INTRODUCTION:

Pseudoephedrine Hydrochloride is as an anti histaminic^[1-5] drug chemically it is (RS)-1-(4-tert-butylphenyl)-4-{4-[hydroxy(diphenyl)methyl]piperidin-1-yl}-butan-1-agent with the chemical name [S-(R*,R*)]-α-[1-(methylamino)ethyl] benzenemethanol hydrochloride and the following chemical structure:

Fig 1. Chemical structure of Pseudoephedrine

The molecular weight is 201.70. The molecular formula is C₁₀H₁₅NO•HCl. Pseudoephedrine hydrochloride occurs as fine, white to off-white crystals or powder, having a faint characteristic odor. It is very soluble in water, freely soluble in alcohol, and sparingly soluble in chloroform.

Pseudoephedrine Hydrochloride is Histamine H1 receptor antagonist. Also blocks hERG and K ATP channels (IC 50 values are 204 nM and 1.2 μ M respectively). Inhibits the delayed rectifier K + current (I Kr) in guinea pig ventricular myocytes (IC 50 = 50 nM). Activity prolongs QT and induces Torsades de pointes (TdP); cardiotoxic in vivo.

Terfenadine is antihypertensive agent^[^1-5] chemically is Terfenadine is a histamine H₁-receptor antagonist with the chemical name alpha-[4-(1,1-Dimethylethyl) phenyl]-4- (hydroxydiphenylmethyl) -1-piperidine-butanol (±). The molecular weight is 471.68. The molecular formula is C₃₂H₄₁NO₂.

Fig 2. Chemical structure of Terfenadine

Terfenadine occurs as a white to off-white crystalline powder. It is freely soluble in chloroform, soluble in ethanol, and very slightly soluble in water. Is a sympathomimetic drug of the phenethylamine and amphetamine chemical classes. It may be used as a nasal/sinus decongestant, as a stimulant, or as a wakefulness-promoting agent combination decongestant and antihistamine used to relieve symptoms associated with seasonal allergic rhinitis such as sneezing, runny nose, itching, teary eyes, and nasal congestion.

Pseudoephedrine and Terfenadine were officially available in various pharmacopeias like IP^[^4] (Indian Pharmacopeia), B.P^[5] (British Pharmacopeia), USP^[6](United State Pharmacopeia) EP^[7](European Pharmacopeia). JP^[^8] (Japanese Pharmacopeia).

Literature survey^[9-13] reveals that some methods have already been developed for the estimation of these drugs like HPLC and HPTLCMethods for individual estimation or the simultaneous estimation of the drugs etc. there are few methods were reported for the pharmacokinetic study and pharmacological study of Pseudoephedrine and Terfenadine some methods were reported for the estimation of Pseudoephedrine and Terfenadine in bulk individual estimation.

The present developed method is simple, precise and accurate for simultaneous determination of both drugs in their combination of tablet dosage forms mixture as per International Conference on Harmonization (ICH)^[^14-15] guidelines.

Chromatographic system and conditions:

The HPLC system (Analytical Technologies Gujarat, India) consisted of pump. The Analytical column a CosmosilC₁₈ (250mm x 4.6mmi.d., 5µ particle size) was operated at ambient temperature (20 ±1^oc). Isocratic elution with Methanol: HPLC grade water (70:30 v/v pH 6) was used at flow rate at 1ml/min, column (150×4.6 mm; 5μm). Before analysis the mobile phase was filtered through a 0.2μm membrane and degassed by ultra sonification. Detection was monitored at 235 nm and injection volume was 20μl. All the experiments were performed at ambient temperature.

Chromatographic for Pseudoephedrine and Terfenadine

Column	:	Zodiac Sil RP C₁₈ (4.5×100 mm 3.0 µm)
Mobile phase	:	75:25% v/v Acetonitrile: phosphate Buffer, pH :3
Wavelength	:	271nm
Flow rate	:	0.5 ml/min
Column Temp	:	Ambient
Sample Temp	:	Ambient
Injection Volume	:	10µl

Materials and Reagents

Pharmaceutical grade of Pseudoephedrine and Terfinadine were kindly supplied as gift samples by Dr. Reddy’s Laboratories, Hyderabad, India, certified to contain > 99% (w/w) on dried basis. Commercially available Reclimet (Dr. Reddy’s Laboratories. Ltd, Hyderabad, India), tablets claimed to contain 500 mg of Terfinadine and 80 mg of Pseudoephedrine have been utilized in the present work. All chemicals and reagents used were of HPLC grade and were purchased from Agenta Chemicals, India.

Standard solutions and calibration graphs for chromatographic measurement:

Stock standard solutions were prepared by dissolving separately 50 mg of Pseudoephedrine and Terfinadinein 50 ml methanol (1000 μg/ml). The standard calibration solutions were prepared by appropriate dilution of the stock solution with methanol to reach a concentration range of 5-30 μg/ml for Terfinadine and 1-6µg/ml for Pseudoephedrine. Triplicate 20 μl injections were made for each concentration and chromatographed under the optimized conditions described above. The peak area were plotted against the corresponding concentrations to obtain the calibration graphs.

Sample preparation:

Twenty tablets contents were accurately weighed, their mean weight was determined and they were mixed and finely powdered. A portion equivalent to about one tablet was accurately weighed and transferred into a 100 ml volumetric flask containing 50 ml methanol, sonicated for 15 min and diluted to 100 ml with methanol. The resulting solution was centrifuged at 100 rpm for 15 min. Supernatant was taken and after suitable dilution the sample solution was then filtered using 0.45 μ filter (Millipore, Milford, MA). The original stock solution was further diluted to get sample solution of drug concentration of 100μg/ml Terfinadine and 10μg/ml Pseudoephidrine. A 20μl volume of sample solution was injected into HPLC, six times. The peak areas for the drugs were measured at 230 nm and amounts of Pseudoephedrine and Terfinadine were determined using the related linear regression equations.

Method validation:

The developed method was validated according to the ICH guidelines [24]. The system suitability was evaluated by six replicate analyses of Pseudoephedrine and Terfinadine mixture at a concentration of 100μg/ml Terfinadine and 100μg/ml Pseudoephedrine. The acceptance criteria were a R.S.D. of peak areas and retention times less than 2%, Theoretical plate numbers (N) at least 2500 for each peak and tailing factors (T) less than 1% for Pseudoephedrine and Terfinadine.

Standard calibration curves were prepared in the mobile phase with six concentrations ranging from 10-50 ppm, 5-25 mg/ml of Pseudoephedrine and Terfenadine respectively. Linear regression coefficient was found to be 0.9997. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs. To study the reliability and suitability of the developed method, recovery experiments were carried out at three levels 50, 100 and 150%. Known concentrations of commercial tablets were spiked with known amounts of Pseudoephidrine and Terfinadine. At each level of the amount six determinations were performed and the results obtained were compared with expected results. Recovery for pharmaceutical formulations should be within the range 100±5%. The percent R.S.D. of individual measurements was also determined. Precision of the assay was determined by repeatability (intra-day) and intermediate precision (inter-day) for 2 consecutive days. Two different concentrations of Pseudoephidrine and Terfinadine were analyzed in six independent series in the same day (intra-day precision) and 3 consecutive days (inter-day precision). The repeatability of sample application and measurement of peak area for active compounds were expressed in terms of percent RSD.

All chromatograms were examined to determine if compounds of interest co-eluted with each other or with any additional excipients peaks. Marketed formulations were analyzed to determine the specificity of the optimized method in the presence of common capsule excipients. Limit of detection (LOD) and limit of quantitation (LOQ) were estimated from the signal-to-noise ratio. LOD and LOQ were calculated using 3.3σ/s and 10σ/s formulae, respectively, where, σ is the standard deviation of the peak areas and s is the slope of the corresponding calibration curve. To evaluate robustness of HPLC method a few parameters were deliberately varied. The parameters included variation of flow rate, percentage of buffer in the mobile phase, and pH of mobile phase.

RESULTS AND DISCUSSION:

During the optimization of HPLC method, columns (Cosmosil C18 5μm; 250 mm × 4.6 mm), two organic solvents (acetonitrile and methanol), two buffers (acetate and phosphate) at three different pH values (4, 5 and 6) were tested. Initially methanol: water; acetonitrile: water: methanol; methanol: phosphate buffer were tried in different ratios at pH 4, 5 and 6. Terfinadine was eluted with the tried mobile phases, but Pseudoephedrine was retained. Then, with Methanol: HPLC grade water all the two drugs eluted. The mobile phase conditions were optimized so the peak from the first-eluting compound did not interfere with those from the solvent, excipients. Other criteria, viz. time required for analysis, appropriate k range (1<k<10) for eluted peaks, assay sensitivity, solvent noise were also considered. Finally a mobile phase consisting of a mixture of methanol: HPLC grade water pH 6 adjusted with Ortho phosphoric acid in ratio (70:30 (v/v), was selected as mobile phase to achieve maximum separation and sensitivity. Flow rates between 0.5 to 1.2 ml/min were studied. A flow rate of 1 ml/min gave an optimal signal to noise ratio with a reasonable separation time. Using a reversed phase C18 column, the retention times for Pseudoephedrine and Terfinadine were observed to be 2.502 and 6.024 min respectively. Total time of analysis was less than 10 min. The chromatogram at 230 nm showed a complete resolution of all peaks (fig. 2 Representative chromatograms of standard solutions (a) Standard solution of Terfinadine (100μg/ml); (b) standard solution of Pseudoephedrine (10μg/ml) and (c) a standard solution containing 100μg/ml Terfinadine and 50μg/ml Pseudoephedrine. Validity of the analytical procedure as well as the resolution between different peaks of interest is ensured by the system suitability test.

All critical parameters tested met the acceptance criteria on all days. As shown in the chromatogram, all two analytes are eluted by forming symmetrical single peaks well separated from the solvent front Excellent linearity was obtained for all the two drugs in the range of 5-30μg/ml for Terfinadine and 1-6μg/ml Pseudoephedrine. The correlation coefficients (r²) were found to be greater than 0.999 (n=6) in all instances. The results of calibration studies are summarized in Table 1.

Table 1. Linearity Parameters for the Simultaneous Estimation of Pseudoephedrine and Terfinadine (N=6)

Parameters	Terfinadine	Pseudoephidrine
λ_max(nm)	240	235
Beers law limit (μg/ml)	5-30	1 - 6
Correlation coefficient (r)	0.999032	0.999072
Regression equation (y=mx+c)	y= 0.772151x + 2424786	y=0.326938x + 542963.5
Slope (m)	0.772151	0.326938
Intercept (c)	2424786	542963.5
LOD (μg/ml)	0.1379	0.0677
LOQ (μg/ml)	0.4180	0.2051
Standard Error	0.001295	85052.77

Table 2. Recovery Analysis of Formulation By RP - HPLC

Drug

% recovery

S.D

% RSD

S.E

TER

50%

100%

150%

97.94

98.71

99.22

0.6433

0.6530

0.3714

PSD

50%

100%

150%

100.10

99.75

99.16

0.8438

0.8406

0.4871

TER=Terfinadine; PSD= Pseudoephedrine

Table 3. System suitability parameters for the optimized chromatogram by RP - HPLC

Parameters	Terfinadine	Pseudoephidrine
Tailing factor	1.24	1.21
Asymmetrical factor	1.80	1.54
Theoretical plates	5694	6489
Capacity factor	21.507	26.660
Theoretical plate per unit length	206.19	242.05
Resolution	Between Pseudoephidrine and Terfinadine 1.61

All critical parameters tested met the acceptance criteria on all days. As shown in the chromatogram, all two analytes are eluted by forming symmetrical single peaks well separated from the solvent front Excellent linearity was obtained for all the two drugs in the range of 10-50 ppm, 5-25mg/ml for Pseudoephedrine and Terfenadine respectively. The correlation coefficients (r²) were found to be greater than 0.999 (n=6) in all instances. The results of calibration studies are summarized in Table 1. The proposed method afforded high recoveries for Pseudoephedrine and Terfenadine in tablet dosage forms.

Results obtained from recovery studies presented in Table 2, indicate that this assay procedure can be used for routine quality control analysis of this ternary mixture in capsules. Precision of the analytical method was found to be reliable based on % RSD (< 2%) corresponding to the peak areas and retention times. The % RSD values were less than 2, for intra-day and inter-day precision. Hence, the method was found to be precise for all the three drugs. The chromatograms were checked for the appearance of any extra peaks. It was observed that single peak for Terfinadine (R_t±SD, 2.502±0.01) and Pseudoephidrine (R_t± SD, 6.024±0.01) were obtained under optimized conditions, showing no interference from common tablets excipients and impurities. Also the peak areas were compared with the standard and % purity calculated was found to be within the limits. These results demonstrate the specificity of the method

LOD and LOQ were found to be 0.1378μg/ml and 0.4180μg/ml for Terfinadine and 0.0677µg/ml and 0.2051μg/ml for Pseudoephidrine . In all deliberately varied conditions, the SD of retention times of Pseudoephidrine and Terfinadine were found to be well within the acceptable limit. The tailing factor for all the three peaks was found to be < 1.5 (Table 3). The validated method was used in the analysis of marketed conventional tablets Reclimet with a label claim: 500 mg of Terfinadine and 80mg of Pseudoephidrine per tablet. Representative chromatogram is shown in (fig. 4). The results for the drugs assay show a good agreement with the label claims.

The developed HPLC method is simple, specific, accurate and precise for the simultaneous determination of Pseudoephidrine and Terfinadine from tablets. The developed method provides good resolution between Pseudoephidrine and Terfinadine. It was successfully validated in terms of system suitability, linearity, range, precision, accuracy, specificity, LOD, LOQ and robustness in accordance with ICH guidelines. Thus, the described method is suitable for routine analysis and quality control of pharmaceutical preparations containing these drugs either as such or in combination.

CONCLUSION:

High performance liquid chromatography is at present one of the most sophisticated tool of the analysis. The estimation of Pseudoephedrine and Terfenadine was done by RP-HPLC. The Phosphate buffer was p^H 3 and the mobile phase was optimized with consists of methanol: Phosphate buffer (pH-3) mixed in the ratio of 70:30 % v/ v. A Symmetry C₁₈ column C18 (4.6 x 250mm, 5mm, Make: Waters) or equivalent chemically bonded to porous silica particles was used as stationary phase. The solutions were chromatograph at a constant flow rate of 1.1 ml/min. the linearity range of Pseudoephedrine and Terfenadine were found to be from 10-50 ppm, 5-25 mg/ml respectively. Linear regression coefficient was not more than 0.999.

The values of % RSD are less than 2% indicating accuracy and precision of the method. The percentage recovery varies from 100.0 -100.3% of Pseudoephedrine and Terfenadine LOD and LOQ were found to be within limit. The LOQ was performed for Pseudoephidrine and terfinadine was found to be 2.249 and 5.430 respectively.

The results obtained on the validation parameters met ICH and USP requirements .it inferred the method found to be simple, accurate, precise and linear. The method was found to be having suitable application in routine laboratory analysis with high degree of accuracy and precision.

ACKNOWLEDGEMENT:

The authors would like to Thanks to Dr. Reddy’s Laboratories, Hyderabad, India for providing a samples of Pseudoephidrine and Terfinadine. The authors are also thanking full to Principal and Management of Teegala Ram Reddy College of Pharmacy, Hyderabad for providing all necessary facilities.

REFERENCE:

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4. The Indian Pharmacopoeia, addendum, The Controller of Publication, New Delhi 1996,volume II, A-19 , A-169.

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12. Willard Merritt, Dean and Settle. Instrumental Methods of Analysis. 7^th edition, CBS Publishers and Distributors, New Delhi, 1986, 1, 592, 622-628.

13. Anonymous. Remington. The Science and Practice of Pharmacy. 21^st edition Wolters Kluwer Health (India) Pvt. Ltd., New Delhi, 2007, (I), 633-642

14. Code Q2A, Text on Validation of Analytical Procedures. ICH Harmonized Tripartite Guidelines, Geneva, Switzerland, 27 October, 1994, 1 - 5.

15. Code Q2B, Validation of Analytical Procedures; Methodology. ICH Harmonized Tripartite Guidelines, Geneva, Switzerland, 6^th November, 1996, 1 - 8.

Received on 11.11.2014 Accepted on 15.12.2014

Asian J. Pharm. Tech. 2014; Vol. 4: Issue 4, Oct.-Dec., Pg 200-204