100

24467

24897

24632

0.8794

300

62368

62181

62551

0.2966

500

97515

97067

97113

0.2534

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 pazufloxacin mesylate were found to be 0.005 ng/mL and 10 ng/mL respectively, fig. 15 & 16.

Fig. 15 Limit of detection

Fig. 16: Limit of quantification

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 reanalysed by the proposed method.

Recovery studies carried out at 80, 100 and 120% levels. The percentage recovery and its %RSD were calculated, table 9.

Table 9: Recovery studies

%Recovery

% RSD*

80%

100%

120%

80%

100%

120%

98.67

100.98

99.84

0.6321

0.5467

0.6849

*RSD of six determinations

Stability

Sample solution of pazufloxacin mesylate 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 8 hours and under refrigeration up to 24 hours.

System suitability parameters

The system suitability parameters were calculated from the standard chromatogram and shown in table 10.

Table 10: System suitability parameters

Tailing factor

Plate count (N)

Resolution

Asymmetric factor

1.23

2841

7.6

1.41

Robustness

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

± 5 units of %B in mobile phase

± 0.2 units in pH of buffer

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

ANALYSIS OF FORMULATION

Preparation of standard solution

Standard stock solution of pazufloxacin mesylate (100 µg/mL) was prepared in methanol. Suitable aliquots of drug solutions were transferred in 10 mL standard flask and diluted with methanol: water (50: 50, v/v) to get concentration ranging from 100 - 1000 ng/mL.

Preparation of sample solution

Volume equivalent to 10 mg of pazufloxacin mesylate was taken from the infusion strength 500 mg/100 mL and transferred to a 100 mL volumetric flask and made up to volume with methanol (100 µg/mL). From the above solution, suitable aliquot of formulation solution was prepared in methanol: water (50: 50, v/v).

Recording of chromatograms

A steady baseline was recorded with the fixed chromatographic conditions, standard and sample drug solutions were injected and chromatograms were recorded, (fig. 17-23). Retention time was found to be 5.6 minutes for pazufloxacin mesylate. This was followed by injection of sample solution obtained from the formulation, fig. 24.

The results of formulation analysis are given in table 11.

Table 11: Analysis of formulation

Formulation

Amount of drug (mg/infusion)

% label claim

% RSD*

Labeled

Estimated

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500

499.84

99.24

0.6382

*RSD of six determinations

CHROMATOGRAM OF STANDARDS

Fig. 17: Pazufloxacin mesylate - 100 ng/mL

Fig. 18: Pazufloxacin mesylate - 200 ng/mL

Fig. 19: Pazufloxacin mesylate - 300 ng/mL

Fig. 20: Pazufloxacin mesylate - 500 ng/mL

Fig. 21: Pazufloxacin mesylate - 700 ng/mL

Fig. 22: Pazufloxacin mesylate - 900 ng/mL

Fig. 23: Pazufloxacin mesylate - 1000 ng/mL

Fig. 24: Chromatogram of formulation (700 ng/mL)

FORCED DEGRADATION STUDIES OF PAZUFLOXACIN MESYLATE IN PHARMACEUTICAL DOSAGE FORM

The chromatographic method developed above was utilized for stability indicating assay of pazufloxacin mesylate. The fixed chromatographic conditions followed are given below,

Fixed chromatographic parameters

Stationary phase : LichroCART, C18 column (250 mm X

4.0 mm, 5 µm)

Mobile phase : 0.1% orthophosphoric acid: methanol

Ratio : 20:80%, v/v

Flow rate : 0.9mL/min

Operating temperature : Room temperature

Detection wavelength : 241nm

Procedure

The stress testing was conducted as per ICH guidelines. Forced degradation for the drug substance was carried out under acid/ base/ neutral hydrolysis, photolytic, oxidative stress conditions 28-30.

Drug at a concentration of 1 mg/mL was used in all degradation studies.

In each study, blank and control (0 hrs sample) were used to compare and calculate the percentage degradation.

There were 4 samples prepared in each stress test 31,

Blank solution stored under normal condition

Blank solution subjected to stress like the drug

Zero time sample containing the drug which is stored under normal condition (control) and

Drug solution subjected to stress.

HYDROLYTIC STUDIES

Acidic condition

Solution was prepared by dissolving the drug substance in water and the drug was subjected to accelerated degradation under acidic condition by refluxing with 0.1N HCL at 70°C and the sampling was done at every half an hour till sufficient degradation was achieved. The resulting solution was neutralized, appropriately diluted and chromatograms were recorded.

Alkaline condition

Drug was subjected to accelerated degradation under alkaline condition by refluxing with 0.1N NaOH at 70°C and the sampling was done at every half an hour till sufficient degradation was achieved. The resulting solution was neutralized, appropriately diluted and chromatograms were recorded.

Oxidative studies

Initial oxidation was performed using 3% H2O2 at room temperature for 10 hours, subsequently the drug was exposed to 30% H2O2 at room temperature and analyzed periodically. The resulting solution was appropriately diluted and chromatograms were recorded.

Neutral condition

For force degradation study in neutral condition, drug dissolved in water was heated at 80° C; samples were withdrawn at appropriate time intervals and subjected to HPLC analysis after suitable dilutions.

Photolytic studies

For photolytic stress studies, the drug product was exposed to artificial light under laboratory condition for 10.5 hours. Samples were withdrawn at appropriate time intervals and subjected to HPLC analysis after suitable dilutions.

Appropriates controls were also prepared and injected for each degradation studies.

RESULTS AND DISCUSSION

HPLC studies of samples obtained on stress testing of pazufloxacin mesylate under different conditions using 0.1% orthophosphoric acid: methanol (20: 80, v/v) as mobile solvent system suggested the following degradation behaviour.

A chromatogram of zero time sample containing the drug stored under normal condition is shown in, fig. 25.

Hydrolytic studies

Acidic conditions

It was observed that around 27% of the drug degraded on heating it in 0.1 N HCL for 7 hours at 70° C forming degradation product at retention time at 3.4 minutes, (fig. 26). Rate of hydrolysis was faster when compared to that of alkali and water.

Alkaline condition

For alkaline hydrolysis it was found that 25% of the drug degraded on heating it in 0.1N NaOH for 9 hours at 70° C forming degradation product at retention time at 3.4 minutes fig. 27.

Neutral condition

In neutral stress condition, about 25% of the degradation of the drug was achieved after heating the drug at 80° C for 10.5 hours forming degradation product at retention time at 3.4 minutes fig. 28.

Oxidative studies

In oxidative stress condition, about 22% degradation of drug was obtained after exposure to 30% H2O2 for 9 hours, fig. 29.

Photolytic studies

Here sufficient degradation was achieved by exposing the drug product to artificial light under laboratory condition for 10.5 hours, fig. 30.

In degradation studies except, acid hydrolysis, alkaline hydrolysis and neutral hydrolysis there was no corresponding formation of degradation products when compared to the zero time sample solution of the drug. This indicated that, may be the drug degraded to low molecular weight non-chromophoric compounds. The drug showed extensive degradation in acid hydrolytic condition. The blank chromatogram of all stress test were studied, its show there is no interference between the drug and stress solutions, fig. 31-35. Chromatographic peak purity data was obtained from the spectral analysis report and peak purity index value of 0.999 indicated a homogeneous peak thus establishing the specificity of the assay method. Hence, the developed RP-HPLC method was stability indicating, rapid, simple, specific, accurate and precise and can be employed successfully for the determination of pazufloxacin mesylate in presence of degradant products.

Chromatograms of forced degradation samples of pazufloxacin mesylate

Fig. 25: Zero time sample containing pazufloxacin mesylate (10µg/mL)

Fig. 26: Sample subjected to acid hydrolysis

Fig. 27: Sample subjected to alkali hydrolysis

Fig. 28: Sample subjected to neutral condition

Fig. 29: Sample subjected to oxidative degradation

Fig. 30: Sample subjected to photolytic degradation

CHROMATOGRAM OF BLANK UNDER NORMAL CONDITION

Fig. 31: Acid blank

Fig. 32: Base blank

Fig. 33: Oxidative blank

CHROMATOGRAM OF BLANK UNDER STRESS CONDITION (LIKE DRUG)

Fig. 34: Acid blank

Fig. 35: Base blank