INTRODUCTION:

Paroxetine hydrochloride is a potent and selective serotonin reuptake inhibitor¹. Paroxetine hydrochloride is indicated for the treatment of depression, obsessive-compulsive disorder, panic disorder and social phobia². Chemically Paroxetine hydrochloride is (-)-trans-4R-(4'-fluorophenyl)-3S-[(3',4'-methylenedioxyphenoxy)methyl] piperidine hydrochloride hemihydrate (Fig. 1)³. Paroxetine acts by potentiation of serotonergic activity in the central nervous system resulting from inhibition of neuronal reuptake of serotonin (5-hydroxy-tryptamine, 5-HT)⁴. A few Electroanalytical⁵, Spectrophotometric^6-8, HPTLC^9,10, HPLC^11-14, LC-MS^15-17and GC-MS^18,19methods were reported earlier for the estimation of Paroxetine in bulk and pharmaceutical dosage forms. In the present study the authors report a rapid, sensitive, accurate and precise HPLC method for the estimation of Paroxetine in bulk drug and in tablet dosage forms.

Fig. 1: Chemical structure of Paroxetine hydrochloride

MATERIALS AND METHODS:

Chromatographic conditions:

The analysis of the drug was carried out on a Waters HPLC system equipped with a reverse phase Inertsil ODS C18 column (150 x 4.6 mm I.D., 5 μm particle size), a 2695 binary pump, a 20 μL injection loop, auto sampler and a 2487 dual absorbance DAD or UV detector and running on Waters Empower software.

Chemicals and solvents:

The reference sample of Paroxetine was provided as gift sample from Pharma Train Lab, Hyderabad, India. Paroxetine tablets were purchased from local market. HPLC grade acetonitrile was purchased from E.Merck (India) Ltd., Mumbai, India. Sodium dihydrogen phosphate and orthophosphoric acid of AR Grade was obtained from S.D. Fine Chemicals Ltd., Mumbai, India. HPLC grade water obtained from Milli-Q water purification system was used throughout the study.

Preparation of Phosphate buffer pH 2.6:

2.5 grams of sodium dihydrogen phosphatewas weighed into a 1000 ml beaker, dissolved in HPLC water. Diluted to 1000 ml with Milli-Q water and pH adjusted to 2.6 with orthophosphoric acid.

Preparation of mobile phase and diluents:

700 ml of the phosphate buffer was mixed with 300 ml of acetonitrile. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45 μm filter under vacuum. The same mobile phase was used as diluent.

Preparation of standard stock solution:

Accurately weigh and transfer 10 mg of Paroxetine working standard into a 10 mL volumetric flask add about 7 mL of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. Further pipette 0.1 mL of the above stock solution into a 10 mL volumetric flask and dilute up to the mark with diluent. Mixwell and filter through 0.45 μm filter.

Preparation of sample solution:

Weigh 20 Paroxetine tablets and calculate the average weight. Accurately weigh and transfer the sample equivalent to 10 mg of Paroxetine into a 10 mL volumetric flask. Add about 7 mL of diluent, sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45 μm filter. Further pipette 1 mL of the above stock solution into a 10 mL volumetric flask and dilute up to the mark with diluent .Mix well and filter through 0.45 μm filter.

Calibration plot:

About 10 mg of Paroxetine was weighed accurately, transferred into a 10 mL volumetric flask and dissolved in 7 mL of a 70:30 v/v mixture of phosphate buffer and acetonitrile. The solution was sonicated for 15 min and the volume made up to the mark with a further quantity of the diluent to get a 1000 μg/mL solution. From this, a working standard solution of the drug (100 μg/mL) was prepared by diluting with the above solution to 10 mL in a volumetric flask. Further dilutions ranging from 25-150 μg/mL were prepared from the solution in 10 mL volumetric flasks using the above diluent. 20 μL of each dilution was injected six times into the column at a flow rate of 1.0 mL/min and the corresponding chromatograms were obtained. From these chromatograms, the average area under the peak of each dilution was computed. The calibration graph constructed by plotting concentration of the drug against peak area (Fig. 2) was found to be linear in the concentration range of 25-150 μg/mL of the drug. The relevant data are furnished in Table 1. The regression equation of this curve was computed. This regression equation was later used to estimate the amount of Paroxetine in tablet dosage forms.

Table 1. Calibration data of the method

Concentration in μg/mL	Area
25	459229
50	907787
75	1341459
100	1815794
125	2226065
150	2621867

Procedure:

A mixture of phosphate buffer and acetonitrile in the ratio of 70:30 v/v was found to be the most suitable mobile phase for ideal separation of Paroxetine. The solvent mixture was filtered through 0.45 μm membrane filter and sonicated before use. It was pumped through the column at a flow rate of 1.0 mL/min. The column was maintained at ambient temperature. The pump pressure was set at 800 psi. The column was equilibrated by pumping the mobile phase through the column for at least 30 min prior to the injection of the drug solution. Inject 20 μL of the standard, sample solutions into the chromatographic system and measure the area for the Paroxetine peak. The detection of the drug was monitored at 211 nm. The run time was set at 7 min. Under these optimized chromatographic conditions the retention time obtained for the drug Paroxetine was 3.276 min. A typical chromatogram showing the separation of the drug is given in Fig. 3.

Fig. 2: Calibration curve of Paroxetine

Fig. 3: Typical chromatogram of Paroxetine

Validation of the proposed method:

The specificity, linearity, precision, accuracy, limit of detection, limit of quantification, robustness and system suitability parameters were studied systematically to validate the proposed HPLC method as per the ICH guidelines for the estimation of Paroxetine²⁰. Solution containing 100 μg/mL solution of Paroxetine was subjected to the proposed HPLC analysis to check method precision and intermediate precision of the method and the results are furnished in Table 2. The accuracy of the HPLC method was assessed by analyzing solutions of Paroxetine at 50, 100 and 150 % concentration levels by the proposed method. The results are furnished Table 3. The system suitability parameters are given in Table 4.

Table 2. Precision data of the proposed HPLC method:

Concentration of Paroxetine (100 μg/mL)	Peak area
Concentration of Paroxetine (100 μg/mL)	Method precision	Intermediate precision
Injection-1	1765828	1706935
Injection-2	1764847	1711914
Injection-3	1759459	1715004
Injection-4	1787757	1762101
Injection-5	1753973	1697939
Injection-6	1760933	1712139
Mean	1765466	1717672
Standard deviation	11714.3	22580.8
% RSD	0.66	1.3

Table 3. Accuracy studies:

% Concentration (at specification level)	Amount added (μg)	Amount found (μg)	% Recovery	% Mean recovery
50 %	50	49.07	99.41%	99.73%
100%	100	99.93	99.93%
150%	150	149.78	99.85%

Table 4. System suitability parameters:

S. No.	System suitability	Results
1	Linearity range (μg/mL)	25-150
2	Correlation coefficient	0.999
3	Theoretical plates (N)	2840
4	Tailing factor	1.28
5	LOD (μg/mL)	0.06
6	LOQ (μg/mL)	0.18

Estimation of Paroxetine in tablet dosage forms:

Commercial formulations of Paroxetine tablets were chosen for testing the suitability of the proposed method to estimate Paroxetine in tablet formulations. Twenty tablets were weighed and powdered. An accurately weighed portion of this powder equivalent to 10 mg of Paroxetine was transferred into a 10 mL volumetric flask and dissolved in 7 mL of a 70:30 v/v mixture of phosphate buffer and acetonitrile. The contents of the flask were sonicated for 15 min and further 3 mL of the diluent was added, the flask was shaken continuously for 15 min to ensure complete solubility of the drug. The volume was made up with the diluent and the solution was filtered through a 0.45 μm membrane filter. Further pipette 1 mL of the above stock solution into a 10 mL volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45 μm filter. This solution containing 100 μg/mL of Paroxetine was injected into the column six times. The average peak area of the drug was computed from the chromatograms and the amount of the drug present in the tablet dosage form was calculated by using the regression equation obtained for the pure drug. The relevant results are furnished in Table 5.

Table 5. Assay studies

S. No.	Formulation	Label claim	Amount found	%Assay
1	PAXIDEP CR	12.5mg	12.46mg	99.79%

RESULTS AND DISCUSSION:

In the proposed method, the retention time of Paroxetine was found to be 3.276 min. Quantification was linear in the concentration range of 25-150 μg/mL. The regression equation of the linearity plot of concentration of Paroxetine over its peak area was found to be (y=9463+17859x (r²=0.999), where x is the concentration of Paroxetine (μg/mL) and y is the corresponding peak area. The %RSD for method precision and intermediate precision of Paroxetine was 0.66 and 1.3. The percentage recovery of the drug Paroxetine was 99.41 % to 99.93 % from the tablet formulation. The high percentage of recovery indicates that the proposed method is highly accurate. The number of theoretical plates calculated was 2840, which indicates efficient performance of the column. The limit of detection and limit of quantification were found to be 0.06 μg/mL and 0.18μg/mL respectively, which indicate the sensitivity of the method. The use of phosphate buffer and acetonitrile in the ratio of 70:30 v/v resulted in peak with good shape and resolution. The %assay of Paroxetine in commercial formulations was 99.79%. No interfering peaks were found in the chromatogram of the formulation within the run time indicating that excipients used in tablet formulations did not interfere with the estimation of the drug Paroxetine by the proposed HPLC method.

CONCLUSION:

The proposed HPLC method is rapid, sensitive, accurate and precise for the determination of Paroxetine and can be reliably adopted for routine quality control analysis of Paroxetine in its tablet dosage forms.

REFERENCES:

1. O.J.M. Neil. The Merck Index, An Encyclopedia of Chemicals Drug and Biologicals, 14^th Ed., Merck Research Laboratories, Division of Merck and Co. Inc., White House Station, NJ. 2006, 1215.

2. M. Bourin, P. Chue and Y. Guillon. Paroxetine: a review. CNS Drug Reviews, 2001, 7(1), 25-47.

3. M. Bourin. Use of Paroxetine for the treatment of depression and anxiety disorders in the elderly: a review. Human Psychopharmacology, 2003, 18(3), 185-190.

4. P. Tucker, R. Zaninelli, R. Yehuda, L. Ruggiero, K. Dillingham and C.D. Pitts. Paroxetine in the treatment of chronic posttraumatic stress disorder: results of a placebo-controlled, flexible-dosage trial. Journal of Clinical Psychiatry, 2001, 62(11), 860-868.

5. H.P.A. Nouws, C. Delerue-Matos, A.A. Barros and J.A. Rodrigues. Electroanalytical determination of Paroxetine in pharmaceuticals. Journal of Pharmaceutical and Biomedical Analysis, 2006, 42(3), 341-346.

6. A. Onal, S.E. Kepekci and A. Oztunc. Spectrophotometric methods for the determination of the antidepressant drug Paroxetine hydrochloride in tablets. Journal of AOAC International, 2005, 88(2), 490-495.

7. A.D. Ibrahim, H.A. Heba, M.A. Sawsan and I.A. Lama. Simple spectrophotometric method for determination of Paroxetine in tablets using 1,2-naphthoquinone-4-sulphonate as a chromogenic reagent. International Journal of Analytical Chemistry, 2009, 1-8.

8. M.I. Walash, F. Belal, N. El-Enany and H. El-Mansi. Spectrophotometric determination of the antidepressants Sertraline and Paroxetine HCl using 2,4-dinitrofluorobenzene. International Journal of Biomedical Science, 2010, 6(3), 252-259.

9. S. Robert, M. Genowefa and K. Marcin. Determination of Fluoxetine and Paroxetine in pharmaceutical formulations by densitometric and videodensitometric TLC. Journal of Planar Chromatography-Modern TLC, 2003, 16(1), 19-22.

10. A. Venkatachalam and V.S. Chatterjee. Stability-indicating high performance thin layer chromatography determination of Paroxetine hydrochloride in bulk drug and pharmaceutical formulations. Analytica Chimica Acta, 2007, 598(2), 312-317.

11. J. Knoeller, R. Vogt-Schenkel and M.A. Brett. A simple and robust HPLC method for the determination of Paroxetine in human plasma. Journal of Pharmaceutical and Biomedical Analysis, 1995, 13(4-5), 6358.

12. N. Erk and J. Biryol. Voltammetric and HPLC techniques for the determination of Paroxetine hydrochloride. Pharmazie, 2003, 58(10), 699-704.

13. I.A. Zainaghi, V.L. Lanchote and R.H.C. Queiroz. Determination of Paroxetine in geriatric depression by high-performance liquid chromatography. Pharmacological Research, 2003, 48(2), 217-221.

14. M. Lakshmi Surekha, G. Kumaraswamy and G.L. Ashwini. Validated RP-HPLC method for the estimation of Paroxetine hydrochloride in bulk and tablet dosage forms. Journal of Pharmacy Research, 2012, 5(3), 1768-1770.

15. Z.Zhuand L. Neirinck. High-performance liquid chromatography-mass spectrometry method for the determination of Paroxetine in human plasma. Journal of Chromatography B, 2002, 780(2), 295-300.

16. P. Massaroti, N.M. Cassiano and L.F. Duarte. Validation of a selective method for determination of Paroxetine in human plasma by LC-MS/MS. Journal of Pharmacy and Pharmaceutical Sciences, 2005, 8(2), 340-347.

17. O.H. Jhee, H.K. Seo and M.H. Lee. Determination of Paroxetine in plasma by liquid chromatography coupled to tandem mass spectrometry for pharmacokinetic and bioequivalence studies. Arzneimittel-Forschung, 2007, 57(7), 455-461.

18. C.B. Eap, G. Bouchoux, M. Amey, N. Cochard, L. Savary and P. Baumann. Simultaneous determination of human plasma levels of Citalopram, Paroxetine, Sertraline and their metabolites by gas chromatography-mass spectrometry. Journal of Chromatographic Science, 1998, 36(7), 365-371.

19. J.L. Hans, W. Werner and F. Gunter. Improved sample preparation for the quantitative analysis of Paroxetine in human plasma by stable isotope dilution negative ion chemical ionisation gas chromatography-mass spectrometry. Journal of Chromatography B, 2002, 779(2), 353-357.

20. ICH Harmonised Tripartite Guideline, Q2(R1), Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonisation, Geneva. 2005, 1-13.

Received on 14.02.2014 Modified on 12.03.2014

Asian J. Research Chem. 7(4): April 2014; Page 397-400