INTRODUCTION:

Paracetamol designed chemically as {N-(4-Hydroxyphenyl) acetamide}, it is an analgesic, antipyretic agent. Caffeine designed 1, 3, 7-Trimethyl-3, 7-dihydro-1H-purine-2,6-dione, it is a Central nervous system stimulant. Pseudoephedrine Hydrochloride designed chemically as (1S, 2S)-2-methylamino-1-phenyl-1-propanol hydrochloride, it is a novel Sympathomimetic. Dextromethorphan Hydrobromide chemically designed as {ent-3-methoxy-9a- methyl morphinan Hydrobromide monohydrate}, it is a Cough suppressant. Loratadine designed chemically as 4-(8-chloro-5,6-dihydro-11H-benzo[5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidine-carboxylic acid ethyl ester}, Loratadine acts as Anti-allergic agent, Antihistamines, Antipruritics¹.

(Paracetamol)

(Caffeine)

(Pseudoephedrine HCl)

(Dextromethorphan HBr)

(Loratadine)

Various HPLC methods were reported for Paracetamol^2-7, Pseudoephedrine Hydrochloride^{5, 6}, Dextromethorphan Hydrobromide⁷, and Caffeine⁸. But no HPLC method was reported for simultaneous determination of Paracetamol, Caffeine, Pseudoephedrine Hydrochloride, Dextromethorphan Hydrobromide and Loratadine in combination.

MATERIALS AND METHOD:

Apparatus:

Instruments used in present study were Agilent 1200 series and Shimadzu LC-2010A_HT.Liquid chromatographic system equipped with UV- Vis detector and data analyzed by using Chromeleon 6.2 version software.

Materials:

Paracetamol, Caffeine, Pseudoephedrine Hydrochloride, Dextromethorphan Hydrobromide and Loratadine working standards and marketed drugs formulation Cofdex tablet were procured from Okasa Pharma Pvt. Ltd., Satara, (Maharashtra).

Methanol, Acetonitrile and Water used was of HPLC grade. 1- Heptane Sulfonic Acid Sodium Salt was used in mobile phase A and Orthophosphoric acid was used for P^H adjustment.

Chromatographic conditions:

Chromatographic separation was performed on an Inertsil ODS 3V, 4.6 mm x 5 cm, 3 µm. column. The analysis was resolved by using a mobile phase (Sodium heptane sulphonate buffer solution and acetonitrile in the ratio of 95:5) at a flow rate of 1ml/min for gradient program shown in (Table No.1). The injection volume was 10µl and ambient at temperature. The mobile phase was filtered through a 0.45 μ membrane filter and sonicated. Analysis was performed at ambient temperature. Detection was carried out a 205 nm. Retention time were found to be for Paracetamol (3.5 mins), Caffeine (5.5 mins), Pseudoephedrine HCl (8mins), Dextromethorphan HBr (14 mins), Loratadine (15 mins), within run time of 25 mins.

Preparation of solutions:

Diluent: Purified water, acetonitrile and methanol in the ratio of 25:25:50.

Standard preparation:

Weigh accurately and transfered about 21 mg of Dextromethorphan Hydrobromide working standard and 61 mg of Caffeine working standard into a 50 ml volumetric flask. Add 30 ml of diluent. Sonicated to dissolve, cool and diluted upto the volume with diluent. (Solution A). Weigh accurately and transfered about 26 mg of Loratadine working standard into a 100 ml volumetric flask. Add 60 ml of diluent. Sonicated to dissolve, cool and diluted upto the volume with diluent. (Solution B). Weigh accurately and transferd about 25 mg of Paracetamol working standard into a 25 ml volumetric flask. Add 10 ml of diluent. Sonicated to dissolve, cool and dilute upto the volume with diluent. (Solution C). Weigh accurately and transfered about 24 mg of Pseudoephedrine Hydrochloride working standard into a 100 ml volumetric flask. Add 60 ml of diluent, sonicated to dissolve, cool and add 10 ml of solution A, 5 ml of solution B and 10 ml of solution C. Diluted upto the volume with diluent.

Sample preparation:

For Loratadine, Pseudoephedrine Hydrochloride, Dextromethorphan Hydrobromide and Caffeine:

Weigh accurately 20 tablets and triturated with glass mortar and pestle. Weigh accurately and transfered 0.900 gm of sample (equivalent to 1 tablet) into a 250 ml volumetric flask. Add 170 ml of diluent. Sonicated for 30 mins, cool and dilute upto the volume with diluent. Filtere through 0.45 µm syringe filter. (Solution A)

For Paracetamol:

Pipette out 5 ml solution A, transfered into a 100 ml volumetric flask. Add 70 ml of diluent. Shake and diluted upto the volume with diluent. Filter through 0.45 µm Syringe filter.

Method development:

The method for estimation of Paracetamol, Caffeine, Pseudoephedrine Hydrochloride, Dextromethorphan Hydrobromide and Loratadine were developed using different mobile phase concentration and wavelength of detection. The mobile phase concentration for gradient program was shown in (Table No.1) and wavelength of detection for Caffeine, Pseudoephedrine Hydrochloride, Dextromethorphan Hydrobromide and Loratadine at 236nm and for Paracetamol at 205nm. Finally, 205nm was found to be ideal wavelength of detection for Caffeine, Pseudoephedrine Hydrochloride, Dextromethorphan Hydrobromide and Loratadine.

Table 1: Gradient Program of Mobile Phase

Time (min.)	Mobile Phase A	Mobile Phase B
0.01	95	5
3	90	10
4.5	80	20
10	80	20
11	65	35
15	50	50
16	50	50
17	90	10
25	95	5

RESULT AND DISCUSSION:

Analytical method used for assay of Paracetamol, Caffeine, Pseudoephedrine Hydrochloride, Dextromethorphan Hydrobromide and Loratadine used in Cofdex Tablet by using High performance liquid chromatography technique was validated. Validation was carried out on Shimadzu LC 2010A HPLC System and Agilent-1200 HPLC System with Chromeleon software (6.2). The validation of the method was assessed by establishing validation criteria such as Specificity and System Suitability, Linearity and Range, Precision (repeatability and intermediate precision), Accuracy, Solution Stability and Robustness study.

Validation of method:^{9, 10}

Specificity:

Specificity was carried out to monitor interference from blank and to monitor system suitability. Standard solutions were injected into the chromatograph in six replicates. The % RSD for peak area response and retention were found within limit (Not more than 2.00% for peak area response and not more than 1.00% for retention time). The system suitability parameters like theoretical plates and tailing factor were found within limits.

Linearity, LOD and LOQ:

Linearity and Range were carried out over a range of 50 to 150% of working level concentration. The linearity regression correlation coefficient, % Y- intercept and % RSD for peak area response and retention time for lower and higher range were calculated. The linearity regression correlation coefficient for the component was found within limit (Not less than 0.999).The % Y- intercept for the component was found within the limit. (Not more than + 2.0). The %RSD for the peak area response and retention time for lower and higher range was found within limit. The % RSD for Response factor was found within the limit (NMT 5.00%). LOD and LOQ were calculated by STEYX method.

Accuracy: (% recovery)

Accuracy levels were prepared by 50, 100, and 150 % of working level concentration, prepared in triplicate for each levels and the percentage recovery were calculated for each levels separately. The percentage recoveries observed for the levels were found well within the limit set for the accuracy study (Not less than 98.0% and not more than 102.0%), shown that the content was recovered and hence is accurate.

Figure No.1: (STD mixture)

Figure No.2: (Sample -Caffeine, Pseudoephedrine HCl, Dextromethorphan HBr, Loratadine)

Figure No.3: (Sample-Paracetamol)

Table 2: Summary System Suitability

Parameters	Theoretical Plates	Tailing Factor	Similarity Factor	%RSD of STD A for Area	%RSD of STD A for RT
Paracetamol	2817	0.9	0.99	0.12%	0.09%
Caffeine	25699	1.1	1.0	0.19%	0.05%
Pseudoephedrine HCl	33926	1.4	0.99	0.15%	0.03%
Dextromethorphan HBr	301825	1.3	0.99	0.16%	0.01%
Loratadine	211434	1.2	0.99	0.18%	0.01%

Figure No.4: Standard Mix. (6 Replicates) for Specificity

Table 3: Summary of Linearity, LOD, LOQ

Name	%Y Intercept	Correlation Coefficient	Response Factor (%RSD)	LOD	LOQ
Paracetamol	0.6	1.000	1.2%	0.083616	0.253382
Caffeine	1.4	1.000	1.2%	0.234738	0.711326
Pseudoephedrine HCL	2.0	1.000	1.6%	0.047135	0.142834
Dextromethorphan HBr	1.5	0.999	1.4%	0.026874	0.081438
Loratadine	-1.1	1.000	0.4%	0.005663	0.01716

Figure 4: Linearity of Paracetamol

Figure 5: Linearity of Caffeine.

Figure 6: Linearity of Pseudoephedrine HCl

Figure 7: Linearity of Dextromethorphan HBr

Figure 8: Linearity of Loratadine

Intermediate Precision (Ruggedness):

Sample was reanalyzed by another analyst on another system on another day by using another column for six times. Results were calculated. The results of intermediate precision study along with repeatability study were compared and found well within the limits set for the intermediate precision study.

Table 4: Summary of Accuracy

Parameters	Recovery (50%)	Recovery (100%)	Recovery (150%)	% RSD of Recovery
Paracetamol	101.7	98.3	98.7	0.33
Caffeine	101.3	101.3	98.8	0.40
Pseudoephedrine HCl	100.9	100.8	98.9	0.26
Dextromethorphan HBr	101.6	100.6	99.8	0.33
Loratadine	101.2	100.7	100.5	0.30

Table 5: Summary of Intermediate Precision (Ruggedness)

Parameters	%RSD of Assay	% Variation	Similarity Factor
Paracetamol	0.9	0.9	1.00
Caffeine	0.4	0.4	1.00
Pseudoephedrine HCl	0.5	1.1	1.01
Dextromethorphan HBr	0.6	0.4	1.01
Loratadine	0.7	1.1	1.00

Solution stability:

The solution stability was monitored to check the stability of solution. A sample solution was preserved at 2-8ºC. Solution stability Checked at after each time interval (2, 4, 8,12,16,20 and 24 hrs) and analyzed after the specified time interval. The results of initial analysis and the results of analysis after preservation for content was compared and found well within the set limit for solution stability study for Paracetamol, Caffeine, Pseudoephedrine Hydrochloride, Dextromethorphan Hydrobromide and Loratadine content in sample upto 24 hours.

Robustness:

The robustness of method was carried out by changing the different chromatographic conditions (one at a time) such as:

Ø Change in flow rate of mobile phase from 1.0 to 0.9 ml/min.

Ø Change in flow rate of mobile phase from 1.0 to 1.1 ml/min.

Ø Change in pH from 3.0 to 2.8

Ø Change in pH from 3.0 to 3.2

Table 6: Robustness

Parameter	Flow rate		Change in pH
Changes in parameter	0.9 mi/min	1.1 ml/min	Low pH (2.8)	High pH (3.2)
% LABLE CLAIM
Paracetamol	99.7	99.1	99.8	99.8
Caffeine	101.2	102.5	101.4	102.4
Pseudoephedrine HCl	99.8	101.2	99.2	100.4
Dextromethorphan HBr	103.7	103.9	104.1	104.6
Loratadine	99.9	100.3	97.6	100.8

The results of robustness study along with precision study were compared and found well within the limits set for the robustness study. Hence the method is robust.

CONCLUSION:

The method provides selective quantification of Paracetamol, Caffeine, Pseudoephedrine HCl, Dextromethorphan HBr and Loratadine without interference from blank affirming its stability- indicating nature. The proposed method was found to be highly sensitive, reproducible, specific and rapid. The method was completely validated showing satisfactory data for all the method validation parameters tested. The developed method was robust in the separation and quantification of Paracetamol, Caffeine, Pseudoephedrine HCl, Dextromethorphan HBr and Loratadine. This method can be used for the routine analysis of production samples. The information presented herein could be very useful for quality monitoring of bulk samples and as well employed to check the quality during stability studies.

ACKNOWLEDGEMENT:

We are thankful to Okasa Pharma Pvt. Ltd., Satara (Maharashtra) and also thankful to Dr. A. D. Taranalli Principal, KLEU’s College of Pharmacy, Belgaum (Karnataka), for providing all facilities during research work.

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Received on 18.04.2011 Modified on 22.05.2011

Asian J. Research Chem. 4(7): July, 2011; Page 1141-1147