Analytical Method Development and Validation for the Simultaneous Estimation of Desvenlafaxine Succinate and Clonazepam in Bulk and Tablet Dosage Form by RP –HPLC Method

Abstract

A simple, precise, accurate, and robust reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantitative estimation of Desvenlafaxine succinate and Clonazepam in bulk drug and pharmaceutical dosage forms. Chromatographic separation was achieved on a C18 column using a mobile phase consisting of buffer, methanol and acetonitrile in the ratio of 30:20:50, at a flow rate of 1.0 mL/min. Detection was carried out at a wavelength of 222 nm and the retention times of Desvenlafaxine succinate and Clonazepam was found to be 2.510 min and 4.397 min respectively. The method was validated according to ICH Q2(R1) guidelines for parameters such as linearity, accuracy, precision, specificity, robustness, and system suitability. Linearity was observed in the concentration range of 60-140 µg/mL for Desvenlafaxine succinate and 0.6-1.4 µg/mL for Clonazepam, with a correlation coefficient (R²) of 0.999 for both the drugs. The percentage recovery ranged between 98–102%, indicating the accuracy of the method. The precision studies showed %RSD values less than 2%, confirming the reproducibility. The method was found to be specific for Desvenlafaxine succinate and Clonazepam with no interference from formulation excipients. This validated method can be successfully applied for routine quality control analysis of Desvenlafaxine succinate and Clonazepam in bulk and pharmaceutical formulations.

Keywords

Introduction

Desvenlafaxine is synthetically 4-[2-(dimethylamino)-1-(1-hydroxycyclohexyl)ethyl] phenol butanedioic acid hydrate. Desvenlafaxine (O-desmethylvenlafaxine) is the 0-demetyhlated active metabolite of venlafaxine. Desvenlafaxine is also an antidepressant belonging to the class of serotonin-norepinephrine reuptake inhibitor (SNRI) class. It was approved by the FDA in 2008 for the treatment of adults with major depressive disorder. It works by increasing the amount of natural chemicals called serotonin and norepinephrine in the brain (1-16).

Figure 1: Chemical structure of Desvenlafaxine

Clonazepam is a long-acting benzodiazepine used to treat panic disorders, severe anxiety, and seizures. A benzodiazepine used to treat various seizures, including myotonic or atonic seizures, photosensitive epilepsy.  It acts by binding to the benzodiazepine site of the GABA receptors, which enhances the electric effect of GABA binding on neurons, resulting in an increased influx of chloride ions into the neurons. This further result in an inhibition of synaptic transmission across the central nervous system (17-18).

 

Figure 2: Chemical structure of Clonazepam

MATERIALS AND METHODS

Acetonitrile HPLC grade, potassium dihydrogen orthophosphate and orthophosphoric acid AR grade were procured from Qualigens fine chemicals, Mumbai. Water HPLC grade was procured from S. D. Fine Chemicals. Reference standard of Desvenlafaxine and Clonazepam were procured from Alembic Pharmaceuticals Ltd., Gujrat, India and Welfeed Remedies Pvt Ltd Ahmedabad, India (1-14). Chromatographic separation was performed on a Shimadzu liquid chromatographic system equipped with a LC-10 VP solvent delivery system (Pump), Shimadzu UV-Visible Spectrophotometer, Rheodyne injector with 20 µl loop volume. Spinchrom software was applied for data collecting and processing. A Zodiac C18 (4.6 x 250 mm, 5μ) column was used for the separation. The mobile phase was a mixture of Buffer: Methanol: Acetonitrile (30:20:50 v/v/v). It was filtered through a 0.45 µ membrane filter and degassed. A flow rate of 1.0 ml/min and a detection wavelength of 222 nm were used.

Preparation of standard stock solution: An accurately weighed quantity of Desvenlafaxine succinate (50 mg) and Clonazepam (0.5 mg) were transferred into a separate 50 ml volumetric flasks and dissolved and diluted to the mark with Mobile phase to obtain standard solutions having concentration of Desvenlafaxine succinate (1000μg/ml) and Clonazepam (10μg/ml). The 100 percent mixed standard solution of Desvenlafaxine succinate and Clonazepam was prepared by transferring 5 ml of Desvenlafaxine succinate (1000 µg/ml) and 1 ml of Clonazepam (10 µg/ml) to the 50 ml volumetric flasks and made up to the mark with mobile phase to get 100 µg/ml of Desvenlafaxine succinate and 1µg/ml Clonazepam.

Preparation of the sample solution: 20 Tablet contents were weighed and triturated to fine powder. Accurately weighed and transferred the quantity of powder equivalent to 50 mg Desvenlafaxine succinate and 0.5 mg Clonazepam into a 50 ml volumetric flask. To that 10 ml mobile phase is added to dissolve and then the volume is making up with mobile phase. Then it is filtered with 0.45 micron filter and it is sonicated. From this stock solution 5 ml is pipette out and transferred to 50 ml volumetric flask and volume is makeup to 50 ml to prepare 100μg/ml and 1μg/ml final mixed concentrations of Desvenlafaxine succinate and Clonazepam respectively.

Preparation of buffer: 4.6g of potassium dihydrogen phosphate was accurately weighed and dissolved in 1000ml of distilled water. The pH of the buffer was adjusted to 3.0 with ortho phosphoric acid.

RESULTS AND DISCUSSION

Method Validation: Validation parameters includes specificity, linearity, range, accuracy, precision, repeatability, intermediate precision, limit of detection, limit of quantification, robustness.

Specificity: Specificity is the ability to assessing equivocally the analyte in the presence of components which may be expected to be present. Typically these components include impurities, degradants, matrix etc. Blank solution and standard solutions of Desvenlafaxine succinate (100μg/ml) and Clonazepam (1μg/ ml) were injected into the HPLC system. The peak purity data of Desvenlafaxine succinate and Clonazepam were compared. There should not be any interference at the retention time of the main peaks.

Linearity: Linearity for the drugs Desvenlafaxine succinate and Clonazepam was determined by preparing the standard solutions at five concentrations levels in the range of 60-140μg/ml for Desvenlafaxine succinate and 0.6-1.4μg/ml for Clonazepam from stock solution. The linearity charts of Desvenlafaxine succinate and Clonazepam was shown in the figure no 5&6. The correlation coefficient of Desvenlafaxine succinate and Clonazepam was found to be 0.999 and 0.998 respectively. Linearity results were tabulated in table 2 (19-23).

Accuracy: Accuracy was performed by spiking known amounts of standard solution to sample solution at three different concentrations levels (85%, 105%, 125%) and there by analyzed for  %RSD which should not be more than 2.0.The % recovery was calculated and the results was reported in table no. 3 & 4.

Precision: The precision of the analytical method was studied by injecting six replicates of standard containing 100μg/ml of Desvenlafaxine succinate and 1μg/ml of Clonazepam which were injected into HPLC system. The % RSD was calculated and the results were reported in the table no.5.

Limit of Detection (LOD) and Limit of Quantification (LOQ) (24-28):

The limit of detection was defined as the concentration which yields a signal - to – noise ratio 3:1 where as the limit of quantification was calculated to be the lowest concentration that could be measured with signal - to – noise ratio10:1. LOD and LOQ were calculated from slope and standard deviation. The results were tabulated in table no. 6.

Robustness: The smallest deliberate changes in method like change in flow rate and change in wavelength are made but there were no predictable changes in the results and are in the range as per ICH guidelines. Conditions like decrease in flow rate (0.8 ml/min), increase in flow rate (1.2 ml/min), decrease in wavelength (220nm) and increase in wavelength (224nm) were maintained and sample were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. % RSD was found to be within the limits and results were tabulated in table no. 7 & 8.

Assay: Assay was conducted on marketed formulation and mean % assay was found. The results were tabulated in table no. 9.

Figure 3: Overlay spectra of Desvenlafaxine Succinate and Clonazepam (Isobestic point at 222nm)

Table 1: Chromatographic conditions for optimized method

Equipment	High performance liquid chromatography equipped with UV detector
Column	Zodiac C18 (4.6 x 250 mm, 5μ)
Mobile phase	Buffer: Methanol: Acetonitrile (30:20:50 v/v/v)
Flow rate	1.0 ml/ min
Wavelength	222 nm
Injection volume	20 ml
Run time	6 min
Retention time	Desvenlafaxine succinate - 2.510min, Clonazepam - 4.397min

Figure 4: Optimized Chromatogram

Table 2: Linearity results of Desvenlafaxine Succinate and Clonazepam

Desvenlafaxine succinate		Clonazepam
Conc (µg/ml)	Peak area	Conc(µg/ml)	Peak area
60	1345.991	0.6	117.759
80	1843.264	0.8	157.325
100	2232.682	1	195.218
120	2697.195	1.2	227.674
140	3106.06	1.4	259.795

Figure 5: Calibration curve of Desvenlafaxine succinate

Figure 6: Calibration curve of Clonazepam

CONCLUSION                    

The developed RP-HPLC method for the simultaneous estimation of Desvenlafaxine succinate and Clonazepam in combined dosage forms is accurate, precise, linear, robust, simple and rapid. Hence the present RP-HPLC method is suitable for the quality control of the raw materials and formulations.

ACKNOWLEDGEMENTS: Authors are thankful to the management of Viswanadha Institute of Pharmaceutical Sciences (VNIPS) for providing facilities and support to carry out this work.

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