INTRODUCTION:

Pseudoephidrine sulphate (PSEUDO) is an orally active direct and indirect sympathomimetic upper respiratory tract decongestant drug. It is widely used in allergic rhinitis, cold, coughs, and sinusitis and to prevent otisis barotrauma. Chemically the drug is (1S, 2S) -2- methylamino-1- phenylpropan-1-ol sulphate. Several methods such as HPLC^[1-2], HPTLC³, packed column supercritical fluid chromatography⁴, and spectrophotometry^5-8 have been reported in the literature.

Desloratidine (DES) is 8- Chloro-6; 1- dihydro-11 (-4 piperidinylidine) -5H- benzo [5, 6] Cycloheptal [1, 2-b] pyridine, descarboethoxyloratidine. C₁₉H₁₉ClN₂ ^{9, 10}having molecular weight 310.82. It is not yet official in any Pharmacopoeia. It is non sedating peripheral histamine H₁ receptor antagonist, active metabolite of loratidine. A survey of literature reveals that DES is estimated by HPLC^11-12, HPTLC¹³, spectrophotometry¹⁴, special data analysis¹⁵ . There is no single method for this combination. The present HPLC method was validated as per ICH guidelines¹⁶.

EXPERIMENTAL:

Reagents and Chemicals:

Acetonitrile (HPLC grade) and Methanol (HPLC grade) was purchased from Merck specialties pvt. Ltd. (Worli, Mumbai, India) and Water (HPLC grade) was purchased from Loba Chemie (Mumbai, India). Ammonium acetate was purchased from Sisco research Laboratories Pvt. Ltd. (Mumbai, India).

All other reagents used were of HPLC grade. Working standard of Pseudoephidrine sulphate and Desloratidine were provided by Glenmark Ltd., Mumbai, India and Nabumetone was obtained from Micro Labs Ltd., Hosur.

(c) Pharmaceutical formulation:

Commercial tablets, each containing Pseudoephidrine sulphate (120mg) and Desloratidine (5 mg); were procured from the local market.

Method Development:

Different mobile phases containing methanol, water, Acetonitrile, and different buffers in different proportion were tried and finally of Acetonitrile: Methanol: Ammonium acetate 45:40:15 v/v was selected as moile phase which gave good resolution and acceptable peak parameters for both Pseudoephidrine sulphate and Desloratidine.

System Suitability Studies:

The resolution, number of theoretical plates and peak asymmetry were calculated for the standard solutions and is as shown in Table 1. The values obtained demonstrated the suitability of the system for the analysis of these drugs in combinations. The typical chromatogram of standard solution is as shown in Fig.1.

Apparatus and chromatographic Conditions:

Chromatographic separation was performed on a Jasco chromatographic system equipped with a Jasco PU-2080 plus HPLC pump, Jasco UV-2075 plus UV / VIS detector and Rheodyne injector with 20 ml loop volume. HiQ SiL C18 (250mm x 4.6 mm i.d) was used for the separation; mobile phase of a mixture of Acetonitrile: Methanol: Ammonium acetate was delivered at a flow rate of 1.5 ml/min with detection at 265nm. The mobile phase was filtered through a 0.2 m membrane filter and degassed. The injection volume was 20 ml; analysis was performed at ambient temperature.

Table 1: System Suitability Studies

Sr. Parameters Pseudoephidrine Desloratidine

No. sulphate

1. Theoretical plate/ meter 3540.11 3168.22

2. Resolution Factor 2.81 5.33

3. Asymmetry 1.10 1.47

4. LOD (µg/ml) 0.2649 0.08449

5. LOQ (µg/ml) 0.802 0.256

Preparation of Standard Solutions:

Standard stock solutions of strength 0.5 mg/ml of Pseudoephidrine sulphate, 0.1 mg/ml Desloratidine and 0.1 mg/ml of Nabumetone were prepared separately using acetonitrile. From Standard stock solution of each drug, mixed standard solution was prepared in mobile phase to contain 60mg/ml of Pseudoephidrine sulphate, 1.25 mg/ml of Desloratidine and 5mg/ml Nabumetone as an internal standard.

Calibration Curve:

Linearity of the system was investigated by serially diluting the stock solutions to give concentrations in the range of 60 mg/ml to 480 mg/ml for Pseudoephidrine sulphate and 1.25 to 10 mg/ml for Desloratidine. An aliquot (20 ml) was injected using mixture Acetonitrile: Methanol: Ammonium acetate (45:40:15) v/v, as mobile phase. Calibration curves were obtained by plotting the response factor vs. concentration. The response factor is calculated as area of the drug peak divided by area of peak for internal standard. The calibration curves are as shown in Fig.2 and Fig.3 for Pseudoephidrine sulphate and Desloratidine respectively. The equations of the regression lines are

For Pseudoephidrine sulphate

y =0.0086X + 0.00371 (R²= 0.999)

For Desloratidine

y = 0.9458X+ 0.0044(R²= 0.9999)

Fig. 1: Chromatogram of Pseudoephidrine sulphate (2.067 min), Desloratidine 3.050 min), Nabumetone (3.750 min) respectively.

Fig.2: Calibration curve for Pseudoephidrine sulphate.

Assay:

Preparation of Sample Solutions:

Twenty Tablets, each containing 120 mg for Pseudoephidrine sulphate and 2.5mg Desloratidine were weighed and finely powdered. A quantity of powder equivalent to 50mg Pseudoephidrine sulphate was weighed and transferred to 25 ml volumetric flask. To this, acetonitrile was added and sonicated for 10 min; the volume was made up to 25 ml with acetonitrile to get solution of 500 mg/ml. The solution was filtered using whatmann filter paper. From the filtrate appropriate dilutions were made to obtain concentration in the range of 60 to 480mg/ml for Pseudoephidrine sulphate and 1.25 to 10 mg/ml Desloratidine respectively. Nabumetone was added to each sample dilution at 5mg/ml as internal standard.

With the optimized chromatographic conditions, a steady baseline was recorded, the mixed standard solution was injected and the chromatogram was recorded. The retention time of Pseudoephidrine sulphate, Desloratidine, and Nabumetone were found to be 2.067, 3.050, and 3.750 min, respectively. This procedure was repeated for the sample solution obtained from the formulation. The proposed method was found to be specific and no interference from common tablet excipents like lactose, starch etc was observed. The response factor (peak area ratio of standard peak area

Table 2: Recovery Studies of Pseudoephidrine sulphate and Desloratidine:

Level of % Recovery	% Mean Recovery*		Standard Deviation		% R.S.D.^†
Level of % Recovery	PSEUDO	DESLOR	PSEUDO	DESLOR	PSEUDO	DESLOR
80	100.37	100.33	0.1137	0.01528	0.06993	0.3388
100	99.87	99.416	0.08185	0.02021	0.04553	0.4066
120	99.82	98.23	0.0.1514	0.01528	0.0766	0.28283

Table 3: Intraday and Interday Precision Studies (System precision)

Conc. (mg/ml)	Pseudoephidrine sulphate		Conc. (mg/ml)	Desloratidine
	% RSD			% RSD
	Intra- day	Inter- day		Intra- day	Inter- day
60	0.16	0.22	1.25	0.53	0.62
120	0.29	0.30	2.5	0.69	0.76
240	0.2	0.27	5.0	0.31	0.36
360	0.31	0.25	7.5	0.92	1.5
480	0.32	0.37	10	0.13	0.2

RSD = Relative Standard Deviation (n = 3).

Table4: Intraday and Interday Precision Studies

(Method precision)

Conc. (mg/ml)	Pseudoephidrine sulphate		Conc. (mg/ml)	Desloratidine
	% RSD			% RSD
	Intra- day	Inter- day		Intra- day	Inter- day
60	0.36	0.52	1.25	0.45	0.63
120	0.42	0.48	2.5	0.61	0.76
240	0.56	0.43	5.0	0.35	0.39
360	0.36	0.21	7.5	1.2	1.33
480	0.21	0.34	10	0.9	0.37

RSD = Relative Standard Deviation (n = 3).

and internal standard peak area) of the standard solution and sample solution were calculated. The assay was calculated from the equation of regression line for each drug. The percentage of individual drugs found in formulations was calculated and presented in table 2. The results of analysis shows that the amounts of drugs were in good agreement with the label claim of the formulations.

Method Validation:

As per the ICH guidelines, the method validation parameters checked were linearity, accuracy, precision, limit of detection, limit of quantitation and robustness.

Linearity and Range:

The linearity of the method was determined for the formulation at five concentration levels ranging from 60 to 480 mg/ml for Pseudoephidrine sulphate and 1.25 to 10 mg/ml Desloratidine. The equation for regression line was y = 0.0073X + 0.0089 for Pseudoephidrine sulphate and y = 0.41X + 0.0376 for Desloratidine. The results show that an excellent correlation exists between response factor and concentration of drugs within the concentration range indicated above.

Accuracy and Precision:

The accuracy of the method was determined by recovery experiments. The recovery studies were carried out at three levels of 80, 100 and 120% and the percentage recovery was calculated and presented in Table 2. Recovery was within the range of 100 ± 2% which indicates accuracy of the method.

The precision of the method was demonstrated by inter day and intra day variation studies. In the intra day studies, 3 repeated injections of standard and sample solutions were made in a day and the response factor of drug peaks and percentage RSD were calculated and presented in Table 3. In the inter day variation studies, 3 repeated injections of standard and sample solutions were made on 3 consecutive days and response factor of drugs peaks and percentage RSD were calculated and presented in Table 3. The data obtained, %RSD not more than 1.5%, indicates that the developed RP-HPLC method is precise.

Limit of Detection and Limit of Quantification:

The Limit of Detection (LOD) is the smallest concentration of the analyte that gives the measurable response. LOD was calculated using the following formula

LOD = (3.3 x standard deviation)/ Slope of calibration curve

The LOD for Pseudoephidrine sulphate and Desloratidine were found to be 0.32 mg/ml and 0.045 mg/ml, respectively.

The Limit of Quantification (LOQ) is the smallest concentration of the analyte, which gives response that can be accurately quantified. LOQ was calculated using the following formula

LOQ = (10 x standard deviation) / Slope of calibration curve.

The LOQ was 0.97 mg/ml and 0.13 mg/ml for Pseudoephidrine sulphate and Desloratidine respectively.

Robustness:

Robustness is checked by making slight deliberate change in the experimental procedures. In the present method a deliberate change of room temperature and pH was made and the effects were noted. The method was found to be robust with respect to change in room temperatures.

Fig.3: Calibration curve for Desloratidine.

RESULT AND DISCUSSION:

The proposed method was found to be simple and linear in the concentration range of 60 to 480 mg/ml for Pseudoephidrine sulphate and 1.25 to 10 mg/ml Desloratidine respectively. The method was found to be accurate and precise as indicated by recovery studies and % RSD not more than 1.5. Moreover LOD and LOQ for Pseudoephidrine sulphate were found to be 0.032 mg/ml and 0.97 mg/ml, respectively and for Desloratidine were 0.045 mg/ml and 0.13 mg/ml, respectively. Thus the method is specific and sensitive.

CONCLUSION:

The proposed RP-HPLC method for the simultaneous estimation of for Pseudoephidrine sulphate and Desloratidine in combined dosage forms was found to be sensitive, accurate, precise, simple and rapid. Hence the present RP –HPLC method may be used for routine analysis of the raw materials and formulations.

ACKNOWLEDGEMENT:

The author wish to thank e to M/s Glen mark Pharmaceuticals Ltd., Nasik, India, for providing Working standard of Pseudoephidrine sulphate and Desloratidine and also wish to express their gratitude to M/s torrent pharmaceutical Ltd., Indrad, Gujarat, India, for providing Working standard of Nabumetone.

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Received on 25.03.2009 Modified on 12.04.2009

Asian J. Research Chem. 2(2): April.-June, 2009 page 139-142

Pravara Rural college of Pharmacy, Pravaranagar, Dist. Ahemadnagar 413 736. (MS) India

*Corresponding Author E-mail: godgerk1rediffmail.com