Determination of Curcumin in Rat Plasma by Liquid–liquid
Extraction using LC–MS/MS with Electrospray Ionization:
Assay Development, Validation and Application to a
Pharmacokinetic Study
Pharmacokinetics and Metabolism Division, Central Drug Research Institute, Council of Scientific and Industrial Research (CSIR), Lucknow 226001, Uttar Pradesh, India
*Corresponding author:
Dr. Wahajuddin
Pharmacokinetics and Metabolism Division
Central Drug Research Institute
CSIR, Lucknow 226001
Uttar Pradesh, India Tel: +91-522-2612411-18 (Ext-4377) Fax: +91-522-2623405 E-mail: wahajuddin@cdri.res.in, wahajuddin@gmail.com
Received July 23, 2010; Accepted August 23, 2010; Published August 23, 2010
Citation: Singh SP, Wahajuddin, Jain GK (2010) Determination of Curcumin
in Rat Plasma by Liquid–liquid Extraction using LC–MS/MS with Electrospray
Ionization: Assay Development, Validation and Application to a Pharmacokinetic
Study. J Bioanal Biomed 2: 079-084. doi:10.4172/1948-593X.1000027
A simple, specific and rapid LC–MS/MS method has been developed and validated for the estimation of curcumin
in rat plasma, using biochanin as internal standard (IS). The assay procedure involved liquid–liquid extraction of
curcumin and IS from rat plasma. The recovery of curcumin and IS in rat plasma were 87.62 and 88.25%, respectively.
The resolution of peaks was achieved with 0.01 M ammonium acetate (pH 5.5):acetonitrile (10:90, v/v) on a Supelco
Discovery C18 column. The total chromatographic run time was 4 min. Specificity and matrix effect on ionization was
determined and found that method was specific and there was no significant matrix effect. The method was proved to
be accurate and precise at linearity range of 10–2000 ng/mL with a correlation coefficient (r) of =0.995. The MS/MS
ion transitions monitored were 367?217 for curcumin and 283?268 for IS. The intra- and inter-day assay precision
ranged from 2.79 to 8.20% and 4.15 to 7.76%, respectively; and intra- and inter-day assay accuracy was between
92.83–107.83% and 93.92–104.26%, respectively. Practical utility of this LC–MS/MS method was demonstrated in a
pilot pharmacokinetic study in male Sprague–Dawley rats following intravenous administration of curcumin.
Keywords
Curcumin; Rat plasma; Validation; LC–MS/ MS;
Pharmacokinetics
Introduction
Curcuma spp. contain turmerin, essential oils, and curcuminoids,
including curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-
heptadiene-3,5-dione] which is a phenolic substance derived
from dietary spice herb Curcuma longa. It is regarded as the most
biologically active constituent of the spice turmeric and it comprises
2-8% of most turmeric preparations (Bisht and Maitra, 2009; Sharma
et al., 2007). Interests in this dietary polyphenol has grown in
recent years due to its vast array of beneficial pharmacological
effects including antioxidant, anti-inflammatory, anticarcinogenic,
hypocholesterolemic, antibacterial, wound healing, antispasmodic,
anticoagulant, antitumor, anti-angiogenic and hepatoprotective
activities (Aggarwal and Harikumar, 2009; Pari et al., 2008). With
regard to considerable public and scientific interest in the use of
phytochemicals derived from dietary components to combat or
prevent human diseases, curcumin is currently a leading agent. For
these reasons, turmeric has been widely used as a food additive,
condiment, and health food. Further, data obtained in multiple
preclinical models, as well as in preliminary clinical trials, have
documented minimal toxicity of curcumin, even at relatively high
doses. However, the clinical advancement of this promising molecule
has been hindered by its poor water solubility, short biological
half-life, and low bioavailability after oral administration (Sharma
et al., 2007). Efficient first-pass metabolism and some degree of
intestinal metabolism, particularly glucuronidation and sulfation of
curcumin, might explain its poor oral systemic availability (Anand
et al., 2007; Sharma et al., 2007). The absorption, metabolism, and
tissue distribution of curcumin after oral administration of 400, 80
and 10 mg of curcumin in rats has been studied (Ravindranath and
Chandrasekhara, 1981). Due to its wide spectrum nutritional as well
as therapeutic effects, a lot of preclinical multi-disciplinary evaluation
work is going on with curcumin to advocate its clinical utility.
Development of validated assay procedure in preclinical
animal species is essential at various stages of drug discovery and development. So far, several assay methodologies have been reported
for determination of curcuminoids using gas chromatography,
capillary electrophoresis(Lechtenberg et al., 2004), HPLC (Asakawa et
al., 1981; Heath et al., 2005; Pak et al., 2003) and LC-MS/MS (Marczylo
et al., 2009; Yang et al., 2007). However, there is an ever-increasing
need to have a battery of improved, faster, reproducible and reliable
bio-analytical methods which ultimately may lead to variety of choices
before the analysts. Therefore, we developed and validated a rapid,
specific and robust LC-ESI-MS/MS method for the quantification of
curcumin in rat plasma. The speed of sample preparation and analysis,
selectivity and sensitivity proved to be satisfactory. In addition,
using the method described above, the pharmacokinetic profile of
curcumin in rats has been determined following intravenous bolus
administration.
Experimental
Chemicals and reagents
Curcumin and biochanin (IS) were purchased from Sigma Aldrich
Ltd (St Louis, USA). Chemical structure of curcumin and biochanin
is shown in Figure 1. HPLC grade acetonitrile, methanol, ethyl
acetate and dichloromethane were purchased from Sisco Research
Laboratories (SRL) Pvt. Limited (Mumbai, India). Dimethyl sulfoxide
was purchased from Thomas baker chemicals Pvt. Limited (Mumbai, India). Ammonium acetate and glacial acetic acid (GAA) AR were
purchased from E Merck Limited (Mumbai, India). Ultra pure water
was obtained from a Sartorious Arium 611 system. Heparin sodium
injection I.P. (1000 IU/mL, Biologicals E. Limited, Hyderabad, India)
was purchased from local pharmacy. Blank, drug free plasma samples
were collected from adult, healthy male Sprague–Dawley rats at
Division of Laboratory Animals (DOLA) of Central Drug Research
Institute (Lucknow, India). Plasma was obtained by centrifuging the
heparinised blood (25 IU/mL) at 2000×g for 10 min. Prior approval
from the Institutional Animal Ethics Committee (IAEC) was sought
for maintenance, experimental studies, euthanasia and disposal of
carcass of animals.
Figure 1: Structural representation of curcumin and biochanin.
Instrumentation and chromatographic conditions
HPLC system consists of Series 200 pumps and auto sampler
with temperature controlled Peltier-tray (Perkin- Elmer instruments,
Norwalk, USA) was used to inject 10 µL aliquots of the processed
samples on a Supelco Discovery C18 column (4.6 × 50 mm, 5.0 µm).
The system was run in isocratic mode with mobile phase consisting
of 0.01 M ammonium acetate (pH 5.5) and acetonitrile in the ratio
of 10:90 (v/v) at a flow rate of 0.5 mL/min. Mobile phase was duly
filtered through 0.22 µm Millipore filter (Billerica, USA) and degassed
ultrasonically for 15 min prior to use. Separations were performed
at room temperature. Auto-sampler carry-over was determined by
injecting the highest calibration standard then a blank sample. No
carry-over was observed, as indicated by the lack of curcumin and
biochanin peaks in the blank sample.
Mass spectrometric detection was performed on an API 4000
mass spectrometer (Applied Biosystems, MDS Sciex Toronto, Canada)
equipped with an API electrospray ionization (ESI) source. The ion
spray voltage was set at -5500 V. The instrument parameters viz.,
nebulizer gas, curtain gas, auxillary gas and collision gas were
set at 40, 10, 20 and 8, respectively. Compounds parameters viz.,
declustering potential (DP), collision energy (CE), entrance potential
(EP) and collision exit potential (CXP) were -60, -15, -10, -10 V and
-92, -12, -10, -8 V for curcumin and IS, respectively. Zero air was used
as source gas while nitrogen was used as both curtain and collision
gas. The mass spectrometer was operated at ESI negative ion mode
and detection of the ions was performed in the multiple reaction
monitoring (MRM) mode, monitoring the transition of m/z 367 precursor ion [M–H]– to the m/z 217 product ion for curcumin and m/z 283 precursor ion [M–H]– to the m/z 268 product ion for IS. Data
acquisition and quantitation were performed using analyst software
version 1.4.1 (Applied Biosystems, MDS Sciex Toronto, Canada).
Preparation of stock and standard solutions
The primary stock solution of the curcumin was prepared by dissolving
5 mg in 0.2 mL of dimethyl sulfoxide and diluting with methanol to
a final concentration of 1000 µg/mL. The IS primary stock solution of
1000 µg/mL was prepared in methanol. Appropriate dilutions were
made in methanol for curcumin to produce working stock solution
of 20, 10, 5, 2, 1, 0.5, 0.2, 0.1 µg/mL and on the day of analysis this
set of stocks was used to prepare standards for the calibration curve.
Another set of working stock solutions of curcumin was made in
methanol at 16, 8, 0.4 and 0.1 µg/mL for preparation of QC samples.
Individually QC and CC working stock solutions of curcumin were
spiked into blank plasma for QC and CC samples. A working stock
solution of IS (1 µg/mL) was prepared in methanol from primary stock
solution of 1000 µg/mL in methanol. Calibration standards and QC
samples were prepared by spiking 90 µL of control pooled rat plasma
with the appropriate working solution of curcumin (10 µL) and IS (10
µL) on the day of analysis. Samples for the determination of precision
and accuracy were prepared by individually spiking control rat plasma
at four concentration levels [10 ng/mL (lower limit of quantitation,
LLOQ), 40 ng/mL (QC low), 800 ng/mL (QC medium) and 1600 ng/mL
(QC high)] and stored at –80 ± 10°C until analysis.
Recovery
The recovery of curcumin and IS, through liquid–liquid extraction
procedure, was determined by comparing the peak areas of the
analytes extracted from replicate QC samples (n = 6) with the peak
areas of analytes from post-extracted plasma blank samples spiked
at equivalent concentrations (Dams et al., 2003; Singh et al., 2008; Wahajuddin et al., 2009). Recoveries of curcumin were determined
at QC low, QC medium and QC high concentrations viz., 40, 800,
and 1600 ng/mL, whereas the recovery of the IS was determined at a
single concentration of 100 ng/mL.
Sample preparation
A simple liquid–liquid extraction method was followed for extraction
of curcumin from rat plasma. To 100 µL of plasma in a tube, 10 µL of
IS solution (biochanin at 1 µg/mL in methanol), was added and mixed
for 15 sec on a cyclomixer (Spinix Tarsons, Kolkata, India). Then 2
mL of dichloromethane/ethyl acetate (1/1, v/v) was added and the
mixture was vortexed for 3 min, followed by centrifugation for 5 min
at 2000×g on Sigma 3-16K (Frankfurt, Germany). The organic layer
(1.6 mL) was separated and evaporated to dryness under vaccum in
speedvac concentrator (Savant Instrument, Farmingdale, USA). The
residue was reconstituted in 200 µL of the mobile phase and 10 µL
was injected onto analytical column.
Validation procedures
A full validation according to the FDA guidelines was performed
for the assay in rat plasma (US DHHS, FDA, CDER. 2001).
Specificity and selectivity: The specificity of the method was
evaluated by analyzing rat plasma samples collected from six
different rats to investigate the potential interferences at the LC peak
region for analyte and IS using the proposed extraction procedure
and chromatographic-MS conditions.
Matrix effect: The effect of rat plasma constituents over the ionization of curcumin and IS was determined by comparing the
responses of the post-extracted plasma standard QC samples (n = 6)
with the response of analytes from neat standard samples (10 µL of
required working stock sample spiked into 90 µL of methanol instead of blank plasma) at equivalent concentrations (Dams and others 2003; Singh and others 2008; Wahajuddin and others 2009) [13-15]. The
matrix effect for curcumin was determined at QC low, QC medium
and QC high concentrations, viz., 40, 800 and 1600 ng/mL whereas the matrix effect over the IS was determined at a single concentration
of 100 ng/mL.
Calibration curve: The calibration curve was acquired by plotting
the ratio of peak area of curcumin to that of IS against the nominal
concentration of calibration standards. The final
concentrations of calibration standards obtained for plotting the
calibration curve were 10, 20, 50, 100, 200, 500, 1000, 2000 ng/
mL. The results were fitted to linear regression analysis using 1/X2
as weighting factor. The calibration curve had to have a correlation
coefficient (r) of 0.995 or better. The acceptance criteria for each
back-calculated standard concentration were ±15% deviation from
the nominal value except at LLOQ, which was set at ±20% (US DHHS,
FDA, CDER. 2001).
Precision and accuracy: The intra-day assay precision and accuracy
were estimated by analyzing six replicates at four different QC levels,
i.e., 10, 40, 800 and 1600 ng/mL. The inter-day assay precision was
determined by analyzing the four levels QC samples on three different
runs. The criteria for acceptability of the data included accuracy within ±15% standard deviation (S.D.) from the nominal values and a
precision of within ±15% relative standard deviation (R.S.D.), except
for LLOQ, where it should not exceed ±20% of accuracy as well as
precision (US DHHS, FDA, CDER. 2001).
Stability experiments: All stability studies were conducted at two
concentration levels, i.e. QC low and QC high, using six replicates at
each concentration levels. Replicate injections of processed samples
were analyzed up to 20 h to establish autosampler stability of analyte
and IS. The peak areas of analyte and IS obtained at initial cycle
were used as the reference to determine the stability at subsequent
points. The stability of curcumin in the biomatrix during 4 h exposure
at room temperature in rat plasma (bench top) was determined at
ambient temperature (25 ± 2°C). Freeze/thaw stability was evaluated
up to three cycles. In each cycle samples were frozen for at least
12 h at –80 ± 10°C. Freezer stability of curcumin in rat plasma was
assessed by analyzing the QC samples stored at –80 ± 10°C for at
least 15 days. Samples were considered to be stable if assay values
were within the acceptable limits of accuracy (i.e., ±15% S.D.) and
precision (i.e., ±15% R.S.D.).
Application to a pharmacokinetic study in rats
A pharmacokinetic study was performed to show the applicability
of newly developed and validated bioanalytical method. Study was
performed in male Sprague–Dawley rats (n = 3, weight range 200–
220 g). Curcumin was administered intravenously at a dose of 10 mg/
kg. Blood samples were collected from the retro-orbital plexus of rats
under light ether anesthesia into microfuge tubes containing heparin
as an anti-coagulant at 5, 15, 30, 45, 60, 120, 180 and 240 min postdosing.
Plasma was harvested by centrifuging the blood at 2000×g for
5 min and stored frozen at –80 ± 10°C until analysis. Plasma (100µL)
samples were spiked with IS, and processed as described above.
Along with the plasma samples, QC samples at low, medium and high
concentration were assayed in duplicate and were distributed among
calibrators and unknown samples in the analytical run.
Results
Liquid chromatography
Feasibility of various mixture(s) of solvents such as acetonitrile
and methanol using different buffers such as ammonium acetate,
acetic acid and formic acid with variable pH range of 4.5–6.5, along
with altered flow-rates (in the range of 0.4–0.8 mL/min) were tested
for complete chromatographic resolution of curcumin and IS (data
not shown). Mobile phase comprising of 0.01 M ammonium acetate
(pH 4.5):acetonitrile (10:90, v/v) was delivered at a flow rate of 0.5
mL/min was found to be suitable during LC optimization and enabled
the determination of electrospray response for curcumin and IS.
Experiments were also performed with different C18 columns and
found that chromatographic resolution, selectivity and sensitivity
were good with Supelco Discovery C18 column (4.6 × 50 mm, 5.0
µm).
Mass spectrometry
In order to optimize ESI conditions for curcumin and IS,
quadrupole full scans were carried out in negative ion detection
mode. During a direct infusion experiment, the mass spectra for
curcumin and IS revealed peaks at m/z 367 and 283, respectively as
deprotonated molecular ions [M–H]–. The product ion mass spectrum
for curcumin shows the formation of characteristic product ions at m/z 149.2, 172.9 and 217.1 (Figure 2). Following detailed optimization
of mass spectrometry conditions (provided in instrumentation and
chromatographic conditions section), the m/z 367 precursor ion to
the m/z 217 was used for quantification for curcumin. Similarly, for
IS m/z 283 precursor ion to the m/z 268 was used for quantification
purpose.
Recovery
The results of the comparison of pre extracted standards versus
post-extracted plasma standards were estimated for curcumin at 40,
800 and 1600 ng/mL and the absolute mean recovery was 87.62%. The
absolute recovery of IS at 100 ng/mL was 88.25%.
Validation procedures
Matrix effect, specificity and selectivity: In this study, the matrix
effect was evaluated by analyzing QC low (40 ng/mL), QC medium
(800 ng/mL) and QC high samples (1600 ng/mL). Average matrix effect
values obtained were 2.53, 3.24 and 2.88% at QC low, QC medium
and QC high, respectively. Matrix effect on IS was found to be 0.33%
at tested concentration of 100 ng/mL.
In the present study, the specificity and selectivity has been
studied by using independent plasma samples from six different rats.
Figure 3 shows a typical overlaid chromatogram for the control
rat plasma (free of analyte and IS), rat plasma spiked with curcumin at
LLOQ and IS and an in vivo rat plasma sample obtained at 5 min after
intravenous administration of curcumin. No significant interference
at the retention time of the drug or IS was found. The retention time
of curcumin and IS were 2.02 and 2.05 min, respectively. The total
chromatographic run time was 4 min.
Calibration curve: The calibration curve was acquired by plotting
the ratio of peak area of curcumin to that of IS against the nominal
concentration of calibration standards. The calibration
curve was prepared by determining the best fit of peak-area ratios
(peak area analyte / peak area IS) versus concentration, and fitted to
the y = mx + c using weighing factor (1/X2). The average regression
(n = 3) was found to be ≥ 0.997. The lowest concentration with
R.S.D. < 20% was taken as LLOQ and was found to be 10 ng/mL. The
% accuracy observed for the mean of back-calculated concentrations
for three calibration curves was within 88.70–108.33; while the %
precision values ranged from 0.71–5.97 (Table 1).
Accuracy and precision: Accuracy and precision data for intra- and
inter-day plasma samples are presented in Table 2 and Table 3. The
assay values on both the occasions (intra- and inter-day) were found
to be within the accepted variable limits.
Stability: The predicted concentrations for curcumin at 40 and
1600 ng/mL samples deviated within the nominal concentrations in
a battery of stability tests, viz., in-injector (20 h), bench-top (4 h),
repeated three freeze/thaw cycles and at –80 ± 10°C for at least
for 15 days (Table 4). The results were found to be within the assay
variability limits during the entire process.
Application of the method
The rat plasma samples generated following intravenous
administration of curcumin were analyzed by the newly developed
and validated method along with QC samples. All the QCs met the
acceptance criteria (data not shown). The sensitivity and specificity
of the assay were found to be sufficient for accurately characterizing
the pharmacokinetics of curcumin in rats. The mean concentration
of curcumin versus the time profile is show
n in Figure 4. The half-life
(t½) of curcumin was 2.29 h, while the AUC(0-α) was 429.15 ng.h/mL.
Figure 2: MS/MS spectra of curcumin and biochanin showing prominent precursor to product ion transitions.
Figure 3: Typical MRM chromatograms of curcumin (left panel) and IS (right panel) in (a) rat blank plasma, (b) rat plasma spiked with curcumin at LLOQ (10 ng/mL) and
IS (c) a 5 min in vivo plasma sample showing curcumin peak obtained following intravenous administration of curcumin.
Figure 4: Mean ± S.D. plasma concentration-time profile of curcumin in rat
plasma following intravenous administration of curcumin (n = 3).
Table 1: Precision and accuracy data of back-calculated concentrations of
calibration samples for curcumin in rat plasma (n = 3).
Table 2: Intra-day assay precision and accuracy for curcumin in rat plasma (n = 6).
Table 3: Inter-day assay precision and accuracy for curcumin in rat plasma.
Table 4: Stability of curcumin in rat plasma.
Conclusion
In conclusion, we have developed and validated a simple, specific,
accurate and reproducible LC–MS/MS assay to quantify curcumin
using commercially available IS from small volumes of rat plasma.
From the results of all the validation parameters and applicability of the assay, we can conclude that the present method can be useful
for preclinical pharmacokinetic studies of curcumin with desired
precision and accuracy along with high throughput.
Acknowledgements
The authors are thankful to Director, CDRI for his constant encouragement
and support. We also acknowledge Council of Scientific and Industrial Research
(CSIR) for providing research fellowship to S. P. Singh.
US DHHS, FDA, CDER (2001) Guidance for Industry: Bioanalytical Method
Validation. US Department of Health and Human Services, Food and Drug
Administration, Center for Drug Evaluation and Research, Center for Veterinary
Medicine. Available at: http://www/fda.gov/cder/guidance/index.htm
