INTRODUCTION:

Chemically bupropion (Figure 1) is 2-(tert-butylamino)-1-(3-chlorophenyl) propan-1-one formerly known as amfebutamone which is an atypical antidepressant and smoking cessation aid. The drug is a non-tricyclic antidepressant and differs from most commonly prescribed antidepressants such as SSRIs. It also acts as a nicotinic acetylcholine receptor antagonist. Bupropion belongs to the chemical class of aminoketones and is similar in structure to stimulants cathinone and diethylpropion, and to phenethylamines in general.^[1]

Literature survey revealed that several chromatographic methods were reported for the estimation of bupropion hydrochloride in human plasma using its formulation.^[2-4] There exist reports on LC^[5-7], LC-MS-MS^[8-10], GC^[11-12], TLC^[13] and HPTLC^[14]for analysis of the drug. There also exists report on chiral separation of bupropion using a chiral AGP column.^[15] No method is published so far for the determination of bupropion in bulk drugs. The main purpose of the present work was to develop simple, rapid and accurate method which could be applied to analyze bupropion HCl in bulk form and to validate the method as per ICH (Q2R1) guideline.^[16]

MATERIALS AND METHODS:

Chemicals and Reagents:

Pure sample of bupropion hydrochloride was obtained as gratis from Wockhardt Ltd. (Aurangabad, India) and was used as without further purification. HPLC grade methanol was procured from Merck specialties Pvt. Ltd. (Mumbai, India). Analytical reagents (AR) grade ortho phosphoric acid and potassium dihydrogen phosphate were procured from Qualigens fine chemicals Pvt. Ltd. (Mumbai, India). Ultra pure water obtained from Millipore water purification system (Molsheim, France) was used throughout the study.

Equipments:

UV-visible spectrophotometer (PerkinElmer, Shelton, CT, USA) was used for the determination of detection wavelength. A high-performance liquid chromatography (HPLC) system from PerkinElmer (Shelton, CT, USA) was used for the analysis of samples, which consisted of online degasser, sample injector (Rheodyne sample loop 20 μL), UV-visible detector (Series 200), pump (Reciprocating, series 200) and computer system loaded with Total Chrome Navigator (version 6.3.1) software. Other equipment used were, pH meter (Labindia, Mumbai, India), weighing balance (Shimadzu, AUX220, Kyoto, Japan) and a micro-pipette (Erba Biohit, Mannheim, Germany). All the samples were analyzed by using Kromasil C-18 column 250mm x 4.6mm i.d., particle size 5μm; (Eka Chemicals AB, Bohus, Sweden).

Preparation of sample for HPLC analysis:

Stock solution of 1000 μg mL^-1was prepared by dissolving 10 mg bupropion HCl in 10 mL methanol. Sample solution was prepared by dissolving stock solution mobile phase as per the required concentrations.

Fig.1. Structure of Bupropion Hydrochloride

Selection of detection wavelength:

Appropriate dilutions of the bupropion HCl were prepared using water as diluent. Solution was scanned using double beam UV-visible spectrophotometer between the ranges of 400 to 200 nm. The detection wavelength was found to be 252 nm.

Preparation of buffer:

Potassium dihydrogen orthophosphate buffer (10 mM) was prepared by dissolving 1.36 gm of potassium dihydrogen ortho phosphate in 1000 ml of Milli-Q water and the pH was adjusted to 4.0 with ortho phosphoric acid.

HPLC method development and validation:

Detection wavelength for the drug was found to be 252 nm and hence methanol was used as an organic phase. UV spectra of bupropion HCl is shown in Figure 2. Various trials were conducted to achieve adequate peak symmetry and response factor by varying the concentration of organic phase and pH of the buffer system. Finally methanol and buffer (phosphate buffer 10 mM) with pH 4.0 was selected to analyze bupropion HCl samples by using gradient HPLC mode. The chromatogram of the drug is shown in Figure 3. The flow rate, injection volume and detection wavelength were 1 mL min^-1, 20 μL and 252 nm, respectively.

The developed method was validated with respect to various parameters outlined in the International Conference on Harmonization (ICH) guidelines Q2 (R1). A stock solution containing 1 mL min^-1 drug was prepared in MeOH and the linearity was established by using the concentration in the range of 20-120 μg mL^-1. The solutions were prepared in triplicate and analyzed by injecting 20 μL into HPLC. The intra-day and inter-day precision were established by analyzing 40 μg mL^-1, 60 μg mL^-1 and 80 μg mL^-1 drug solutions three times on the same day and the next day, respectively. Accuracy was determined by spiking three known concentrations of the drug, viz., 80%, 100% and 120% in a 10 μg mL^-1 of measured standard stock solution in triplicate and then determining the percent recovery of the added drug. Limit of detection and limit of quantitation were assessed by determining the signal to noise ratio of the injected concentration of the analyte. Robustness of the developed method was also determined by with respect to different parameters.

Fig.2: UV chromatogram of Bupropion Hydrochloride

RESULT AND DISCUSSION:

Method development and validation:

An acceptable separation was achieved by using buffer of pH 4.0 along with the organic phase in a gradient mode shown in Table 1. The developed method was validated with respect to linearity, precision, accuracy, LOD, LOQ and robustness.

Table 1: Gradient program of mobile phase

Steps	Time	Flow	%A	%B
0	0.0	1.0	30	70
1	5.0	1.0	50	50
2	7.0	1.0	70	30
3 4	2.0 5.0	1.0 1.0	30 30	70 70

Fig.3: HPLC chromatogram of Bupropion HCl

Method validation:

Linearity:

As shown in Figure 4, The response for the drug was found to be linear in the investigated concentration range. The values of slope and correlation coefficient (R²) were 79966 and 0.997, respectively. The linearity data are shown in Table 2.

Fig. 4: Linearity of Bupropion Hydrochloride

Table: 2 Linearity parameter of Bupropion Hydrochloride

Parameter	Bupropion Hydrochloride
Slope	81088
Correlation coefficient	0.9976

Precision:

Inter-day and intra-day precision data are enlisted in Table 3. The RSD values ranging from 1.52% to 1.71% for intra-day precision and from 1.09% to 1.70% for inter-day precision studies respectively, confirmed that the method was sufficiently precise.

Table: 3 Precision parameter of Bupropion Hydrochloride

Concentration (µg mL^-1)	Intra-day		Inter-day
Concentration (µg mL^-1)	Mean	%RSD	Mean	%RSD
40	3062875.94	1.67	3072722.94	1.70
60	4510928.48	1.52	4502759.11	1.29
80	6163267.00	1.71	6148113.78	1.09

Accuracy:

The accuracy was expressed in term of recovery. In accuracy the recovery studies was carried out by adding known amount drug with preanalyse sample solution and content was reanalyzed by the proposed method. The accuracy was carried out by injecting the bupropion HCl solution three time at three different concentration solution of 80%, 100%, 120% of the active ingredient by adding known amount of bupropion HCl standard to sample of known concentration. As shown in Table 4, the percent recovery ranged between 99.49% to 101.14%.

Table 4: Accuracy parameter of Bupropion Hydrochloride

Concentration (µg mL^-1)	Amount added (µg)	Amount found (µg)	% recovered	% RSD
100	80	79.59	99.49	1.68
100	100	101.14	101.14	1.43
100	120	118.16	98.48	1.96

Limit of detection (LOD) and Limit of quantitation (LOQ):

The LOD and LOQ was calculated with standard deviation of response and slope of calibration curve. The LOD and LOQ for bupropion HCl was found to be 4.50 ng mL^-1 and 8.50 ng mL^-1, respectively.

Robustness:

In robustness, method was checked by changing the chromatographic condition like flow rate (+0.1 and -0.1ml/min) and absorbance wavelength (+5 and -5nm). After changes it was observed that the peak symmetry and peak response was found to be adequate. The data obtained in robustness study are shown in Table 5, indicating that the test method was robust in variable condition.

Table: 5 Robustness parameter of Bupropion hydrochloride

Parameter change	Flow rate		Wavelength
Parameter change	0.9 mL min^-1	1.1 mL min^-1	+2nm	-2nm
Mean area	7859230.70	7844542.74	7879902.88	7865917.76
SD	110264.14	87206.47	86939.09	104021.59
% RSD	1.40	1.11	1.10	1.32

CONCLUSION:

The developed method was found to be rapid, accurate, precise and reproducible. The method was linear over a wide concentration range, economical and utilizes a mobile phase which can be easily prepared. All these factors make this method suitable for the estimation of bupropion HCl. The developed method can be used for the routine analysis and assay of bupropion HCl in quality control laboratories.

REFERENCES:

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16. International conference on harmonization (1996) Validation of analytical procedures: text and methodologies.

Received on 05.05.2011 Modified on 06.06.2011

Research J. Pharm. and Tech. 4(9): Sept.2011; Page 1480-1482