New chiral normal phase UFLC method for determination of venlafaxine hydrochloride enantiomers in pharmaceutical formulations
ABSTRACT
Aim: A simple, specific, precise, sensitive and rapid normal phase-UFLC method was developed for determination of venlafaxine hydrochloride enantiomers in pharmaceutical formulation.
Methodology: The method was developed on a Lux amylase 2 column (150 x 4.0 mm I.D., particle size 5 μ); the mobile phase was n-hexane and ethanol (97:3 v/v); in 0.1%diethyamine using UV detector was fixed at 254 nm with a flow rate was 1 mL/min.
Results: The retention time (tR) of R- venlafaxine hydrochloride and S- venlafaxine hydrochloride were found to be 4.5±0.2 min and 5.3±0.3 min, respectively. The linearity over the concentration range of 5-30 µg mL-1 for venlafaxine. The intra-day and inter-day coefficient of variation of the assay method were found to be 0.293 to 1.760 and 0.319 to 0.210 respectively, with high accuracy and precision results. The proposed NP-UFLC method is suitable for analysis of venlafaxine hydrochloride enantiomers in pharmaceutical dosage forms.
Conclusion: The validated NP-UFLC method was developed for the quantification of venlafaxine in tablet dosage form.
Keywords: R-venlafaxine hydrochloride, S-venlafaxine hydrochloride, enantiomers, NP-UFLC, Validation
INTRODUCTION
Venlafaxine is a second-generation antidepressant drug marketed as a racemic mixture (Figure 1). The R-enantiomer exhibits dual presynaptic inhibition of serotonin and noradrenaline uptake, whereas the S-enantiomer is a serotonine reuptake inhibitor. Thus, the drug is the first and most commonly used serotonin and noradrenaline reuptake inhibitor. Its synthesis and that of several analogues were described many years ago. The synthetic routes are similar and vary according to the nature of the aromatic substituents. However, the final products are racemic mixtures, and they were crystallized as hydrochlorides.[1] Although the disposition of venlafaxine in humans was originally found not to be stereoselective.[2] In view of the near expiration date (June 2008) of the first patent for the racemic compound and of these recent clinical findings, venlafaxine appears to be a good candidate for a chiral switch.[3-4]
Figure 1: Molecular structure of Venlafaxine
The trend toward single enantiomer drugs is clear and the number of racemic drugs that reach the market as new chemical entities is decreasing.[5] The relevance of chirality in antidepressant drugs was highlighted several years ago and many examples are illustrated in a recent very complete review.[6-7] In the previously cited research on the resolution of venlafaxine, the enantiomers were separated by either of two general approaches. The first is the classical method of diastereoisomeric salt formation and fractional crystallization and the second approach uses analytical enantioselective electro driven methods. In the latter cases, either cyclodextrinsin capillary electrophoresis.[8] There is only one literature report where an HPLC baseline separation of the enantiomers of venlafaxine extracted was achieved using a CSP and normalphase mode.[9] From an analytical point of view, enantioselective chromatography offers the advantages of a method that can be developed on a semipreparative or preparative scale for the isolation of single enantiomers, which then become available for pharmaceutical testing strategies and requirements for enantioselective.[10]
In the present research work, a simple, sensitive and accurate normal phase UFLC method to separate R and S-enantiomer of venlafaxine in bulk drugs and tablets using Lux amylase 2 column column has been reported for first time. The method was also validated to ensure the compliance in accordance with the ICH guidelines.
MATERIALS AND METHODS
Chemicals and Reagents:
Venlafaxine hydrochloride enantiomers were a gift sample from R N FINE CHEMICALS BANGALURU, India. The solvents like n-hexane and ethanol diethylamine used was of HPLC grade (Merck, India). Commercially available racemic venlafaxine hydrochloride tablets claimed to contain 25mg of drug were procured from local market.
Instrumentation:
Quantitative NP-UFLC was performed on gradient high pressure liquid chromatography (Shimadzu) auto sampler consisting of a LC–20HT solvent module, SPD– 10A, and an PDA detector with LC software. The column used was LUX amylase 2 chiral column(150 x 4.0 mm ) particle size 5 μ.
UFLC conditions:
The composition of the mobile phase was n-hexane and ethanol in the ratio of 97:03 v/v. They were filtered before use through a 0.2 mm membrane filter, degassed in a bath sonicator for 10 min. The mobile phase was pumped from the solvent reservoir to the column at a flow rate of 1mL/min, which yielded a column backpressure of 96 kg/cm2. The run time was set at 20 min and column temperature was ambient. The volume of injection loop was 20 mL. prior to injection of drug solutions, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. The eluents were monitored at 254 nm and data was acquired, stored and analyzed with the LC 10 software.
REAGENTS USED
Mobile phase
n-hexane and ethanol of HPLC grade was taken as mobile phase in the ratio of 97:3 % (v/v).
Preparation of standard stock solution
Standard stock solution (100 µg mL-1) of Venlafaxine hydrochloride was prepared by weighing exactly 10 mg of drug dissolved in isopropanol and diluted to 100 mL with same solvent.
Preparation of calibration curve
Aliquots of Venlafaxine hydrochloride ranging from 0.5-3 mL (each mL contains 100 µg mL-1) were pipetted into as a series of 10mL volumetric flasks. The volume was made up to the mark at with isopropanol. Aliquoets of 10µL was injected (six time) into HPLC. The elution of the drug measured at 254.0 nm. The amount of venlafaxine hydrochloride present in the sample solution was computed from its calibration curve and it was constructed by plotting peak area of chromatogram against the concentration of Venlafaxine hydrochloride. The blank chromatogram and standard drug chromatogram were shown in figure 2 and 3 respectively. Linearity was 5.0-30 µg mL-1 for Venlafaxine hydrochloride was shown in figure 4.
Figure 2: Blank chromatogram
Figure 3: Standard Chromatogram of venlafaxine enantiomer
Figure 4: Calibration curve of venlafaxine hydrochloride
ANALYSIS OF TABLET DOSAGE FORM
Five tablets (EFFEROX), each containing 25 mg of venlafaxine hydrochloride were weighed and finely powdered. Powder equivalent to 125 mg of venlafaxine hydrochloride was weighed and transferred to a standard volumetric flask. The contents were mixed thoroughly and filtered through a 0.45 μm membrane filter. 10 μL of the sample was injected in to UFLC system for the analysis. The peak profile and peak purity of both enantiomers are shown in Fig. 5, 6, 7 and 8.
Figure 5: Peak Profile Enantiomer 1
Figure 6: Peak Profile Enantiomer 2
Figure 7: Peak Purity Enantiomer 1
Figure 8: Peak Purity Enantiomer 2
RESULTS AND DISCUSSION
Validation of the method
The developed method for the assay of venlafaxine has been validated as per the current ICH Q2 (R1) guidelines.[11]
Analytical parameters
The development of NP-UFLC method for the determination of enantiomers has received a considerable attention in recent past because of its importance in the quality control of drugs and drug products. The assay of venlafaxine hydrochloride enantiomers was resolved with good accuracy. The retention time (tR) of R- venlafaxine hydrochloride and S- venlafaxine hydrochloride were found to be 4.5±0.2 min and 5.3±0.3 min, respectively. A typical chromatogram of R-Venlafaxine hydrochloride and S- venlafaxine hydrochloride is shown in Figure 3. Tailing factor for both R-venlafaxine hydrochloride and S-venlafaxine hydrochloride was found to be 1.1 and 0.8 respectively. The calibration curve was constructed by plotting the peak areas against the concentration of R-and S-venlafaxine hydrochloride in 5-30 µg mL-1 were shown in the Figure 4. It was found to be linear with a correlation coefficient of 0.9971 for R-venlafaxine hydrochloride and 0.9992 for S-venlafaxine hydrochloride, the representative linear regression equation being y = 10507X +2467.1 and y = 10654X +2065.8 for both the enantiomers respectively. The slope, y-intercept, and their standard deviations evaluated are presented in Table 1.
Table 1: Regression and sensitivity parameters of enantiomer-1 and enantiomer-2
Parameters
Enantiomer-1
Enantiomer-2
Retention time
4.5±0.2
5.3±0.3
Plate count
9175
6894
Tailing factor
1.1
0.8
Resolution
—
3.664
Beer’s range (µg mL-1)
5-30
5-30
Slope (m)
10470
10629
Y Intercept
2636
2382
Correlation coefficient (r2)
0.997
0.999
LOD (µg mL-1)
0.06
0.05
LOQ (µg mL-1)
0.20
0.197
Accuracy and precision
The amount of venlafaxine hydrochloride enantiomers in the matrix was calculated using following formula.
% Recovery = T-A /S×100
T–total amount of drug estimated, A-initial amount of drug in the tablet powder and S- amount of pure drug added. The results revealed (Table 2), high recovery of Venlafaxine hydrochloride enantiomers, indicating that the proposed method for the determination of venlafaxine hydrochloride enantiomers in the tablet is highly accurate. The intraday and inter day percentage relative standard deviation values were shown in Table 3. These values were within the standard limits.
Table 2: Accuracy data of enantiomer-1 and enantiomer-2
Sample
Amount added
(µg mL-1)
Amount found
(µg mL-1)
%Recovery
%RSD
Enantiomer-1
5
4.941
98.82
1.149
10
10.096
100.96
0.405
15
15.062
100.41
0.193
Enantiomer-2
5
5.080
101.6
1.423
10
10.09
100.9
1.983
15
14.931
99.54
0.652
Mean value of six determinations
Table 3: Precision data of enantiomer-1 and enantiomer-2
Theoretical concentration
(µg mL-1)
Intraday(n=6)
Found (µg mL-1)
%RSD
Inter day(n=6)
Found(µg mL-1)
%RSD
Enantiomer-1
5
5.008
1.003
5.052
0.276
10
10.047
0.293
10.059
1.766
15
15.092
0.119
15.092
0.122
Enantiomer-2
5
5.064
1.524
5.078
1.002
10
10.078
0.319
10.020
0.210
15
15.092
0.119
15.092
0.122
Limit of detection and limit of quantification
Limit of detection can be calculated using the following equation according to ICH guidelines: LOD = 3.3 x N/S
LOQ = 10 x N/S
where N is the standard deviation of peak areas of the drug and S is the slope of the corresponding calibration curve. The results are shown in Table 1.
Assay of the drug
The chiral NP-HPLC method developed in the present investigation was used to quantify venlafaxine hydrochloride enantiomers in tablet dosage forms. The obtained results are given in Tables 4. The average drug content was found to be 10.047 mg for R-venlafaxine hydrochloride and 9.978 mg for S-venlafaxine hydrochloride of the labelled amount in 25mg of racemic venlafaxine hydrochloride, respectively.
Table 4: Assay of Venlafaxine
Sample
Injected (µg mL-1)
Amount found (µg mL-1)
% Amount found
%RSD
Enantiomer-1
(EFFEROX 25mg tablet)
10
10.047
100.47
0.286
Enantiomer-2
(EFFEROX 25mg tablet)
10
9.978
99.78
0.329
Robustness of the method and stability of the solution
The robustness of an analytical procedure has been defined by the ICH as a “measure of its capacity to remain unaffected by small, but deliberate variations in method parameters. The most important aspect of robustness is to develop methods that develop methods that allow for expected variations in method parameters. According to ICH guidelines, robustness should be considered early in the development stage of a method. The typical variations studied under this parameter are flow rate, wavelength and mobile phase composition. The results are tabulated in Table 5.
Table 5: Robustness data of enantiomer-1 and enantiomer-2
Parameters
%RSD
Flow Rate
0.98 mL/min
1.02 mL/min
1.45
Wavelength
250
258
0.838
Mobile phase composition
97:3
99:1
0.914
CONCLUSION
A simple, rapid and normal phase chiral UFLC method has been developed and validated for the enantiomeric separation of venlafaxine in tablet formulation. This method is precise, accurate, robust, and specific. Satisfactory results were obtained from the validation of the method. The short retention time (4.5 min for enantiomer 1 and 5.3 for enantiomer 2) obtained provides rapid determination of venlafaxine, which is significant for its routine analysis in quality control. The method exhibits an excellent performance in terms of sensitivity and robust. The experimental results of the present study showed that the proposed NP-UFLC method is simple, specific, precise, sensitive, rapid and accurate and is useful for separation of venlafaxine hydrochloride enantiomers in its pharmaceutical formulation.
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