INTRODUCTION:

In this communication the present work proposes UV spectrophotometric method for assay of amoxicillin and carbocisteine from combined pharmaceutical dosage form i.e. tablet.

Amoxicillin tri-hydrate is described chemically as 6 - (D - 4 hydroxy phenyl glycyl amino) penicillin acid tri-hydrate. It is semi- synthetic penicillin that belongs to the class of β – lactam antibiotics. It is generally used as antibacterial. Amoxicillin tri-hydrate is official in USP ¹, IP ² and BP ³.

Carbocisteine is described chemically as (2R)-2-amino-3-[(carboxy-methyl) sulphanyl] propanoic acid. Carbocisteine is a mucolytic drug, which breaks down mucus in the body so that it can be more easily cleared from the body. In chronic obstructive pulmonary disease (COPD) symptoms involve the over secretion of mucus, mucolytic have great potential for treatment of this disease. Additional characteristics of COPD include airflow limitation oxidative, stress and airway inflammation. Carbocisteine is official in British Pharmacopoeia¹ and European Pharmacopoeia². In literature survey HPLC^4-6 methods were reported for validation of combined dosage form

In this communication a new simple UV spectrophotometric method is reported for simultaneous determination of amoxicillin tri-hydrate and carbocisteine in combination dosage form. This simple method can also be used for the routine analysis of this combination formulation. In the proposed work development, optimization and validation of the method are presented.

MATERIALS AND METHODS:

Instrument and reagents:

Spectral scan was made on a Shimadzu UV-spectrophotometer, model 1800 (Shimadzu, Japan) with spectral band width of 0.5 nm with automatic wavelength corrections by using a pair of 10 mm quartz cells. All spectral measurements were done by using UV-Probe 2.42 software. Reference standards of amoxicillin and carbocisteine were obtained from reputed firm with certificate analysis.

Preparation of standard drug solution:

A 100 mg standard amoxicillin tri-hydrate was weighed accurately and transferred to a 100 ml volumetric flask and sonicated with 30 ml of 0.1N hydrochloric acid for 15 minutes. The volume was made up to the mark with 0.1 N HCl to give a stock solution of concentration 1000 μg /ml. From this solution, 10 ml of solution was pipetted out and transferred into 100 ml volumetric flask. The volume was made up to mark with 0.1 N HCl to give a working standard solution of concentration 100 μg/ml.

A 100 mg standard carbocisteine was weighed accurately and transferred to a 100 ml volumetric flask and sonicated with 30 ml of 0.1 N HCl for 15 minutes. The volume was made up to the mark with 0.1 N HCl to give a stock solution of concentration 1000 μg /ml. From this solution, 10 ml of solution was pipetted out and transferred into 100 ml volumetric flask. The volume was made up to mark with 0.1 N HCl to give a working standard solution of concentration 100 μg/ml.

Preparation of sample solution:

EXPERIMENTAL:

Method: First order derivative method:

(a) For amoxicillin:

For the selection of analytical wavelength, 100 μg/ml solution of amoxicillin was scanned in the spectrum mode from 400 nm to 190 nm by using 0.1 N hydrochloric acid as blank. The first order derivative spectrum was obtained by using derivative mode by UV probe 2.42 software. From the spectrum, the amplitude of the derivative spectrum was measured at 271.2 nm.

(b) For carbocisteine:

For the selection of analytical wavelength, 100 μg/ml solution of carbocisteine was scanned in the spectrum mode from 400 nm to 190 nm by using 0.1 N hydrochloric acid as blank. The first order derivative spectrum was obtained by using derivative mode by UV probe 2.42 software. From the spectrum, the amplitude of the derivative spectrum was measured at 219 nm.

Preparation of calibration curves:

Series of solutions containing 20 – 100 µg/ ml of amoxicillin and 10 -100 µg/ ml of carbocisteine were used to determine linearity of the proposed method respectively. Solutions were scanned in the spectrum mode and absorbance spectra were converted to first order derivative spectra. The overlain spectrum of amoxicillin and carbocisteine were given in Fig. 1(a), 1(b) respectively.

Fig. 1(a): Overlay spectra of first order derivative of amoxicillin in the concentration range of 20 – 100 µg/ ml.

Fig. 1(b): Overlay spectra of first order derivative of carbocisteine in the concentration range of 10 – 100 µg/ ml.

After observing the overlain first order derivative spectra of amoxicillin and carbocisteine, the zero crossing points of both drugs were selected for analysis of other drug. The first wave length selected was 271.2 nm, the zero crossing point of carbocisteine where amoxicillin showed considerable absorbance. The second wavelength was 219 nm, the zero crossing point of amoxicillin, where carbocisteine showed considerable absorbance. The calibration curves were plotted of dA/ dλ against concentrations [Fig. 2 (a), 2(b)].

Fig.2 (a): Calibration curve of amoxicillin in the concentration range of 20-100 µg/ml.

Fig.2 (b): Calibration curve of carbocisteine in the concentration range of 10-100 µg/ml.

Results of the analysis are given in table 1.

Table 1: Values of results of optical and regression of drugs

Parameter	Amoxicillin	Carbocisteine
Detection Wavelength (nm)	271.2	219
Beer Law Limits (µg/ml)	20-100	10-100
Correlation coefficient(r2)	0.9991	0.9999
Regression equation (y=b+ac)
Slope (a)	0.0004	-0.0001
Intercept (b)	0.0001	0.00004

Estimation from capsules:

Powdered from twenty capsules were collected and weighed accurately and average weight of powder from each capsule was determined. Powder equivalent to 25 mg of amoxicillin and 15 mg of carbocisteine was weighed and transferred in 100 ml of volumetric flask. A 30 ml of 0.1N hydrochloric acid was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with 0.1N hydrochloric acid to give concentration as 250 μg /ml of amoxicillin and 150 μg /ml of carbocisteine respectively. Such solutions were scanned in the range of 190-400 nm against 0.1 N hydrochloric acid as blank. The absorbance spectra were converted to first order derivative spectra. Calculations were done as per the equations. The concentrations of amoxicillin and carbocisteine present in capsules were calculated by substituting the values of absorbance in linearity equations.

(a) For amoxicillin Y = 0.0004x + 0.0001

(b) For carbocisteine Y = -0.0001x + 0.00004

Method Validation:

These methods were validated according to ICH guidelines.

Accuracy:

To ascertain the accuracy of proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%). Percentage recovery for amoxicillin and carbocisteine was found in the range of 100.06 % to 100.74 %. (Table 2).

Table 2: Statistical evaluation of the data subjected to accuracy

Level of % Recovery	Amount present Inµg/ml		Amount added in µg/ml		Amount found in µg/ml		% Recovery		Mean % recovery
	Amox	Carbo	Amox	Carbo	Amox	Carbo	Amox	Carbo	Amox	Carbo
80%	25	15	20	12	45.220	17.071	100.5	100.46	100.16	100.26
	25	15	20	12	44.892	17.147	99.76	100.87
	25	15	20	12	45.103	16.906	100.23	99.45
100%	25	15	25	15	50.225	30.111	100.45	100.37	100.36	100.06
	25	15	25	15	50.390	29.868	100.78	99.56
	25	15	25	15	49.930	30.072	99.860	100.24
120%	25	15	30	18	55.676	33.184	101.23	100.56	100.74	100.15
	25	15	30	18	55.478	32.854	100.87	99.56
	25	15	30	18	55.077	33.108	100.14	100.33
Mean									100.42	100.156

Amox = amoxicillin, Carbo= carbocisteine

Linearity:

The linearity of measurement was evaluated by analyzing different concentration of the standard solutions of amoxicillin and carbocisteine. For both the drugs concentration range was found to be 20-100 µg/ml for amoxicillin and 10-100 µg/ml for carbocisteine.

Precision:

The method precision was established by carrying out the analysis of powder blend from capsules containing 250 mg of amoxicillin and 150 mg of carbocisteine. The assay was carried out for the drugs by using proposed analytical method in six replicates. The values of relative standard deviation were well within limits 2.569 to 6.235 % for amoxicillin and 0.7487 to 3.549 % for carbocisteine respectively indicating the sample repeatability of the method. The results obtained are tabulated in table 3.

Table 3: Statistical evaluation of the data subjected to method of precision

Sr.No.	Sample No.	% Assay
		Amoxicillin	Carbocisteine
1	1	100.23	100.37
2	2	100.45	99.56
3	3	100.78	100.24
4	4	100.87	99.87
5	5	100.54	100.84
6	6	99.56	99.67
Mean % assay		100.405	100.091
%R.S.D.		0.4717	0.4830

Intra-day precision was estimated by assaying tablets powder blend containing 250 mg of amoxicillin and 150 mg of carbocisteine. The assay was carried out for the drugs by using proposed analytical method in six replicates. The results were average for statistical evaluation.

Inter-day precision was estimated by assaying tablets powder blend containing 250 mg of amoxicillin and 150 mg of carbocisteine for three consecutive days (i.e. 1st, 3rd and 5th days). The statistical validation data for intra and inter day precision is summarized in table 4.

Table 4: Summary of validation parameter for intra-day and inter-day

Sr. No.

Parameters

Amoxicillin

carbocisteine

Intra-day precision

(N=3) amount found ± % R.S.D.

99.60%

0.24847

99.45%

0.03446

Inter-day precision

(N=3) amount found ± % R.S.D.

98.484

0.13607

98.762%

0.00768

Both intra- day and inter-day precision variation found to be less in % RSD values. It indicates high degree of precision of the method.

RESULT AND DISCUSSION:

The developed first order derivative spectrophotometric method for simultaneous determination of amoxicillin and carbocisteine in tablet formulation was found to be simple and convenient for the routine analysis of two drugs. The method is used to eliminate the spectral interference from one of the two drugs while estimating the other drug by selecting the zero crossing point on the derivative spectra of each drug as the selected wavelength. Simultaneous equation and absorbance ratio methods were not used due to maximum spectral overlap and more difference in the absorbance. The proposed method is accurate, precise and reproducible. It is confirmed from validation data as given in tables 1 to 4. The % RSD was found to be less than 1, which indicates validity of method. Linearity was observed by linear regression equation method for amoxicillin and carbocisteine in different concentration range. The correlation coefficient of these drugs was found to be close to 1.00, indicating good linearity figure 2 (a) and 2 (b).

The assay results obtained by proposed method is shown in table 2 are in good agreement. Hence proposed method can be used for routine analysis of these two drugs in combined dosage form. Method is simple, accurate, precise, reliable, rapid, sensitive, reproducible and economical. It is validate as per ICH guidelines.

CONCLUSION:

The proposed method is simple, precise, accurate and rapid for the determination of amoxicillin and carbocisteine in combined dosage form. The method does not require any ratio of first order derivative as suggested in literature [8]. The amplitude of first order derivative can be directly used to assay of formulation. This method can be adopted as an alternative to the existing methods. It can be easily and conveniently adopted for routine quality control analysis.

ACKNOWLEDGEMENT:

Authors express sincere thanks to the principal of D.G. Ruparel College, Dr. Tushar Desai, for encouragement and providing laboratory facilities.

REFERENCES:

[1] The United States Pharmacopeia. United States Pharmacopeia convention Inc, Rockville 2008, USP 29 NF 24, Vol no.30 (4), 1208.

[2] Indian Pharmacopeia, Controller of Publication, Delhi, 2007, Vol- 1, II, III.

[3] British pharmacopoeia. Licensing division HMSO, Norwich, 2003, 357.

[4] Archana Nadiminti, Ashwini Gunda, Karnaker Reddy Tupally,Abbaraju Prasanna Lakshmi, Kedarnath Jakka, Aravind Sai Nagubandi , Simultaneous estimation of Amoxicillin Trihydrate and Carbocysteine drug present informulation by RP-HPLC method and its Validation. Journal of Pharmacy Research. 5(4); 2012:1889-1895.

[5] Sonali K. Sarode, Sujata T. Pise, P. Dhore, D.R. Mundhada, Shyamala Bhaskaran. Development of analytical method for simultaneous estimation of Amoxicillin and Carbocisteine in solid dosage form by RP-HPLC. BioMedRx. 1(4); 2013:360-362.

[6] Vishal A. Patil, Anil C. Vispute, Sanjay B. Bari, Dilip A. Patil. RP-HPLC methods for simultaneous estimation of amoxicillin and carbocisteine in dosage form. Trade Science Inc. 13(5); 2013.

Received on 02.02.2014 Modified on 05.03.2014

Asian J. Research Chem. 7(4): April 2014; Page 412-416