1. INTRODUCTION:

Diethylcarbamazine citrate (N,N diethyl-4-methyl piperazine-1 carboxamide dihydrogen citrate)¹ is an anthelmintic drug belonging to class of piperazine having highly selective effect on microfilaria and also for tropical eosinophilia². Cetirizine hydrochloride (2-(4-(4 chlorophenyl) phenyl methyl)1-piperazinyl) ethoxy acetic acid³ is second generation anti histamine belonging to class of piperazines.It is used in upper respiratory allergies and also inhibits eosinophil chemotaxis². The structures of Diethylcarbamazine citrate and Cetirizine hydrochloride are shown in the figure-1.The literature review reveals that some individual works of both drugs were carried out in HPLC. The chromatographic conditions for the analysis of DECC are by using C₈column with phosphate buffer and Acetonitrile (1:9 v/v) at pH-3.2 at 210nm with a flow rate of 1.5ml/min⁴ and Cetirizine was done at 205nm with Acetonitrile and water in the ratio (1:1 v/v) by using C₁₈column⁵. The present paper describes a simple, sensitive, validated and economic method for simultaneous estimation of DECC and Cetirizine.

2. EXPERIMENTAL:

2.1 Materials and methods:

Reagents:

Reference standards of Diethylcarbamazine citrate and Cetirizine hydrochloride were obtained from SIGMA-ALDRICH, USA. Acetonitrile (HPLC grade, Merck) potassium dihydrogen phosphate and orthophosphoric acid AR grade were procured from local sources. Formulation was obtained from local market.

2.2 Instrumentation:

The HPLC (Shimadzu LC 10 AT VP) instrument was equipped with UV-Visible SPD 10 Avp detector, rheodyne injector with a 20µl loop and C₁₈Column (250 × 4.6mm, 5µm).

2.3 Chromatographic condition:

The mobile phase consisting of Acetonitrile and mixed phosphate buffer 60:40(%v/v) (pH – 3.0 with 10% orthophosphoric acid) was used at a flow rate of 1.0ml/min.The wavelength was set at 220nm.Analysis was performed at ambient temperature.

2.4 Preparation of 0.05 M phosphate buffer solution:

1.625 gm of potassium dihydrogen phosphate and 300mg of Di potassium hydrogen phosphate were weighed and made upto 550ml with water. The solution was sonicated for 20 minutes and membrane filteration was carried out. pH is adjusted with 10% orthophosphoric acid to 3.

(a) Diethylcarbamazine citrate

(b) Cetirizine hydrochloride

Figure -1 Structures of Diethylcarbamazine citrate and Cetirizine hydrochloride

2.4.1 Preparation of Standard stock solution:

30mg of Diethylcarbamazine citrate and 1mg of Cetirizine Hydrochloride were transferred to 25 ml volumetric flask, 10ml of mobile phase was added and sonicated for 15 min and volume was made up with mobile phase. From this 0.2,0.4,0.6,0.8,1,1.2ml were drawn into 10ml flask and made upto volume to get a concentration of 24 -144µg/ml(DECC) and 0.8-4.8µg/ml of Cetirizine.This solution is used for the total work.

2.4.2 Preparation of sample solution:

For estimating the tablet dosage form, twenty tablets (CETRI –PLUS) were accurately weighed and finely powdered. Tablet powder equivalent to 300mg of DECC and 10mg of Cetirizine was transferred into 100ml volumetric flask.15ml of mobile phase was added and subjected to sonication for 10min with intermediate shaking for complete extraction of drugs. The solution was made up to the mark with mobile phase. The sample was centrifuged in tight closure for 10min at 2000rpm.Then 0.8ml of clear solution was transferred into 10ml volumetric flask and diluted with mobile phase .Then the solution is injected into HPLC system for analysis.

3. METHOD VALIDATION:

The method was validated in terms of linearity, range, specificity, accuracy, precision, limit of detection (LOD) and limit of Quantitation (LOQ).

3.1 Linearity and Range:

Six different concentrations of mixture of two drugs were prepared for linearity studies. The responses were measured as peak area. The calibration curve obtained by plotting area against concentration showed linearity in the concentration range of 24-144 µg/ml and 0.8- 4.8 µg/ml for both the drugs. The linear regression equations for DECC and Cetirizine were found to be Y = 8.423x + 16.20 and Y = 31.76x + 1.065 respectively. The regression co-efficient values (r²) were found to be 0.998 and 0.997 respectively.

3.2 Specificity:

The Specificity studies states the absence of interference, since none of the peaks of Diethylcarbamazine citrate and Cetirizine appeared at same retention time. It was carried out by using different conditions like treating with acid, base, heat etc.

3.3 Precision:

System precision and Method precision were determined in the following manner.

3.3.1 System Precision:

From standard stock solutions (100%), six replicate injections were injected into rheodyne injector, peak areas were obtained and %RSD was determined from the peak areas.

3.3.2 Method Precision:

From sample solution (100%), six replicate injections were injected into rheodyne injector, peak areas were obtained and %RSD was calculated from the peak areas.

3.4 Assay:

One Standard and three samples solutions were injected into the system. Peak areas were obtained and %recovery was found out.

3.5 Accuracy:

Recovery studies were carried out at 80%, 100% and 120%.The amount recovered and percentage recovery was found out at each level, by injecting six replicate injections. From the results obtained it can be concluded that the method was accurate.

3.6 Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ were separately determined on the basis of standard calibration curve.LOD is the lowest concentration that can be detected where as LOQ is lowest concentration that can be quantified. The LOD is calculated by using the formula LOD = 3.3 x SD/S and LOQ is calculated by LOQ = 10 x SD/S, where SD is standard deviation and S is slope of calibration curve.

4. RESULTS AND DISCUSSION:

Method development:

The drugs DECC and Cetirizine were very soluble in water, sparingly soluble in alcohol, practically insoluble in acetone, chloroform and ether. Then it was chequed for different dilutions of Acetonitrile and phosphate buffer .The spectrum was scanned over a range of 200 – 400 nm. DECC was detected at 216nm and Cetirizine at 230nm, the overlapping spectra showed that 220nm is optimum wavelength for both drugs and hence analysis is carried out at this wavelength. The spectra is given in the Figure – 3.

Figure -3 Overlapping spectra of DECC and Cetirizine.

Different mobile phase ratios and pH were tried to resolve the peaks of DECC and Cetirizine, because of different reasons like peak shape, tailing factor, less retention time they were not selected.The optimum mobile phase containing Acetonitirile and mixed phosphate buffer 60:40(%v/v) was selected because of good resolution 3.518 with a retention time of 3.050 for DECC and 3.967 for Cetirizine. The pH was adjusted to 3.0 with orthophosphoric acid.

Method Validation:

Linear regression data showed a good relationship over a concentration range of 24 -144 µg/ml and 0.8 - 4.8 µg/ml for DECC and CTH. The correlation co-efficients (r²) were found to be 0.998 and 0.997 respectively. The %RSD for System and Method precision for Diethylcarbamazine citrate is 0.143 and 0.112 and for Cetirizine is 0.344 and 0.513 respectively. The lower value of %RSD indicates that the method is precise. Analysis of marketed tablets (CETRI-PLUS) was carried out using optimized chromatographic condition. The % drug content obtained by the proposed method was found to be between 98-101%.The results are given in the table -1.

Table -1 Assay of Marketed Formulations

S. No	Area of DECC	Area of CTH	% of DECC	% of CTH
1	864.225	108.283	98.03	98.33
2	873.379	108.833	99.07	98.83
3	873.614	110.503	99.09	100.3
%RSD	0.615	1.057	1.009	1.032

To study accuracy of developed method, recovery studies was carried out at three different levels 80%,100% and 120%.The percentage recovery was showed in the Table -2.

Table -2 Recovery Studies

Injection sample	Spike level	Amount present	Amount recovered	% Recovery
DECC	80%	96 mcg	95.58	99.56%
DECC	100%	120 mcg	119.49	99.58%
DECC	120%	144 mcg	144.49	100.43%
CTH	80%	3.2 mcg	3.18	99.52%
CTH	100%	4.0 mcg	3.94	98.53%
CTH	120%	4.8 mcg	4.78	99.58%

The limit of detection and limit of Quantitation for DECC was found to be 0.62 and 1.89 µg/ml and for Cetirizine was 0.105 and 0.32 µg/ml for Cetirizine.

System suitability parameters:

For system suitability parameters, the standard solution was injected and parameters like resolution, tailing factor, number of theoretical plates and retention time were calculated. The results are shown in the table -3.

Table -3 System suitability parameters

System suitability parameters	Diethylcarbamazine citrate	Cetirizine hydrochloride
Resolution	-	3.518
Tailing factor	1.987	1.879
Number of theoretical plates	2509	3133
Retention time	3.050	3.967

The Method was validated as per ICH guidelines in terms of Linearity, Accuracy, Specificity, precision, LOD, LOQ and the results are shown in the table -4.

Table -4 Validation parameters

S. No	Parameters	DECC	Cetirizine
1	Linearity and Range	0.998	0.997
2	Specificity	No interference	No interference
3	System Precision	0.143	0.344
4	Method Precision	0.1124	0.513
5	Accuracy	99.56% -100.4%	99.52% - 99.58%
6	Limit of Detection	0.62µg/ml	0.105µg/ml
7	Limit of Quantitation	1.89µg/ml	0.32µg/ml

5. CONCLUSION:

The proposed study describes an RP-HPLC method for the Simultaneous estimation of Diethylcarbamazine citrate and Cetirizine hydrochloride. The method was simple and precise because of commonly used buffer, easier extraction procedure and shorter run time. It is accurate and cost effective that can be applicable over the range of 24-144 µg/ml and 0.8-4.8µg/ml. High percentage of recovery shows that the method was free from the interferences of the excepients used in the formulations. The low standard deviation value and %RSD value indicate good precision. The method relies upon use of simple, cheap chemicals and showed correlation coefficient of 0.998 and 0.997.

Hence this method can be used as an alternative for rapid and routine determination of DECC and Cetirizine in bulk samples and formulation.

6. REFERENCES:

1. Indian pharmacopoeia 2007 and 2010, pp 1030.

2. Martindale, The complete drug reference – 34^thedition, 2005, pp-97, 104.

3. British pharmacopoeia 2008, vol –I, page no 463.

4. Nisha Mathew and Kalyanasundaram.M, “High performance liquid chromatographic method for the estimation of Diethylcarbamazine content in medicated salt samples”, Acta Tropica, 2001: 97-102.

5. Najma sultana, M.saeed Arayne and Farhan Ahmed Siddiqui “Determination and quantification of Cetirizine Hydrochloride in dosage formulations by RP – HPLC”, Pakistan journal of pharmaceutical sciences, 2005, vol-18:7-11.

6. Birajdar A.S “simultaneous analysis of Ambroxol Hydrochloride with Cetirizine Hydrochloride and of Ambroxol hydrochloride with Levocetirizine dihydrochloride in solid dosage form by RP –HPLC”, Acta chromatographica, volume 20, september 2008, issue 3:411-421.

Received on 23.03.2011 Modified on 02.05.2011

Asian J. Research Chem. 4(7): July, 2011; Page 1064-1066