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/MS^{4, 5,
6}, HPLC^7,8method 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 3/4^th 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 (t_R)	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
_S.No	_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 data_{of 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, 6^th 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, 2^nd Edition.

Received on 17.04.2018 Accepted on 05.07.2018

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

DOI: 10.5958/2231-5675.2018.00028.5