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International Journal of Recent Advances in Pharmaceutical Research
April 2012; 2(2): 78-83
____________________________________________________________________________________________________________________________________________ Simultaneous Estimation Of Amoxicillin, Tinidazole And Omeprazole In
Microsphere Formulation By RP-HPLC
VIKAS KASNIA,*M. SENTHIL KUMAR, N. MAHADEVAN Nanomedicine Research Centre, Department of Pharmaceutics, Rajendra Institute of Technology and Sciences, Abstract
A simple isocratic reverse phase high performance liquid chromatography (RP-HPLC) method has been developed and
subsequently validated for estimation drugs (amoxicillin, tinidazole and omeprazole) in combined dosage form. The three
compounds were monitored at 230 nm using an isocratic mode on HPLC of Younglin prominence consist of SP930D
solvent delivery pump on inertsil ODS C18 column (250 × 4.6 mm, 5 µ) with flow rate of 1.0 ml/min and a mobile phase
consisting of methanol: acetonitrile: water in a ratio of 49: 49: 2 % v/v/v. The retention time for amoxicillin, tinidazole
and omeprazole was found to be 3.4 min., 5.0 min., and 5.7 min., respectively. Thus, the proposed method could be applied
for simultaneous estimation of amoxicillin, tinidazole and omeprazole in routine analysis.
Keywords: Amoxicillin; Tinidazole; Omeprazole; HPLC.
Helicobacter pylori has identified as a human The current management of H. pylori pathogen causing active gastritis and peptic ulcer infections relies on antibiotic therapy, consisting of [1]. H. pylori are gram negative bacterium that can a combination with two different antibiotics infect the mucosal lining of the stomach and together with a proton-pump inhibitor with or duodenum. Spiral-shaped, gram negative bacterium without colloidal bismuth, which in most cases is H. pylori found in colonized gastric mucosa or successful in eradicating the bacteria [6]. adherent to the epithelial linings of the stomach [2]. H. pylori infects 25-50% of individuals in the bacteriolytic, β-lactam antibiotic used to treat developed countries and up to 70–90% of individuals in the developing countries [3]. microorganisms [2]. Amoxicillin[(2S,5R,6R)- 6- combination of antimicrobial agents, typically acetyl]amino}- 3,3-dimethyl- 7-oxo- 4-thia- 1- combined with antisecretory agents or bismuth [4]. azabicyclo[3.2.0] heptane- 2-carboxylic acid] is an Combination therapies including proton pump oral semi-synthetic penicillin structurally related to inhibitors are preferred, especially for patients with ampicillin [7] (Figure 1). The presence of a benzyl
ulcer, as they provide rapid symptomatic ring in the side chain extends the antibacterial improvement as well as having a bacteriostatic effect [5]. Triple combination therapy, using two antibacterial antibiotics and a proton pump inhibitor, achieved a high eradication rate. ____________________________________________________________ *Correspondence Dr. M. Senthil Kumar Professor & Head Department of Pharmaceutics Nanomedicine Research Center, Institute of Pharmacy Rajendra Institute of Technology and Sciences (RITS) Figure 1: Structure of Amoxicillin
Email Address: _____________________________________________________________________________________ Vikas Kasnia et al. Int J Recent Adv Pharm Res, 2012;2(2):78-83 ISSN: 2230-9306;
International Journal of Recent Advances in Pharmaceutical Research
April 2012; 2(2): 78-83
____________________________________________________________________________________________________________________________________________ It is usually the drug of choice within the class because it is better absorbed, following oral administration, than other β-lactam antibiotics. Amoxicillin is susceptible to degradation by β-lactamase-producing bacteria, about 20 % is bound to plasma proteins in the circulation and plasma half-life of 1 to 1.5 hours has been reported [9, 10]. benzimidazole compound and a prototype anti- secretory agent [11] (Figure 2). It is very effective
in the treatment of Zollinger–Ellison syndrome. It is
Figure 3: Structure of Tinidazole
the first “proton pump inhibitor” widely used for symptomatic gastro-oesophageal reflux and also for The combination of amoxycillin along with prophylaxis [12]. It acts by interacting with H+/K+ tinidazole is available in the market in the Tablet ATPase in the secretory membranes of the parietal A review of the literature revealed that several high performance liquid chromatographic (HPLC) methods have been reported for the individual determination of amoxicillin, tinidazole and omeprazole. However, no HPLC method has been developed for the simultaneous determination of amoxicillin, tinidazole and omeprazole. In this paper, we report an isocratic reversed-phase HPLC method to assay amoxicillin, tinidazole, and omeprazole using a C18 SPE extraction cartridge and UV detection at 230 nm. This combination of Figure 2: Structure of Omeprazole
SPE and UV detection results in a method with high recoveries and good linearity, accuracy, and precision. It is a lipophilic, weak base and can be degraded unless it is protected against acid 2.0. EXPERIMENTAL
conditions. OPZ contains a tri-coordinated sulphur 2.1. Chemical and reagents
atom in a pyramidal structure and because of this structure omeprazole exists in two different Amoxicillin, Tinidazole and Omeprazole was optically active forms, (S)- and (R)-omeprazole. OPZ obtained as gift samples. Acetonitrile (ACN) and was first approved as a racemic mixture, but the (S) methanol used were of HPLC grade and obtained isomer was recently introduced on the market [13]. from RFCL Ltd. Millipore water of HPLC grade was used for the study. Tinidazole is a 5-nitro imidazole derivative and has antimicrobial actions as that of 2.2. Instrumentation
metronidazole. It is 1-(2-ethylsulfonylethyl)-2- A quaternary HPLC was performed on an Isocratic methyl-5-nitro-imidazole and is used similarly in HPLC of Younglin prominence consist of SP930D the treatment of susceptible protozoal infections, in solvent delivery pump pump on Inertsil ODS C18 the treatment and prophylaxis of anaerobic column (250 × 4.6 mm, 5 µ) attached to UV/VIS dual bacterial infections (Figure 3).
absorbance detector. The control of HPLC system _____________________________________________________________________________________ Int J Recent Adv Pharm Res, 2012;2(2):78-83 ISSN: 2230-9306;
International Journal of Recent Advances in Pharmaceutical Research
April 2012; 2(2): 78-83
____________________________________________________________________________________________________________________________________________ and data collection was performed by Autochro- with n‐hexane and dried in vacuum at room 2.3. Chromatographic conditions
2.6. Method validation
The process was carried out on C18 column using As per ICH guidelines, the method was validated the mobile phase consisting of ACN, methanol and and the parameters checked were accuracy, Millipore water 49:49:2 (% v/v/v). The mobile precision, linearity and range, limit of detection phase was degassed under vacuum by filtration (LOD), limit of quantification (LOQ), specificity, through a Polytetrafluoroethylene (PTFE) filter ruggedness, robustness and stability. For all the (0.22 µm) and sonicated to remove air bubbles by parameters percentage relative standard deviation bath sonicater prior to use. The mobile phase flow rate was 1 mL/min and the detection wavelength 3.0 RESULT AND DISCUSSION
was set at 230 nm. Sample preparation was also filtered through PTFE (0.22µm) filter paper and 3.1. Method optimization
injected through Rheodyne injector. The mobile In order to develop a suitable and robust HPLC phase was prepared fresh daily and sonicated method for the determination of amoxicillin, tinidazole and omeprazole, different mobile phases 2.4. Preparation of stock and standard working
and columns were employed to achieve the best solutions
phosphate buffer, acetonitrile was tried as mobile Stock solutions of amoxicillin, tinidazole and phase, in which satisfactory peak were not obtained, then different ratios of acetonitrile, methanol and appropriate amounts of each drug in mobile phase to obtain final drug concentrations of 100 mg/ml in satisfactory. Finally, the mobile phase consisting of 100 ml volumetric flask. Stock solutions were acetonitrile, methanol and water (49:49:2 % v/v/v) stored at 4°C. The standard working solution of at a flow rate of 1.0 ml/min, using a Inertsil ODS amoxicillin, tinidazole and omeprazole were C18 column (250 × 4.6 mm, 5 µ) column was found prepared separately by further it was diluted with to be appropriate, allowing good separation of the mobile phase. Each standard solution (20µl) was given drugs (Figure 4).
injected into the column after filtration using 0.2 micron membrane filter. 2.5. Preparation of mucoadhesive microsphere

Microspheres were prepared by a solvent evaporation acetone/liquid paraffin was used. Agglomeration of microspheres was prevented by using 0.75% w/v Span80. Eudragit RL 100 was used as core of microspheres and carbopol 934P was coated to produce mucoadhesion. Eudragit RL 100 was dissolved in acetone and weighed quantity of drugs, Carbopol 934P were dispersed in it. This homogeneous final dispersion was cooled to 5°C and poured slowly with stirring (700 rpm) into liquid paraffin containing 1% w/v span 80, which was previously also cooled to 5 °C. The obtained emulsion was stirred at 40 °C for 40 min. The Figure 4: A typical chromatogram of Amoxicillin,
microspheres in liquid paraffin was filtered, washed Tinidazole and Omeprazole
_____________________________________________________________________________________ Int J Recent Adv Pharm Res, 2012;2(2):78-83 ISSN: 2230-9306;
International Journal of Recent Advances in Pharmaceutical Research
April 2012; 2(2): 78-83
3.2. Method Validation
and diluted with mobile phase to get final concentrations in range 10-60 µg/ml of amoxicillin 3.2.1. Specificity
and tinidazole, and 50-300 µg/ml of omeprazole. The specificity is the ability of the analytical method Calibration graph was plotted between the mean to measure accurately and specifically the analyte in peak area v/s respective concentration and the presence of the other component that might be regression equation was derived. The linear expected to be present in the sample. Condition of regression equations of the lines for amoxicillin Y= HPLC method like percentage of organic solvent in 42.63x+342.3, (r2= 0.991), for tinidazole Y= 73.87x- mobile phase, ionic strength, flow rate etc, was 37.61, (r2= 0.993), for omeprazole Y= 7.903x+201.6, changed. In spite of above changes no additional peaks were found, although there were shift 3.2.4. LOD and LOQ Determination
retention times or little changes in peak shapes. Limit of detection can be calculated by using 3.2.2. Accuracy and Precision
The accuracy of the proposed method was checked by analyzing different concentration of the drugs in pure form within the calibration range in triplicate (n = 3). The precision of the developed method was based on standard deviation of the response and the calculated by determining the inter-day and intra- day coefficient of variation. The inter-day precision was calculated by analyzing samples for three consecutive days. The intra-day precision was Where σ = Standard deviation of the Y intercept of calculated by analyzing the three different concentration of the drug in triplicate and three times in day. The percentage recovery of amoxicillin, tinidazole and omeprazole were The Limit of Detection (LOD) were found to between 97.2 to 99.6 with % RSD of 1.19 to 1.94 be 6.93 µg/ml for the amoxycillin, 8.71 µg/ml for (Table 1).
tinidazole and 6.54 µg/ml for omeprazole while Limit of Quantitation (LOQ) were found to be 19.36 3.2.3. Linearity and range
µg/ml for amoxycillin, 21.40 µg/ml for tinidazole To establish the linearity, aliquots of standard stock Omeprazole were taken in 10 ml volumetric flasks Table 1: Results of accuracy and precision study of Amoxicillin, Tinidazole and Omeprazole
_____________________________________________________________________________________ Int J Recent Adv Pharm Res, 2012;2(2):78-83 ISSN: 2230-9306;
International Journal of Recent Advances in Pharmaceutical Research
April 2012; 2(2): 78-83
3.2.5. Robustness
Characterization of Amoxicillin Trihydrate Robustness of the method was determined by Gastric Retention. International Journal Of Pharmaceutical Sciences and Drug Research wavelength, pH and flow rate and ratio of mobile phases. Different wavelengths (220, 230, 240, 250, 260, 225, and 235 nm) were tested for yielding the [3] Suman Ramteke, N. Ganesh, S. Bhattacharya best peak shapes. At 235 nm drugs peak appeared but not sharp, at 225 nm the peak shapes were good but peak area was lower. Using 230 nm, the Targeted Nanoparticles for The Treatment of chromatogram yielding a bigger area count and H. Pylori. Journal of Drug Targeting, 2009; sharp peak. Hence, 230 nm has been opted as wavelength of choice. It was observed that there [4] Graham D. Y, Graham, F. Hammoud, H. M. T. were no marked changes in the chromatogram. It El-Zimaity, J. G. Kim, M. S. Osato & H. B. El- suggests that the developed method is robust. Serag. Meta-Analysis: Proton Pump Inhibitor 4.0. CONCLUSION
or H2-Receptor Antagonist for Helicobacter Pylori Eradication. Aliment Pharmacol Ther The reported RP-HPLC method was proved to be simple, rapid and reproducible. The validation data indicate good precision, accuracy and reliability of [5] Venkateswaramurthy N, Sambathkumar R, the method. The developed method offers several Vijayabaskaran M, Perumal P. Formulation advantages in terms of simplicity in mobile phase, and In Vitro Evaluation of Furazolidone isocratic mode of elution, easy sample preparation Mucoadhesive Microspheres. International steps and comparative short run time which makes the method specific and reliable for its intended use in simultaneous determination of Amoxycillin, [6] Satoshi Ishizone, Fukuto Maruta, Kazufumi Tinidazole and Omeprazole. This chromatographic assay fulfilled all the requirements for being a reliable and feasible method, including accuracy, linearity, recovery and precision. It is a highly Hiroyoshi Ota. In vivo bactericidal activities of specific and precise analytical procedure. Therefore, Japanese rice-fluid against H. pylori in a this HPLC method can be used as a routine sample Mongolian gerbil model. International Journal Acknowledgements
[7] Arindam Basu, Nandita Saha, Inder Singh We express our gratitude to Dr. Rajendar Singh Sra, Chairman, and Shri. Om Parkash, Director, Rajendra Amoxycillin and Tinidazole In Tablet Dosage Institute of Technology and Sciences, Sirsa, India, Form. International Journal of Pharmaceutical REFERENCES
[8] Luis Renato Pires De Abreu, Rodrigo Agustin Mas Ortiz Hplc Determination of Amoxicillin [1] Suman Ramteke, N. Ganesh, S. Bhattacharya & Volunteers after a Single Dose Administration. Targeted Nanoparticles for the Treatment of J Pharm Pharmaceut Sci 2003; 6(2): 223-230. Helicobacter Pylori. Journal of Drug Targeting, November 2008; 16(9): 694–705. [9] British Pharmacopoeia. London Stationary [2] Shiva Kumar Yellanki, Jeet Singh, Jawad Ali _____________________________________________________________________________________ Int J Recent Adv Pharm Res, 2012;2(2):78-83 ISSN: 2230-9306;
International Journal of Recent Advances in Pharmaceutical Research
April 2012; 2(2): 78-83
____________________________________________________________________________________________________________________________________________ [10] Martindale. The Complete Drug Reference. [13] Espinosa Bosh M, Ruiz Sanchez A.J., Sanchez 32nd Ed. Pharmaceutical Press, 1999; Pp. Methodologies for the Determination of Omeprazole: An Overview. Journal of [11] Cristina Iuga, Marius Bojiţă, Sorin E. Leucuţa. Development of a Validated RP-HPLC Method for Separation and Determination of Process-Related Impurities of Omeprazole In Bulk [14] Venkateswaramurthy N, Sambathkumar R, [12] Zarna Dedania, Ronak Dedania, Vaishali Mucoadhesive Microsphere for Helicobacter Karkhanis, G Vidya Sagar, Meeta Baldania and Pylori Eradication Therapy. International Nr Sheth. RP-HPLC Method for Simultaneous Estimation of Omeprazole and Ondansetron in Combined Dosage Forms. Asian J. Research Chem. 2009; 2(2): 108-111. _____________________________________________________________________________________ Int J Recent Adv Pharm Res, 2012;2(2):78-83 ISSN: 2230-9306;


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