INTRODUCTION:

Amoxicillin is chemically (2S.5R.6R)-6-{[(2R)-2-amino-2-(4-hydroxyphenyl)-acetyl] amino}-3, 3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0] heptane-2-carboxylic acid^[1]. Amoxicillin is amino Penicillin with spectrum similar to that of Ampicillin ^[2]. Amoxicillin is a moderate–spectrum bacteriolytic β-lactum antibiotic used to treat bacterial infections caused by susceptible micro organisms. It is usually the drug of choice within the class because it is better absorbed following oral administration. Amoxicillin acts by inhibiting the synthesis of bacterial cell wall^[3]. It is official drug in Indian Pharmacopoeia^[4], British Pharmacopoeia^[5]. The molecular formula is C₁₆H₁₉N₃O₅S and Molecular weight is 365.1 g/mol^[^6].

Fig-1: Structure of Amoxicillin

Tinidazole is chemically 1‐(2‐ Ethyl Sulfonyl Ethyl) –2 – methyl – 5‐nitroimidozole ^[^7].Tinidazole is a prodrug and antiprotozoal agent. The nitro group of tinidazole is reduced in Trichomonas by a ferredoxin-mediated electron transport system and used for Metronidazole-Resistant Vaginal Trichomoniasis^[^8]. The free nitro radical generated as a result of this reduction is believed to be responsible for the antiprotozoal activity. It is suggested that the toxic free radicals covalently bind to DNA, causing DNA damage and leading to cell death^[9]. Tinidazole is the subject of monograph in each of the BP and the USP. British Pharmacopoeia ^[^10]describes the non-aqueous titration method using perchloric acid as titrant and malachite green as indicator for the assay of tinidazole. The molecular formula is C₈H₁₃N₃O₄S and Molecular weight is 247.2 g/mol^[^11].Both the drugs are used to treat Gastro intestinal infectious diseases and upper respiratory tract infections.

Fig 2: Structure of Tinidazole

Literature survey revealed that a number of methods have been reported for estimation of Amoxicillin^[^12-20] and Tinidazole^[^21-27] individually or in combination with other drugs.Only one spectophotometric method^[^28] and one LC estimation^[^29] method have been reported for the quantitative estimation of Amoxicillin trihydrate and tinidazole in pharmaceutical dosage form. Hence an attempt has been made to develop new HPLC method which is simple, rapid, reproducible and economical method for simultaneous estimation of Amoxicillin trihydrate and Tinidazole in tablet dosage form. This method has been successfully used for quality-control analysis of drugs and for other analytical purposes.

MATERIAL AND METHODS:

Chemicals

Standard bulk drug samples of Amoxicillin and tinidazole were provided by Drugs India private Ltd (Hyderabad, India). Pharmaceutical dosage form used in this study was TINIMOX^® labeled to contain 500 mg of Amoxicillin trihydrate and 300mg of Tinidazole (manufactured by Dr. Reddy’s Laboratories, Hyderabad, India). Acetonitrile (HPLC grade) and ortho phosphoric acid was obtained from Merck Specialties Private limited, Mumbai, India. Water (HPLC grade) was obtained from milli‐Q system; Potassium dihyrdrogen ortho phosphate purified was obtained from S.D. Fine Chemicals Ltd, Mumbai, India.

Instrumentation and chromatographic conditions

HPLC method development and validation was done on a Shimadzu (Japan) Liquid chromatograph equipped with (LC-20 AD pump), LC-20A UV/Vis detector, (Rheodyne) 7725i injector with 20µl loop and LC-Solutions software. Stationary phase used was Luna 5µ C18 (2) 100A 250 × 4.60mm Column. Membrane filter of size 0.45µ (Rankem Nylon membranes, New Delhi, India) was used. All weighing were done on Shimadzu electronic balance, BL-220 H (Shimadzu Corporation, Japan).

Preparation of mobile phase

Acetonitrile : 25mM Potassium dihydrogen phosphate (adjusted to pH 4.5 by Ortho-phosphoric acid (10% aqueous) (60:40 v/v)was prepared, filtered through 0.45 μm membrane filter and sonicated on ultra sonic bath.

Preparation of Standard Stock Solution

The standard stock solution 1mg/ml of Amoxicillin trihydrate and Tinidazole were prepared separately by dissolving 10 mg of each drug in 100 ml mixture of acetonitrile and water (60:40 v/v). From the standard stock solution, mixed standard solution was prepared to contain 100 µg/mL of Amoxicillin trihydrate and 100 µg/mL of Tinidazole.

Preparation of sample solution

Twenty tablets were weighed and powder equivalent to 10 mg of Amoxicillin trihydrate was weighed accurately and transferred to a 100ml volumetric flask and extracted with mixture of Acetonitrile and water (60:40 v/v). The combined extracts were filtered and transferred to a volumetric flask and the volume adjusted to 100 ml with Acetonitrile and water. From this solution, 3 ml was pipetted and transferred to 10 ml volumetric flask and made volume upto the mark with acetonitrile and water to get the concentration 30µg/ml of Amoxicillin trihydrate and 30 µg/ml of Tinidazole.

Validation of HPLC method

The method was validated for the parameters like system suitability, specificity, linearity, accuracy precision, robustness, LOD and LOQ.

System suitability testing was done by six replicate analyses of standard solution at a 100% level to verify resolution and reproducibility of the chromatographic system. Parameters such as theoretical plates, tailing factor were determined and compared against the specifications.

For specificity studies standard solutions of AMOX and TZ were injected first. Then solutions containing mixed components, commercial product and excipient solution were run. The chromatograms of Amoxicillin and Tinidazole were analysed for RT and peak area.

Linearity studies were done by constructing calibration curves of Amoxycillin and Tinidazole standard solutions with concentrations ranging from 5-50 ug/ml each and correlation coefficients were determined.

To study accuracy of the method, recovery studies were carried out by addition of standard drug solution to sample at 3 different levels 50%, 100% and 150% of the test concentration (test concentration is 20µg/ml for Amoxicillin trihydrate and 20 µg/ml for Tinidazole) and the contents were reanalyzed by the proposed method.

Precision of the method was checked with respect to repeatability and Ruggudness. The % RSD was determined to evaluate the repeatability. Repeatability was determined by six replicate injections of same standard solution on the same day, under same experimental conditions. Ruggudness was assessed by injecting the same standard solution on different days. The relative standard deviation (% RSD) was determined in order to assess the precision of the method.

Robustness of the method was determined by changing the experimental conditions like_{mobile
phase ratio and pH
of}mobile phase. The mobile phase ratio was changed by ±2 units for both acetonitrile and buffer, pH of mobile phase was changed by 4.5±0.2

Limit of Detection (LOD) and Limit of Quantitation(LOQ) were determined by injecting progressively low concentrations of the standard solutions.

LOD and LOQ were calculated using following formulae

LOD =3.3*F/S

LOQ =10 *F/S

Where

F = Standard deviation of the response

_{S = Slope of the calibration curve}

RESULTS AND DISCUSSION:

Method Development

The solutions of Amoxicillin trihydrate and Tinidazole working standards were injected into the HPLC system and run in different solvent systems as mobile phases. Different mobile phases containing Acetonitrile, Buffers (Triethanolamine, phosphate) in different proportions were tried and finally Acetonitrile : 25mM potassium dihydrogen phosphate adjusted to pH4.5 with ortho phosphoric acid (60:40 v/v) was selected as an appropriate mobile phase which gave good resolution and acceptable peak parameters for both Amoxicillin trihydrate and Tinidazole. Representative chromatogram of mixed standard of Amoxicillin trihydrate and Tinidazole is shown in Figure 3.

From the standard stock solution further dilutions(Amoxicillin trihydrate 30 µg/ml and Tinidazole 30 µg/ml) were done using mobile phase and scanned over the range of 200-400 nm and the spectra were overlain. It was observed that at 238 nm both Amoxicillin trihydrate and Tinidazole showed considerable absorbance and therefore it was selected as detection wavelength. Overlain spectra of both drugs are shown in Fig.4.

Method Validation

The method was validated as per ICH guidelines [30].

System suitability: System suitability tests were carried out to evaluate the reproducibility of the system for the analysis. Table 2 and 3 represents the system suitability results of this method. The system was found to be suitable in respect of theoretical plate count and tailing factor.

Specificity: Specificity is the ability of the method to assess the analyte in presence of excipients, impurities,degraded products, matrix etc. peaks from sample solution and mixed srandard solution were found on same time. Excipients did not change the retention times. So the method is specific for simultaneous estimation of Amoxicillin and Tinidazole.

Linearity: The linear relationship was observed between the peak area and concentration over the range of 5-50µg/ml for both the drugs. The method was proven to be linear in the concentration range of 5- 50ug/ml as the correlation coefficient_{was 0.999 for Amoxicillin trihydrate and}0.999 for Tinidazole. Correlation coefficient, y- intercept, slope of regression line is shown in Figure 5 and 6.

Fig 5: Calibration Curve for Amoxicillin trihydrate

Fig 6: Calibration Curve for Tinidazole

Table 1: Optimized Chromatographic Conditions

Parameters	Method
Stationary phase (Column)	Phenomenex^® (Luna 5µ C18(2) 100A (250 × 4.60 mm) Column
Mobile phase	25mM Potassium dihydrogen phosphate (adjusted to pH 4.5 by Ortho-phosphoric acid) and Acetonitrile in the ratio of 40:60 % v/v
Flow rate	1 ml/min
Pressure	110 kgf
Run time	5 min
Column temperature(⁰c)	Ambient
Detection wavelength (nm)	238
Drugs Retention time (min) Amoxicillin trihydrate Tinidazole	2.052 ± 0.14 2.942 ± 0.10

Accuracy: Accuracy is generally assessed by analyzing the samples with known concentration and comparing the measured value with the true value. The measure values were obtained by conducting recovery studies. % recoveries are shown in Table 8.

Precision: The method was found to be precise as % RSD values for both repeatability and ruggudness were less than 2. The results of precision are given in Table 4 and 5.

Robustness: Robustness studies were carried out after deliberate alterations of mobile phase compositions and mobile phase pH. It was observed that the small changes in these operational parameters, did not lead to changes of retention times of peak of interest. Results of robustness studies are shown in Table 6 and 7.

Analysis of Marketed Products: The proposed method was evaluated in the assay of tablet formulation containing Amoxicillin trihydrate and Tinidazole. Five replicate determinations were carried out and % assay found was 98.60 -101.85 for Amoxicillin trihydrate and 99.60-100.60 % for Tinidazole. Results of tablet analysis are shown in Table 9.

Table 2: System suitability parameters for Amoxicillin trihydrate

S.No.	RT	Area	USP Plate count	USP Tailing
1	2.045	837040	3421.260	1.388
2	2.044	834617	3334.017	1.384
3	2.045	834524	3349.321	1.385
4	2.043	837656	3459.209	1.390
5	2.047	837562	3432.664	1.391
Mean		836280
SD		1517
% RSD		0.18

Table 3: System suitability parameters for Tinidazole

S. No.	RT	Area	USP Plate count	USP Tailing
1	2.935	615412	6567.838	1.404
2	2.937	612720	6800.499	1.401
3	2.939	615977	6490.672	1.398
4	2.942	618022	6545.286	1.401
5	2.933	613264	6527.171	1.401
Mean		615079
SD		2146
% RSD		0.34

Table 4: Repeatability (Intraday Precision)

S. No	Concentration		Peak area
S. No	Amoxicillin trihydrate	Tinidazole	Amoxicillin trihydrate	Tinidazole
1	30	25	834512	538442
	30	25	837726	537768
	30	25	834052	538486
	30	25	829526	539018
	30	25	827483	534306
	30	25	837625	538648
% RSD			0.53	0.32

Table 5: Ruggudness (Interday Precision)

S. No	Concentration		Peak area
S. No	Amoxicillin trihydrate	Tinidazole	Amoxicillin trihydrate	Tinidazole
1	30	25	805746	515924
	30	25	807161	509796
	30	25	810133	515856
	30	25	806895	525417
	30	25	809121	525400
	30	25	807692	509974
% RSD			0.196	1.32

Table 6: Robustness Results for variations in pH.

Parameter	Retention Time		Tailing factor
Parameter	Amoxicillin trihydrate	Tinidazole	Amoxicillin trihydrate	Tinidazole
pH 4.3	2.052	2.942	1.390	1.401
pH 4.7	2.047	2.937	1.384	1.401

Table 7: Robustness Results for variations in Mobile phase.

Parameter	Retention Time		Tailing factor
Parameter	Amoxicillin trihydrate	Tinidazole	Amoxicillin trihydrate	Tinidazole
ACN : buffer (62 : 38)	2.045	2.935	1.388	1.404
ACN : buffer (58 : 42)	2.048	2.939	1.385	1.398

Table 8: Recovery Studies of Amoxicillin trihydrate and Tinidazole

Drug		50%	100 %	150%
Amoxicillin Trihydrate	Mean % recovery	98.62	99.54	101.85
Amoxicillin Trihydrate	% RSD	1.5	0.53	1.2
Tinidazole	Mean % recovery	99.62	100.48	100.66
Tinidazole	% RSD	0.29	1.3	1.7

Table 9: Analysis of Formulation

Drug	Amount(mg/tablet)		% Label claim	% RSD
Drug	Labelled	Estimated	% Label claim	% RSD
Amoxicillin trihydrate	500mg	498.16	99.63	0.42
Tinidazole	300 mg	302.02	100.67	0.65

CONCLUSION:

The method described enables the quantification of Amoxicillin Trihydrate and Tinidazole in combined Tablet dosage form. The validation data demonstrates good precision and accuracy, which proves to be reliable of the proposed method. Hence, this HPLC method can be used routinely for simultaneous estimation of both components in solid oral dosage form.

ACKNOWLEDGEMENT:

The authors are thankful to Drugs India Private Ltd, Hyderabad, India for providing gift samples of Amoxicillin Trihydrate and Tinidazole and also to School of Pharmacy, Anurag Group of Institutions, Hyderabad for giving permission to carry out this work.

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Received on 17.11.2012 Modified on 14.12.2012

Asian J. Research Chem. 6(1): January 2013; Page 58-63