Battula Sreenivasa Rao et al.

/ Journal of Pharmacy Research 2012,5(5),2683-2687

Research Article ISSN: 0974-6943

Available online through www.jpronline.info

Validation of Venlafaxine in Pharmaceutical Dosage by Reverse Phase HPLC

Battula Sreenivasa Rao* B.Venkata Kiran, and Som Shankar Dubey Department of Chemistry, GITAM Institute of Technology, GITAM University,Visakhapatnam 530045, Andhra Pradesh, India.

Received on:11-01-2012; Revised on: 17-02-2012; Accepted on:19-04-2012 ABSTRACT

A rapid, specific and accurate isocratic HPLC method was developed and validated for the assay of venlafaxine in pharmaceutical dosage forms. The assay involved an isocratic elution of venlafaxine in ODS- C18 column using mobile phase composition consists of (65:35 v/v) of acetonitrile and sodium acetate with 0.1% tri-ethyl amine. The wavelength of detection is 225nm.The method showed good linearity in the range of 2.0-50.0 mg/mL. The runtime of the method is 5 mins. The proposed method can be used for routine quality control samples in industry in bulk and in finished dosage forms. In present study, a rapid specific precise and validated HPLC method for the quantitative estimation of venlafaxine in pharmaceutical dosage forms has been reported. The developed method can be applied to directly and easily to the analysis of the pharmaceutical tablet p reparations. The percentage recoveries were near 100% for given methods. The method was completely validated and proven to be rugged. The excipients did not interfere in the analysis. The results showed that this method can be used for rapid determination of venlafaxine in pharmaceutical tablet with precision, accuracy and specificity. Key words : Venlafaxine Hcl, Assay, reverse phase, HPLC. INTRODUCTION Venlafaxine Hydrochloride is an established anti-depressant drug. Chemically it is known as [2- (Dimethylamino)-1-(4-methoxyphenyl)ethyl] cyclohexanol hydrochloride (Fig-1). Venlafaxine hydrochloride is a third generation antidepressant 1, 2 . It inhibits the reuptake of serotonin, nor epinephrine and to a lesser extent dopamine3 . It lacks monoamine oxidase activity and more importantly, the adverse effect profile of tricyclic antidepressants. Venlafaxine has no affinity for brain muscarinic, cholinergic, histaminergic or adrenergic receptors6 . On the basis of clinical trials, this drug appears to lack many side effects associated with tricyclic antidepressants. In humans, venlafaxine hydrochloride is absorbed almost completely (92%) after oral intake and undergoes extensive metabolism in the liver. About 1% of VEN is desmethylated to N-desmethylvenlafaxine (NDV);16% becomes O, N- desmethy-lvenlafaxine (DDV) and 56% is metabolized to O-desmethylvenlafaxine (ODV) . Among all these metabolites, ODV is pharmacologically active with higher concentrations and longer half-life than the parent compound (4-9 hrs versus 1113 hrs) and significantly contributes to the therapeutic effects of VEN 7 . Therapeutic plasma levels of VEN usually range from 30 to 200 ng/ml, while the corresponding levels of ODV are in the range of 50500 ng/ ml8 . Figure-1 : Molecular structure of Venlafaxine Hcl Several methods have been reported for the quantitative determination of venlafaxine in bulk, pharmaceutical and biological samples. These methods include HPLC 9-16 , HPLC-MS1 7 - 1 9, Florometric detection 2 0 - 2 2 , UVVisiblespectrophotometric23-24. Literature survey revealed that most of the HPLC methods used hyphenated techniques with detectors such as mass-spectrometry, flourimetry, Electrospray mass spectrometric techniques all these methods have high sensitivity, but most of them highly expensive and are not easily available in quality control laboratories. So authors objective is to develop accurate, simple, reproducible reverse phase HPLC method which is free from extraction techniques, and shorter run time and high sensitivity. Experimental Chemicals and Reagent Venlafaxine (99.89%) pure was gift sample from Corpuscle research solutions. Acetonitrile (HPLC grade) was obtained from Qualigens fine chemicals. Milli-Q water was purchased from Ranbaxy fine chemicals limited (RFCL). All chemicals used were of analytical grade. Instrumentation The HPLC system consisted of a Shimadzu Class VP Binary pump LC10Atvp, SIL-10Dvp Auto sampler, CTO-10Avp column temperature Oven, PDA-UV Detector. All the components of the system are controlled using SCL-10Avp System Controller. Data acquisitions was done using LC-solution software. Mobile phase composition consists of (65:35 v/v) of acetonitrile and sodium acetate with 0.1% of tri-ethyl amine operated on isocratic mode. Analysis was carried out at 225nm. The chromatographic separation of venlafaxine (drug) was carried out using ODS C18 column (50x4.6 mm ID,5 um). The flow rate is 1.0 ml/min .The injection volume is 10L. Diluents consist of 50:50 (v/v) methanol and 0.1% ortho phosphoric acid. Preparation of Solutions Drug stock Solution and Internal Standard Two different Stock solutions of venlafaxine working standard was prepared by dissolving accurately weighed 10mg of drug in 10 ml of acetonitrile, so that final concentration is 1mg/1ml.The prepared stock solution is stored in 40 C-80 C protected from light. Suitable dilutions of drug and internal standard were prepared by using 50:50 5 v/v methanol and 0.1% ortho phosphoric acid as diluents solution.

*Corresponding author.
Battula Sreenivasa Rao Department of Chemistry, GITAM Institute of Technology, GITAM University, Visakhapatnam 530045, Andhra Pradesh, India.

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Figure-4: Linearity Data

Figure-2: Representative chromatogram of LOD Injection

Figure-3: Representative chromatogram of LOQ Injection

Figure-5: Blank a concentration of 20.0 g/ml. The acceptance criterion is 1% for the per cent coefficient of the variation for the peak area and retention times for the drug. Detection and Quantization Limits (Sensitivity) Limits of detection (LOD) (Fig-2) and quantization (LOQ) (Fig-3) were estimated from both linearity calibration curve method and signal to noise ratio method. The detection limit was defined as the lowest concentration level resulting in a peak area of three times the baseline noise. The quantization limit was defined as the lowest concentration level that provided a peak area with signal to noise ratio higher than 10, with precision (%CV) and accuracy with () 10%. Linearity (Calibration Curve) The calibration curve was constructed with eight concentrations ranging from 2.00 to 50.00 g/ml. The linearity was evaluated by linear regression analysis, which was calculated by least square method. It is depicted in ( Fig4). Accuracy and Precision Accuracy of assay method was determined for both intra-day and inter-day variations using triplicate analysis of the QC samples. Precision of the assay was determined by repeatability (intra-day) and intermediate precision (inter-day). Repeatability refers to the use of the analytical procedure within the laboratory over the shorter period of the time that was evaluated by assaying the QC samples during the same day. Intermediate precision was assessed by comparing the assays on different days (3 days).

Calibration Standards and Quality Control Samples An eight point linear calibration curve standards were prepared using diluents solutions in the concentration range of 2.0 to 50.00 g/ml Calibration standards were prepared at the concentration of 2.00, 5.00, 10.000, 15.000, 25.000, 40.00, 45.000, 50.00 g/ml from first standard stock solution. Three quality control samples were at the concentrations of 5.0 g/ml, 20.0g/ml and 40.00 g/ml representing low, medium and high concentration respectively .The quality control samples were prepare from second standard stock solution Sample Preparation Commercially available tablets of venlafaxine are taken from two different brands and tested for assay. Ten tablets of each brand are taken and crushed to powder. A powder equivalent to 50mg of venlafaxine is taken and transferred into a stoppered conical flask to which 25ml of methanol is added. The contents are transferred into a stoppered flask and shaken for 30 mins to extract the drug. Contents are carefully transferred into a centrifuge tube and centrifuged for 3000 rpm for 30mins. The supernatant liquid is taken and diluted with diluents, to obtain approximately final concentration of 40g/ ml. This sample is analyzed in triplicate. The accuracy and concentration is determined using regression equation. Method Validation System Suitability The system suitability was assessed by six replicate analysis of the drug at

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Table-5, Intra-day and Inter-day precision and accuracy of HPLC assay of venlafaxine
Day=1 Mean (n=3) S.D R.S.D Recovery(%) Day=2 Mean(n=3) S.D R.S.D Recovery(%) Day-3 Mean (n=3) S.D R.S.D Recovery(%) Nominal concentration 5.0g/ml 20.0 g/ml 131851.7 2269.65 1.72 99.86 131192.7 683.89 0.52 99.27 130556.7 335.11 0.26 98.73 489408.7 623.42 0.13 99.25 484621 717.01 0.15 98.25 486410.3 751.68 0.15 98.63 40.00 g/ml 985624.7 7690.59 0.78 101.17 972009.3 13131.02 1.35 99.76 982394.7 6383.137 0.65 100.83

Each mean value is a result of triplicate analysis (n=3)

Fig-6 - Typical Chromatogram containing standard. Table-1, System Suitability Study

Drug Inj-01 Inj-02 Inj-03 Inj-04 Inj-05 Inj-06 Mean S.D RSD 429411 428559 429159 428066 424032 427085 427718.7 1987.88 0.46 T.P 5765 5767 5766 5771 5793 5781 5773.8 11.07 0.19 Tailing 1.15 1.14 1.15 1.14 1.14 1.14 1.143 0.005 0.456 RT(Drug) 2.83 2.83 2.83 2.83 2.83 2.83 2.83 0 0

Table-6, Short-term, long term and auto-sampler stability of venlafaxine

Short term stability (12 hrs) Mean (n=3) S.D R.S.D Recovery(%) Auto sampler stability(24 hrs) Mean(n=3) S.D R.S.D Recovery(%) Nominal concentration 5.0g/ml 20.0 g/ml 130490.3 340.58 0.26 98.67 130223.7 599.65 0.46 98.53 484891 713.61 0.15 98.3 484308.7 584.20 0.12 98.18 40.0g/ml 976385.3 5922.20 0.61 100.21 976073 6037.85 0.62 100.18

Each mean value is a result of triplicate analysis (n=3)

Table-7, Effect of Various parameters in assessment of method Table-2, Limit of detection

Injection. No 01 02 03 04 05 06 Mean S.D RSD Drug(Area) 10463 11145 11444 10367 10703 10703 10804.17 413.17 3.82 T.P 4832 4832 4450 5189 4528 5200 4838.5 316.53 6.54 T.F 0.85 0.89 0.89 0.92 0.97 0.97 0.915 0.048 5.252 Parameters Flow rate Column temperature Mobile phase Variation 0.9 ml/min 1.1 ml/min 20 0 C 30 0 C 90% organic 110% organic R.T 2.91 2.72 2.83 2.84 2.92 2.73 T.P 5997 5771 6141 6918 5070 6229 Tailing 1.07 1.21 1.21 1.08 1.22 1.09

Table-8, Results of Venlafaxine in marketed product

Marketed formulation Brand-1 Brand-2 Drug venlafaxine -25 mg venlafaxine -75 mg % Amount obtained 99.05 0.16 98.26 0.11 % RSD 0.16 0.11

Table-3, Limit of Quantification

Injection.No 01 02 03 04 05 06 Mean S.D RSD Drug(Area) 25038 24811 23919 24045 22828 23362 24000.5 839.57 3.50 T.P 4370 4201 4478 4482 4989 4342 4477 271.30 6.06 T.F 0.93 1.03 0.93 0.98 0.96 1.00 0.972 0.0397 4.086

Each value is a result of triplicate analysis.

2)Standard chromatogram (Fig-6). A less than 20% interference of the peak area at the retention time of the drug in the blank sample is taken as acceptance criteria for the analyte. Sample Specificity is also observed in the degradation study of the drug. None of the degraded products must interfere with the quantification of the drug. Stability The stability of the drug is determined by using QC samples for the short term stability by keeping at room temperature up to 12 hours and then analyzing them. Further, auto-sampler stability for up to 24 hrs was studied and established. RESULTS AND DISCUSSION Method Development and Validation The HPLC procedure was optimized with a view to develop a stability

Table-4, Results and regression analysis of linearity data of venlafaxine

Mean S.D(n=3) Slope Intercept Correlation coefficient(R 2 ) 24066 134 11708 735 0.9907 0.0003

Each mean value is a result of triplicate analysis (n=3) Specificity Specificity of the method was determined by injecting 2 samples 1)Blank sample. (Fig-5).

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indicating assay method. Different permutations and combinations, at different pH values ranging from pH 3.0 to pH 11.0 using various columns like Hypersil-BDS-C18, Symmetry C18, Ymc-pack C18, Ymc-pack pro, Sperisorb C18, Phenomenox C18 have been tried with different buffer salts such ammonium acetate, ortho phosphoric acid, di-potassium hydrogen orthophosphate, in combination with acetonitrile, methanol and tetrahydrofuran. However good resolution, less tailing and high theoretical plates are obtained with ODS column C18 50 X 4.6 cm 5. Mobile phase composition consists of (65:35 v/v) of acetonitrile and Sodium acetate with 0.1% of triethyl amine operated on isocratic mode The flow rate of the method is 1.0 ml/ min. Diluents is prepared in the same way as mobile phase which consist of (50:50) methanol and 0.1% orthophosphoric acid. The wavelength of detection is 225nm. The column temperature is maintained at 250 C. At the reported flow rate peak shape was excellent, however increasing or decreasing the flow rate increased the tailing factor and resulting in poor peak shape and also resolution between the drug and internal standard also decreased. Hence 1.0 ml/min was optimized flow rate decreasing the consumption of the mobile phase, which in turn proves to be cost effective for long term routine quality control analysis. There was no interference in the drug and internal standard, from the blank. The peak shape and symmetry were found to be good when the mobile phase composition of 50:50 v/v was used with better resolution of the drug and internal standard. Method Validation System Suitability The % RSD of the peak area and the retention time for both drug and internal standard are within the acceptable the range (Table-1). The efficiency of the column was expressed as the number of theoretical plates for the six replicate injections was around 5774 11 and the USP tailing factor was 1.14 0.0005. Determination and Quantization Limits (Sensitivity) (Fig-2) and (Fig-3) represents the six replicate injections of the limit of detection and limit of quantification. The method is found to be sensitive which can be determined from the data obtained from the (Table-2) and (Table-3). Linearity The calibration curve constructed was evaluated by its correlation coefficient. The peak area ratio of the drug and internal standard was linear, and the range, is 2.00 and 50.00 g/ml. The linearity was determined in three sets, the correlation coefficient (R2 ) was consistently greater then 0.999 (Table-4). From the data in ( Fig-4 and Table-4) regression equation, limit of quantification and limit of detection was determined from the calibration curve method. Regression equation: y = 24066x - 11709 (Equation:1) Accuracy and Precision Accuracy and precision calculated for the QC samples during the intra- and inter day run are given the (Table-5). The intra-day (day-1) and inter-day accuracy ranged from 97.95 to 101.06. The results obtained from intermediate precision (inter-day) also indicated a good method precision .All the data were within the acceptance criteria. Specificity Specificity was determined from Blank (Fig-5) Standard (Fig-6). Stability Stability studies were done for short term stability up to 12 hrs, auto sampler stability up to 24hrs of low QC, medium QC, High QC levels conditions and the mobile phase is stable up to 72 hrs. (Table-6). Robustness study Robustness is the measure of method capacity to remain unaffected by deliberate small changes in the chromatographic conditions. The experimental conditions were deliberately altered to test evaluate the robustness of the method. The impact of flow-rate(1.00.1), column temperature (250 C50 C)changes and effect of mobile-phase composition(10%) was evaluated on the important system suitability factors such as retention time, theoretical plates, and tailing factor, were studied. The experimental results were presented in the (Table-7). Application of the method to dosage forms The HPLC method developed is sensitive and specific for the quantitative determination of venlafaxine. Also the method is validated for different parameters, hence has been applied for the estimation of drug in pharmaceutical dosage forms. Venlafaxine tablets of 25mg, 75mg strength from two different manufacturers were evaluated for the amount of venlafaxine .The amount of venlafaxine in tablet 1 is 99.05 0.16 and tablet 2 is 98.26 0.11 (Table-8) .None of the tablets ingredients interfere with the analytic peak. The spectrum of venlafaxine is extracted from the tablets was matching with that of standard venlafaxine showing the purity of peak of venlafaxine in the tablets. Conclusions The method gave accurate and precise results in the concentration range of 2.00 to 50.00g/mL. The mobile phase composition consists of (65:35 v/v) of Acetonitrile and Sodium acetate with 0.1% Tri-ethyl amine, at the flow rate of 1.0 ml/min. The retention times of the drug are 2.83. The column is a 50 X 4.6mm C18 column with the particle size of 5m.A rapid sensitive and specific method for the determination of Venlafaxine in the pharmaceutical formulations has been developed. Nomenclature : 1)mV : mill volts. 2)nm : Wave length. 3)min : minutes. 4)Fig : Figure. 5)Inj : Injection. REFERENCES 1. Morton WA, Sonne SC and Verga MA. Venlafaxine: a structurally unique and novel antidepressant. Ann 1. Pharmacother, 1995; 29: 387-395. 2. Reis M, Lundmark J, Bjrk H and Bengtsson F. Therapeutic drug monitoring of racemic venlafaxine and its 2. main metabolites in an everyday clinical setting. Ther Drug Monit, 2002, 24, 545-553 3. Charlier C, Pinto E, Ansseau M, Plomteux G. Venlafaxine: the relationship between dose, lasma concentration and clinical response in depressive patients. J Psychopharmacol, 2002; 16: 369379. 4. Liu W, Cai HL, Li HD. High performance liquid chromatographyelectrospray ionization mass spectrum etry (HPLC-MS/ESI) method for simultaneous determination of venlafaxine and its three metabolites in human plasma. J Chromatogr B Analyt Technol. Biomed Life Sci, 2007; 850:405-411. 5. Patel BN, Sharma N, Sanyal M, Shrivastav PS. Liquid chromatography tandem mass spectrometry assay 6. for the simultaneous determination of venlafaxine and O-desmethylvenlafaxine in human plasma and its application to a bioequivalence study. J Pharm Biomed Anal, 2008; 47: 603-611. 6. Vu RL, Helmeste D, Albers L, Reist C. Rapid determination of venlafaxine and O-desmethylvenlafaxine 7. in human plasma by high-performance liquid chromatography with fluorimetric detec-

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7. tion. J Chromatogr B Biomed Sci Appl, 1997; 703: 195-201 Waschgler R, Moll W, Knig P, Conca A. Quantification of venlafaxine and O-desmethylvenlafaxine in 8. human serum using HPLC analysis. Int J Clin Pharmacol Ther, 2004; 42: 724-728. Reis M, Lundmark J, Bjrk H, Bengtsson F. Therapeutic drug monitoring of racemic venlafaxine and its main metabolites in an everyday clinical setting. Ther Drug Monit, 2002; 24: 545-553. Charlier C, Pinto E, Ansseau M, Plomteux G. Venlafaxine: the relationship between dose, plasma concen-tration and clinical response in depressive patients. J Psychopharmacol, 2002; 16: 369379. Mandrioli R, Mercolini L, Cesta R, Fanali S, Amore M and Raggi M A, Analysis of the second generation antidepressant venlafaxine and its main active metabolite O- desmethylvenlafaxine in human plasma by HPLC with spectrofluorimetric detection. J Chromatogr B., 2007, 856(1-2), 88-94. Liu W, Wang F and Li H, Simultaneous stereoselective analysis of venlafaxine and O-desmethylvenlafaxine enantiomers in human plasma by HPLC-ESI/MS using a vancomycin chiral column J Chromatogr B., 2007, 850(1-2), 183-189. Ebenezer B, Asafu-Adjaye, Faustino P J, Tawakkul M A, Anderson L W, Yu L X, Kwon H and Volpe D A, Validation and application of a stability-indicating HPLC method for the in vitro determination of gastric and intestinal stability of venlafaxine J Pharm Biomed Analysis, 2007, 43(5), 1854-1859. Kirchherr H and Kuhnvelten WN, Quantitative determination of forty-eight antidepressants and antipsychotics in human serum by HPLC tandem mass spectrometry: multi-level, single-sample approach J Chromatogr B., 2006, 843,100-113. Juan H, Zhiling Z and Huande L, Simultaneous determination of fluoxetine, citalopram, paroxetine, venlafaxine in plasma by high performance liquid chromatographyelectrospray ionization mass spectrometry (HPLCMS/ESI)J Chromatogr B., 2005, 820, 33-39. Raut B. B., Kolte B.L., Deo A.A., Bagool M. A., Shinde D.B., A rapid and sensitive HPLC method for the determination of velafaxine and Odesmethylvenlafaxine in human plasma with UV detection J. Liq. Chromatogr.Relat. Technol., 2003, 26(8): 12971313. Rahnert C, Rao M L and Grasmader K, Analysis of eighteen antidepressants, four atypical antipsychotics and active metabolites in serum by liquid chromatography: a simple tool for therapeutic drug monitoring , J Chromatogr B., 2003, 794,35-47. Liu W, Cai HL and Li HD. High performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI) method for simultaneous determination of venlafaxine and its three metabolites in human plasma. J Chromatogr B Analyt Technol. Biomed Life Sci, 2007, 850, 405-411. Bhatt J, Jangid A, Venkatesh G, Subbaiah G and Singh S. Liquid chromatography-tandem Mass spectrometry (LC-MS-MS) method for simultaneous determination of venlafaxine and its active metabolite O-desmethyl venlafaxine in human plasma. J Chromatogr B Analy Technol Biomed Life Sci, 2005, 829, 75-81. Patel BN, Sharma N, Sanyal M and Shrivastav P S. Liquid chromatography tandem mass spectrometry assay for the simultaneous determination of venlafaxine and O- desmethylvenlafaxine in human plasma and its application to a bioequivalence study. J Pharm Biomed Anal, 2008, 47, 603-611. Vu RL, Helmeste D, Albers L and Reist C. Rapid determination of venlafaxine and O-desmethylvenlafaxine in human plasma by highperformance liquid chromatography with fluorimetric detection. J Chromatogr B Biomed Sci Appl, 1997, 703, 195-201 Waschgler R, Moll W, Knig P and Conca A. Quantification of venlafaxine and O- desmethylvenlafaxine in human serum using HPLC analysis. Int J Clin Pharmacol Ther, 2004, 42, 724-728. 22 Mandrioli R, Mercolini L, Cesta R, Fanali S, Amore M, Raggi MA. Analysis of the second generation anti-depressant venlafaxine and its main active metabolite O- desmethylvenlafaxine in human plasma by HPLC with spectrofluorimetric detection. J Chromatogr B Analyt Technol Biomed Life Sci, 2007; 856, 88-94 Matoga M, Pehourcq F, Titier K, Dumora F and Jarry C. Rapid high-performance liquid chromatographic measurement of venlafaxine and O-desmethylvenlafaxine in human plasma. Application to management of acute intoxications. J Chrom. B Biomed Sci, Appl, 2001, 760, 213-218. Raut BB, Kolte BL, Deo AA, Bagool MA and Shinde DB. A rapid and sensitive HPLC method for the determination of venlafaxine and O-desmethylvenlafaxine in human plasma with UV detection. J Liq Chrom. Related Technol, 2003, 26, 1297-2001.

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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 5.May 2012

2683-2687