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ORIGINAL ARTICLE
Year : 2010  |  Volume : 2  |  Issue : 1  |  Page : 22-26 Table of Contents     

Development and validation of stability indicating method for the quantitative determination of venlafaxine hydrochloride in extended release formulation using high performance liquid chromatography


1 Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal 576 104, India
2 Analytical Research and Development-New Drug Discovery Research, Ranbaxy Research Laboratories, Gurgaon, Haryana, India

Date of Submission23-Jan-2010
Date of Decision15-Feb-2010
Date of Acceptance25-Feb-2010
Date of Web Publication23-Apr-2010

Correspondence Address:
Abhishek Gupta
Analytical Research and Development-New Drug Discovery Research, Ranbaxy Research Laboratories, Gurgaon, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-7406.62701

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   Abstract 

Objective : Venlafaxine,hydrochloride is a structurally novel phenethyl bicyclic antidepressant, and is usually categorized as a serotonin-norepinephrine reuptake inhibitor (SNRI) but it has been referred to as a serotonin-norepinephrine-dopamine reuptake inhibitor. It inhibits the reuptake of dopamine. Venlafaxine HCL is widely prescribed in the form of sustained release formulations. In the current article we are reporting the development and validation of a fast and simple stability indicating, isocratic high performance liquid chromatographic (HPLC) method for the determination of venlafaxine hydrochloride in sustained release formulations. Materials and Methods : The quantitative determination of venlafaxine hydrochloride was performed on a Kromasil C18 analytical column (250 x 4.6 mm i.d., 5 μm particle size) with 0.01 M phosphate buffer (pH 4.5): methanol (40: 60) as a mobile phase, at a flow rate of 1.0 ml/min. For HPLC methods, UV detection was made at 225 nm. Results : During method validation, parameters such as precision, linearity, accuracy, stability, limit of quantification and detection and specificity were evaluated, which remained within acceptable limits. Conclusions : The method has been successfully applied for the quantification and dissolution profiling of Venlafaxine HCL in sustained release formulation. The method presents a simple and reliable solution for the routine quantitative analysis of Venlafaxine HCL.

Keywords: Dissolution, extended release formulation, high-performance liquid chromatography, method development, venlafaxine hydrochloride, validation


How to cite this article:
Kaur J, Srinivasan K K, Joseph A, Gupta A, Singh Y, Srinivas KS, Jain G. Development and validation of stability indicating method for the quantitative determination of venlafaxine hydrochloride in extended release formulation using high performance liquid chromatography. J Pharm Bioall Sci 2010;2:22-6

How to cite this URL:
Kaur J, Srinivasan K K, Joseph A, Gupta A, Singh Y, Srinivas KS, Jain G. Development and validation of stability indicating method for the quantitative determination of venlafaxine hydrochloride in extended release formulation using high performance liquid chromatography. J Pharm Bioall Sci [serial online] 2010 [cited 2019 Nov 15];2:22-6. Available from: http://www.jpbsonline.org/text.asp?2010/2/1/22/62701

Venlafaxine,1-[2-dimethylamino-1-(4-methoxyphenyl)-ethyl]-cyclohexan-1-olhydrochloride [Figure 1] is a structurally novel phenethyl bicyclic antidepressant, and is usually categorized as a serotonin-norepinephrine reuptake inhibitor (SNRI) [1],[2] but it has been referred to as a serotonin-norepinephrine-dopamine reuptake inhibitor. [2] It weakly inhibits the reuptake of dopamine. [3]

Various methods have been reported for the estimation of venlafaxine hydrochloride in biological matrices such as plasma, which include the use of liquid chromatography (LC) with UV detection, [4] LC with electrospray ionization mass spectrometry, [5] LC with coulometric detection, [6] LC with fluorimetric detection, [7] LC with diode array detection, [8] gas chromatography-mass spectrometery (GC-MS), [9] LC-MS-MS, [10] and for the estimation in serum by using LC. [11] Stability indicating methods have also been reported for its in vitro determination in gastric and intestinal fluids [12] and pharmaceutical formulations. [13]

Both the reported stability indicating methods use acetonitrile and buffer in various proportions for the quantification of venlafaxine hydrochloride. The present study involves development of reverse phase-high performance liquid chromatographic (RP-HPLC) method using simple mobile phase containing methanol and buffer for quantitative estimation of venlafaxine hydrochloride in tablet dosage forms, which is sensitive and requires shorter analysis time of 15 min. The developed method was validated as per International conference on harmonization (ICH) guidelines [14] and was applied to study the dissolution profile of the drug in extended release tablets.


   Materials and Methods Top


Materials

Venlafaxine hydrochloride API (active pharmaceutical ingredient) reference standard was extracted from tablets in-house and was characterized thoroughly and certified to contain 99.29% venlafaxine hydrochloride API. Extended release tablets containing 75 mg of venlafaxine hydrochloride API were obtained from commercial sources within their shelf life period.

All solvents were of HPLC grade and all reagents were of analytical grade. Methanol, sodium hydroxide, hydrochloric acid, phosphoric acid, and hydrogen peroxide were obtained from Qualigen Fine Chemicals (India). Potassium dihydrogen phosphate was obtained from Merck (Germany). Potassium hydroxide was obtained from Rankem (India). Water was purified with Milli-Q Plus, Millipore System (USA). All solvents and solutions were filtered through a membrane filter (Millipore Millex-HV filter units, 0.45 μm pore size) and degassed before use.

Instrumentation

A gradient high performance liquid chromatograph from Waters Alliance HPLC system, equipped with a diode array detector and Empower software data station system for data integration was used. A reversed phase C-18 (250 Χ 4.60 mm i.d, 5 μm particle size) analytical column was used for the present analysis.

Reference standard and sample preparations

Exactly 75 mg of venlafaxine hydrochloride reference standard (99.29%) was accurately weighed and transferred to a 100-ml volumetric flask and dissolved first in methanol, sonicated for 20 min and then 0.1 N HCl was added and the contents were sonicated for another 20 min. The final volume was made with diluent (methanol : 0.1 N HCl, 50 : 50 v/v). Then 4 ml of this solution was diluted to 50 ml with the diluent for the chromatographic method (final concentration 60 μg/ml). The solution so obtained was filtered through 0.45 μm pore size filter units before use.

The required number of tablets under analysis was crushed in a mortar to get homogenous powder. An amount equivalent to 75 mg of venlafaxine hydrochloride, present in tablets was transferred to a 100-ml volumetric flask and the sample was prepared according to the above-defined procedure for standard preparation.

Chromatographic conditions

The samples were chromatographed on a reversed phase C-18 (250 Χ 4.6 mm i.d, 5 μm particle size) column with a flow rate of 1.0 ml/min. All analyses were carried out at a temperature of 402C under isocratic conditions. The mobile phase consisted of a mixture of 0.01 M phosphate buffer (pH 4.5, adjusted with phosphoric acid) : methanol (40 : 60, v/v). The volume of injection was 20 μl and the detection was made at 225 nm.

Optimization of the solvent system

Varying compositions of phosphate buffer (0.01 M, pH 4.5) : methanol viz. 65 : 35, 50 : 50, 40 : 60, and 30 : 70 v/v were evaluated as mobile phase in order to achieve good peak shape, USP plate count and short run time. Finally, phosphate buffer (0.01 M, pH 4.5) : methanol (40 : 60) v/v isocratic method was used.

Stability indicating method

The drug was subjected to forced degradation under acidic (10% hydrochloric acid, 2 ml), basic (10% sodium hydroxide, 5 ml), and oxidative (3% hydrogen peroxide, 2 ml) stress conditions. Samples were treated accordingly and a final concentration of 60 μg/ml was prepared using the diluent and injected.

Method validation

The method used for the determination of venlafaxine hydrochloride in pharmaceuticals was validated for linearity, precision, accuracy, specificity, sensitivity, robustness, and stability according to the International Conference on Harmonization guidelines for analytical procedures validation.

Linearity

The linearity was evaluated by linear regression analysis. The calibration curve was obtained with seven concentrations of reference standard solution ranging from 70 to 130% (42, 48, 54, 60, 66, 72, and 78 μg/ml−1 ) of the target analyte concentrations for the chromatographic method.

Precision

The precision of the procedure was determined by repeatability (intraday). Intraday precision was evaluated by assaying both the reference and the samples at the same concentration and during the same day. Six solutions (60 μg/ml) of the reference were prepared and analyzed for the determination of system precision. Similarly, six solutions (60 μg/ml) of the sample were prepared and assayed for the determination of method precision.

Accuracy

The accuracy of the method was determined by spiking placebo with three concentrations (48, 60, and 72 μg/ml) of reference standard and assaying for the chromatographic method.

Specificity

The specificity was determined by analyzing placebo. The possible interferences were analyzed by the peak purity, which was calculated using software.

Sensibility (Limit of detection and limit of quantification)

For HPLC method, the limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the response and the slope by using three calibration curves.

Robustness

For the HPLC method, the robustness was determined by the analysis of the samples under a variety of conditions making small changes in the buffer pH (4.3 and 4.7), in the percentage of mobile phase compounds (phosphate buffer : methanol in the ratios 38 : 62 and 42 : 58), in the flow rate (0.9 and 1.1 ml/min), in the temperature conditions (35 and 45C), and changing the wavelength (223 and 225 nm).

Stability in analytical solution

The sample was analyzed for 24 h at room temperature, i.e., at 25C.


   Results and Discussion Top


Optimization of the solvent system

The mobile phase phosphate buffer (0.01 M, pH 4.5): methanol, 40 : 60 v/v was found to be a suitable solvent system. [Figure 2] shows a typical chromatogram obtained from the analysis of a reference standard using the proposed method for venlafaxine hydrochloride. As shown in this figure, a symmetrical peak represents venlafaxine hydrochloride. The retention time observed in the assay (4.49 min) associated with the simple sample preparation (for tablets) allowed a rapid determination of the drug in pharmaceutical products. The USP tailing and plate count were found to be 1.62 and 5885, respectively.

Stability indicating method

The drug shows 11.21, 14.30, and 22.16% degradation under acidic, basic, and oxidative conditions, respectively. The method was found to be stability indicating as seen in the purity plots [Figure 3] in which the purity angle (PA) is less than purity threshold (TH).

Validation

Linearity

The calibration curve of analytical method was assessed by plotting concentration versus peak area, which showed suitable linearity in the range 42-78 μg/ml for the HPLC method. In linearity graph, the correlation coefficient (r2 ) was found to be 0.9997 [Figure 4].

The linear range obtained for the procedure applied to formulations by HPLC allows one to assay a dissolution profile of tablets containing 75 mg of venlafaxine hydrochloride.

Precision and accuracy

For the chromatographic method, the relative standard deviation (RSD) values for intraday system and method precision were 0.24 and 1.01%, respectively.

The percent recovery at 80, 100, and 120% accuracy levels was found to be 97.26, 102.69, and 103.95%, respectively [Table 1].

Specificity

For the HPLC method, no interference from the matrix and excipients was found in the placebo of the tablets.

Sensibility

For developed HPLC method, the theoretical LOD was found to be 0.075 μg/ml with RSD of 20.1%, whereas the LOQ was found to be 0.15μg/ml, with a suitable RSD of 9.8%.

Robustness

To prove the reliability of the analytical method during normal usage, some small but deliberate changes were made in analytical method, e.g., flow rate, pH, organic phase, column temperature, and UV detector wave length. Changes in chromatographic parameters, i.e., theoretical plates and tailing factor were evaluated for the studies. In all the deliberately varied chromatographic conditions, the chromatogram for system suitability solution showed satisfactory resolution. (RSD<2%) with no significant changes in chromatographic parameters [Table 2].

Stability in analytical solution

The sample was found to be stable at 25C for 24 h and the overall %RSD was found to be 0.99.

Application

The dissolution data for the six tablets of one batch of venlafaxine hydrochloride are given in the following table [Table 3]. The data reveal that the amount of drug release with time is linear in nature. Thus, the validated HPLC method has been successfully applied to the dissolution study of venlafaxine hydrochloride in extended release formulations tablets.


   Conclusions Top


The developed HPLC method enables a quantitative determination of venlafaxine hydrochloride in extended release formulations. The application of this method in routine analysis can be justified as easy sample preparation steps are involved using simple reagents and solvents. The validation demonstrated that the procedure is suitable for the intended purpose because the method was found to be linear, precise, accurate, stability indicating, and specific. The method is reproducible and selective for the estimation of venlafaxine hydrochloride in pharmaceutical formulations. The application of validated HPLC method to study the dissolution profile of venlafaxine hydrochloride in extended release tablets showed good and linear results.


   Acknowledgment Top


The author would like to thank Ms. Aarti Sharma (Jaipur National University, Jaipur, India) for her contribution in restructuring this article.

 
   References Top

1.Acute effectiveness of additional drugs to the standard treatment of depression. Available from: http://ClinicalTrials.gov. [last retrieved on 2005 June 23].  Back to cited text no. 1      
2.Goeringer KE, McIntyre IM, Drummer OH. Postmortem tissue concentrations of venlafaxine. Forensic Sci Int 2001;121:70-5.  Back to cited text no. 2      
3.Wellington K, Perry CM. Venlafaxine extended-release: A review of its use in the management of major depression. CNS Drugs 2001;15:643-69.  Back to cited text no. 3      
4.Raut BB, Kolte BL, Deo AA, Bagool MA, Shinde DB. A rapid and sensitive high performance liquid chromatographic method for determination of venlafaxine and o-desmethyl venlafaxine in human plasma with UV detection. J Liq Chromatogr Technol 2003;26:1297-313.  Back to cited text no. 4      
5.Juan H, Zhiling Z, Huande L. Simultaneous determination of fluoxetine, citalopram, paroxetine, venlafaxine in plasma by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI). J Chromatogr B Analyt Technol Biomed Life Sci 2005;820:33-9.  Back to cited text no. 5      
6.Clement EM, Odontiadis J, Franklin M. Simultaneous measurement of venlafaxine and its major metabolite, O-desmethylvenlafaxine, in human plasma by high-performance liquid chromatography with coulometric detection and utilisation of solid-phase extraction. J Chromatogr B Biomed Sci Appl 1998;705:303-8.  Back to cited text no. 6      
7.Vu RL, Helmeste D, Albers L, Reist C. Rapid determination of venlafaxine and O-desmethylvenlafaxine in human plasma by high-performance liquid chromatography with fluorimetric detection. J Chromatogr B Biomed Sci Appl 1997;703:195-201.  Back to cited text no. 7      
8.Duverneuil C, de la Grandmaison GL, de Mazancourt P, Alvarez JC. A high-performance liquid chromatography method with photodiode-array UV detection for therapeutic drug monitoring of the nontricyclic antidepressant drugs. Ther Drug Monit 2003;25:567-73.  Back to cited text no. 8      
9.Wille SM, Maudens KE, Van Peteghem CH, Lambert WE. Development of a solid phase extraction for 13 'new' generation antidepressants and their active metabolites for gas chromatographic-mass spectrometric analysis. J Chromatogr A 2005;1098:19-29.  Back to cited text no. 9      
10.Bhatt J, Jangid A, Venkatesh G, Subbaiah G, 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 Analyt Technol Biomed Life Sci 2005;829:75-81.  Back to cited text no. 10      
11.Frahnert C, Rao ML, Grasmδder 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 Analyt Technol Biomed Life Sci 2003;794:35-47.  Back to cited text no. 11      
12.Asafu-Adjaye EB, Faustino PJ, Tawakkul MA, Anderson LW, Yu LX, Kwon H, et al. Validation and application of a stability-indicating HPLC method for the invitro determination of gastric and intestinal stability of venlafaxine. J Pharm Biomed Anal 2007;43:1854-9.  Back to cited text no. 12      
13.Makhija SN, Vavia PR. Stability indicating LC method for the estimation of venlafaxine in pharmaceutical formulations. J Pharm Biomed Anal 2002;28:1055-9.  Back to cited text no. 13      
14.ICH - international conference on harmonization of technical requirements for registration of pharmaceuticals for human use. Validation of Analytical Procedures: Text and Methodology ICH Secretariat, Geneva, Switzerland, 1996.  Back to cited text no. 14      


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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