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),
E-mail address- email@example.com
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.
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briefly withdrawn due to seizures incidences but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
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.
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
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
Standard calibration samples were prepared by
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
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
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 (%)
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
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
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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