Development And Validation Of RP HPLC Method Biology Essay

The chemical nature of the sample provides valuable information for LC separation. The nature of the sample i.e. ionic, non-ionic or impersonal, its molecular weight and solubility plays a major function in the choice of the method. Since the drug, pazufloxacin mesylate is polar in nature, change by reversal stage chromatography was selected for separation.

Choice of wavelength

Good analytical consequences will be obtained merely by careful choice of wavelength used for sensing. This pick requires cognition of soaking up spectra of the sample. An ultraviolet spectrum of pazufloxacin mesylate was recorded and it was found the drug showed maximal optical density at I»max of 241 nanometers and 330 nanometer, ( fig. 1 ) . A wavelength of 241 nanometer was selected as it gave good response.

Fig. 1: Ultraviolet spectrum of pazufloxacin mesylate

Optimization of chromatographic conditions

Choice of nomadic stage

Solvent selectivity ( solvent type ) , solvent strength ( per centum of organic dissolver in the nomadic stage ) , strength of buffer, flow rate etc. were optimized to acquire the chromatographic conditions, that gave the best separation, table 1.

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Initial chromatographic conditions for the separation

Column: Merck – LichroCART, C18 column ( 250 millimeter X 4.0 millimeter, 5 Aµm )

Detection wavelength: 241 nanometer

Flow rate: 1 mL/min

Operating temperature: Room temperature

Mobile stage: 0.1 % orthophosphoric acid: methyl alcohol

For developing LC method, different nomadic stages with different ratios were tried, table 1 & A ; fig. 2-5.

Table 1: Choice of nomadic stage

S.No

Mobile stages

Chromatogram

( Fig. 2 – 5 )

1

Water: methyl alcohol

( 20: 80, v/v )

2

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

3

0.1 % Acetic acid: methyl alcohol

( 20: 80, v/v )

4

0.1 % Orthophosphoric acid: methyl alcohol ( 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: methyl alcohol showed symmetrical extremum with good separation, ( fig. 6-8 ) . Hence 0.1 % was selected as the strength of formic acid for separation, table 2.

Table 2: Consequence of ionic strength

S.No

Ionic strength of

orthophosphoric acid

Chromatogram

( Fig. 6 – 8 )

1

0.05 %

2

0.1 %

3

0.5 %

Mobile stage ratio

A nomadic stage of 0.1 % orthophosphoric acid: methyl alcohol 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 extremum features. Therefore 0.1 % orthophosphoric acid: methyl alcohol in the ratio of 20:80, v/v was selected as the ideal ratio for consecutive stairss, table 3.

Table 3: Consequence of nomadic stage ratio

S.No

1 % orthophosphoric acid: methyl alcohol

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 parametric quantities of nomadic stage system changeless, 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 extremums with good peak country response and therefore selected, table 4.

Table 4: Consequence of flow rate

Flow rate ( mL/ min )

Peak country

Retention clip ( min )

0.9

1908527

5.6

1

1752891

4.8

1.1

1576828

4.0

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

Fixed chromatographic parametric quantities

Stationary stage: LichroCART, C18 column ( 250 millimeter Ten

4.0 millimeter, 5 Aµm )

Mobile stage: 0.1 % orthophosphoric acid: methyl alcohol

Ratio: 20:80, v/v

Flow rate: 0.9 mL/min

Operating temperature: Room temperature

Detection wavelength: 241nm

Validation OF THE METHOD

Specificity

No extra extremums were found with the change in the experimental conditions such as ionic strength, flow rate, ratio etc. , which revealed that the developed method was specific for the drugs. Peak pureness trials were besides done. The peak pureness index of pazufloxacin mesylate was found to be 0.999. Peak pureness index values near to one proves peak pureness of the drug.

One-dimensionality

Adequate dilutions were prepared from the standard stock solution to acquire a concentration runing from for 100 – 1000 ng/mL of pazufloxacin mesylate utilizing methyl alcohol. Peak countries of these solutions were measured at 241 nanometer. The standardization curve was plotted utilizing the average peak country vs. concentration of standard solutions, ( fig. 14 ) . From the graph it was found that pazufloxacin mesylate showed the one-dimensionality scope between 100 – 1000 ng/ml. The standardization informations is shown in table 5. The incline, intercept and correlativity co-efficient values were found to 179204.71, 6944.68 and 0.999, severally.

Table 5: Calibration informations

Concentration ( ng/mL )

Peak country

100

25480

200

41485

300

62637

500

96512

700

132952

1000

185410

Calciferol: BookNewImageHplclnearity1.png

Fig. 14: Calibration graph of pazufloxacin mesylate

( 100-1000 ng/mL )

Preciseness

Repeatability

The on the job solutions of different concentrations in the scope of 100 & A ; 200 ng/mL were injected 3 times and chromatograms were observed for the peak countries and % RSD was calculated, table 6.

Table 6: Repeatability informations

Concentration ( ng/mL )

% RSD*

100

0.2897

200

0.5789

*RSD of six findings

Intra-day preciseness

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

Table 7: Intra-day preciseness informations

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 preciseness

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

Table 8: Inter-day preciseness informations

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 sensing ( LOD ) and bound of quantification ( LOQ )

LOD and LOQ were determined by shooting increasingly lower concentrations of the drug. LOD and LOQ of pazufloxacin mesylate were found to be 0.005 ng/mL and 10 ng/mL severally, fig. 15 & A ; 16.

Fig. 15 Limit of sensing

Fig. 16: Limit of quantification

Accuracy

Recovery surveies were done for finding truth parametric quantity. It was done by blending known measure of standard drug with the analysed sample preparation and the contents were reanalysed by the proposed method.

Recovery surveies carried out at 80, 100 and 120 % degrees. The per centum recovery and its % RSD were calculated, table 9.

Table 9: Recovery surveies

% Recovery

% RSD*

80 %

100 %

120 %

80 %

100 %

120 %

98.67

100.98

99.84

0.6321

0.5467

0.6849

*RSD of six findings

Stability

Sample solution of pazufloxacin mesylate was subjected to stableness surveies under refrigerated and room conditions. Stabilities were studied by looking for any alteration in keeping clip, declaration, peak form, etc. when compared to chromatogram of newly prepared solution. The solution stored under room temperature was stable up to 8 hours and under infrigidation up to 24 hours.

System suitableness parametric quantities

The system suitableness parametric quantities were calculated from the standard chromatogram and shown in table 10.

Table 10: System suitableness parametric quantities

Chasing factor

Plate count ( N )

Resolution

Asymmetric factor

1.23

2841

7.6

1.41

Robustness

In order to show the hardiness of the method, the following optimized conditions were somewhat varied.

A± 5 units of % B in nomadic stage

A± 0.2 units in pH of buffer

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

Analysis OF FORMULATION

Preparation of standard solution

Standard stock solution of pazufloxacin mesylate ( 100 Aµg/mL ) was prepared in methyl alcohol. Suitable aliquots of drug solutions were transferred in 10 mL criterion flask and diluted with methyl alcohol: H2O ( 50: 50, v/v ) to acquire concentration runing from 100 – 1000 ng/mL.

Preparation of sample solution

Volume equivalent to 10 milligram of pazufloxacin mesylate was taken from the extract strength 500 mg/100 milliliter and transferred to a 100 milliliter volumetric flask and made up to volume with methyl alcohol ( 100 Aµg/mL ) . From the above solution, suited aliquot of preparation solution was prepared in methyl alcohol: H2O ( 50: 50, v/v ) .

Recording of chromatograms

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

The consequences of preparation analysis are given in table 11.

Table 11: Analysis of preparation

Formulation

Amount of drug ( mg/infusion )

% label claim

% RSD*

Labeled

Estimated

Pazubid ( Pazufloxacin mesylate )

500

499.84

99.24

0.6382

*RSD of six findings

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 preparation ( 700 ng/mL )

FORCED DEGRADATION STUDIES OF PAZUFLOXACIN MESYLATE IN PHARMACEUTICAL DOSAGE FORM

The chromatographic method developed above was utilized for stableness bespeaking check of pazufloxacin mesylate. The fixed chromatographic conditions followed are given below,

Fixed chromatographic parametric quantities

Stationary stage: LichroCART, C18 column ( 250 millimeter Ten

4.0 millimeter, 5 Aµm )

Mobile stage: 0.1 % orthophosphoric acid: methyl alcohol

Ratio: 20:80 % , v/v

Flow rate: 0.9mL/min

Operating temperature: Room temperature

Detection wavelength: 241nm

Procedure

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

Drug at a concentration of 1 mg/mL was used in all debasement surveies.

In each survey, clean and control ( 0 hrs sample ) were used to compare and cipher the per centum debasement.

There were 4 samples prepared in each emphasis trial 31,

Blank solution stored under normal status

Blank solution subjected to emphasize like the drug

Zero clip sample incorporating the drug which is stored under normal status ( control ) and

Drug solution subjected to emphasize.

HYDROLYTIC STUDIES

Acidic status

Solution was prepared by fade outing the drug substance in H2O and the drug was subjected to accelerated debasement under acidic status by refluxing with 0.1N HCL at 70°C and the sampling was done at every half an hr boulder clay sufficient debasement was achieved. The ensuing solution was neutralized, suitably diluted and chromatograms were recorded.

Alkaline status

Drug was subjected to accelerated debasement under alkalic status by refluxing with 0.1N NaOH at 70°C and the sampling was done at every half an hr boulder clay sufficient debasement was achieved. The ensuing solution was neutralized, suitably diluted and chromatograms were recorded.

Oxidative surveies

Initial oxidization was performed utilizing 3 % H2O2 at room temperature for 10 hours, later the drug was exposed to 30 % H2O2 at room temperature and analyzed sporadically. The ensuing solution was suitably diluted and chromatograms were recorded.

Impersonal status

For force debasement survey in impersonal status, drug dissolved in H2O was heated at 80° C ; samples were withdrawn at appropriate clip intervals and subjected to HPLC analysis after suited dilutions.

Photolytic surveies

For photolytic emphasis surveies, the drug merchandise was exposed to unreal visible radiation under laboratory status for 10.5 hours. Samples were withdrawn at appropriate clip intervals and subjected to HPLC analysis after suited dilutions.

Appropriates controls were besides prepared and injected for each debasement surveies.

RESULTS AND DISCUSSION

HPLC surveies of samples obtained on stress testing of pazufloxacin mesylate under different conditions utilizing 0.1 % orthophosphoric acid: methyl alcohol ( 20: 80, v/v ) as nomadic dissolver system suggested the undermentioned debasement behavior.

A chromatogram of nothing clip sample incorporating the drug stored under normal status is shown in, fig. 25.

Hydrolytic surveies

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 organizing debasement merchandise at keeping clip at 3.4 proceedingss, ( fig. 26 ) . Rate of hydrolysis was faster when compared to that of base and H2O.

Alkaline status

For alkalic hydrolysis it was found that 25 % of the drug degraded on heating it in 0.1N NaOH for 9 hours at 70° C organizing debasement merchandise at keeping clip at 3.4 proceedingss fig. 27.

Impersonal status

In impersonal emphasis status, approximately 25 % of the debasement of the drug was achieved after heating the drug at 80° C for 10.5 hours organizing debasement merchandise at keeping clip at 3.4 proceedingss fig. 28.

Oxidative surveies

In oxidative emphasis status, approximately 22 % debasement of drug was obtained after exposure to 30 % H2O2 for 9 hours, fig. 29.

Photolytic surveies

Here sufficient debasement was achieved by exposing the drug merchandise to unreal visible radiation under laboratory status for 10.5 hours, fig. 30.

In debasement surveies except, acerb hydrolysis, alkalic hydrolysis and impersonal hydrolysis there was no corresponding formation of debasement merchandises when compared to the nothing clip sample solution of the drug. This indicated that, may be the drug degraded to moo molecular weight non-chromophoric compounds. The drug showed extended debasement in acerb hydrolytic status. The clean chromatogram of all stress trial were studied, its show there is no intervention between the drug and emphasis solutions, fig. 31-35. Chromatographic peak pureness informations was obtained from the spectral analysis study and peak pureness index value of 0.999 indicated a homogenous extremum therefore set uping the specificity of the check method. Hence, the developed RP-HPLC method was stableness indicating, rapid, simple, specific, accurate and precise and can be employed successfully for the finding of pazufloxacin mesylate in presence of degradant merchandises.

Chromatograms of forced debasement samples of pazufloxacin mesylate

Fig. 25: Zero clip sample incorporating pazufloxacin mesylate ( 10Aµg/mL )

Fig. 26: Sample subjected to acid hydrolysis

Fig. 27: Sample subjected to alkali hydrolysis

Fig. 28: Sample subjected to impersonal status

Fig. 29: Sample subjected to oxidative debasement

Fig. 30: Sample subjected to photolytic debasement

CHROMATOGRAM OF BLANK UNDER NORMAL CONDITION

Fig. 31: Acid blank

Fig. 32: Base space

Fig. 33: Oxidative space

CHROMATOGRAM OF BLANK UNDER STRESS CONDITION ( LIKE DRUG )

Fig. 34: Acid blank

Fig. 35: Base space