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ORIGINAL ARTICLE
Year : 2015  |  Volume : 6  |  Issue : 1  |  Page : 33-37

Stability indicating high performance thin layer chromatographic method for quantitation of venlafaxine in bulk and pharmaceutical dosage form


Department of Pharmacy, Birla Institute of Technology and Science, Pilani, Rajasthan, India

Date of Web Publication8-Jan-2015

Correspondence Address:
Sunil K Dubey
Department of Pharmacy, Birla Institute of Technology and Science, Pilani, Rajasthan
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2394-2002.148891

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  Abstract 

Background: Venlafaxine (VEN) is a phenethylamine bicyclic compound, chemically, 1-(2-[dimethyl amino]-1-[4-methoxy phenyl] ethyl) cyclo-hexan-1ol hydrochloride. It is a antidepressant. It inhibits the reuptake of serotonin, nor adrenaline and dopamine to a lesser extent at the presynaptic membrane. Aim: A simple, rapid, precise, accurate, and economical high performance thin layer chromatographic (HPTLC) method has been developed and validated for the determination of VEN both as a bulk drug and in formulation. Materials and Methods: The method uses aluminum plates precoated with silica gel 60 F254 as the stationary phase and dichloromethane:acetonitrile:N-hexane:triethylamine: 0.5:0.5:4:0.7 (v/v/v/v) as mobile phase. Results: This system gave compact spots for VEN (R f = 0.46 ± 0.05). Forced degradation studies were done by subjecting VEN to acid and alkali hydrolysis, oxidation, and reduction. The peak of the degradation product was well resolved from that of the pure drug and had significant different R f values. Analysis of VEN was performed in the absorbance mode at 225 nm. The limit of detection and quantification were 12.48 and 37.81 ng/spot respectively. Conclusions: The developed method was validated and found to be simple, specific, accurate and precise and can be used for routine quality control analysis of VEN in bulk and pharmaceutical formulation.

Keywords: Forced degradation, high performance thin layer chromatographic, stability, venlafaxine


How to cite this article:
Dubey SK, Anand A, Saha RN. Stability indicating high performance thin layer chromatographic method for quantitation of venlafaxine in bulk and pharmaceutical dosage form. Drug Dev Ther 2015;6:33-7

How to cite this URL:
Dubey SK, Anand A, Saha RN. Stability indicating high performance thin layer chromatographic method for quantitation of venlafaxine in bulk and pharmaceutical dosage form. Drug Dev Ther [serial online] 2015 [cited 2019 Aug 18];6:33-7. Available from: http://www.ddtjournal.org/text.asp?2015/6/1/33/148891


  Introduction Top


Venlafaxine (VEN) is a phenethylamine bicyclic compound, chemically, 1-(2-[dimethyl amino]-1-[4-methoxy phenyl] ethyl) cyclo-hexan-1ol hydrochloride [Figure 1]. It is a potential antidepressant; it inhibits the reuptake of serotonin, nor adrenaline and dopamine to a lesser extent at the presynaptic membrane. [1] The molecule exists as a racemic mixture of R (+) and S (−) in equal proportions. [2],[3] Enantiomers of VEN inhibit reuptake of nor adrenaline and serotonin, but R (+) −VEN primarily inhibits serotonin reuptake. [4] The unique property of VEN to reduce noradrenergic responsiveness after a single dose led to the suggestion that it might have a more rapid onset of action. VEN and its active metabolite exhibit a linear relationship between dose and plasma concentration for doses ranging from 75 to 450 mg/day. VEN is well absorbed after oral administration. [3]
Figure 1: Structure of venlafaxine

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Nowadays, high performance thin layer chromatographic (HPTLC) is becoming a routine analysis technique due to advantages of low-operating cost, high-sample throughput, need for minimum sample clean up, and several samples can be run simultaneously using a small quantity of mobile phase unlike HPLC, thus lowering analysis time and cost per analysis. [5]

The aim of the present work was to study the degradation behavior of VEN under stress conditions and to develop an accurate, specific, repeatable, and stability indicating HPTLC method for the determination of VEN in the presence of its degradation products. The proposed method was validated according to ICH guidelines [6] and its updated international convention. Advantage of this method is its calibration range (100-1000 ng/band) over reported range of 400-2000 ng/band. [7] Other advantages include a short time of analysis and the lesser amount of solvent used (6 ml per plate). [8] Furthermore, in one of the reported method limit of quantification (LOQ) is 130.89 ng/band but calibration curve range is 100-600 ng/band which means calibration range can not start from 100 ng/ band . write this and remove the line which means LOQ should be 100 or below 100 ng/ band. Therefore, our method is more accurate and sensitive compared to reported methods.


  Materials and Methods Top


Apparatus and instruments

  • Camag, 100 μl sample (Hamilton, Bonaduz, Switzerland) syringe
  • Camag Linomat V (Switzerland) sample applicator
  • Twin trough glass chamber (Camag, Muttenz, Switzerland)
  • Camag TLC scanner III
  • Electronic balance (AG-135, Mettler-Toledo, Germany)
  • pH meter (pH tutor, Eutech Instruments, Singapore)
  • Sonicator (Toshiba, New Delhi)
  • Milli-Q water purification system (Millipore, USA) was used for obtaining high quality HPLC grade water
  • Volumetric flasks: 10, 100, 500, 1000 ml (Borosil)
  • Pipettes: 100, 1 ml (Thermo)
  • Separating funnel
  • Beakers: 100, 200, 500 ml (Borosil).


Reagents and materials

  • Racemic VEN was purchased from TRC, Canada
  • dichloromethane, acetonitrile, N-hexane, methanol and triethylamine used were of analytical grade and purchased from Merck chemicals (Merck, India)
  • Silica gel recoated aluminum plate 60 F254, (with the thickness of 250 μm; E. Merck, Darmstadt, Germany).


Formulation

Venlafaxine capsules (Effexor XR).

High performance thin layer chromatographic condition

Sample application


The samples were spotted in the form of bands of width 4 mm with an on silica gel recoated aluminum plate 60 F254, (with the thickness of 250 μm; E. Merck, Darmstadt, Germany), using a Camag Linomat V (Switzerland) sample applicator. The plates were prewashed with methanol and activated at 110°C for 5 min prior to chromatography.

Mobile phase

Dichloromethane:acetonitrile:n-hexane:triethylamine: 0.5:0.5:4:0.7 (v/v/v/v). Linear ascending development was carried out in 20 cm × 20 cm twin trough glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile phase. The optimized chamber saturation time for the mobile phase was 15 min at room temperature (25°C) at relative humidity of 60%. The length of the chromatogram run was 4 cm. Subsequent to the development, TLC plates were dried at 145°C for 10 min.

Detection wavelength

Densitometric scanning was done at 225 nm on Camag TLC scanner III in the reflectance absorbance mode and operated by Camag WinCAT software.

Sample preparation

Primary stock solution of 1000 μg/ml of VEN was prepared in methanol. Secondary stock solution of 100 μg/ml was prepared from the primary stock solution.

Validation of the method

Validation of the optimized HPTLC method was done with respect to following parameters.

Linearity and range

Linearity was assessed in the range of 100-1000 ng/spot. Average peak area at each level was plotted against concentration and curves were subjected to linear regression analysis by least square method. Regression equation was used to calculate the corresponding predicted concentration.

Precision and accuracy

Precision was determined through repeatability (intra-batch) and intermediate (inter-batch) precision. Study was conducted by quality control standards prepared at lower (150 ng/spot), medium (600 ng/spot) and higher (900 ng/spot) concentration levels. Precision of the method was expressed as percent relative standard deviation (% RSD). Three batches were run for precision study. To determine intra-day precision two batches were run on the same day and one batch was run on the second day to determine inter-day precision. Accuracy was determined by placebo spiking method.

Limit of detection and limit of quantification (sensitivity)

Limit of detection (LOD) and LOQ were calculated on the basis of response and slope of the regression equation. They were calculated from the formula 3.3 to 10 σ/s respectively where "σ" is a standard deviation of the y-intercept of the regression line and "s" is slope of the calibration curve.

Analysis of marketed formulation

Two different lots of commercially available VEN capsules (Effexor XR) were analyzed using the validated HPTLC method.

Forced degradation studies

Forced degradation studies were done by subjecting VEN to acid and alkali hydrolysis, oxidation and reduction.

Acid induced degradation

Acid decomposition studies were performed by refluxing 10 ml of 1 mg/ml drug solution in 3 N HCl at 80°C for 8 h. After 8 h 5 ml of this sample was neutralized with 5 ml of 3 N NaOH and final concentration was made to 100 μg/ml with methanol. The resultant solutions were applied on TLC plate in such a way that final concentration achieved was 900 ng/spot and chromatogram was run as described in HPTLC section.

Base induced degradation

Base decomposition studies were performed by refluxing 10 ml of 1 mg/ml drug solution in 3 N NaOH solution at 80°C for 8 h. After 8 h 5 ml of this sample was neutralized with 5 ml of 3 N HCl and final concentration was made to 100 μg/ml with methanol. The resultant solutions were applied on TLC plate in such a way that final concentration achieved was 900 ng/spot and chromatogram was run as described in HPTLC section.

Oxidation induced degradation

Hydrogen peroxide decomposition studies (oxidation) were performed by keeping 10 ml of 1 mg/ml drug solution in 33% hydrogen peroxide at room temperature for 8 h. After 8 h 5 ml of this sample was diluted to 100 μg/ml with methanol. The resultant solutions were applied on TLC plate in such a way that final concentration achieved was 900 ng/spot and chromatogram was run as described in HPTLC section.

Reduction induced degradation

Reduction was carried out by refluxing the 10 ml of 1 mg/ml drug solution in 3 N HCl containing zinc dust at 80°C for 8 h. After 8 h 5 ml of this sample was neutralized with 5 ml of 3 N NaOH and final concentration was made to 100 μg/ml with methanol. The resultant solutions were applied on TLC plate in such a way that final concentration achieved was 900 ng/spot and chromatogram was run as described in HPTLC section.


  Results and Discussion Top


Optimization of stability indicating high-performance thin layer chromatographic method

The HPTLC procedure was optimized with a view to develop a stability indicating assay method. Initial trial with acetonitrile:toluene:methanol:ammonia: 7:2:0.5:0.5 gave good spot but Rf (0.85) was too high. Similar value of Rf was obtained with acetonitrile: Ammonia: Water 6:0.5:1.5. Rf 0.42 was obtained with dichloromethane:acetonitrile:n-hexane: 0.5:0.5:4 (v/v/v) but peak tailing was present. Therefore, to reduce the peak tailing and improve the compactness of spot triethylamine was added. Optimized mobile phase dichloromethane:acetonitrile:N-hexane:triethylamine: 0.5:0.5:4:0.7 (v/v/v/v). 5 ml of this system was used for each run. The chamber was saturated with mobile phase vapor for 15 min and mobile phase was allowed to run up to 4 cm TLC plate.

Validation of the stability indicating method

Linearity and range


The response was linear in the concentration range of 100-1000 ng/spot [Figure 2] and [Figure 3].
Figure 2: Densitogram of standard venlafaxine (400 ng/spot); peak 1 (Rf: 0.46 ± 0.05), mobile phase: dichloromethane:acetonitrile:n-hexane:triethylamine: 0.5:0.5:4:0.7 (v/v/v/v)

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Figure 3: Three-dimensional representation of the calibration curve

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Precision and accuracy

The results of the repeatability and intermediate precision experiments are shown in [Table 1]. The developed method was found to be precise as the RSD values for repeatability and intermediate precision studies were <2%, respectively as recommended by ICH guideline. Recovery by placebo spiking method is shown in [Table 1].
Table 1: Validation parameter of venlafaxine


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Limit of detection and limit of quantification

The signal:noise ratios of 3:1 and 10:1 were considered as LOD and LOQ, respectively. The LOD and LOQ were found to be 12.48 and 37.81 ng/spot respectively.

Estimation of formulation

Venlafaxine capsules (Effexor XR) were analyzed using the validated HPTLC method. Recovery from two lots of the capsule was 99.07% to 100.96% respectively. Results are shown in [Table 2].
Table 2: Analysis of pharmaceutical formulation


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Stability indicating property

Results of stability studies are mentioned below.

Acid induced degradation

Degradation was observed when drug solution was heated to 80°C for in 3N HCl. The acid degraded product was observed at Rf of 0.61 [Figure 4].
Figure 4: Densitogram of acid-induced degradation; condition: 3N HCl at 80°C for 8 h; peak 1 (venlafaxine, Rf: 0.46) and peak 2 (degraded, Rf: 0.65)

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Base induced degradation

It was found that the drug was found to be highly stable to alkali condition.

Oxidation induced degradation

Negligible degradation was observed in 33% of hydrogen peroxide at room temperature.

Reduction induced degradation

Degradation was observed when drug solution was heated to for 8 h at 80°C in 3N HCl having some amount of zinc dust. The degraded product was observed at R f of 0.61 [Figure 5] and [Table 3].
Figure 5: Densitogram of reduction induced degradation; condition: 3N HCl with zinc dust at 80°C for 8 h; peak 1 (venlafaxine, Rf: 0.46) and peak 2 (degraded, Rf: 0.67)

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Table 3: Summary of forced degradation studies


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  Conclusion Top


A stability indicating HPTLC method was developed and validated as per ICH guidelines. The developed HPTLC technique is precise, specific, and accurate. Statistical analysis proves that the method is suitable for the analysis of VEN as bulk drug and in pharmaceutical formulation without any interference from the excipient. The drug was found to degrade extensively in acidic condition but stable to alkali, oxidative, and photolytic conditions.

 
  References Top

1.
Holliday SM, Benfield P. Venlafaxine. A review of its pharmacology and therapeutic potential in depression. Drugs 1995;49:280-94.  Back to cited text no. 1
    
2.
Howell SR, Husbands GE, Scatina JA, Sisenwine SF. Metabolic disposition of 14C-venlafaxine in mouse, rat, dog, rhesus monkey and man. Xenobiotica 1993;23:349-59.  Back to cited text no. 2
    
3.
Wang CP, Howell SR, Scatina J, Sisenwine SF. The disposition of venlafaxine enantiomers in dogs, rats, and humans receiving venlafaxine. Chirality 1992;4:84-90.  Back to cited text no. 3
    
4.
Klamerus KJ, Maloney K, Rudolph RL, Sisenwine SF, Jusko WJ, Chiang ST. Introduction of a composite parameter to the pharmacokinetics of venlafaxine and its active O-desmethyl metabolite. J Clin Pharmacol 1992;32:716-24.  Back to cited text no. 4
    
5.
Sethi PD. High Performance Thin Layer Chromatography, Quantitative Analysis of Pharmaceutical Formulations. 2 nd ed. New Delhi: CBS Publishers and Distributors; 1996.  Back to cited text no. 5
    
6.
ICH. Q2(R1) - Validation of Analytical Procedures: Text and Methodology International Conference on Harmonization. Geneva, Switzerland: ICH, 2005.  Back to cited text no. 6
    
7.
Shirvi VD, Channabasavaraj KP, Vijaya Kumar G, Tamizh MT. HPTLC Analysis of venlafaxine hydrochloride in the bulk drug and tablets. J Planar Chromatogr 2010;23:369-72.  Back to cited text no. 7
    
8.
Ramesh B, Narayana PS, Reddy AS, Sita Devi P. Stability-indicating HPTLC method for analysis of venlafaxine hydrochloride, and use of the method to study degradation kinetics. J. Planar Chromatogr 2011;24:160-5.  Back to cited text no. 8
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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