Development and Validation of Stability Indicating RP-HPLC Method for Quantitative Estimation of Ornidazole and its Impurities in Ornidazole Injection

 

N. Prasad Babu1, D. Ramachandran2

1Department of Chemistry, V.S.R Government Degree and P.G. College, Movva, Andhra Pradesh, India.

2Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, Andhra Pradesh, India.

*Corresponding Author E-mail: chemistryprasad.n@gmail.com

 

ABSTRACT:

The analysis of improved RP-HPLC method for the separation and quantification of Ornidazole and its impurities are described. Samples are analysed by means of reverse phase (RP-HPLC) using an Symmetry shield RP18, 250 x 4.6 mm, 5m, and the mobile phase consists of phosphate buffer: methanol in the ratio of 70:30 %v/v. The flow rate is 1.0 ml/min. The column temperature was maintained at 25C and sample temperature was maintained at ambient (25C) and wavelength fixed at 318nm UV-detection. It is found that the method of RP-HPLC with UV-detection system for the analysis of Ornidazole impurities are straight forward and applied in qualitative and quantitative analysis. The developed LC method was validated with respect to specificity, precision, linearity, accuracy, solution stability and robustness. Validation study compared as per ICH guideline.

 

KEYWORDS: Ornidazole injection, Estimation of related substances, Liquid chromatography, Validation.

 

 


1.0 INTRODUCTION:

Ornidazole, a 5-nitroimidazole is used in the treatment of protozoal infections and also in the treatment and prophylaxis of anaerobic infections. It has been investigated for use in Crohn's disease after bowel resection. Chemically, it is 1-chloro-3-(2-methyl-5-nitro-1 H-imidazol-1-yl propan-2-ol. It is used either as monotherapy or in combination with cephalosporins and in fluoroquinolone antibiotics1-6. Molecular formula is C7H10ClN3O3 and Molecular weight 219.63 g/mol. The chemical structure of Ornidazole shown in (Fig. 1.1).

 

Figure 1.1: Chemical Structure of Ornidazole

 

1.1 Impurity structure:

 

Figure 1.2: Chemical structure of 2-methyl 5-nitro imidazole

 

A few analytical methods have been reported for the determination of Ornidazole in pure drug, pharmaceutical dosage forms and biological samples using spectrophotometry7,8, Ornidazole in its various drug combinations have been estimated using UV-Spectrophotometry9-16 high performance liquid chromatography17,18, high performance thin layer chromatography18,19. The objective of the present work is to develop a stability indicating HPLC method and validated as per ICH guidelines20 for the estimation of Ornidazolein applied for routine analysis in laboratories and is suitable for the quality control of the raw materials.

 

2.0 EXPERIMENTAL:

2.1 Reagents and chemicals:

Potassium dihydrogen orthophosphate, Methanol was acquired from Merck. Water (Milli-Q).

2.2 Instrumentation:

Chromatographic separation was achieved by using an Agilent-1200, Open-lab software using, Symmetry shield RP18, 250x4.6mm, 5m column and the movable segment consists of phosphate buffer: methanol (70:30 %v/v). The flow rate is 1.0 ml/min. The column temperature was maintained at 25C and sample temperature was maintained at ambient (25C) and wavelength fixed at 318nm UV-detection. The overall run time was 30 minutes. 20 l of test was inserted into the HPLC system. Retention epochs of impurity 4.24 minutes for 2-methyl 5-nitro imidazole and 11.25 minutes for Ornidazole.

 

2.3 Preparation of mobile phase and standard and sample solution:

Preparation of Buffer:

Weighed accurately 1.36g of potassium dihydrogen orthophosphate transferred in to 1000mL beaker dissolved and diluted to volume with 1000mL milli-Q or HPLC grade water. Filter through 0.45μ membrane filter and degas.

 

Mobile phase: Transferred 700mL of buffer and 300mL of methanol into 1000mL beaker mixed well. Filter through 0.45μ membrane filter and degas.

 

Diluent preparation: Mixed buffer and methanol in the proportion of (70:30 v/v).

 

Preparation of Standard stock solution: Accurately weighed and transferred 10mg of Ornidazole standard in to 10mL volumetric flask, added 5ml of diluent sonicated to dissolve. Then volume is made up to the mark with diluent.

 

Standard solution preparation: Taken 5 ml of standard stock solution in to 100 ml volumetric flask and diluted to the mark with diluent. Further diluted 5 ml of this solution in to 50 ml volumetric flask and made up to the mark with diluent.

 

Preparation of Sensitivity solution: Diluted 5 ml of the standard solution to 100 ml with diluent and mixed well.

 

Preparation of System suitability solution: Accurately Weighed and transferred 5.0mg of 2-methyl 5-nitro imidazole into a 100 ml volumetric flask, added 50 ml diluent sonicated to dissolved, then volume is made up to the mark with diluent.

 

Further transferred 5 ml of the above solution in to 50 ml volumetric flask containing 25 ml diluent and then added 5 ml of standard stock solution, volume is made up to the mark with diluent.

 

Preparation of Placebo solution: Transferred 5 ml of the placebo solution into a 50ml volumetric flask and diluted to the volume with diluent and mixed well.

 

Preparation of Test solution: Transferred 5 ml of the sample solution into a 50ml volumetric flask and diluted to the volume with diluent and mixed well.

 

3.0 RESULTS AND DISCUSSION:

3.1 Method optimization parameters:

An understanding of the nature of API (functionality, acidity, or basicity), the synthetic process, related impurities, the possible degradation pathways and their degradation products are needed for successful method development in reverse-phase HPLC. In addition, successful method development should result a robust, simple and time efficient method that is capable of being utilized in manufacturing setting.

 

3.2 Selection of wavelength:

The sensitivity of the HPLC method depends upon the selection of detection wavelength. An ideal wavelength is one that gives good response for related substances and the drugs to be detected. The wavelength for measurement was selected as 318 nm from the absorption spectrum.

 

3.3. Selection of stationary phase:

Proper selection of the stationary phase depends up on the nature of the sample and chemical profile. The drug selected for the present study was polar compound and could be separated either by normal phase chromatography or reverse phase chromatography. From literature survey, it was found that different C18 columns could be appropriately used for the separation of related substances for Ornidazole.

 

3.4. Selection of movable segment:

Different movable segment and immobile segments were employed to develop a appropriate LC technique for the quantitative fortitude of impurities in Ornidazole. A number of column chemistries supplied by different manufacturers and different mobile phase composition were tried to get good peak shapes and selectivity for the impurities present in Ornidazole.

 

Poor peak shape and resolution was observed when Hypersil BDS C18 (250mm x 4.6mm, 5). There was no suitable resolution of impurities and analyte crest and competence of the crest is also not attained and crest meddling are there.

 

In second effort through by Symmetry shield RP18, 250x4.6 mm, 5m column and the mobile phase consists of phosphate buffer: methanol (70:30 %v/v). The resolution of both drug and impurities was achieved. These chromatographic conditions were selected for validation studies.

 

4.0 METHOD VALIDATION:

4.1 Specificity and System suitability:

Blank interference:

Blank was prepared and injected as per test method. It was observed that no blank peaks were interfering with analytical peaks.

 

Placebo interference:

Placebo solutions were prepared in duplicate and injected as per test method. It was observed that no placebo peaks were interfering with analytical peaks.

 

Figure 1.3: typical chromatogram of Blank

 

Figure 1.4: Typical chromatogram of Placebo

 

Figure 1.5: typical chromatogram of impurity

 

Figure 1.6: typical chromatogram of System suitability solution

 

Table 1.1: Impurity interference data

Peak Name

Retention Time

Blank

Placebo

2-methyl 5-nitro imidazole

4.65

No interference

No interference

Ornidazole

11.25

No interference

No interference

 

It was scrutinized that identified impurities are not co-eluting with apiece other and main analyte crest. Ornidazole standard solution preparation and in spiked test preparation was calculated and found to be within the acceptable limit.

 


Table 1.2: Results of system suitability solution

Component Name

Retention Time

Relative retention time (RRT)

Resolution

Asymmetry

Theoretical plates

2-methyl 5-nitro imidazole

4.64

0.41

0

1.1

12559

Ornidazole

11.25

1.0

23.9

1.0

13532

 


Force Degradation studies:

Table 1.3: Forced degradation results

Degradation condition

% Degradation

% Net Degradation

Unstressed Sample

0.025

-

0.1 N HCl / 60C for 2 hours

0.024

0.001

0.005N NaOH / 60C for 1 hours

3.461

3.436

Thermal 105C for 48 hours

0.709

0.684

3% H2O2/ 60C for 30 min

0.32

0.295

UV Light at 254nm for 7 days

0.034

0.009

Significant degradation was observed in alkali stress condition. Hence it can be concluded that Ornidazole injection 5mg/ml is sensitive to alkali.

 

4.2 Precision:

4.2.1 System Precision:

Perform the study of reference solution (Diluted standard) six times and determine the %RSD of crest vicinity of replicate injections of Ornidazole.

 

Table 1.4: System Precision data for Ornidazole

Injection No

Ornidazole

1

4147632

2

4165479

3

4157861

4

4159480

5

4153268

6

4145690

Mean area

4154902

%RSD

0.18

 

The %RSD of peak area for Ornidazole was found to be 0.18% which is below 10.0% indicates that the system gives precise result.

 

4.2.2 Method Precision

Exactitude of the impurities and degradants method was concluded by injecting six sample solutions spiked with impurity (2-methyl 5-nitro imidazole) at specification level. The samples were arranged as per the technique and the result for exactitude study is tabulated in Table 1.5.

 

Table 1.5: Results of method precision

Inj.

No

2-methyl

5-nitro imidazole

Maximum Unknown impurity

Total impurities

1

1.327

0.011

1.346

2

1.320

0.011

1.389

3

1.322

0.011

1.342

4

1.318

0.011

1.337

5

1.322

0.011

1.341

6

1.317

0.011

1.336

Mean

1.321

0.011

1.349

SD

0.004

0.000

0.020

% RSD

0.27

0.000

1.50

 

The %RSD of recovery for known impurities of spiked samples within the acceptance criteria.

 

The %RSD of total impurities and single maximum impurity within the getting criterion.


 

Table 1.7: LOQ for Ornidazole and impurities

Component

Inj-1

Inj-2

Inj-3

Inj-4

Inj-5

Inj-6

Avg.

%RSD

Ornidazole

146143

144692

155020

149433

155712

141176

148696

3.91

2-methyl 5-nitro imidazole

182587

183356

179529

188630

185294

180728

183354

1.79

 


4.3 Limit of detection (LOD):

A solution containing 0.0102 g/ml of Ornidazole standard and 0.0113 g/ml of impurity (2-methyl 5-nitro imidazole). The results are presented in the table 1.6.

 

Table: 1.6 LOD for Ornidazole and impurities

Name

Area

S/N

Ornidazole

45060

4.01

Impurity (2-methyl 5-nitro imidazole)

55565

3.90

 

4.4 Limit of Quantitation (LOQ):

A solution containing 0.0308 g/ml of Ornidazole standard and 0.0343 g/ml of impurity (2-methyl 5-nitro imidazole) was injected six times. The results are presented in the table 1.7.

 

Figure 1.7: typical chromatogram of LOQ solution

 

The limit of quantitation and detection of quantitation assessments acquired for impurity and Ornidazole are within the getting criterion.

 

4.5 Linearity and Range:

The linearity is determined by injecting the solutions in duplicate containing known impurities and Ornidazole and impurities ranging from LOQ to 150% of the specified limit. Results obtained are in the tables and figures show the line of best fit for peak area versus concentration for impurity.

 

Table: 1.8 Linearity of detector response Impurity

Level (%)

Concentration (ppm)

Mean Area

LOQ

0.034

172290

25

1.350

6778341

50

2.701

13577996

75

3.967

20068766

100

5.233

26548139

150

8.102

40943545

Correlation coefficient

0.9999

R2 Value

1.0000

% Y-intercept

-0.10

Slope

5061626.89

Intercept

-27322.87

 

Figure 1.8: linearity of detector response for Impurity (2-methyl 5-nitro imidazole)

 

Table: 1.9 Linearity of detector response Ornidazole

Level (%)

Concentration (ppm)

Mean Area

LOQ

0.034

138993

25

1.350

5214454

50

2.701

10483252

75

3.967

15500876

100

5.233

20480771

150

8.102

31595929

Correlation coefficient

0.9999

R2 Value

1.0000

% Y-intercept

-0.10

Slope

3906164.95

Intercept

-21145.80

 

Figure: 1.9 linearity of detector response for Ornidazole

 

The linearity results for Ornidazole and impurities in the specified concentration range are found satisfactory.

 

4.6 Accuracy:

Recovery of Ornidazole impurities in Ornidazole was performed. The sample was taken and varying amounts of Ornidazole impurities representing LOQ to 150 % of specification level were added to the flasks. The spiked samples were prepared as per the method and the results are tabulated in Table 1.10 and 1.11.

 

Table 1.10: Accuracy study of Ornidazole

S.No.

Accuracy

level (%)

Ornidazole

% Mean Recovery

%RSD

1

LOQ

107.2

4.72

3

50

101.1

0.11

3

100

102.0

0.11

4

150

105.5

0.44

 

Table: 1.11 Accuracy study of Impurity (2-methyl 5-nitro imidazole)

S. No.

Accuracy level (%)

Impurity (2-methyl 5-nitro imidazole)

%Mean Recovery

% RSD

1

LOQ

104.9

1.32

3

50

99.6

0.32

3

100

100.6

0.86

4

150

103.9

0.47

 

4.7 Solution stability of analytical solutions:

Standard and sample solutions were kept for about 24 hrs at room temperature in transparent bottles in auto sampler and in refrigerator 2-8C. The stability of standard and sample solutions was determined by comparison of old prepared standard solutions with freshly prepared standard solutions.

 

Table: 1.12 Results for solution stability of standard

Time Interval

Similarity factor

Room temperature

Refrigerator

Initial

NA

NA

12hrs

0.98

0.99

24hrs

0.98

0.99

 

Table: 1.13 Results for solution stability of test solution at room temperature

Component

Initial Study

After 24Hrs

% Difference

2-methyl 5-nitro imidazole

1.321

1.323

0.2

Maximum Unknown impurity

0.011

0.012

0.1

Total impurities

1.349

1.353

0.4

 

Table 1.14: Results for solution stability of test solution at refrigerator

Component

Initial Study

After 24Hrs

% Difference

2-methyl 5-nitro imidazole

1.321

1.322

0.1

Maximum Unknown impurity

0.011

0.013

0.2

Total impurities

1.349

1.352

0.3

 

4.8 Robustness:

To validate the method robustness the chromatographic performance at changed conditions was evaluated compared to nominal conditions of the method. System suitability solution was injected at each of the following changed conditions:


 

Table 1.15: Results of Robustness

Parameter

Altered condition

Resolution

Asymmetry

Theoretical plates

%RSD of areas

Mobile phase variation in the ratio 70:30 v/v

67: 33 v/v

20.6

1

12768

0.1

73: 27 v/v

25.1

1

13736

0.23

Flow variation 1.0 mL/min

0.8 mL/min

23.4

1

14521

0.29

1.2 mL/min

23.1

1

12588

0.15

Temperature variation (30C)

20C

22.7

1

12824

0.12

30C

23.9

1

14313

0.16

 

 


5.0 RESULTS AND DISCUSSION:

A simple, economic, accurate and precise HPLC method was successfully developed. In this method it was carried out by using Symmetry shield RP18, 250 x 4.6 mm, 5m. Injection volume of 20μl is injected and eluted with the mobile phase phosphate buffer: methanol (700:300 %v/v), which is pumped at a flow rate of 1.0 ml/min. Column temperature 25C and sample temperature 25C. Detection was carried out at 318 nm. The results obtained were accurate and reproducible. The method developed was statistically validated in terms of Selectivity, accuracy, linearity, precision, LOD and LOQ, robustness, and stability of solution.

 

For Selectivity, the chromatograms were recorded for standard and sample solutions of Ornidazole and its related substances. Selectivity studies reveal that the peak is well separated from each other. Therefore the method is selective for the determination of related substances in Ornidazole. There is no interference of diluent at Ornidazole and impurities peaks. The elution order and the retention times of Impurities and Ornidazole obtained from individual standard preparations and mixed standard Preparations are comparable.

 

The limit of detection (LOD) and limit of quantitation (LOQ) for Ornidazole standard 0.0102 and 0.0308 g/mL and impurity-C 0.0113 and 0.0343 g/mL respectively. The linearity results for Ornidazole and the impurities in the specified concentration range are found satisfactory, with a correlation coefficient greater than 0.99.Calibration curve was plotted and correlation co-efficient for Ornidazole and its impurity found to be 0.9999 and 0.9999 respectively.

 

The accuracy studies were shown as % recovery for Ornidazole and its impurities at specification level. The limit of % recovered shown is in the range of 90 and 110% and the results obtained were found to be within the limits. Hence the method was found to be accurate.

 

The relative standard deviation values of recoveries obtained for impurity are in the range of 0.32%-1.32%

 

For exactitude studies six (6) repeat injections were executed. %RSD was concluded from the crest vicinity of Ornidazole and its impurity. The results were found to be within the acceptance limits.

 

The robustness of the method was checked by varying flow rate, mobile phase composition and temperature and found that the system suitability parameters were within the limit at all variable conditions, hence the method was robust.

 

The stability indicating method was checked by subjecting the drug to stress conditions like acidic, basic, oxidative, photolytic and thermal degradation and found that the degradation peaks were well separated from the sample peak and % net degradation was within the limit.

 

Hence, the chromatographic method developed for Ornidazole and its related substances are rapid, simple, sensitive, precise, and accurate. Therefore, the proposed method can be successfully applied for the routine analysis of the active pharmaceutical ingredients for assurance of its quality during its formulation.

 

6.0. CONCLUSION:

The new HPLC method developed and validated for determination of Ornidazole pharmaceutical dosage forms and assured the satisfactory precision and accuracy and also determining lower concentration of drug in its injection formulation form by RP-HPLC method. The method was found to be simple, accurate, economical and rapid and they can be applied for routine analysis in laboratories and is suitable for the quality control of the raw materials.

 

7.0. ACKNOWLEDGMENT:

The authors are grateful to Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur. Andhra Pradesh, India, for providing facilities to carry this research work.

 

8.0. CONFLICT OF INTERESTS:

The authors claim that there is no conflict of interest.

 

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Received on 22.11.2020 Modified on 17.02.2021

Accepted on 21.03.2021 RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(1):82-88.

DOI: 10.52711/0974-360X.2022.00015