Method Development and Validation for Simultaneous Estimation of Omeprazole and Ofloxacin by using RP-HPLC

 

Ganesh Akula1*, P. Rajashekar1, Rangu Nirmala2, K. Kanakaiah3, Dr. A. Jaswanth4

1Department of Pharmaceutical Chemistry, Surabhi Dayakar Rao College of Pharmacy, Rimmanaguda, Gajwel, Siddipet, Telangana-502312.

2Department of Pharmaceutics, Surabhi Dayakar Rao College of Pharmacy, Rimmanaguda, Gajwel, Siddipet, Telangana.

3Department of Pharmacology, Surabhi Dayakar Rao College of Pharmacy, Rimmanaguda, Gajwel, Siddipet, Telangana.

*Corresponding Author E-mail: akulaganesh@gmail.com

 

ABSTRACT:

A simple, Accurate, precise Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) method was developed for the simultaneous estimation of the omeprazole and Ofloxacin in Tablet dosage form. Chromatogram was run through ODS (150X4.6mm, 5µ). Mobile phase containing Buffer and Acetonitrile in the ratio of 45:55 was pumped through column at a flow rate of 0.8 ml/min. Buffer temperature was maintained at 30°C. Optimized wavelength for Omeprazole and Ofloxacin was 220nm. Retention time of Omeprazole and Ofloxacin were found to be 2.16 min and 3.39 min. %RSD of the Omeprazole and Ofloxacin were and found to be 0.62 and 0.74 respectively. %Recover was Obtained as 100.07% and 100.72% for Omeprazole and Ofloxacin. LOD, LOQ values were obtained from regression equations of Omeprazole and Ofloxacin were 0.27ppm, 0.37ppm and 0.83ppm, 1.13ppm respectively. Regression equation of Omeprazole is y=11878x+281.6, and of Ofloxacin is y=14453x+3910.2.

 

KEYWORDS: Omeprazole, Ofloxacin, RP-HPLC, LOD, LOQ.

 

 


 

 

 

INTRODUCTION:

Omeprazole1, (RS)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl) Methylsulfinyl)-1H-benzo [d] imidazole, is a proton pump inhibitor, it suppresses gastric acid secretion by specific inhibition of the H+/K+-ATPase in the gastric parietal cell. By acting specifically on the proton pump, omeprazole blocks the final step in acid production, thus reducing gastric acidity. Few bioanalytical methods by HPLC using human plasma and also spectrophotometric methods using pharmaceutical dosage forms have been reported for the estimation of Omeprazole.

 

 

Fig.1: Structure of Omeprazole

 

Of loxacin2, Chemically (±)-9-Fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido [1,2,3-di]-1,4-benzoxazine-6-carboxylicacid, is a synthetic antibiotic of fluoroquinolone drug class considered to be a second-generationfluoroquinolone. Ofloxacin is a racemic mixture, which consists of 50% levofloxacin (the biologically active component) and 50% of its “mirror image” or enantiomer dextrofloxacin, its mode of action depends on blocking of bacterial DNA replication by binding itself to an enzyme called DNA gyrase, which allows the untwisting required to replicate one DNA double helix into two. Notably the drug has 100 times higher affinity for bacterial DNA gyrase than for mammalian.

 

 

Fig.2: Structure of Ofloxacin

 

Varieties of analytical methods are used for the analysis of drugs in bulk, formulations and bio analytical samples. In Pharmaceutical industry, spectrophotometric and chromatographic methods have gained the significance in recent studies. Hence a RP–HPLC method was developed and validated as per ICH guidelines3. The literature reveals that various methods for the determination of Omeprazole and Ofloxacin in pharmaceutical validations among these methods are LC-MS and LC-MS/MS4, 5, 6, HPLC7,8 method for title compounds, was reported. Anattempt was madeto developa method which is precise, simple, robustand most economic methodsofarfortheirdetermination.

 

MATERIALS AND METHODS:

Materials:

HPLC instrument used was of WATERS HPLC 2965 SYSTEM with Auto Injector and PDA Detector. Software used is Empower 2. Acetonitrile, Phosphate buffer, ammonium acetate buffer, glacial acetic acid, methanol, potassium dihydrogen phosphate buffer, tetra hydrofuran, triethylamine, ortho-phosphoric acid were analytical grade.

 

Methods: Preparation of 0.1% OPA buffer:

1ml of ortho phosphoric acid in a 1000ml of volumetric flask adds about 900ml of milli-Q water added and degas to sonicate and finally make up the volume with water.

 

Standard Preparation:

Accurately Weighed and transferred 25mg of Omeprazole and 10mg of Ofloxacin working Standards into a 10ml clean dry volumetric flask, add 3/4th volume of diluent, sonicated for 5 minutes and make up to the final volume with diluents. 1ml from the above two stock solutions was taken into a 10ml volumetric flask and made up to 10ml.

 

Sample Preparation:

5 ml was transferred into a 50mL volumetric flask, 30mL of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. From the filtered solution 1 ml was pipette out into a 10 ml volumetric flask and made up to 10ml with diluent.

 

Method Development:

There are many trials were done by changing columns and Mobile phases and were reported optimized method below. Drugs were eluted with good resolution, retention time all the parameters were within the limits.

Mobile phase:                Buffer and Acetonitrile (45:55)

Flow rate:                        0.8 ml/min

Column:                          ODS 150 X 4.6 mm, 5m.

Detector wave length:   220nm

Column temperature:    30°C

Injection volume:          10mL

Run time:                        6 min

Diluent:                           first dissolved in Methanol and made up with water

 

 

Fig.3: Optimized chromatogram

 

 

RESULTS AND DISCUSSION:

Systemsuitability:

All the system suitability parameters are within range and satisfactory as per ICH guidelines, results were shown in table-1.

 

Table-1: Systemsuitability studies

Property

Omeprazole

Ofloxacin

Retention time (tR)

2.16±0.3 min

3.39±0.3min

Theoretical plates(N)

3188 ± 163.48

5704±163.48

Tailingfactor(T)

1.58±0.117

1.35±0.117

Peak area

282342

1503692

 

 

Fig.4: Typical chromatogram of Omeprazole and Ofloxacin.

 

Linearity:

Linearity solutions are prepared such that 0.25, 0.5, 0.75, 1, 1.25, 1.5ml from the Stock solutions of Omeprazole and Ofloxacin are taken in to 6 different volumetric flasks and diluted to 10ml with diluents to get 62.5ppm, 125ppm, 187.5ppm, 250ppm, 312.5ppm and 375ppm of Omeprazole and 25ppm, 50ppm, 75ppm 100ppm, 125ppm, 150ppm of Ofloxacin.Regression equation of the Omeprazole and Ofloxacin are found to be, y=11878x+281.6, and y=14453x+3910 and the regression co-efficient was 0.999. Linearity results were shown in table-2.

 

 

Fig 5: CalibrationcurveofOmeprazole

Table-2: Linearity Calibrationdata ofOmeprazole andOfloxacin.

S.No

Omeprazole

Concentration (µg/ml)

Response

Ofloxacin

Concentration (µg/ml)

Response

1

62.5

776358

25

379357

2

125

1500879

50

748453

3

187.5

2184123

75

1057693

4

250

2929388

100

1431042

5

312.5

3692826

125

1802617

6

375

4508415

150

2195967

 

 

 

Fig6: Calibrationcurve of Ofloxacin

 

Intraday precision:

Intraday Precision was performed and % RSD for Omeprazoleand Ofloxacin were found to be 0.62% and 0.74% respectively those are below 2. Results were shown in table-3.

 

Interday precision:

Inter day precision was performed with 24 hrs time lag and the %RSD Obtained for Omeprazole and Ofloxacin were 0.12% and 0.03%. Results were shown in table-3.

 

Table-3: Precision results

S.No

Inter day

Intraday

Omeprazole

Ofloxacin

Omeprazole

Ofloxacin

1

2849235

1502705

2823421

1503692

2

2868588

1490595

2851485

1501755

3

2848014

1497287

2876717

1529532

4

2833690

1503932

2858841

1521086

5

2851740

1508829

2844759

1507129

6

2845264

1501256

2858256

1519037

Mean

2847759

1500963

2852247

1513705

Std.Dev.

3528.46

414.36

17697.2

11132.9

%RSD

0.12

0.03

0.62

0.74

 

Accuracy:

Threeconcentrations 50%, 100%, 150%, were injected in a triplicate manner and amount Recovered and % Recovery were displayed in Table 4 and the recovery was within the range 98-102%.

 

 

 

Table-4: Accuracyresults

Sample

Amount added (µg/ml)

Amount Recovered (µg/ml)

% Recovery

% RSD

Omeprazole

125

124.62

99.7

0.60

250

248.42

99.37

0.93

375

373.87

99.70

1.44

Ofloxacin

50

50.31

100.62

0.46

100

100.8

100.80

0.19

150

149.25

99.5

0.22

 

LOD:

Limit of detection was calculated by intercept Omeprazole and Ofloxacin method and LOD for Omeprazolewas found to be 0.01 and Ofloxacin was 0.01respectively.

 

LOQ:

Limit of Quantification was calculated by intercept Omeprazole and Ofloxacin method and LOQ for Omeprazole and Ofloxacin were found to be 0.05 and 0.04respectively.

 

Robustness:

Small deliberatechanges in method like Flow rate, mobile phase ratio, and temperature are made but there were no recognized change in the result and are within range% RSD below 2 as per ICH Guide lines.

 

Table-5: Robustness dataof Omeprazole and Ofloxacin.

S.No.

Robustness  Condition

Omeprazole

%RSD

Ofloxacin

%RSD

1

Flow minus

0.40

0.27

2

Flow Plus

1.29

0.66

3

Mobile phase minus

0.3

0.4

4

Mobile phase Plus

1.29

1.36

5

Temperature minus

0.3

0.1

6

Temperature Plus

0.2

0.4

 

Assay:

Standard preparations are made from the API and Sample Preparations are from Formulation. Both sample and standards are injected six homogeneous samples. Drug in the formulation was estimated by taking the standard as the reference. The Average % Assay was calculated and found to be 100.07% forOmeprazole and 100.72 for Ofloxacin.

 

Table-6: Assay Results

S.No

Omeprazole

Ofloxacin

1

99.06

100.06

2

100.04

99.93

3

100.93

101.78

4

100.30

101.22

5

99.81

100.29

6

100.28

101.08

Mean

100.07

100.72

Std.Dev.

0.6209

0.7408

%RSD

0.62

0.74

 

 

 

CONCLUSION:

A simple, Accurate, precise method was developed for the simultaneous estimation of the Omeprazole and Ofloxacin in Tablet dosage form. Retention time of Omeprazole and Ofloxacin were found to be 2.16 min and 3.39 min. %RSD of the Omeprazole and Ofloxacin were and found to be 0.62 and 0.74 respectively. % Recovery was Obtained as 100.07% and 100.72% for Omeprazole and Ofloxacin respectively. LOD, LOQ values were obtained from the regression equations of Omeprazole and Ofloxacin were 0.27ppm, 0.037ppm and 0.083ppm, 1.13ppm respectively. Regression equation of Omeprazole is y=11878x+281.64, and of Ofloxacin is y=14453x+3910.2.Retention times are decreased and that run time was decreased so the method developed was simple and economical that can be adopted in regular Quality control test in Industries.

 

REFERENCE:

1.       R.S.Satoskar, S.D.Bhandarkar and S.S.Ainapure. “Pharmacology and Pharmacotherapeutics”, 17th edition, Popular Prakashan, Mumbai, India, 2001.

2.       “Goodman and Gilman’s The Pharmacological Basis of Therapeutics”, 9th edition, McGraw-Hill health professions division, New york, 1996.

3.       International Conference on Harmonization, Validation of Analytical Procedures; Methodology. Federal Register, Nov. 1996, 1-8.

4.       Ludwig Huber, Validation and qualification in analytical laboratories, First Edition, 1999, 107-140.

5.       ICH Harmonized Tripartite Guidelines, Validation of analytical procedure methodology, 6th November, 1996.

6.       ICH Harmonized Tripartite Guidelines, Text on Validation of analytical procedure step-4 Q2A, October, 1994.

7.       HPLC Methods for pharmaceutical Analysis, Vol-2 By, George Lunn, 2000, John wileyand Sons.

8.       Lloyd R. Synder, Joseph J. Kirland, Joseph L. Glajch. Practical HPLC method development, 2nd Edition.

 

 

 

 

Received on 17.04.2018       Accepted on 05.07.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(3): 153-156.

DOI: 10.5958/2231-5675.2018.00028.5