INTRODUCTION:

Venlafaxine 1-[(1RS)-2-(Dimethyl amino)-1-(4-methoxyphenyl) ethyl] cyclohexanol hydrochloride¹ is official in European Pharmacopoeia² and British Pharmacopoeia³. It is white powder freely soluble in water and methanol. It is a bicyclic phenylethylamine derivative, of anti-depressant, structurally differs from other currently available anti-depressants^1-4. Venlafaxine and its active metabolite, O-desmethylvenlafaxine, inhibit the neuronal uptake of norepinephrine, serotonin, and, to a lesser degree, dopamine, but have no monoamine oxidase inhibitory activity and a low affinity for brain muscarinic, cholinergic, histaminergic, or alpha-adrenergic receptors^5-9. Hence, it lacks the adverse anti-cholinergic, sedative, and cardiovascular effects of tricyclic anti-depressants³. Venlafaxine used for the treatment of depressive disorders^10-13.

It has been proposed that anti-depressants with a dual action of inhibiting the re-uptake of both noradrenalin and serotonin (5-hydroxytryptamine, 5-HT) may be more effective than drugs acting on a single monoamine (e.g., selective serotonin re-uptake inhibitors, SSRIs). Venlafaxine is the first drug to be marketed that inhibits both noradrenalin and 5-HT re-uptake without actions in other receptors^14-16.

Venlafaxine is available in immediate-release (IR) formulation and half-life = 4 h, so this drug can be given in two divided doses. An extended-release (XR) preparation is also available in 37.5, 75, and 150 mg doses: Once-a-day XR dosing achieves bioavailability equivalent to that of twice-a-day dosing with IR formulation⁵.The XR preparation is also associated to a better compliance and demonstrates both anti-depressant and, after 2–3 weeks of treatment, also a good anxiolytic effect^17-18. Metabolism of venlafaxine occurs primarily via O-demethylation (mediated by cytochrome P450 [CYP] 2D6) and, to a lesser extent, by N-demethylation (mediated by CYP3A4)^19-20.

Figure 1. Structure of Venlafaxine Hydrochloride

MATERIALS AND METHOD:

Chemical and reagents:

Venlafaxine hydrochloride was gifted by pharma company, Hyderabad, Telangana, India. VENTAB XL75 was procured from the local market. HPLC grade methanol was procured from Rankem chemicals limited, New Delhi, India. Ammonium acetate buffer was used.

Instrument:

Shimadzu HPLC (LC-20AD Multi-solvent delivery system, rheodyne injector with 20µL fixed loop, SPD-20A UV-Visible detector, LC solution software), CONTECH analytical weighing balance, LABOTECH Sonicator (LMUC-2, L8486)

Chromatographic conditions:

The chromatographic separation was performed using Phenomenex C₁₈ (250mm×4.6mm×5µ) at ambient temperature. The mobile phase consisted of ammonium acetate buffer: methanol (60:40) with pH adjusted to 5 with glacial acetic acid and was isocratically delivered at a flow rate of 1ml/min. The mobile was filtered using membrane filters and degassed before use. The injection volumes were 20µL. The elution was monitored at 227nm.

Preparation was Standard Stock Solution:

Standard stock solution of venlafaxine hydrochloride was prepared by dissolving 10mg of venlafaxine HCl in 10ml of HPLC grade water in order to produce 1000 µg/ml.

Preparation of Working Standard Stock Solution:

From the standard stock solution, 1ml was pipetted into a 10ml volumetric flask and the volume was made up to the mark with HPLC grade water to prepare a concentration of 100µg/ml.

Preparation of Working Standard Solution:

From the working standard stock solution, 5ml was pipetted into a 10ml volumetric flask and the volume was made up to the mark with HPLC grade to prepare a concentration of 50µg/ml.

RESULT:

Optimization of the solvent system:

The pH and composition of mobile phase was varied to optimize the system varying composition of methanol and water, ammonium acetate buffer and methanol were evaluated as the mobile phase. Finally, ammonium acetate buffer and methanol in the ratio of 60:40(v/v) in the isocratic mode was used because of the good peak shape, higher USP and acceptable tailing factor.

Table 1. Optimized Chromatographic Conditions

Column	Phenomenex C₁₈250mm x 4.6 mm, 5µ.
Mobile phase	Ammonium acetate buffer : methanol (60:40) pH 5 adjusted with glacial acetic acid
Flow rate	1.0 ml/min
Injection Volume	20.0μL
Detector	UV 227nm
Pump mode	Isocratic
Retention time	2 min
Run time:	10min

Figure 2: Standard optimized chromatogram of 50µg/ml Venlafaxine Hydrochloride solution

Method validation:

Validation is the process of “establishing documented evidence” which provides high degree of assurance that a specific activity will consistently produce desired results or product meeting its predetermined specifications and quality.

Specificity:

The ability to assessunequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products and matrix components.

No peaks were observed at the retention time of venlafaxine hydrochloride when blank was injected as shown in figure 3 and chromatogram with standard in figure 3a.

Linearity:

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

Linearity was evaluated by injecting venlafaxine hydrochloride standard stock solution in the range of 15 to 165 µg/ml. The calibration curve was prepared by plotting Area v/s Concentration. The method was found to be linear with coefficient linear 0.9997

Table 2. Linearity data of RP-HPLC Method

Concentration (µg/ml)	R_t (min.)	Area	Tailing factor
15	2.001	1005240	1.240
30	2.00	1961745	1.190
45	2.00	2777393	1.157
60	2.001	3750572	1.185
75	2.00	4732409	1.028
90	2.00	5565994	1.164
105	2.001	6487516	1.150
120	2.001	7387516	1.108
135	2.004	8425558	1.113
150	2.001	9413663	0.911
165	2.01	10302329	0.746

Figure 4: calibration curve of Venlafaxine Hydrochloride

Precision:

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.

Precision was assessed by injecting venlafaxine hydrochloride 75µg/ml concentration of 6 replicate injections into HPLC system. %RSD was calculated.

Table 3. Precision data of RP-HPLC Method

Injections	Area	Ret. time	Tailing factor
1	4732409	2. 004	1.028
2	4732409	2.002	1.028
3	4732409	2.00	1.028
4	4732411	2.002	1.028
5	4732402	2.0004	1.028
6	4732419	2.00	1.028
Average	4732410
SD	5.455884
%RSD	0.000115

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between conventional true value or an accepted reference value and the value found.

The accuracy of the method was determined by preparing different concentrations that is 50%, 100%, 150% in which the amount of marketed formulation was kept constant and the amount of pure drug was varied that is 5µg/ml, 10µg/ml, 15µg/ml for 50%, 100%and 150%. it is indicated by % recovery.

Table 4: Accuracy data of proposed method

Levels	Fixed conc.	Conc. Added	Combined area	Sample area	Area recovered	% recovery
50%	15	15	1997947	992702	1957988	98.7
	15	15	1999588	992702	1977592	101.1
	15	15	1999745	992702	1978389	100.4
100%	15	30	2924447	992702	2877655	98.4
	15	30	2935202	992702	2905849	99
	15	30	2922278	992702	2872599	98.3
150%	15	45	3750572	992702	3821832	101.9
	15	45	3796946	992702	3777662	99.5
	15	45	3739103	992702	3694233	98.8

Table 5 Robustness data [Change in the mobile phase composition and flow rate(±10%)]

Concentration (µg/ml)	Mobile phase composition	Peak area	Flow rate (ml/min.)	Peak area
75	Ammonium acetate buffer : Methanol (54:46)	4489474	0.9	4612251
75	Ammonium acetate buffer : Methanol (60:40)	4444862	1	4615087
75	Ammonium acetate buffer : Methanol (66:36)	4615081	1.1	4732409
Average		4516472.3		4653249
S.D		88262.73		68569.235
% RSD		1.9542405		1.4735776

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

The robustness of the proposed method was evaluated by injecting three replicates injections of 75µg/ml venlafaxine hydrochloride solution at varied flow rate and mobile phase ratio. Peak areas and retention time changes were studied.

Limit of detection:

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. The detection limit (L.O.D) may be expressed as:

L.O.D=3.3 σ/S

Where:

σ = Standard deviation of the response

S = Slope of the calibration curves the slope S may be estimated from

the calibration curve of the analyte.

L.O.D=3.3× 3489.78595/61980

= 185ng/ml

Limit of quantification:

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The Quantitation limit (QL) may be expressed as:

L.O.Q=10 σ /S

Where:

σ = Standard deviation of the response

S = Slope of the calibration curves, the slope S may be estimated from

the calibration curve of the analyte.

L.O.Q = 10×3489.78595/61980

= 563ng/ml

Assay:

Preparation of Sample Stock Solution (1000µg/ml):

10 tablets were accurately weighed and average weight of each tablet was calculated. Then the tablets were powered, the tablet powder equivalent to 10mg was taken in 10ml volumetric flask. Then 3/4^th of the diluent was added and sonicated for 10min, filtered and made up to the mark with diluent which produced 1000µg/ml venlafaxine hydrochloride solution.

Preparation of Sample Working stock Solutions (100µg/ml):

From the above filtered sample stock, 1ml of solution was pipette out and transferred to 10ml volumetric flask and the volume was made up to the mark with diluent which gave 100µg/ml of venlafaxine hydrochloride solution.

Preparation of Sample Working Solutions (75µg/ml):

From the above solution 7.5ml was pipetted and taken into 10ml volumetric flask and made up to the mark with the diluent. The amount of the drug present was calculated using following formula.

%Assay = (sample area/standard area)*100

= 4695087/4732409×100

=99.2%

Table 6: Summary of RP-HPLC Method

Parameters	Results
Diluent	HPLC grade water
Mobile phase	HPLC grade Ammonium acetate buffer: Methanol (60:40) adjusted pH 5 with glacialacetic acid
Calibration range(mcg/ml)	15-165µg/ml
Optimized wavelength	227nm
Retention time	2.004
Regression equation(y)	Y=61980x + 40900
Correlation coefficient(r²)	0.9997
Precision (%RSD)	0.000115
%recovery	99.5
Limit of detection	185 ng/ml
Limit of quantification	563 ng/ml

CONCLUSION:

The developed chromatographic method is simple, precise, accurate, linear, and reproducible for the estimation of venlafaxine hydrochloride without any interference from the excipients. It can be successfully applied for the routine analysis of venlafaxine hydrochloride in pharmaceutical dosage form.

CONFLICT OF INTEREST:

The authors declares that they have no conflict of interest.

ACKNOWLEDGEMENT:

I want to acknowledge our beloved principal Prof. M. Sumakanth RBVRR Women’s College of Pharmacy for giving me the opportunity for performing the research work.

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Received on 09.07.2021 Modified on 29.01.2022

Research J. Pharm. and Tech 2023; 16(2):524-528.

DOI: 10.52711/0974-360X.2023.00089