INTRODUCTION:

Venlafaxine is an example of synthetic novel anti-depressant¹ drug, which acts by inhibiting the reuptake of serotonin and noradrenaline². It is chemically 1-[(1 RS)-2-(dimethyl amino)-1-(4 methoxy phenyl) ethyl] cyclohexanol hydrochloride³.

Fig. No. 1 Venlafaxine Hydrochloride

It is official in European Pharmacopoeia⁴. It is prescribed for the treatment of clinical depression and anxiety disorders. Molecular basis of Venlafaxine reveals that it is a complex molecule for the estimation by UV method and the –OCH3, Tertiary amine, alcoholic groups are the responsible for its therapeutic activity and quality control parameters⁵. A few analytical methods have been reported for its quantitative estimation in pharmaceutical formulations, which includes few HPLC methods^6-11 and spectrophotometric methods^12-16.

METHOD DEVELOPMENT:

Solubility Test

Solubility test for the drug Venlafaxine was performed by using various solvents. The solvents include Ethanol, Dist. water, Acetic Acid, 0.1 N Hydrochloric Acid, 0.1 N Sodium Hydroxide and Chloroform. However, distilled water was chosen as a solvent for developing the method.

Preparation of Stock Solution

The standard stock solution of 100 μg/mL of venlafaxine was prepared by weighing 50 mg of the drug, taken in 50 mL volumetric flask and was dissolved in distilled water up to the mark. Further 1 ml of above solution was pipette out and diluted up to 10 ml with dist. Water to produce 100 μg/mL.

Preparation of Working Standard Solution

5 ml of stock solution was taken in 10 ml volumetric flask and was diluted with water. Further 1 ml above resulting solution was taken and diluted up to 10 ml with distilled water to produce final concentration of 5μg/mL. Further dilutions were made with distilled water to obtain concentrations ranging from 05-25 μg/mL.

Determination of λ max

By appropriate dilution of standard solutions with distilled water, solutions containing 5 μg/mL of venlafaxine were scanned in the range of 200-800 nm to determine the wavelength of maximum absorbance for the drugs. Venlafaxine showed absorbance maxima at 225 nm.

Fig. No.2 λmax of venlafaxine at 225 nm

Assay of Venlafaxine Tablet

Accurately weighed and powdered 20 tablets. Venlafaxine equivalent to 50 mg was taken and transferred to a 50 mL of volumetric flask by adjusting the final volume up to 50 mL with dist. water. Further 1 ml of above solution was taken and diluted to 10 ml with dist. Water. From this solution 1 ml sample was again diluted up to 10 ml with dist. water. Finally absorbance of this resulting solution was noted at selected wavelength of 225 nm.

Table No 1: Assay of Venlafaxine

Brand Name	Label Claim	Assay
VENTAB XR	75 mg	96.05 %

METHOD VALIDATION:

Validation is a process of establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics^17,18. The method was validated for different parameters like Linearity, Accuracy, Precision, LOD, LOQ, Robustness, Ruggedness.

Linearity and Range

Various aliquots were prepared form the stock solution ranging from 5-25 μg/ml. The samples were scanned in UV-VIS Spectrophotometer using dist. water as a blank. It was found that the selected drug shows linearity between the range 5-25 μg/ml (Table 2 and 3).

Accuracy

The accuracy of the method was determined by preparing solutions of different concentrations that is 80%, 100% and 120% in which the amount of marketed formulation (10mg) was kept constant and the amount of pure drug was varied that is 8mg, 10mg and 12mg for 80%, 100% and 120% respectively. The solutions were prepared in triplicates and the accuracy was indicated by % recovery (Table 2 and 4)

Precision

Precision of the method was demonstrated by intraday and interday variation studies. In intraday variation study, 6 different solutions of same concentration that is 10μg/ml were prepared and analyzed in a day and the absorbance were noted. The result was indicated by % RSD (table no.6, & table no.7). In the interday variation study, solutions of same concentration 20μg/ml were prepared and analyzed for three consecutive days and the absorbances were noted. The result was indicated by % RSD (Table 2 and 5).

Ruggedness

Ruggedness of the method was determined by analyzing same sample (different batches) by different analysts at different conditions and the respective absorbance’s were noted.( Table 7)

Robustness

Robustness of the method was determined by carrying out the analysis at two different wavelengths i.e. at (+2nm and -2nm). (Table 2 and 8)

Limit of Detection (LOD)

The limit of detection (LOD) was determined by preparing solutions of different concentrations ranging from 0.1-1μg/ml. The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected but not necessarily quantitated as an exact value (Table 2 ).

Limit of Quantification (LOQ)

The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOQ was calculated using the formula involving standard deviation of response and slope of calibration curve (Table 2 ).

RESULTS AND DISCUSSION:

The developed method was found to be precise as the %RSD values for intra-day and inter-day were found to be less than 2%. Good recoveries (98% to 101%) of the drug were obtained at each added concentration, indicating that the method was accurate. The method was also found to be specific indicated by the % recoveries ranging from 99.8% to 101.2%. The method was also found to be robust and rugged as indicated by the % RSD values which are less than 2%. The results of Assay showed that the amount of drug was in good agreement with the label claim of the formulation as indicated by % recovery (100.03%). Summary of validation parameters of proposed spectrophotometric method is shown in table 2.

Table No. 2 Validation Summery

Parameters	Results
Linearity indicated by correlation coefficient	0.999
Precision indicated by %RSD	0.976
Accuracy indicated by %Recovery	99.3
Range	5-25 μg/ml
Linear regression equation	y = 0.019x
Limit of Detection	0.79 μg/ml
Limit of Quantification	2.59 μg/ml
Robustness indicated by %RSD	0.757
Assay indicated by % Recovery	96.05%

VALIDATION:

Linearity

Table No. 3 Linearity Table of Venlaflaxine hydrochloride in Working Standard

Cocⁿ(μg/ml)	Absorbance	Calculation
5	0.102	SD= 0.1502 RSD=0.5110 Slope=0.0191 C.C. =0.999 Intercept=-0.007
10	0.200
15	0.296
20	0.385
25	0.487

Fig. No. 3 Linearity curve of Venelafaxine.HCl

Accuracy

Table No.4 Accuracy of Venlafaxine Hydrochloride

No. of Prepn	Cocn (μg/ml)		% Recovery	Statistical Results
No. of Prepn	Formulation	Pure Drug	% Recovery	Mean	SD	%RSD
S1 – 80%	10	8	98	98.22	0.251	0.256
S2 – 80%	10	8	98.2
S3 – 80%	10	8	98.5
S4 – 100%	10	10	101	101.16	0.378	0.374
S5 – 100%	10	10	101.6
S6 – 100%	10	10	100.9
S7 – 120%	10	12	99.8	100.46	0.702	0.699
S8 – 120%	10	12	100.4
S9 – 120%	10	12	101.2

Precision

Table No 5: Intraday precision

Sr. No.	Conc.( μg/ml)	Abs
1	10	0.198
2	10	0.198
3	10	0.201
4	10	0.200
5	10	0.201
6	10	0.203
Avg.		0.200
SD		0.0019
%RSD		0.976%

Table No 6 : Interday precision

Sr. No.	Concn. (ppm)	Day –I	Day-II	Day III
1	20	0.385	0.387	0.391
2	20	0.389	0.389	0.389
3	20	0.385	0.390	0.387
4	20	0.386	0.387	0.388
5	20	0.383	0.386	0.388
6	20	0.381	0.384	0.385
Mean		0.384	0.387	0.388
SD		0.00286	0.00214	0.00126
%RSD		0.286	0.554	0.326

Ruggedness

Table No. 7 Ruggedness of Venlafaxine hydrochloride

Sr. No.	Parameter	Set I	Set II
1	System	Bioera	Schimadzu 1700
2	Sample	Batch No-X	Batch.No- Y
3	Day	Saturday	Monday
4	Time	11:00 am	3:30pm
5	Lab	Analysis	Chemistry
6	Analyst	73/12	76/12
7	Sample	10 μg/ml	10 μg/ml
8	Assay	99.68 %	99.88%

Robustness

Table No. 8 Robustness of Venlafaxine hydrochloride

Sr. No.	Cocⁿ(μg/ml)	Wavelength(nm)	Absorbance	Calculations
1	15	227	0.281	Mean = 0.280 S. D. = 0.0012 %RSD = 0.428
			0.279
			0.281
2	15	223	0.279	Mean = 0.276 S. D. = 0.003 %RSD = 1.086
			0.273
			0.276

Limit of Detection (LOD)

The LOD for Venlafaxine was found to be 0.79 μg/ml μg/ml.

Limit of Quantitation (LOQ)

The LOQ for Venlafaxine was found to be 2.59μg/ml

CONCLUSION:

The proposed method development and validation of UV-Vis Spectrophotometric method was to determine Venlafaxine Hydrochloride. The developed method was validated in distilled water according to ICH guideline and shown to be accurate, precise and cost effective. It do not require expensive or sophisticated and chemicals in contrast with chromatographic method. It can be used for the routine Q. C. analysis and quantification of the drug in the formulations.

ACKNOWLEDGEMENT:

The authors are wish to thanks Principal and Management of Shivnagar Vidya Prasarak Mandals College of Pharmacy, Malegaon (Bk), Tal- Baramati, Dist-Pune and Mr. Prabhatkumar Jain, Scan Research Bioanalytical Laboratories, Bhopal, for providing required lab facilities with enthusiastic environment.

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Received on 24.06.2015 Accepted on 28.07.2015

Asian J. Pharm. Ana. 5(3): July- Sept. 2015; Page 142-146

DOI: 10.5958/2231-5675.2015.00023.X