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U.V. SPECTROPHOTOMETRIC METHOD FOR ESTIMATION OF VENLAFAXINE HCL IN BULK AND FORMULATION 


Daswadkar S.C. *, Gajale V. N., Shevkar C. D., Phadtare G. A., Sonawane C. S.
 Department of Chemistry, Dr. D. Y. Patil College of Pharmacy, Akurdi, Pune, India.

 ABSTRACT

Three simple, precise and economical UV methods have been developed for estimation of Venlafaxine HCl in single component. Venlafaxine HCl has the absorbance maximum in zero order spectrum (Method A) is 273.5nm. First Order derivative (Method B) was applied for the analysis of Venlafaxine HCl at 263.5nm. Area under curve method (Method C) was applied in the wavelength range of 268.5nm-278.5nm. Drug followed Beer-Lambert’s law in the concentration range of 10-80(µg/ml) for zero order, first order and area under curve method.

The results of the analysis were validated in terms of specificity, limit of detection, limit of quantification, linearity, precision and accuracy As per ICH guidelines and were found to be satisfactory. The high recovery and low relative standard deviation confirm the suitability of these methods can be employed for the routine analysis of tablet containing Venlafaxine HCl.

 Key Words: Venlafaxine, UV Spectrophotometer, Recovery, Validation

INTRODUCTION

 Venlafaxine HCl (1-[2-dimethylamino)-1-(4-methoxy phenyl) ethyl] Cyclohexanol) is an antidepressant of the serotonin and nor epinephrine reuptake inhibitor (SNRI) class [1]. Venlafaxine hydrochloride is a third generation antidepressant. It inhibits the reuptake of serotonin, nor epinephrine and to a lesser extent dopamine. 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 receptors [2].

 Venlafaxine is not official in any of Pharmacopoeias and only listed in the Merck index [3], and Martindale [4], the complete drug references. Literature survey reveals that there are few methods like UV [5-12], RP-HPLC [12-14], UPLC-MS [15], HPTLC [16-17] has been reported for estimation of Venlafaxine HCl, but at different wavelength and use of different solvent system hence the objective of work is to develop simple precise, accurate, sensitive and rapid cheap UV-Visible spectrophotometer in bulk and pharmaceutical formulation.

 

 Figure no. 1. Venlafaxine HCL structure

MATERIALS AND METHOD

Venlafaxine HCl was obtained as gift sample from Gain Pharmaceuticals Pvt. Ltd, Bhosari, Pune. Distilled water was used as the solvent. Tablets of brand Venlafaxine HCl30 (225mg) were obtained from the local market for assay and Recovery studies.

 Instruments:

Shimadzu 1700 Electronic model UV Spectrophotometer with 1cm matched quartz cells was used as the instrument for data collection and analysis. Analytical balance with 0.01 mg was used.

 Experimental

 

Preparation of drug stock solution

Weigh accurately about 10mg of Venlafaxine HCl and transferred to 100ml volumetric flask. To it 20ml of distilled water was added to dissolve the drug completely with vigorous shaking then the volume was made up with distill water upto mark to give the drug stock solution of concentration 100µg/ml.

 

Preparation of analytical standard solutions of Clonazepam

Analytical standard solutions of Venlafaxine were prepared from stock solution of Venlafaxine (100µg/ml) using distilled water, pipetting out aliquots of 1, 2, 3, 4…8ml and transferred in 10ml volumetric flasks. The dilutions were made by using distilled water gives the samples of concentrations 10, 20, 30, 40…. 80ug/ml. These samples were analyzed against standard calibration curve in Quantitation mode.

 

Method A: Zero order method

In this method, measured the absorbance of all analytical standard solutions at 273.5nm against water as blank solvent. A linear graph of absorbance vs. concentration was obtained. The concentration range over which the drugs obeyed Beer – Lambert’s law was found to be 10 to 80µg/ml for Venlafaxine HCl.

 

Method B: First order method

In this method, measured the absorbance of all analytical standard solutions at 263.5 nm against water as blank solvent. A linear graph of absorbance vs. concentration was obtained.  The concentration range over which the drugs obeyed Beer – Lambert’s law was found to be 10 to 80µg/ml for Venlafaxine HCl.

 

Method C: Area under the Curve (AUC) Method

In area under the curve method, the absorbance measured for all analytical standard solutions between wavelengths 268.5 nm to 278.5 nm were selected for the calculation of the linearity and it was obtained within these areas with good reproducibility of results.

Analysis of tablet formulation

Twenty tablets were weighed accurately. The tablet content was weighted and triturated to fine powder. Tablet powder equivalent to 5mg of Venlafaxine HCl was weighed and transferred to 100 ml volumetric flask and dissolved in 20 ml of distilled water. It was kept for ultrasonification for 15 min. finally the volume was made up to the mark with distilled water. Further pipette out 1ml from above solution and dilute up to 10 ml with distilled water this was then filtered through whatman filter paper no. 41 to get tablet stock solution of concentration of 100 μg/ml. various dilution of the tablet solution were prepared and analyzed for six times and concentration was calculated by using the calibration curve for two method (Table 1).

 

Table 1: Analysis of Tablet formulation

Sr. No. Tablet sample Label Claim

(Mg/tab)

Label Claim Found

(Mg/tab)

%

mean

S.D* C.O.V.* S.E.M*
A T1 75 74.99 99.98 0.836 0.679 0.343
B T1 75 74.97 99.97 0.835 0.680 0.342
C T1 75 74.99 99.98 0.837 0.678 0.344

Validation

The developed method was validated according to ICH guidelines. Different standard solutions were prepared by diluting standard stock solution with double distilled water in the concentration range 10-80µg/ml. The absorbance was plotted against corresponding concentrations to obtain the calibration graph. Specificity is the ability of a method to discriminate between the analyte of interest and other components that may present in the sample. The specificity of the method was evaluated to ensure separation of Venlafaxine. Precision of analytical method was expressed in relative standard deviation (RSD) of a series of measurements. The intra-day and inter-day precisions of the proposed methods were determined by estimating the corresponding responses (i.e. three concentrations/ three replicates each) of the sample solution on the same day and on three different days respectively. The signal-to-noise ratio (S/N) method was adopted for the determination of limit of detection and limit of quantification. The limit of detection was estimated as three times the S/N ratio and the limit of quantification was estimated as ten times the S/N ratio. To check the accuracy of the proposed method, recovery studies were carried out by standard addition method. A known amount of standard Venlafaxine corresponding to 80, 100 and 120% of the label claim was added to pre-analyzed sample of tablet. The recovery studies were carried out in triplicate at each level.

 RESULT AND DISCUSSION

 

Selectivity: In the zero order method, Venlafaxine shows the maximum absorbance at wavelength 273.5nm (Figure no. 2), in first order method the maximum absorbance shows at the wavelength 263.5nm (figure no. 3) and in area under the curve method the maximum absorbance shows between the wavelengths 268.5 nm to 278.5 nm. (Figure no. 4)

           

Figure no. 2: zero order spectra                                                      Figure no. 3:  First order spectra

                                 

Figure no. 4: Area under curve spectra

Linearity: 

Linearity was obtained in a concentration range from 10-80ug/ml for Venlafaxine HCl. The response of the drug was found to be linear in the investigation concentration range and the linear regression equation with correlation coefficient for both the methods were shown in (Table 2)                          

Table 2: Statistical parameters of Venlafaxine HCl

Parameters Method A Method B Method C
λmax (nm) 273.5nm 263.5 263.5-273.5nm

 

Beer’s – Lambert’s range (µg/ml) 10-80µg/ml 10-80µg/ml 10-80 µg/ml
Coefficient of correlation (r2 ) 0.9992 0.9997 0.9997
Regression equation : Y = mx + c 0.0049x+0.0003 0.0002x+0.00 0.047x+0.021
a – Slope (m) 0.0049 0.0002 0.047
b – Intercept (c) 0.0003 0.0 0.021
LOD µg/ml 0.26 1.65 0.0006
LOQ µg/ml 0.81 5 0.002
Molar absorptivity 0.165×10-3 0.062×10-3

 

1.57×10-3

Precision: Precision of the analytical method is ascertained by carrying out the analysis as per the procedure and as per normal weight taken for analysis. Repeat the analysis six times. Calculate the % assay, mean assay, % Deviation and % relative standard deviation and %RSD. The developed method was found to be precise as the %RSD values for the repeatability and intermediate precision studies were <0.98% and <0.79%, respectively (Table 3).

Table 3: Evaluation data of precision study

Sample No. % Assay
SET Intraday Interday
1 101.2 99.3
2 99.89 98.2
3 100.1 99.0
4 99.98 99.0
5 100.6 98.6
6 99.6 100.5
Mean 100.22 99.1
SD 0.99 0.78
%RSD 0.98 0.79

 Accuracy:

Recovery studies were carried out at two different level i.e. 80%, 100%, 120% by adding the pure drug (8, 10, and 12 mg respectively) to previously Analyzed tablet powder sample from the amount of drug found percentage recovery was calculated (Table 4). 

Table 4: Accuracy Study Data

Sr. No. Tablet sample Level of recovery (%) Percentage (mean) R.S.D.* C.O.V.* S.E.*
1  

T1

80 100.17 0.5948 0.01 0.3434
2 100 100.64 0.03055 0.07751 0.01764
3 120 100.60 0.03055 0.0245 0.01765
4  

T2

80 100.17 0.5948 0.207 0.3434
5 90 100.64 0.02055 0.146 0.01755
6 100 100.60 0.03055 0.112 0.01785
7  

T3

80 100.17 0.5948 0.200 0.3440
8 100 100.64 0.03045 0.140 0.01745
9 120 100.60 0.03090 0.114 0.01785

Solution stability study: (Table 5) shows the results obtain in the solution stability study at different time intervals for test preparation. It was concluded that the test preparation solution was found stable up to 4 hr at room temperature, as during this time the result was not decrease below the minimum percent.

 Table 5: Evaluation data of solution stability study

Time (Hrs.) Standard Sample
0 0.723 0.723
2 0.72 0.721
4 0.717 0.717
6 0.716 0.717
8 0.716 0.716
Limit at (2hr) 0.414 0.276
Limit at (4hr) 0.829 0.829
Limit at (6hr) 0.835 0.832
Limit at (8hr) 0.842 8.041

 CONCLUSION

 The present analytical method was validated as per ICH Q2 (R1) guideline and it meets to specific acceptance criteria. It is concluded that the analytical method was specific, precise, linear, accurate, robust and having stability indicating characteristics. The present analytical method can be used for its intended purpose.                 

 

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