Research Article

Received: 10 April 2013, Accepted: 26 April 2013

ISSN: 2321-2969

Int. J. Pharm. Biosci. Technol.

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International Journal of Pharma Bioscience and Technology; Volume 1, Issue 1, May 2013, Pg 20-26

Journal home page: www.ijpbst.com

BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF BUPROPION HYDROCHLORIDE IN RAT PLASMA BY RP-HPLC
Dhaval S. Thakar, Alice Varghese*
Shobhaben Pratapbhai Patel, School of Pharmacy & Technology Management, SVKMs NMIMS, Vile Parle (W), Mumbai-400056, India. Corresponding Author* E-mail address- alice.varghese@nmims.edu

ABSTRACT: A novel, rapid, sensitive, accurate and specific HPLC assay with UV-Visible detection (250 nm) was developed and validated for the determination of bupropion hydrochloride in rat plasma. Phenacetin was used as internal standard (IS). The plasma proteins were precipitated by a single step protein precipitation extraction procedure using methanol (acidic pH). Chromatographic separation was achieved with a combination of acetonitrile and 0.01 M potassium dihydrogen phosphate (pH 3.0 adjusted with orthophosphoric acid) in the gradient mode on a C18 (250 mm 4.6 mm, 5 m) analytical column. Mean recovery of bupropion hydrochloride from rat plasma was around 55 % for 2.5-50 g/ml concentrations. The assay exhibited good linear relationship with an r2 of 0.9999. Lower limit of Quantification limit (LLOQ) was 1.84 g/ml of bupropion hydrochloride and accuracy and precision were over the concentration range 2.5-50 g/ml. The method was validated with good sensitivity, accuracy, precision and recovery. The assay can be applied successfully to pharmacokinetic studies. Key words: Bupropion HCl, HPLC, Bioanalysis, Rat plasma, UV detection. INTRODUCTION Bupropion was first patented in 1974[1] and released onto the world market in 1985. It was briefly withdrawn due to seizures incidences but reintroduced in 1989 after the daily recommended dose was reduced to lower seizure likelihood. Bupropion is a dopamine and norepinephrine reuptake inhibitor [2].It is about twice as potent an inhibitor of dopamine reuptake than of norepinephrine reuptake. Besides reuptake inhibition of dopamine and noradrenaline, bupropion also causes the release of dopamine and noradrenaline [3]. Bupropion has numerous therapeutic indications including, depression[4], smoking cessation[5], sexual dysfunction[6], obesity[7], attention deficit hyperactivity disorder[8] and seasonal affective disorder[9]. It has recently been shown to have antiinflammatory properties [10]. In 2007 it was the fourth-most prescribed antidepressant in the USA. Bupropion is the water soluble hydrochloride salt of an aminoketone[11], with a pKa of 7.9[12]. It is Varghese et al also known with the generic name of amfebutamone hydrochloride. Bupropion is a second-generation antidepressant agent that is also used in the management of smoking cessation [13]. CYP2B6 is a polymorphic hepatic enzyme[14]of potential importance in the metabolism of drugs such as Bupropion[15], efavirenz[16] and cyclophosphamide [17]. Wide interindividual variability in the hepatic expression of CYP2B6 has been reported.[18, 19] In humans, bupropion is extensively metabolized to three principal metabolites (Fig.1.) such as hydroxyl-bupropion or morphinol, erythrohydrobupropion, and threo-hydro-bupropion. The pharmacologically active metabolite hydroxyl-bupropion appears to be the major metabolite, since the plasma levels of hydroxybupropion greatly exceeds with respect to those of the parent drug. The cytochrome P450 (CYP) enzyme system, especially CYP2B6, has an important role in bupropion hydroxylation. Also Pg. 20

Int. J. Pharm. Biosci. Technol. product labeling have indicated that bupropion or hydroxybupropion inhibits CYP2D6. The the present study the in vitro hydroxylation of bupropion by the CYP enzyme system was investigated. CYP2B6 was identified to have the major role in hydroxybupropion formation. In addition, we have also investigated the possibility of CYP2D6 inhibition by bupropion or hydroxybupropion [15]. dihydrogen phosphate (pH adjusted to 3.0 with orthophosphoric acid) (Table 1.). Before using the mobile phase, it was filtered through a 0.45 m filter and the filtrate was degassed by using bath sonicator. The peaks were determined using a UV detector set at a wavelength of 250 nm. Bupropion HCL showed a maximum wavelength of 250 nm. All the procedures were performed at ambient temperature. Preparation of stock solution Stock solution of bupropion was prepared in acetonitrile at a concentration of 1 mg/ml and was kept at 2-8C. Stock solution was diluted with acetonitrile to obtain the concentrations of 500, 250, 100, 50, 25, g/ml. Bupropion working solutions in rat plasma were in the range of 2.5 g/ml to 50 g/ml. The internal standard was prepared by dissolving 2.5 mg of phenacetin in 1 ml acetonitrile. Phenacetin was weighed accurately in a micro centrifuge tube and to this 1 ml acetonitrile was added using a micropipette. Samples for the accuracy, precision and recovery were prepared by spiking standard bupropion concentrations in rat plasma to yield concentrations of 2.5, 5, 10, 25 and 50 g/ml and stored at 2-8 C till analysis. Fig. 1. Principal Metabolites of bupropion in humans MATERIALS AND METHODS Bupropion hydrochloride was a gift sample from IPCA labs ltd, Mumbai. Phenacetin was obtained from Sigma-Aldrich chemicals, Mumbai, India. Methanol (HPLC grade), Acetonitrile (HPLC grade) was obtained from J.T.Baker and orthophosphoric acid (AR grade) was obtained from Fisher scientific. Doubled distilled water for analytical purpose and rat plasma were collected from healthy male wistar rats (using EDTA as the anticoagulant). The experiment was performed as per the guidelines of Institutional Animal Care Committee constituted as per the guidelines of the CPCSEA and the protocol [Protocol no. CPCSEA/IAEC/SPTM/P-59/211] was duly approved by the Institutional Animal Ethics Committee. Chromatographic condition The chromatographic system consisted of Perkin Elmer series 200 LC pump, Perkin Elmer LC 200 Auto sampler and series 200 EP diode array detector. The chromatographic separation of bupropion and internal standard (phenacetin) was done using a 2504.6 mm, 5 m, Kromasil C18 analytical column. The mobile phase was a gradient of acetonitrile (A) and 0.01 M potassium Varghese et al Table 1. Gradient conditions for HPLC Time (min) 0 5 10 15 18 Acetonitrile 0.01 N potassium dihydrogen phosphate, pH 3.0 90 70 55 45 90

10 30 45 55 10

Method Development Plasma stability The stability of Bupropion HCl in rat plasma was determined by incubating Bupropion HCl in rat plasma at 37C for 1 hour. Stability study was carried out at a concentration of 25 g/mL of Bupropion HCl. The stability was determined by taking aliquots of spiked plasma at 0, 15, 30 and 60 min. Samples were run in duplicate. Trials of Extraction of drug from rat plasma Extractions were tried with methanol, acetonitrile and ethyl acetate. Acetonitrile and ethyl acetate gave very poor recovery and broad peak Methanol gave better recovery but very broad peak. Peak sharpness and recovery was improved by modifying the pH of the extracting solvent. Using methanol made acidic with 0.05 N HCl gave Pg. 21

Int. J. Pharm. Biosci. Technol. good recovery and peak shape of both Bupropion HCl and Phenacetin with no interferences. Final Extraction Procedure In a micro centrifuge tube, 10 l phenacetin (250 g/ml), 90 L blank rat plasma and 10 l of 10X Bupropion HCl solution were co-spiked and vortexed. The spiked samples were precipitated with 1 ml of methanol (made acidic with 0.05 N HCl) and vortexed for 5 min. The resulting solutions were then centrifuged at 4000 rpm for 10 min. Resulting supernatant (900 l) was evaporated under a gentle stream of nitrogen at 400C. The dried samples were reconstituted with 100 l mobile phase, vortexed, centrifuged and injected in HPLC. All samples were processed in the similar manner as mentioned above. Bioanalytical method validation The RP-HPLC assay validation was done as per ICH Q2A and Q2B guidelines [20, 21]. Linearity in rat plasma Standard calibration samples were prepared by making serial dilution from the stock solution of bupropion (1mg/ml). Calibration curve of concentration versus peak area ratio was plotted at concentration range 2.5, 5, 10, 25 and 50 g/ml. Limit of detection quantification and lower limit of analyzing the spiked standards and extracted samples on two different days. Each concentration was run in duplicate. After concentrations were calculated by re-fitting peak area standard solutions, % RSD was determined at each ratio obtained with different standards solutions into a derived regression equation from the set of these concentrations of the standard solutions from their average value and SD. Recovery The absolute recovery was calculated by comparing the peak area ratio of extracted and unextracted samples containing bupropion HCl and phenacetin. Each measurement was made in triplicates. The % recovery was calculated using following formula. Mean peak area ratio of extracted samples % Recovery = ______________________ Mean peak area ratio of Un-extracted samples System suitability The purpose of system suitability to define asset of parameters that are measured prior to each experiment that will tell the analyst if the system is performing adequately or not. The suitability parameters that are evaluated for HPLC method includes peak area reproducibility and retention time. RESULTS & DISCUSSIONS Chromatography Sensitive, rapid, specific and reproducible HPLC method has been developed and validated for quantitative determination of bupropion HCl in rat plasma samples. After the pre-treatment with a rapid single protein precipitation step, the rat plasma containing bupropion HCL was separated by reverse phase HPLC with UV detection at 250 nm. The representative chromatograms of bupropion HCl in rat plasma is shown in Fig. 2. The retention time of bupropion HCL and phenacetin were 11.81 and 13.68 min., respectively. There was good baseline separation of bupropion HCl. Fig. 2. shows a representative chromatogram of bupropion HCl and phenacetin (IS) in rat plasma. Linearity in rat plasma Plasma stability studies revealed that the drug was found to be stable in rat plasma after incubation for 1 hr at 370 C. Linearity in rat plasma was measured at concentrations of 2.5, 5, 10, 25, 50 Pg. 22 100

The limit of detection (LOD) and lower limit of quantification (LLOQ) were measured according to the FDAs guidance for bioanalytical method validation.[22] The limit of detection was defined as the lowest concentration of bupropion resulting in a peak height greater or equal to three times from background noise (S/N 3.3). The quantification limit was established by assessing the signal to noise ratio level in proportion of 10:1 for each signal. The analyte response at the LLOQ should be at least 5 times the response compared to blank response. Analyte peak (response) should be identifiable, discrete, and reproducible with a precision of 20% and accuracy of 80-120%. Precision and Accuracy The precision and accuracy were determined by analyzing spiked standard and extracted samples at different concentrations ranging from 2.5, 5, 10, 25 and 50 g/ml. The precision of an HPLC method was determined as the coefficient of variation (%RSD) of intra- and inter-day. The intra-day precision was determined by analyzing the spiked standard and extracted samples prepared within a day. The inter-day precision was determined by Varghese et al

Int. J. Pharm. Biosci. Technol. and 100 g/ml of Bupropion HCl. Peak area ratio of Bupropion HCl and phenacetin was calculated. Plot of peak area ratio versus plasma concentration (g/ml) was plotted on Microsoft Excel 2007. The regression equation of the calibration curve was y= 0.024 x + 0.0186 and correlation coefficient, r2 = 0.999, where y is the peak area ratio of Bupropion HCL and phenacetin and x is the concentration of bupropion HCl in g/mL. This result demonstrated a good linearity between peak area ratio and concentration. Overlay chromatograms of all concentrations is shown in Fig. 3. the linearity

Limit of detection and Limit of quantification The limit of detection (LOD) and limit of quantification (LOQ) was found to be 0.75 g/ml (S/N3) and 2.27 g/ml respectively.

Fig. 2. Representative chromatogram of Bupropion HCl and Phenacetin in rat plasma

Fig. 3. Overlay chromatogram of 2.5, 5, 10, 25 and 50 g/ml of Bupropion HCl in rat plasma.

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Int. J. Pharm. Biosci. Technol. Precision and Accuracy The precision of the assay method was validated by the determination of the intra-day and inter-day coefficient of variation (% RSD) and percentage deviation. The intra-day and inter-day precision was evaluated over the concentration range of 2.5 g/ml-50 g/ml. The average % RSD of intra-day and inter-day precision was 7.99% and 5.04% respectively. All % RSD are less than 15%. The accuracy of the method was verified by comparing the concentrations measured for bupropion HCL spiked from extracted sample with actual added concentrations. The intra-day and inter-day accuracy data expressed as percentage deviation of bupropion HCL assay and the data is shown in Table 2. The bioanalytical method was accurate in the range of 2.5 50 g/mL in rat plasma. Table 2. Accuracy and Precision of bupropion hydrochloride bioanalytical method
Spiked conc (g/ml) Calculated concentration (g/ml, mean SD, n=5) R.S.D (%) Deviation (%)

average extraction efficiency were found to be 55.28%. Table 3. Recovery of bupropion hydrochloride from rat plasma sample Concentration (g/ml) 2.5 5 10 25 50 System suitability The % RSD for area response of the drug was 1.16%, which is within the acceptance value 2%. The %RSD for retention time for the drug was 2 %, which is within the acceptance range. The present bioanalytical method for the determination of bupropion HCl in rat plasma samples is novel, sensitive, rapid, specific, accurate and reproducible. Acidification of the extraction solvent (methanol) improved the recovery and peak shape of Bupropion. The excellent separation is demonstrated in the chromatograms and no interfering peaks were observed. The calibration curve was linear and the method was suitable for the analysis of plasma samples over the range of 2.5 to 50 g/ml. The accuracy of the method was in compliance with the proposed limits and the precision of the method is satisfactory. This method shows the system suitability parameters are within the accepted limits. CONCLUSION The bio analytical method for quantification of bupropion HCl is novel, since recovery of analyte was enhanced by acidification of the plasma. This in turn led to a sensitive, accurate and reproducible bioanalytical method. The method can be applied for use in pre-clinical & clinical studies. Another application of the method could be to evaluate any drugs interaction potential with CYP2B6 (since Bupropion hydrochloride is a recommended substrate for CYP2B6). In conclusion, the HPLC method described here can be successfully applied for pharmacokinetic study of bupropion HCl. ACKNOWLEDGEMENTS The authors would like to thank IPCA Labs, for the gift sample of Bupropion hydrochloride Dr. R. S. Concentration of samples after extraction 2.13404058 4.70256172 8.48484085 23.7059055 49.5458636 Recovery (%) 44.98 53.27 52.60 62.97 62.58

Intra-day (n=5) 2.5 5 10 25 50 2.13 0.002 4.67 0.005 10.92 0.020 25.86 0.029 50.67 0.082 6.12 6.36 10.63 6.90 10.01 1.64 4.06 -0.09 -0.03 -0.01

Inter-day (n=5) 2.5 5 10 25 50 Recovery The recovery of bupropion HCl after protein precipitation procedure was evaluated at five concentrations of 2.5, 5, 10, 25 and 50 g/ml. Absolute recovery was calculated by comparing the peak area ratio for bupropion HCL and phenacetin in methanol with those obtained by methanol extracted plasma samples containing same amount of bupropion HCl and phenacetin. Table 3. shows the recovery efficiency of bupropion HCl from rat plasma samples and the 2.16 0.002 5.16 0.002 10.73 0.018 25.36 0.004 50.84 0.039 5.62 2.61 10.48 1.13 4.84 13.21 -3.20 -7.34 -1.46 -1.68

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Int. J. Pharm. Biosci. Technol. ___________________________________________ How to cite this article APA style Thakar, D. S., & Varghese, A. (2013). Bioanalytical method development and validation of bupropion hydrochloride in rat plasma by RP-HPLC. International Journal of Pharma Bioscience and Technology, 1(1), 20 26. Elsevier Harvard style Thakar, D.S., Varghese, A., 2013. Bioanalytical method development and validation of bupropion hydrochloride in rat plasma by RPHPLC. Int. J. Pharm. Biosci. Technol. 1, 2026. Vancouver Style Thakar DS, Varghese A. Bioanalytical method development and validation of bupropion hydrochloride in rat plasma by RP-HPLC. Int. J. Pharm. Biosci. Technol. 2013; 1(1):2026. To receive bibliographic information in RIS format (For Reference Manager, ProCite, EndNote): Send request to: ijpbst@yahoo.com ___________________________________________

substrate specificity, and role in procarcinogen activation. Drug Metabolism& Disposition:the biological fate of chemicals. 1997; 25(8): 985-993. 19. Mimura M, Baba T, Yamazaki H, Ohmori S, Inui Y, Gonzalez FJ, Guengerich FP, Shimada T.Characterization of cytochrome P-450 2B6 in human liver microsomes. Drug Metabolism & Disposition:the biological fate of chemicals.1993; 21(6): 1048-1056. 20. ICH topic Q2A, validation of Analytical Methods: Definitions and Terminology, Step 5, CPMP /ICH/381/ 95. 21. ICH topic Q2B, validation of Analytical Procedures: Methodology, Step 4, CPMP /ICH/281/ 95. 22. http:www.fda.gov/cder/guidance/index.htm.

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