Venlafaxcine Third Paper

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

Battula Sreenivasa Rao et al. / Journal of Pharmacy Research 2012,5(5),2683-2687

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Research ArticleISSN: 0974-6943

Available online throughwww.jpronline.info

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

Validation of Venlafaxine in Pharmaceutical Dosage by Reverse Phase HPLCBattula 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

ABSTRACTA rapid, specific and accurate isocratic HPLC method was developed and validated for the assay of venlafaxine in pharmaceutical dosage forms. The assayinvolved an isocratic – elution of venlafaxine in ODS- C18 column using mobile phase composition consists of (65:35 v/v) of acetonitrile and sodium acetatewith 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 themethod 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. Thedeveloped method can be applied to directly and easily to the analysis of the pharmaceutical tablet p reparations. The percentage recoveries were near100% for given methods. The method was completely validated and proven to be rugged. The excipients did not interfere in the analysis. The results showedthat 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.

INTRODUCTIONVenlafaxine Hydrochloride is an established anti-depressant drug. Chemi-cally it is known as [2- (Dimethylamino)-1-(4-methoxyphenyl)ethyl]cyclohexanol hydrochloride (Fig-1). Venlafaxine hydrochloride is a thirdgeneration antidepressant 1, 2. It inhibits the reuptake of serotonin, nor epi-nephrine and to a lesser extent dopamine3. It lacks monoamine oxidaseactivity and more importantly, the adverse effect profile of tricyclicantidepressants. Venlafaxine has no affinity for brain muscarinic, cholin-ergic, histaminergic or adrenergic receptors6. On the basis of clinical trials,this drug appears to lack many side effects associated with tricyclic antide-pressants. In humans, venlafaxine hydrochloride is absorbed almostcompletely (92%) after oral intake and undergoes extensive metabolismin the liver. About 1% of VEN is desmethylated to N-desmethylvenlafaxine(NDV);16% becomes O, N- desmethy-lvenlafaxine (DDV) and 56% ismetabolized to O-desmethylvenlafaxine (ODV) . Among all these metabo-lites, ODV is pharmacologically active with higher concentrations and

Literature survey revealed that most of the HPLC methods used hyphenatedtechniques with detectors such as mass-spectrometry, flourimetry, Electro-spray mass spectrometric techniques all these methods have high sensitivity,but most of them highly expensive and are not easily available in qualitycontrol laboratories. So author’s objective is to develop accurate, simple,reproducible reverse phase HPLC method which is free from extraction tech-niques, and shorter run time and high sensitivity.

Experimental

Chemicals and ReagentVenlafaxine (99.89%) pure was gift sample from Corpuscle research solu-tions. Acetonitrile (HPLC grade) was obtained from Qualigens fine chemi-cals. 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 LC-10Atvp, SIL-10Dvp Auto sampler, CTO-10Avp column temperature Oven,PDA-UV Detector. All the components of the system are controlled usingSCL-10Avp System Controller. Data acquisitions was done using LC-solu-tion software. Mobile phase composition consists of (65:35 v/v) of acetoni-trile and sodium acetate with 0.1% of tri-ethyl amine operated on isocraticmode. Analysis was carried out at 225nm. The chromatographic separationof venlafaxine (drug) was carried out using ODS C18 column (50x4.6 mmID,5 um). The flow rate is 1.0 ml/min .The injection volume is 10µL. Diluentsconsist 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 preparedby dissolving accurately weighed 10mg of drug in 10 ml of acetonitrile, sothat final concentration is 1mg/1ml.The prepared stock solution is stored in40C-80C protected from light. Suitable dilutions of drug and internal standardwere prepared by using 50:50 5 v/v methanol and 0.1% ortho phosphoricacid as diluents solution.

Figure-1 : Molecular structure ofVenlafaxine Hcl

longer half-life than the parent com-pound (4-9 hrs versus 11–13 hrs)and significantly contributes tothe therapeutic effects of VEN 7 .Therapeutic plasma levels of VENusually range from 30 to 200 ng/ml,while the corresponding levels ofODV are in the range of 50–500 ng/ml8.

Several methods have been reported for the quantitative determination ofvenlafaxine in bulk, pharmaceutical and biological samples. These methodsinclude HPLC 9-16 , HPLC-MS1 7 - 1 9, Florometric detection 20-22 , UV-Visiblespectrophotometric23-24.



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Calibration Standards and Quality Control Samples An eight point linear calibration curve standards were prepared using diluentssolutions in the concentration range of 2.0 to 50.00 µg/ml Calibration stan-dards were prepared at the concentration of 2.00, 5.00, 10.000, 15.000,25.000, 40.00, 45.000, 50.00 µg/ml from first standard stock solution. Threequality control samples were at the concentrations of 5.0 µg/ml, 20.0µg/mland 40.00 µg/ml representing low, medium and high concentration respec-tively .The quality control samples were prepare from second standardstock solution

Sample Preparation Commercially available tablets of venlafaxine are taken from two differentbrands and tested for assay. Ten tablets of each brand are taken and crushedto powder. A powder equivalent to 50mg of venlafaxine is taken and trans-ferred into a stoppered conical flask to which 25ml of methanol is added. Thecontents are transferred into a stoppered flask and shaken for 30 mins toextract the drug. Contents are carefully transferred into a centrifuge tube andcentrifuged for 3000 rpm for 30mins. The supernatant liquid is taken anddiluted with diluents, to obtain approximately final concentration of 40µg/ml. This sample is analyzed in triplicate. The accuracy and concentration isdetermined using regression equation.

Method Validation

System Suitability The system suitability was assessed by six replicate analysis of the drug at

a concentration of 20.0 µg/ml. The acceptance criterion is ± 1% for the percent coefficient of the variation for the peak area and retention times for thedrug.

Detection and Quantization Limits (Sensitivity) Limits of detection (LOD) (Fig-2) and quantization (LOQ) (Fig-3) wereestimated from both linearity calibration curve method and signal to noiseratio method. The detection limit was defined as the lowest concentrationlevel resulting in a peak area of three times the baseline noise. The quantiza-tion limit was defined as the lowest concentration level that provided a peakarea with signal to noise ratio higher than 10, with precision (%CV) andaccuracy with (±) 10%.

Linearity (Calibration Curve)The calibration curve was constructed with eight concentrations rangingfrom 2.00 to 50.00 µg/ml. The linearity was evaluated by linear regressionanalysis, which was calculated by least square method. It is depicted in (Fig-4).

Accuracy and PrecisionAccuracy of assay method was determined for both intra-day and inter-dayvariations using triplicate analysis of the QC samples. Precision of the assaywas determined by repeatability (intra-day) and intermediate precision (in-ter-day). Repeatability refers to the use of the analytical procedure withinthe laboratory over the shorter period of the time that was evaluated byassaying the QC samples during the same day. Intermediate precision wasassessed by comparing the assays on different days (3 days).

Figure-2: Representative chromatogram of LOD Injection

Figure-3: Representative chromatogram of LOQ Injection

Figure-4: Linearity Data

Figure-5: Blank



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Fig-6 - Typical Chromatogram containing standard.

Table-1, System Suitability StudyDrug T.P Tailing RT(Drug)

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

Table-2, Limit of detectionInjection. No Drug(Area) T.P T.F

01 10463 4832 0.8502 11145 4832 0.8903 11444 4450 0.8904 10367 5189 0.9205 10703 4528 0.9706 10703 5200 0.97Mean 10804.17 4838.5 0.915S.D 413.17 316.53 0.048RSD 3.82 6.54 5.252

Table-3, Limit of QuantificationInjection.No Drug(Area) T.P T.F

01 25038 4370 0.9302 24811 4201 1.0303 23919 4478 0.9304 24045 4482 0.9805 22828 4989 0.9606 23362 4342 1.00Mean 24000.5 4477 0.972S.D 839.57 271.30 0.0397RSD 3.50 6.06 4.086

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

Mean ± S.D(n=3)

Slope 24066 ± 134Intercept 11708 ± 735Correlation coefficient(R2) 0.9907 ± 0.0003

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

Table-5, Intra-day and Inter-day precision and accuracy of HPLC assayof venlafaxine

Nominal concentrationDay=1 5.0µg/ml 20.0 µg/ml 40.00 µg/ml

Mean (n=3) 131851.7 489408.7 985624.7S.D 2269.65 623.42 7690.59R.S.D 1.72 0.13 0.78Recovery(%) 99.86 99.25 101.17Day=2Mean(n=3) 131192.7 484621 972009.3S.D 683.89 717.01 13131.02R.S.D 0.52 0.15 1.35Recovery(%) 99.27 98.25 99.76Day-3Mean (n=3) 130556.7 486410.3 982394.7S.D 335.11 751.68 6383.137R.S.D 0.26 0.15 0.65

Recovery(%) 98.73 98.63 100.83


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

Short term stability (12 hrs) 5.0µg/ml 20.0 µg/ml 40.0µg/ml

Mean (n=3) 130490.3 484891 976385.3S.D 340.58 713.61 5922.20R.S.D 0.26 0.15 0.61Recovery(%) 98.67 98.3 100.21Auto sampler stability(24 hrs)Mean(n=3) 130223.7 484308.7 976073S.D 599.65 584.20 6037.85R.S.D 0.46 0.12 0.62Recovery(%) 98.53 98.18 100.18


Table-7, Effect of Various parameters in assessment of methodParameters Variation R.T T.P Tailing

Flow rate 0.9 ml/min 2.91 5997 1.071.1 ml/min 2.72 5771 1.21

Column temperature 200C 2.83 6141 1.21300C 2.84 6918 1.08

Mobile phase 90% organic 2.92 5070 1.22

110% organic 2.73 6229 1.09

Table-8, Results of Venlafaxine in marketed product

Marketed formulation Drug % Amount obtained % RSD

Brand-1 venlafaxine -25 mg 99.05 ± 0.16 0.16Brand-2 venlafaxine -75 mg 98.26 ± 0.11 0.11

Each value is a result of triplicate analysis.

Specificity Specificity of the method was determined by injecting 2 samples1)Blank sample. (Fig-5).

2)Standard chromatogram (Fig-6).

A less than 20% interference of the peak area at the retention time of the drugin the blank sample is taken as acceptance criteria for the analyte. SampleSpecificity is also observed in the degradation study of the drug. None of thedegraded products must interfere with the quantification of the drug.

StabilityThe stability of the drug is determined by using QC samples for the shortterm stability by keeping at room temperature up to 12 hours and thenanalyzing them. Further, auto-sampler stability for up to 24 hrs was studiedand established.

RESULTS AND DISCUSSION

Method Development and ValidationThe HPLC procedure was optimized with a view to develop a stability



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indicating assay method. Different permutations and combinations, at differ-ent pH values ranging from pH 3.0 to pH 11.0 using various columns likeHypersil-BDS-C18, Symmetry C18, Ymc-pack C18, Ymc-pack pro,Sperisorb C18, Phenomenox C18 have been tried with different buffer saltssuch ammonium acetate, ortho phosphoric acid, di-potassium hydrogen or-thophosphate, in combination with acetonitrile, methanol and tetrahydrofu-ran. However good resolution, less tailing and high theoretical plates areobtained with ODS column C18 50 X 4.6 cm 5µ. Mobile phase compositionconsists of (65:35 v/v) of acetonitrile and Sodium acetate with 0.1% of tri-ethyl 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 detec-tion is 225nm. The column temperature is maintained at 250 C. At the re-ported flow rate peak shape was excellent, however increasing or decreasingthe flow rate increased the tailing factor and resulting in poor peak shape andalso resolution between the drug and internal standard also decreased. Hence1.0 ml/min was optimized flow rate decreasing the consumption of the mo-bile phase, which in turn proves to be cost effective for long term routinequality control analysis. There was no interference in the drug and internalstandard, from the blank. The peak shape and symmetry were found to begood when the mobile phase composition of 50:50 v/v was used with betterresolution of the drug and internal standard.

Method Validation

System SuitabilityThe % RSD of the peak area and the retention time for both drug and internalstandard are within the acceptable the range (Table-1). The efficiency of thecolumn was expressed as the number of theoretical plates for the six replicateinjections 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 ofdetection and limit of quantification. The method is found to be sensitivewhich can be determined from the data obtained from the (Table-2) and(Table-3).

LinearityThe calibration curve constructed was evaluated by its correlation coeffi-cient. The peak area ratio of the drug and internal standard was linear, and therange, is 2.00 and 50.00 µg/ml. The linearity was determined in three sets, thecorrelation coefficient (R2) was consistently greater then 0.999 (Table-4).From the data in (Fig-4 and Table-4) regression equation, limit of quantifica-tion and limit of detection was determined from the calibration curve method.Regression equation: y = 24066x - 11709 (Equation:1)

Accuracy and PrecisionAccuracy and precision calculated for the QC samples during the intra- andinter –day run are given the (Table-5). The intra-day (day-1) and inter-dayaccuracy ranged from 97.95 to 101.06. The results obtained from intermedi-ate precision (inter-day) also indicated a good method precision .All the datawere within the acceptance criteria. SpecificitySpecificity was determined from Blank (Fig-5) Standard (Fig-6).

StabilityStability studies were done for short term stability up to 12 hrs, auto sam-pler stability up to 24hrs of low QC, medium QC, High QC levels conditionsand the mobile phase is stable up to 72 hrs. (Table-6).

Robustness studyRobustness is the measure of method capacity to remain unaffected bydeliberate small changes in the chromatographic conditions. The experimen-tal conditions were deliberately altered to test evaluate the robustness of themethod. The impact of flow-rate(1.0±0.1), column temperature(250C±50C)changes and effect of mobile-phase composition(±10%) wasevaluated on the important system suitability factors such as retention time,theoretical plates, and tailing factor, were studied. The experimental resultswere presented in the (Table-7).

Application of the method to dosage formsThe HPLC method developed is sensitive and specific for the quantitativedetermination of venlafaxine. Also the method is validated for different pa-rameters, hence has been applied for the estimation of drug in pharmaceuticaldosage forms. Venlafaxine tablets of 25mg, 75mg strength from two differentmanufacturers were evaluated for the amount of venlafaxine .The amount ofvenlafaxine 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 spec-trum of venlafaxine is extracted from the tablets was matching with that ofstandard venlafaxine showing the purity of peak of venlafaxine in the tablets.

Conclusions The method gave accurate and precise results in the concentration range of2.00 to 50.00µg/mL. The mobile phase composition consists of (65:35 v/v)of Acetonitrile and Sodium acetate with 0.1% Tri-ethyl amine, at the flowrate of 1.0 ml/min. The retention times of the drug are 2.83. The column is a50 X 4.6mm C18 column with the particle size of 5µm.A rapid sensitive andspecific method for the determination of Venlafaxine in the pharmaceuticalformulations has been developed.

Nomenclature :1)mV : mill volts.2)nm : Wave length.3)min : minutes.4)Fig : Figure.5)Inj : Injection.

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

tion. J Chromatogr B Biomed Sci Appl, 1997; 703: 195-2017. Waschgler R, Moll W, König P, Conca A. Quantification of

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9. Charlier C, Pinto E, Ansseau M, Plomteux G. Venlafaxine: therelationship between dose, plasma concen-tration and clinical re-sponse in depressive patients. J Psychopharmacol, 2002; 16: 369-379.

10. Mandrioli R, Mercolini L, Cesta R, Fanali S, Amore M andRaggi M A, Analysis of the second generation antidepressantvenlafaxine and its main active metabolite O- desmethylvenlafaxinein human plasma by HPLC with spectrofluorimetric detection. JChromatogr B., 2007, 856(1-2), 88-94.

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18. Bhatt J, Jangid A, Venkatesh G, Subbaiah G and Singh S. Liquidchromatography-tandem Mass spectrometry (LC-MS-MS) methodfor simultaneous determination of venlafaxine and its active me-tabolite O-desmethyl venlafaxine in human plasma. J ChromatogrB Analy Technol Biomed Life Sci, 2005, 829, 75-81.

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20. Vu RL, Helmeste D, Albers L and Reist C. Rapid determination ofvenlafaxine and O-desmethylvenlafaxine in human plasma by high-performance liquid chromatography with fluorimetric detection. JChromatogr B Biomed Sci Appl, 1997, 703, 195-201

21. Waschgler R, Moll W, König P and Conca A. Quantification ofvenlafaxine and O- desmethylvenlafaxine in human serum usingHPLC analysis. Int J Clin Pharmacol Ther, 2004, 42, 724-728.

22. 22 Mandrioli R, Mercolini L, Cesta R, Fanali S, Amore M, RaggiMA. Analysis of the second generation anti-depressant venlafaxineand its main active metabolite O- desmethylvenlafaxine in humanplasma by HPLC with spectrofluorimetric detection. J ChromatogrB Analyt Technol Biomed Life Sci, 2007; 856, 88-94

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