BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF BUPROPION HYDROCHLORIDE IN RAT PLASMA BY RP-HPLC

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BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF BUPROPION HYDROCHLORIDE IN RAT PLASMA BY RP-HPLC Powered By Docstoc
					Research Article                                                                                     ISSN: 2321-2969
Received: 10 April 2013, Accepted: 26 April 2013                                      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

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        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- 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 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[1] 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
                                                                 [13]. CYP2B6 is a polymorphic hepatic
dose was reduced to lower seizure likelihood.
                                                                 enzyme[14]of potential importance in the
Bupropion is a dopamine and norepinephrine
                                                                 metabolism of drugs such as Bupropion[15],
reuptake inhibitor [2].It is about twice as potent an
                                                                 efavirenz[16] and cyclophosphamide [17]. 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 [3]. Bupropion has numerous                    to three principal metabolites (Fig.1.) such as
therapeutic indications including, depression[4],                hydroxyl-bupropion or morphinol, erythro-
smoking cessation[5], sexual dysfunction[6],                     hydrobupropion, and threo-hydro-bupropion.
obesity[7],    attention      deficit  hyperactivity
                                                                 The     pharmacologically     active   metabolite
disorder[8] and seasonal affective disorder[9]. It
                                                                 hydroxyl-bupropion appears to be the major
has recently been shown to have anti-
                                                                 metabolite, since the plasma levels of
inflammatory properties [10]. 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[11], with a pKa of 7.9[12]. 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 [15].                                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.[22] 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.           Biological
providing support and necessary facilities.             Psychiatry. 2005; 58(8): 658-667.

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                                                    ___________________________________________
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                                                    Thakar, D.S., Varghese, A., 2013. Bioanalytical
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20. ICH topic Q2A, validation of Analytical
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                                                    HPLC. Int. J. Pharm. Biosci. Technol. 1, 20–26.
    CPMP /ICH/381/ 95.
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21. ICH topic Q2B, validation of Analytical         Thakar DS, Varghese A. Bioanalytical method
    Procedures: Methodology, Step 4, CPMP           development and validation of bupropion
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22. http:www.fda.gov/cder/guidance/index.htm.
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Varghese et al                                                                              Pg. 26

				
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Description: Key words: Bupropion HCl, HPLC, Bioanalysis, Rat plasma, UV detection.