VIEWS: 0 PAGES: 7 CATEGORY: Research POSTED ON: 5/17/2013
Key words: Bupropion HCl, HPLC, Bioanalysis, Rat plasma, UV detection.
Research Article ISSN: 2321-2969 Received: 10 April 2013, Accepted: 26 April 2013 Int. J. Pharm. Biosci. Technol. To cite this Article: Click here 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, SVKM’s NMIMS, Vile Parle (W), Mumbai-400056, India. Corresponding Author* E-mail address- firstname.lastname@example.org 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 µm) analytical column. Mean recovery of bupropion hydrochloride from rat plasma was around 55 % for 2.5-50 µg/ml concentrations. The assay exhibited good linear relationship with an r2 of 0.9999. Lower limit of Quantification limit (LLOQ) was 1.84 µg/ml of bupropion hydrochloride and accuracy and precision were over the concentration range 2.5-50 µg/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 also known with the generic name of Bupropion was first patented in 1974 and amfebutamone hydrochloride. Bupropion is a released onto the world market in 1985. It was second-generation antidepressant agent that is briefly withdrawn due to seizures incidences but also used in the management of smoking cessation reintroduced in 1989 after the daily recommended . CYP2B6 is a polymorphic hepatic dose was reduced to lower seizure likelihood. enzymeof potential importance in the Bupropion is a dopamine and norepinephrine metabolism of drugs such as Bupropion, reuptake inhibitor .It is about twice as potent an efavirenz and cyclophosphamide . Wide inhibitor of dopamine reuptake than of interindividual variability in the hepatic norepinephrine reuptake. Besides reuptake expression of CYP2B6 has been reported.[18, 19] inhibition of dopamine and noradrenaline, bupropion also causes the release of dopamine In humans, bupropion is extensively metabolized and noradrenaline . Bupropion has numerous to three principal metabolites (Fig.1.) such as therapeutic indications including, depression, hydroxyl-bupropion or morphinol, erythro- smoking cessation, sexual dysfunction, hydrobupropion, and threo-hydro-bupropion. obesity, attention deficit hyperactivity The pharmacologically active metabolite disorder and seasonal affective disorder. It hydroxyl-bupropion appears to be the major has recently been shown to have anti- metabolite, since the plasma levels of inflammatory properties . In 2007 it was the hydroxybupropion greatly exceeds with respect fourth-most prescribed antidepressant in the USA. to those of the parent drug. The cytochrome P450 Bupropion is the water soluble hydrochloride salt (CYP) enzyme system, especially CYP2B6, has an of an aminoketone, with a pKa of 7.9. It is important role in bupropion hydroxylation. Also Varghese et al Pg. 20 Int. J. Pharm. Biosci. Technol. product labeling have indicated that bupropion or dihydrogen phosphate (pH adjusted to 3.0 with hydroxybupropion inhibits CYP2D6. The the orthophosphoric acid) (Table 1.). Before using the present study the in vitro hydroxylation of mobile phase, it was filtered through a 0.45 µm bupropion by the CYP enzyme system was filter and the filtrate was degassed by using bath investigated. CYP2B6 was identified to have the sonicator. The peaks were determined using a UV major role in hydroxybupropion formation. In detector set at a wavelength of 250 nm. Bupropion addition, we have also investigated the possibility HCL showed a maximum wavelength of 250 nm. of CYP2D6 inhibition by bupropion or All the procedures were performed at ambient hydroxybupropion . temperature. Preparation of stock solution Stock solution of bupropion was prepared in acetonitrile at a concentration of 1 mg/ml and was kept at 2-8ºC. Stock solution was diluted with acetonitrile to obtain the concentrations of 500, 250, 100, 50, 25, µg/ml. Bupropion working solutions in rat plasma were in the range of 2.5 µg/ml to 50 µg/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 µg/ml and stored at 2-8º C till analysis. Fig. 1. Principal Metabolites of bupropion in Table 1. Gradient conditions for HPLC humans Time Acetonitrile 0.01 N potassium MATERIALS AND METHODS (min) dihydrogen phosphate, pH 3.0 Bupropion hydrochloride was a gift sample from IPCA labs ltd, Mumbai. Phenacetin was obtained 0 10 90 from Sigma-Aldrich chemicals, Mumbai, India. 5 30 70 Methanol (HPLC grade), Acetonitrile (HPLC 10 45 55 grade) was obtained from J.T.Baker and 15 55 45 orthophosphoric acid (AR grade) was obtained 18 10 90 from Fisher scientific. Doubled distilled water for analytical purpose and rat plasma were collected Method Development from healthy male wistar rats (using EDTA as the Plasma stability anticoagulant). The experiment was performed as per the guidelines of Institutional Animal Care The stability of Bupropion HCl in rat plasma was Committee constituted as per the guidelines of the determined by incubating Bupropion HCl in rat CPCSEA and the protocol [Protocol no. plasma at 37ºC for 1 hour. Stability study was CPCSEA/IAEC/SPTM/P-59/211] was duly carried out at a concentration of 25 µg/mL of approved by the Institutional Animal Ethics Bupropion HCl. The stability was determined by Committee. taking aliquots of spiked plasma at 0, 15, 30 and 60 min. Samples were run in duplicate. Chromatographic condition Trials of Extraction of drug from rat plasma The chromatographic system consisted of Perkin Elmer series 200 LC pump, Perkin Elmer LC 200 Extractions were tried with methanol, acetonitrile Auto sampler and series 200 EP diode array and ethyl acetate. Acetonitrile and ethyl acetate detector. The chromatographic separation of gave very poor recovery and broad peak bupropion and internal standard (phenacetin) was Methanol gave better recovery but very broad done using a 250×4.6 mm, 5 µm, Kromasil C18 peak. Peak sharpness and recovery was improved analytical column. The mobile phase was a by modifying the pH of the extracting solvent. gradient of acetonitrile (A) and 0.01 M potassium Using methanol made acidic with 0.05 N HCl gave Varghese et al Pg. 21 Int. J. Pharm. Biosci. Technol. good recovery and peak shape of both Bupropion analyzing the spiked standards and extracted HCl and Phenacetin with no interferences. samples on two different days. Each concentration was run in duplicate. After concentrations were Final Extraction Procedure calculated by re-fitting peak area standard solutions, % RSD was determined at each ratio In a micro centrifuge tube, 10 µl phenacetin (250 obtained with different standards solutions into a µg/ml), 90 µL blank rat plasma and 10 µl of 10X derived regression equation from the set of these Bupropion HCl solution were co-spiked and concentrations of the standard solutions from their vortexed. The spiked samples were precipitated average value and SD. with 1 ml of methanol (made acidic with 0.05 N HCl) and vortexed for 5 min. The resulting Recovery solutions were then centrifuged at 4000 rpm for 10 min. Resulting supernatant (900 µl) was The absolute recovery was calculated by evaporated under a gentle stream of nitrogen at comparing the peak area ratio of extracted and 400C. The dried samples were reconstituted with unextracted samples containing bupropion HCl 100 µl mobile phase, vortexed, centrifuged and and phenacetin. Each measurement was made in injected in HPLC. All samples were processed in triplicates. The % recovery was calculated using the similar manner as mentioned above. following formula. Mean peak area ratio Bioanalytical method validation of extracted samples The RP-HPLC assay validation was done as per ICH Q2A and Q2B guidelines [20, 21]. % Recovery = ______________________ × 100 Mean peak area ratio of Linearity in rat plasma Un-extracted samples Standard calibration samples were prepared by System suitability making serial dilution from the stock solution of bupropion (1mg/ml). Calibration curve of The purpose of system suitability to define asset of concentration versus peak area ratio was plotted parameters that are measured prior to each at concentration range 2.5, 5, 10, 25 and 50 µg/ml. experiment that will tell the analyst if the system is performing adequately or not. The suitability Limit of detection and lower limit of parameters that are evaluated for HPLC method quantification includes peak area reproducibility and retention time. The limit of detection (LOD) and lower limit of quantification (LLOQ) were measured according to RESULTS & DISCUSSIONS the FDA’s guidance for bioanalytical method validation. The limit of detection was defined Chromatography as the lowest concentration of bupropion resulting Sensitive, rapid, specific and reproducible HPLC in a peak height greater or equal to three times method has been developed and validated for from background noise (S/N ≥ 3.3). The quantitative determination of bupropion HCl in rat quantification limit was established by assessing plasma samples. After the pre-treatment with a the signal to noise ratio level in proportion of 10:1 rapid single protein precipitation step, the rat for each signal. The analyte response at the LLOQ plasma containing bupropion HCL was separated should be at least 5 times the response compared by reverse phase HPLC with UV detection at 250 to blank response. Analyte peak (response) nm. The representative chromatograms of should be identifiable, discrete, and reproducible bupropion HCl in rat plasma is shown in Fig. 2. with a precision of 20% and accuracy of 80-120%. The retention time of bupropion HCL and phenacetin were 11.81 and 13.68 min., Precision and Accuracy respectively. There was good baseline separation of bupropion HCl. Fig. 2. shows a representative The precision and accuracy were determined by chromatogram of bupropion HCl and phenacetin analyzing spiked standard and extracted samples (IS) in rat plasma. at different concentrations ranging from 2.5, 5, 10, 25 and 50 µg/ml. The precision of an HPLC method Linearity in rat plasma was determined as the coefficient of variation (%RSD) of intra- and inter-day. The intra-day Plasma stability studies revealed that the drug was precision was determined by analyzing the spiked found to be stable in rat plasma after incubation standard and extracted samples prepared within a for 1 hr at 370 C. Linearity in rat plasma was day. The inter-day precision was determined by measured at concentrations of 2.5, 5, 10, 25, 50 Varghese et al Pg. 22 Int. J. Pharm. Biosci. Technol. and 100 µg/ml of Bupropion HCl. Peak area ratio Overlay chromatograms of all the linearity of Bupropion HCl and phenacetin was calculated. concentrations is shown in Fig. 3. Plot of peak area ratio versus plasma concentration (µg/ml) was plotted on Microsoft Limit of detection and Limit of quantification Excel 2007. The regression equation of the The limit of detection (LOD) and limit of calibration curve was y= 0.024 x + 0.0186 and quantification (LOQ) was found to be 0.75 µg/ml correlation coefficient, r2 = 0.999, where y is the (S/N≥3) and 2.27 µg/ml respectively. peak area ratio of Bupropion HCL and phenacetin and x is the concentration of bupropion HCl in µg/mL. This result demonstrated a good linearity between peak area ratio and concentration. Fig. 2. Representative chromatogram of Bupropion HCl and Phenacetin in rat plasma Fig. 3. Overlay chromatogram of 2.5, 5, 10, 25 and 50 µg/ml of Bupropion HCl in rat plasma. Varghese et al Pg. 23 Int. J. Pharm. Biosci. Technol. Precision and Accuracy average extraction efficiency were found to be 55.28%. The precision of the assay method was validated by the determination of the intra-day and inter-day Table 3. Recovery of bupropion hydrochloride coefficient of variation (% RSD) and percentage from rat plasma sample deviation. The intra-day and inter-day precision was evaluated over the concentration range of 2.5 Concentration Concentration Recovery µg/ml-50 µg/ml. The average % RSD of intra-day (µg/ml) of samples (%) after extraction and inter-day precision was 7.99% and 5.04% respectively. All % RSD are less than 15%. The 2.5 2.13404058 44.98 accuracy of the method was verified by 5 4.70256172 53.27 comparing the concentrations measured for bupropion HCL spiked from extracted sample with 10 8.48484085 52.60 actual added concentrations. The intra-day and 25 23.7059055 62.97 inter-day accuracy data expressed as percentage deviation of bupropion HCL assay and the data is 50 49.5458636 62.58 shown in Table 2. The bioanalytical method was accurate in the range of 2.5 – 50 µg/mL in rat System suitability plasma. The % RSD for area response of the drug was Table 2. Accuracy and Precision of bupropion 1.16%, which is within the acceptance value ± 2%. hydrochloride bioanalytical method The %RSD for retention time for the drug was 2 %, Spiked Calculated R.S.D Deviation which is within the acceptance range. The present bioanalytical method for the determination of conc concentration (%) (%) bupropion HCl in rat plasma samples is novel, (µg/ml) (µg/ml, mean ± sensitive, rapid, specific, accurate and SD, n=5) reproducible. Acidification of the extraction solvent (methanol) improved the recovery and Intra-day (n=5) peak shape of Bupropion. The excellent separation 2.5 2.13 ± 0.002 6.12 1.64 is demonstrated in the chromatograms and no interfering peaks were observed. The calibration 5 4.67 ± 0.005 6.36 4.06 curve was linear and the method was suitable for 10 10.92 ± 0.020 10.63 -0.09 the analysis of plasma samples over the range of 2.5 to 50 µg/ml. The accuracy of the method was in 25 25.86 ± 0.029 6.90 -0.03 compliance with the proposed limits and the 50 50.67 ± 0.082 10.01 -0.01 precision of the method is satisfactory. This method shows the system suitability parameters Inter-day (n=5) are within the accepted limits. 2.5 2.16 ± 0.002 5.62 13.21 CONCLUSION 5 5.16 ± 0.002 2.61 -3.20 The bio analytical method for quantification of 10 10.73 ± 0.018 10.48 -7.34 bupropion HCl is novel, since recovery of analyte 25 25.36 ± 0.004 1.13 -1.46 was enhanced by acidification of the plasma. This in turn led to a sensitive, accurate and 50 50.84 ± 0.039 4.84 -1.68 reproducible bioanalytical method. The method can be applied for use in pre-clinical & clinical Recovery studies. Another application of the method could be to evaluate any drug’s interaction potential with The recovery of bupropion HCl after protein CYP2B6 (since Bupropion hydrochloride is a precipitation procedure was evaluated at five recommended substrate for CYP2B6). In concentrations of 2.5, 5, 10, 25 and 50 µg/ml. conclusion, the HPLC method described here can Absolute recovery was calculated by comparing be successfully applied for pharmacokinetic study the peak area ratio for bupropion HCL and of bupropion HCl. phenacetin in methanol with those obtained by methanol extracted plasma samples containing ACKNOWLEDGEMENTS same amount of bupropion HCl and phenacetin. Table 3. shows the recovery efficiency of The authors would like to thank IPCA Labs, for the bupropion HCl from rat plasma samples and the gift sample of Bupropion hydrochloride Dr. R. S. Varghese et al Pg. 24 Int. J. Pharm. Biosci. Technol. Gaud; Dean of SPP-SPTM, NMIMS, Mumbai for treatment with bupropion XL. 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