Reverse Phase High Performance Liquid Chromatographic Method for the Simultaneous Determination of Clarithromycin, Tinidazole and Rabeprazole in combined Formulations

 

Sri Datla VVSSN. Raju*, A. Manikandan

Department of Chemistry, Bharath Institute of Higher Education and Research,

Selaiyur, Chennai, Tamilnadu, India.

 

ABSTRACT:

A simple reproducible stability indicating RP-HPLC method was developed for the simultaneous determination of clarithromycin, tinidazole and rabeprazole in their combined dosage forms, and for drug dissolution studies. A using Phenomenex C18, 5μm, 250mm x4.6 mm i.d. columnand a mobile phase of Phosphate buffer (30mM) pH-4.5, Acetonitrile and Methanol in the ratio of 30: 50: 20%v/v mixture were used for separation and quantification of clarithromycin, tinidazole and rabeprazole. The present drug analytes were run at a flowrate of 1.0ml/min at ambient temperature. The injection volume was 10μL and with ultraviolet detection at 260nm. Under these conditions, cclarithromycin, tinidazole and rabeprazole were eluted at 3.393min, 4.576minand 6.319min, respectively with a total run time shorter than 10min. The developed method was validated according International Conference on Harmonization (ICH) guidelines. The developed RP-HPLC method was applied successfully for quality control assay of clarithromycin, tinidazole and rabeprazole in their combination drug product and in vitro dissolution studies.

 

 

 

INTRODUCTION:

Clarithromycin[1-3],{(3R,4S,5S,6R,7R,9R,11R,12R, 13S,14R)-6-{[(2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy}-14-ethyl-12,13-dihydroxy-4-{[(2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyloxan-2-yl]oxy}-7-methoxy-3,5,7,9,11,13-hexamethyl-1-oxacyclotetradecane-2,10-dione}^1-4 is a semisynthetic macrolide antibiotic [Fig.1.a] used to treat pharyngitis, tonsillitis, acute maxillary sinusitis, acute bacterial exacerbation of chronic bronchitis, pneumonia and skin infections.

 

Fig.1.a. Chemical Structure of Clarithromycin

 

Tinidazole^4-6 [Fig.1.b] 1-[2-(ethanesulfonyl)ethyl]-2-methyl-5-nitro-1H-imidazoleis a nitroimidazole antitrichomonal agent effective against Trichomonas vaginalis, Entamoeba histolytica, and Giardia lamblia infections. It is used to treat amebiasis, giardiasis, trichomoniasis, and vaginosis.

 

Fig.1.b.Chemical Structure of Tinidazole

 

Rabeprazole^7,8 2-[[[4-(3-methoxypropoxy)-3-methyl-2-pyridinyl]-methyl] sulfinyl]-1H-benzimidazole [Fig.1(c)] a new class of substituted benzimidazole proton pump inhibitor is used to treat heartburn and gastroesophageal reflux disease, ulcers, bacterial Infection due to Helicobacter pylori and Zollinger-Ellison Syndrome.

 

 

Fig.1.c. Chemical Structure of Rabeprazole

 

These three drugs are marketed as combined dose tablet kit in the brand name of Combi-Kit (Label claim 250mg,500mg,20mg of clarithromycin, tinidazole and rabeprazole) designated for the treatment of hypertension and lowering blood pressure. At the time this examination no soundness showing RP-HPLC strategy was accounted for in the writing for the measure of clarithromycin, tinidazole and rabeprazole in combinational structure. It was in this way, thought worth-while by the creator to build up dependability showing chromatographic strategy for the concurrent examine of these three medications in their joined measurements structures. The present research paper describes the development and validation of selective reverse phase HPLC assay procedure for the analysis of clarithromycin, tinidazole and rabeprazole in combined dosage formulation in accordance with the ICH guidelines.

 

EXPERIMENTAL:

a. Instrumentation:

The HPLC method development and validation procedures was performed on The Shimadzu HPLC system consisted of LC 10ATvp/ LC 10ADvp pump, Rheodyne injector and SPD 10 UV detector. The chromatographic separations were performed using Phenomenex C18, 5μm, 250mm x 4.6mm i.d. column, maintained at ambient temperature. Data integration was performed using Empower-1 software.

 

 

b. Reagents and Chemicals:

Pure samples of clarithromycin, tinidazole and rabeprazole were made available from SRS Pharma labs, Hyderabad. Methanol and Acetonitrile were of HPLC grade purchased from Merck chemicals Ltd, India. Ortho-phosphoric acid, hydrogen peroxide, Hydrochloric acid and sodium hydroxide were obtained from Rankem (India). Commercially available dosage form in the brand name of Rabemac-Kit (Label claim 500mg, 250mg, 20mg of clarithromycin, tinidazole and rabeprazole) was procured from local pharmacy store. All chemicals and reagents used were of HPLC grade and were purchased from Merck Chemicals, India. Milli-Q water was used throughout the analysis. All solvents and solutions were filtered through a membrane filter (Millipore Millex -HV filter units, 0.45µm pore size; nylon) and degassed before use.

 

c. Preparation of mobile phase:

The mobile phase used was a mixture of Phosphate buffer (30mM) pH-4.5, Acetonitrile and Methanol in the ratio of 30:50: 20% v/v in isocratic mode at a flow rate of 1.0ml/min respectively. The buffer was prepared by weighing accurately 6.8gms of KH₂PO₄ and dissolve with 500ml of HPLC grade water than make up to 1000ml with HPLC grade water then adjust the pH: 4.5 with ortho phosphoric acid or sodium hydroxide Prior to the assay the mobile phase components were filtered through 0.45μ membrane filter prior to use.

 

c. Preparation of diluent:

The same mobile phase prepared above was used as diluent in the present study.

 

e. Preparation of standard solutions:

Accurately weigh and transfer 500mg of Clarithromycin working standard and 500mg of tinidazole and 20mg rabeprazole working standard standard powders (pure) into a 100ml volumetric flask individually containing 10ml of methanol and sonicated to dissolve it completely and make the volume up to the mark with the methanol. Further, pipette out 5.0ml of clarithromycin, tinidazole and rabeprazole from the above stock solution into a 100ml volumetric flask and dilute up to the mark with diluent respectively. From the above stock solution six working standard concentrations ranging from 50-250μg/ml for clarithromycin, 50-500μg/ml fort inidazole, 2.0-24μg/ml for rabeprazole were prepared and injected in triplicate into the LC system.

 

f. Preparation of sample solution [assay of pharmaceutical dosage form]:

Twenty tablets of  Combi-Kit (Acme formulation pvt. Ltd; Label claim 500mg, 500mg, 20mg of clarithromycin, tinidazole and rabeprazole) procured from local pharmacy store were weighed to get the average weight and finely powdered in a mortar. An amount of powder equivalent to 500mg, 500mg, 20mg of clarithromycin, tinidazole and rabeprazole tablet powder was accurately weighed and transferred  into a 100ml volumetric flask containing methanol and sonicated to dissolve it completely, and filter the solution through 0.45μ filter paper and make the volume up to the mark with the same solvent. Further, pipette 5.0ml of above stock solution into a 20ml volumetric flask and dilute up to the mark with diluent.

 

RESULTS AND DISCUSSION:

i. Method Development:

Various chromatographic parameters were optimized to develop HPLC method for simultaneous estimation of clarithromycin, tinidazole and rabeprazole with acceptable resolution respectively. Initially, studies were made by selecting appropriate column. For this purpose, the author used X Bridge C18 (150mm x 4.6mm, 5µm), Phenomenex C18(5μm, 250mm x4.6 mm) and Inertsil ODS C18(150x4.60)mm,5μ columns. Out of these the HPLC columns, Phenomenex C18, 5μm, 250mm x 4.6 mm i.d. column was found to be better as it gave the peaks with better gaussian shape for the three drugs.

 

To improve the shape and width of the obtained peaks for the above column a suitable mobile phase was examined using mobile phase mixtures of different polarity. Various combinations of mobile phases were screened and finally, the mobile phase consisting of Phosphate buffer (30mM) pH-4.5, Acetonitrile and Methanol in the ratio of 30: 50: 20%v/v was preferred to obtain symmetric peaks of clarithromycin, tinidazole and rabeprazole respectively.

 

The best sensitivity and selectivity were obtained by online wavelength switching at 260nm, which allowed the analysis of these three drugs in a single run as the isoabsortive point of clarithromycin, tinidazole and rabeprazole selected was 260nm and this wavelength was chosen for further analysis. Further, the flow rates of the mobile phase between 0.5 and 1.5ml/min were studied. From these studies it is revealed that the flow rate of 1.0ml/min gave an optimal signal to noise ratio with a reasonable separation time of clarithromycin, tinidazole and rabeprazole respectively.

 

Finally, the simultaneous determination of clarithromycin, tinidazole and rabeprazole was carried out by gradient elution with a flow rate of 1.0 mL/min on Phenomenex C18, 5μm, 250mm x 4.6mm at ambient temperature.

 

 

 

Fig. 2. The standard chromatogram of  clarithromycin, tinidazole and rabeprazole

 

The standard chromatogram so obtained was shown in Figure. 2 The system suitability parameters were shown in Table. 2. The retention times of clarithromycin, tinidazole and rabeprazole were found to about 3.393min, 4.576min and 6.319min, respectively.

 

ii. Forced Degradation Studies:

Intentional degradation was attempted to stress conditions exposing it to acid (0.1N Hydrochloric acid), alkali (0.1N NaOH), hydrogen peroxide (5%) and heat (110°C) to evaluate the ability of the proposed method to separate clarithromycin, tinidazole and rabeprazole from its degradation products

 

Preparation of stock solution:

An amount of tablet powder equivalent to 500mg, 500mg, 20mg of clarithromycin, tinidazole and rabeprazole tablet powder respectively into a 250ml volumetric flask individually add methanol and sonicate to dissolve it completely and make volume up to the mark with the same solvent.

 

a. Acid degradation condition:

Pipette 5.0ml of the above stock solution into a 20ml volumetric flask and 3.0ml of 0.1N HCl was added. Then, the volumetric flask was kept at 90ºC for 1 hour and then neutralized with 0.1 N NaOH and make up to 20ml with diluent. Filter the solution with 0.45 microns syringe filters and place in vials.

 

b. Alkali degradation condition:

Pipette 5.0ml of the above stock solution into a 20ml volumetric flask and add 3.0ml of 0.1N NaOH was added in 20ml of volumetric flask. Then, the volumetric flask was kept at 70ºC for 2 hour and then neutralized with 0.1N HCl and make up to 20ml with diluent. Filter the solution with 0.45 microns syringe filters and place in vials.

 

 

 

 

c. Oxidative degradation:

Pipette 5.0ml of the above stock solution into a 20ml volumetric flask 3.0ml of 3.0% w/v of hydrogen peroxide added in 20ml of volumetric flask and the volume was made up to the mark with diluent. The volumetric flask was then kept at room temperature for 1hr min. Filter the solution with 0.45 microns syringe filters and place in vials.

 

d. Thermal degradation:

The sample was taken in petri dish and kept in hot air oven at 110^oC for 24 hours. Then, the sample was taken and diluted with diluents and injected into the above described HPLC system and analyzed.

 

Table.1. Results of % degradation studies of clarithromycin, tinidazole and rabeprazole

 

From the results of degradation studies it was observed considerable degradation was not observed in the chromatograms of clarithromycin, tinidazole and rabeprazole samples,(Figs.3.a- d) under the prescribed different stress conditions such as acid (0.1N NaOH), base (0.1N HCl) hydrogen peroxide (3.0%)  and thermal degradation confirming the stability indicating nature of the developed RP-HPLC method. The summary of forced degradation studies is given in Table.1.

 

 

Fig 3a. chromatogram of acid degradation

 

 

Fig 3b. chromatogram of alkali degradation

 

Fig 3c. chromatogram of oxidative degradation

 

 

Fig 3d. chromatogram of thermal degradation

 

iii. Method Validation:

The proposed HPLC method was validated for parameters, such as system suitability, specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, robustness and ruggedness, according to International Conference on Harmonization (ICH) guidelines.

 

a. System Suitability:

After optimization of the proposed method the efficiency of a chromatographic separation was monitored by applying the following system suitability tests: capacity factor, tailing factor and theoretical plates. The chromatographic parameters such as resolution, selectivity and peak asymmetry were satisfactory for these compounds (Table.2) and the retention values were found to be 3.3934.576 and 6.139 minutes clarithromycin, tinidazole and rabeprazole respectively.

 

Table.2. System suitability results of clarithromycin, tinidazole and rabeprazole

 

b. Selectivity:

The selectivity of the proposed method was evaluated by preparing solution of analytical blank and placebo solutions that were run in the instrument one after another. The results of these tests proved that the components other than the drug did not produce any detectable signal at the retention time of clarithromycin, tinidazole and rabeprazole which confirmed the specificity of the method.

 

c. Linearity:

Linearity for clarithromycin, tinidazole and rabeprazole was established by analyzing six working standard concentrations ranging from 50-250μg/ml for clarithromycin 50-500μg/ml for tinidazole and rabeprazole, 2.0-24μg/ml for rabeprazole were prepared and injected in triplicate into the LC system and their respective chromatograms were recorded respectively. The calibration graphs for the above three drugs were obtained by plotting peak area verses the concentration data was further treated by least-squares linear regression analysis. The linearity plots of clarithromycin, tinidazole and rabeprazole were depicted in Figures. 4. a, b and c and the linearity results of both the drugs were given Table. 3. Excellent linearity was obtained for compounds between the peak areas and concentrations of 50-250μg/ml with r2 = 0.9992 for clarithromycin, 50-500μg/ml with r2 = 0.9993 for tinidazole and 2.0-24μg/ml with r2 = 0.9995 for rabeprazole respectively revealing the good linearity of the proposed RP-HPLC method.

 

Table.3. Results of linearity studies of clarithromycin, tinidazole and rabeprazole

 

 

Fig 4a Linearity plot of clarithromycin

 

 

Fig 4b Linearity plot of Trinidazole

 

Fig 4c Linearity plot of Rabeprazole

 

 

d. LOD and LOQ:

The LOD and LOQ values were found to be 2.5μg/ml and 10μg/ml for clarithromycin, 5.0μg/ml and 20μg/ml for tinidazole and 0.2μg/ml and 0.80μg/ml for rabeprazole respectively which indicated the good sensitivity of the proposed method for clarithromycin, tinidazole and rabeprazole respectively (Table. 3).

 

 

e. Precision:

In the present study the author carried out both repeatability that was carried out for short time interval under the same chromatographic conditions for clarithromycin, tinidazole and rabeprazole. One fixed standard solution of clarithromycin, tinidazole and rabeprazole at 100% concentration were injected for 6 times into the above said column under controlled experimental conditions and the corresponding peak areas for all the six replicates were recorded and the mean and % relative standard deviation (%RSD) were calculated respectively. The results of above precision studies[repeatability] for clarithromycin, tinidazole and rabeprazole were 0.284,1.92 &0.678 and found to be precise as (%RSD <2) value for repeatability and were within acceptable limits.

 

f. Accuracy:

The accuracy of the present developed method was determined by standard addition method which was performed at three concentration levels of 50%, 100% and 150%. For this a known amount of standard drug powder was added to the fixed amount of pre-analyzed tablet solution and were analyzed in triplicate at each level as per the proposed method and the % recovery at each level was calculated and are presented in Table. 5. The results showed the best recovery i.e 99.9-100.8% for clarithromycin,100.1-100.9% for, tinidazole and 99.9.3-100.7% % for rabeprazole respectively indicating developed RP-HPLC method was accurate.

 

g. Robustness:

The robustness study of the proposed method was performed by slight modification in flow rate of the mobile phase and detection wavelength. The change was made in the flow rate ±0.2mLand detection wavelength by ±5nm. The samples of clarithromycin, tinidazole and rabeprazole were analyzed under these changed experimental conditions and the data results and their corresponding chromatograms were represented in Table. 4 and was found that there were no significant changes in the chromatographic patterns for clarithromycin, tinidazole and rabeprazole, concluding that the proposed RP-HPLC method was robust.

 

Table.4. Results of robustness studies of clarithromycin, tinidazole and rabeprazole

 

h. Ruggedness:

The ruggedness of the HPLC method was evaluated by carrying out the analysis using the standard working solution of clarithromycin, tinidazole and rabeprazole, the same chromatographic system and the same column on different days. The prepared mixtures of standards were injected 6 times as a test sample. The % RSD for peak areas of hydrochlorothiazide, amlodipine and valsartan were calculated and the experimental results 0.79, 0.98 & 1.43 (%RSD <2). and were within the limits indicating that the developed RP-HPLC method was found to be rugged.

 

i. Assay of tablet dosage forms: 

Analysis of marketed tablets Combi-Kit (Label claim 500mg, 500mg, 20mg of clarithromycin, tinidazole and rabeprazole) was carried out using the proposed RPHPLC method using the optimized HPLC conditions. The experimental results (Table.5) of the amount of clarithromycin, tinidazole and rabeprazole in dosage form were expressed as a percentage of label claim (99.97% for clarithromycin, 99.94% for tinidazole and 100.2% for rabeprazole) and were in good agreement with the label claims thereby suggesting that there is no interference from any of the excipients which are normally present.

 

Table.5. Results of assay of clarithromycin, tinidazole and rabeprazole [Combi-kit]

*Average of three determinations

 

CONCLUSIONS:

The proposed RP-HPLC strategy was seen as straightforward, explicit, exact, precise and affordable for synchronous estimation of clarithromycin, tinidazole and rabeprazole in tablet dose structure. The proposed strategy expected to have gigantic pertinence in routine quality control investigation based on simple example planning steps are engaged with basic reagents and solvents were utilized tentatively. As far as anyone is concerned, the created RP-HPLC technique is the principal detailed strategy for synchronous assurance of clarithromycin, tinidazole and rabeprazole in their blend tranquilize item.

 

ACKNOWLEDGMENTS:

The creators express gratitude toward Acme formulation pvt. Ltd, India, for giving blessing test of gauges clarithromycin, tinidazole and rabeprazole and Department of chemistry, Bharath Institute of Higher Education and Research, Selaiyur, Chennai, Tamilnadu, for giving offices to accommodating their help and allowing this exploration work to convey for distribution.

 

REFERENCES:

1.      Clarithromycin. International Drug Price Indicator Guide. 2015.

2.      Clarithromycin Side Effects in Detail - Drugs.com. Drugs.com. 2017.

3.      USP monograph, Pharmacopeial Forum: Volume No. 30(4)1179.

4.      Ebel K, Koehler H, Gamer AO, Jackh,R. Imidazole and Derivatives. In Ullmann’s Encyclopedia of Industrial Chemistry.2002.

5.      Edwards, David I. Nitroimidazole drugs - action and resistance mechanisms. I. Mechanism of action" Journal of Antimicrobial Chemotherapy. 1993; 31: 9-20.

6.      Dekkers CPM, Beker JA, Thjodleifsson B.Europeanrabeprazole sodium study group, comparison of rabeprazole sodium 20mgvs.omeprazole 20mg in the treatment of active gastric ulcer European multicentre study. Aliment Pharmacol Ther. 1998; 12: 789-795.

7.      Ma JY, Song YH, Sjostrand SE, Rask L, Mardh. cDNA cloning of the beta-subunit of the human gastric H K-ATPase. Biochem Biophys Res Commun.1991; 180(1): 39-45.

8.      Janssen-Cilag. Rabeprazole Sodium. Pariet. 1999; 1-3.

9.      ICH, International Conference on Harmonization, IFPMA, Geneva, 2006.

 

 

 

Accepted on 10.05.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(3):1333-1338.