Simultaneous estimation of naltrexone and bupropion in pharmaceutical dosage form by using UV spectroscopy

A sensitive and validated method has been developed for simultaneous estimation of Naltrexone and Bupropion in pharmaceutical dosage form by using UV Spectroscopy, without prior separation, by four different techniques (Simultaneous Equation, Absorbance Ratio method, Dual Wavelength Method and Derivative Spectroscopic Method). The work was carried out on Shimadzu electron UV1800 double beam UV-Visible spectrophotometer. The absorption spectra of reference and test solutions were carried out in 1 cm matched quartz cell over the range of 200 400 nm. The linearity ranges for Naltrexone and Bupropion were 2-10 μg/ml. The results of the analysis have been validated statistically and by recovery studies. The proposed procedures are rapid, simple, require no preliminary separation steps and can be used for routine analysis of both drugs in quality control laboratories.

Chemically, Bupropion (Figure 1) is 2-(tert-butylamino)-1- (3-chlorophenyl) propan-1-one [2]. It is a norepinephrine/dopamine-reuptake inhibitor. It is used most commonly for the management of Major Depressive Disorder, Seasonal Affective Disorder and as an aid for smoking cessation. Thus, the two drugs have effects on two separate areas of the brain involved in the regulation of food intake: the hypothalamus (appetite regulatory center) and the mesolimbic dopamine circuit (reward system) and combinational intake of these two medicines helps in chronic weight management [3].
The aim of this paper was to explore the possibility of using techniques of simultaneous equation method, dual wavelength method, isosbestic point method and derivative spectroscopic method for quantifying Naltrexone and Bupropion simultaneously in their mixture forms. The proposed methods are simple, convenient, precise, accurate, and economical than the reported method and validated as per ICH guidelines [7].

Instrumentation
To develop a UV spectroscopic method for simultaneous estimation of Naltrexone and Bupropion, Shimadzu electron UV1800 double beam UV-Visible spectrophotometer was used. The instrument is equipped with Silicon photodiode detector.

Chemicals and solvents
The working standards of Naltrexone and Bupropion were provided as gift samples from Spectrum Pharma Research Solutions, Hyderabad, India. Combined Naltrexone and Bupropion tablets were purchased from Online International Pharmacy Market.

Preparation of standard stock and working standard solutions of naltrexone
100 mg of naltrexone was weighed in to 100 ml volumetric flask and dissolved in distilled water and then dilute up to the mark with distilled water to get a concentration of 1000 µg/ml. The solution was diluted accordingly to get a concentration of 100 µg/ml and was kept as the stock solution. The prepared stock solution was diluted with distilled water solution to get working standard solutions of concentrations 2-10 µg/ml.

Preparation of standard stock and working standard solutions of bupropion
100 mg of Bupropion was weighed and transferred in to 100 ml volumetric flask and dissolved in distilled water and then make up to the mark with distilled water to get a concentration of 1000 µg/ml. The solution was diluted accordingly to get a concentration of 100 µg/ml and was kept as the stock solution. The prepared stock solution was diluted with distilled water solution to get working standard solutions of concentrations 2-10 µg/ml.

Simultaneous equation method (Method-I)
For multi-component system consisting of two components X and Y, each of which absorbs at the λmax of the other, λ 1 is being the wavelength of maximum absorbance of X (λmax) and λ 2 being the wavelength of maximum absorbance of Y (λmax). In such cases, it can be possible to determine both the components by simultaneous equation method. Standard stock solutions (1 mg/ml) of Naltrexone and Bupropion were prepared by dissolving 25 mg of each in 25 ml distilled water, which was further diluted with distilled water to get the working standard solution (100 μg/ml) of Naltrexone and Bupropion. From this, suitable aliquots are taken and diluted with distilled water to get 10 μg/ml of Naltrexone and Bupropion. The absorption spectra of all the solutions were recorded between 200 and 400 nm. The absorbances were measured for Naltrexone and Bupropion at 204.2 nm (λ1) (maximum absorbance of Naltrexone) and 210 nm (λ2) (maximum absorbance of Bupropion), respectively. Wavelengths 204.2 nm and 210 nm were selected for the formation of simultaneous equation (Figure 2). The absorbances were measured at the selected wavelengths. Marketed formulation of Naltrexone and Bupropion were procured. The absorbance of final sample solution was measured against distilled water as blank at 204.2 nm and 210 nm for quantisation of Naltrexone and Bupropion, respectively.
The amount of Naltrexone and Bupropion present in the sample solutions were determined by solving the following simultaneous equations.

Figure 2
Overlain spectra of Naltrexone and Bupropion

Dual Wavelength Method
The utility of dual wavelength data processing programme is to calculate the unknown concentration of a component of interest present in a mixture containing both the component of interest and an unwanted interfering component by the principle of difference in the absorbance between two points on the mixture spectrum. This is directly proportional to the concentration of the component of interest, independent of the interfering components. The prerequisite for dual wavelength method is the selection of two such wavelengths where the interfering component shows same absorbance whereas the component of interest shows significant difference in absorbance with concentration. Based on this criterion, two wavelengths 233.4 nm and 264 nm were selected as λ1 and λ2 for the estimation of Naltrexone as Bupropion shows the same absorbance at these wavelengths. Similarly, wavelengths 257 nm and 283.4 nm were selected as λ3 and λ4 for the estimation of Bupropion as Naltrexone shows the same absorbance at these wavelengths.
For calibration curve, the standard stock solutions of these drugs were diluted in the concentration range of 2-10 µg/ml (2, 4, 6, 8 and 10 µg/ml) for Naltrexone and Bupropion. Absorbances were recorded at selected wavelengths. Absorbances were recorded at selected wavelengths.

Absorbance ratio method (Q-analysis method)
The absorbance ratio method is a modification of the simultaneous equation procedure. It depends on the property that for a substance, which obeys Beer's law at all wavelength, the ratio of absorbance at any two wavelengths is constant value independent of concentration or path length e.g. two dilutions of the same substance give the same absorbance ratio A1/A2. In the USP, this ratio is referred to as Q value. In the quantitative assay of two components in mixture by the absorbance ratio method, absorbance is measured at two wavelengths, one being the λmax of one of the components (λ2) and the other being a wavelength of equal absorptivity of the two components (λ1), i.e., an iso-absorptive point. Qm= A2 / A1 Qx = ax2 / ax1 and Qy= ay2 /ay1

Derivative Spectroscopic Method
Derivative spectrophotometry involves the conversion of a normal spectrum (fundamental, zero th order or D spectrum) to its first, second or higher derivative spectrum by differentiating absorbance of a sample with respect to wavelength λ for higher accuracy.
[ The amplitude (D) is directly proportional to the concentration of analyte provided Beer's law is obeyed by D° spectrum.
In first order derivative spectroscopy, zero crossing point for both drugs is found and the wavelengths are selected in a manner such that at the zero crossing of one drug, the other drug should show substantial absorbance.
The obtained zero order absorption spectra of Naltrexone and Bupropion were converted to first order derivative spectra ( Figure 3) by using transformation mode. After observing the first order derivative spectra, zero crossing points of drugs were selected for the analysis of other drugs. The first wavelength selected was 264 nm for Naltrexone (zero crossing) and second wavelength selected was 310 nm for Bupropion (zero crossing).

Figure 3 First derivative overlay spectrum of Naltrexone and Bupropion
Calibration curve for Naltrexone at 310 nm (zero Crossing for Bupropion) and Bupropion at 264 nm (zero Crossing for Naltrexone) was developed to calculate the concentration of sample.

Linearity
A stock solutions were prepared by dissolving 50 mg of the drugs in 50 ml of mobile phase. Then from these stock solutions dilutions of various concentration from 2 to 10 μg/ml were prepared for Naltrexone and Bupropion respectively. Each dilution was analysed in series to construct the calibration curves. Absorbance of each dilution was noted and plotted against the concentration of each dilution.

Accuracy
Accuracy was determined by calculating %recovery of Naltrexone and Bupropion by standard addition method. The pre-analyzed sample solutions (4 μg/ml of Naltrexone and Bupropion) were spiked with standard drug solutions at three different levels-50, 100 and 150 %. The resulting mixtures were reanalyzed using the proposed method. The experiment was conducted in triplicates accuracy was reported as % recovery.

Precision
Precision of the proposed method was calculated by conducting intermediate precision.

Intra-Day Precision
The intra-day precision was determined by estimating the corresponding absorbance of the drug solution (in triplicates) three times on the same day.

Inter-Day Precision
The inter-day precision was established by analysing the drug solution (in triplicates) on three different days.

Analyst-Analyst
The analyst to analyst precision was established by analysing the drug solution by different analyst.
The standard deviation, %relative standard deviation and estimated concentrations based on standard curve were reported for each set of data.

Robustness
Robustness of the developed method was determined by analysing the drug solution in triplicates by varying the wavelength (±2). Robustness is reported in %RSD.

LOD and LOQ
Detection limit and Quantitation limit of the drug is calculated by using the calibration curve standards. Detection limit and Quantitation limit were calculated from the equation 3.3σ/S and 10σ/S respectively, where σ is the standard deviation of y-intercept and S is the slope of the calibration curve.

Specificity
The specificity of the developed method was seen by analyzing solutions containing excipients and pure drug and demonstrating that the result is unaffected by the presence of the excipients present in it.

Linearity
The calibration curves (Figure 4) drawn by using the proposed method were found to be linear in the range (2-10 μg/ml Naltrexone and Bupropion). Table 1 shows the calibration data with regression coefficient and %RSD was found to be less than 2.

Figure 4 Calibration curves of analytes
Calibration curves of A. Naltrexone and B. Bupropion

Accuracy (recovery)
Accuracy of the methods was determined by using standard addition method and %recovery was found in the range 96-101% and %RSD was within the range for both drugs. Table 1 shows the results of accuracy studies.

Limit of Detection and Limit of Quantification
LOD and LOQ were calculated from the calibration curves of the drugs itself and the results are shows in Table 1.

Assay
%Assay of the formulation was calculated by three different techniques and %recovery was found in the range 96-101% and %RSD was within the range for both drugs. Table 1 shows the results of %assay studies.

Precision
Intra-day precision was assessed by analyzing the drug at 3 different times in the same day. The method passed the test as the %RSD was found to be less than 2. Inter-day precision was assessed by analyzing the drug for three different days. The method passed the test as the %RSD was found to be less than 2. Analyst to analyst Precision was conducted by two different analysts at the same experimental conditions. Results are shown in Table 1.

Robustness
Robustness studies were performed by varying the detection wavelength (±2). The method was found to be robust. Results are shown in Table 2.

Result of specificity
Specificity studies were performed by spiking the formulation and standard drug using two tailed unpaired t-test.

Conclusion
The proposed methods based on simultaneous equation, dual wavelength, absorption ratio and first derivative methods can be used for the simultaneous estimation of Naltrexone and Bupropion in their bulk and pharmaceutical dosage form. The proposed methods are accurate, reproducible, repeatable, linear, precise, selective, reliable and simple to perform. Also, no separation step is required. These results indicate that the proposed method may find practical applications as a quality-control tool in the simultaneous analysis of the two drugs in combined dosage forms in quality-control laboratories.