Research Article

Received: 10 December 2008, Revised: 7 March 2009, Accepted: 9 March 2009 Published online in Wiley Interscience: 1 June 2009

(www.interscience.wiley.com) DOI 10.1002/bmc.1244

Biomed. Chromatogr. 2009; 23: 1201–1207 Copyright © 2009 John Wiley & Sons, Ltd.

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John Wiley & Sons, Ltd.

A rapid and simple HPLC method for the determination of curcumin in rat plasma: assay development, validation and application to a pharmacokinetic study of curcumin liposomeQuantitation of curcumin in rat plasma

Ji Li,a–c† Yunyun Jiang,a–c† Jun Wen,a–c Guorong Fan,a–c* Yutian Wua–c and Chuan Zhanga–c*

ABSTRACT: This paper describes a sensitive, specific and rapid high-performance liquid chromatography (HPLC) method for thedetermination of curcumin in rat plasma. After a simple step of protein precipitation in 96-well format using acetonitrile con-taining the internal standard (IS), emodin, plasma samples were analyzed by reverse-phase HPLC. Curcumin and the IS emodinwere separated on a Diamonsil C18 analytical column (4.6 ×××× 100 mm, 5 μμμμm) using acetonitrile–5% acetic acid (75:25, v/v) asmobile phase at a flow rate of 1.0 mL/min. The method was sensitive with a lower limit of quantitation of 1 ng/mL, with goodlinearity (r2 ≥ 0.999) over the linear range 1–500 ng/mL. All the validation data, such as accuracy and precision, were within therequired limits. A run time of 3.0 min for each sample made high-throughput bioanalysis possible. The assay method wassuccessfully applied to the study of the pharmacokinetics of curcumin liposome in rats. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: curcumin; HPLC; 96-well protein precipitation; pharmacokinetics

Introduction

Curcumin [1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3,

5-dione; Fig. 1] is a phenolic substance derived from the root of

the plant Curcuma longa L., and is used as a dietary spice and a

natural coloring agent for foods. In recent years, it has attracted

interest because of its antioxidant, anti-inflammatory and potential

cancer chemopreventive activities (Sharma, 1976; Ammon and

Wahl, 1991; Conney et al., 1997; Surh, 2003; Payton et al., 2007).

Curcumin is also a potent scavenger of various reactive oxygen

species (ROS) including superoxide anions and hydroxyl radicals

(Ruby et al., 1995). The anti-cancer effect of curcumin may be related

to the inhibition of nuclear factor-κB signaling pathway (Belakavadi

and Salimath, 2005) and activation of apoptotic mechanisms

(Atsumi et al., 2005). In addition, it has been suggested that cur-

cumin may be used for prevention and treatment of Alzheimer

disease by reducing oxidative damage and plaque burden and

suppressing specific inflammatory factors (Lim et al., 2001; Pak

et al., 2003).

Several studies have suggested poor bioavailability of curcumin

in both rodents and humans despite the promising biological

effects that have been observed. The suspected cause may be

Figure 1. Chemical structures of: (a) curcumin, (b) demethoxycurcumin,

(c) bisdemethoxycurcumin, (d) tetrahydrocurcumin and (e) emodin (IS).

* Correspondence to: Guorong Fan, Second Military Medical University,

School of Pharmacy, 325 Guohe Road, Shanghai 200433, People’s Republic

of China. E-mail: guorfan@yahoo.com.cn

Chuan Zhang, School of Pharmacy, Second Military Medical University, No.

325 Guohe Road, Shanghai 200433, People’s Republic of China. E-mail:

zhangchuan@smmu.edu.cn

† Ji Li and Yunyun Jiang contributed equally to this work.

a Department of Pharmaceutical Analysis, School of Pharmacy, Second

Military Medical University, No. 325 Guohe Road, Shanghai 200433, People’s

Republic of China

b Shanghai Key Laboratory for Pharmaceutical Metabolite Research, No. 325

Guohe Road, Shanghai 200433, People’s Republic of China

c Shanghai Research Centre for Drug (Chinese Materia Medica) Metabolism,

No. 325 Guohe Road, Shanghai 200433, People’s Republic of China

Abbreviations used: LLOQ, lower limit of quantitation; MRT, mean residence

time; SD, Sprague–Dawley.

Contract/grant sponsor: Platform on Research of Metabolism Technology of

Traditional Chinese Medicine founded by Science and Technology Depart-

ment of Shanghai, P. R. China; Contract/grant number: 04DZ19216.

J. Li et al.

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poor absorption due to its extremely low aqueous solubility

and/or extensive pre-systemic metabolism (Pak et al., 2003). An

investigation found that it was difficult to detect curcumin in

plasma following a 180 mg oral dose (Sharma et al., 2001). A novel

formulation of curcumin–phospholipid complex was developed

to improve the oral absorption and liver protection of curcumin

in rats (Maiti et al., 2007). In that paper, serum concentration of

curcumin obtained from the curcumin–phospholipid complex

was higher than pure curcumin and the complex maintained

effective concentration of curcumin for a longer period of time

in rat serum. In our study, in order to enhance water solubility

and oral absorption of curcumin, a new preparation of curcumin

liposome was used for intravenous and oral administration to

study the pharmacokinetics of curcumin in rats. The result of

our experiment suggested that the absolute bioavailability of

curcumin was much higher than the reported data (Yang et al.,2007).

The analytical methods used to determine curcumin concen-

trations in biological samples include liquid chromatography

(LC) with UV (Ireson et al., 2001; Pak et al., 2003; Heath et al.,2003; Ma et al., 2007) and tandem mass spectrometric detection

(Liu et al., 2006; Yang et al., 2007). A HPLC-UV assay was developed

by Pak et al. (2003) using three methods of plasma sample prep-

aration in order to quantitate curcumin. The most sensitive sample

pretreatment provided a lower limit of quantitation (LLOQ) of

2.5 ng/mL. However, this sensitive method needed a large sample

volume of 2 mL and the extraction solvent was chloroform, which

is quite toxic. Ma et al. (2007) and Heath et al. (2003) also developed

HPLC-UV analytical methods for determination of curcumin in

biological samples with LLOQs of above 20 ng/mL and the run

times were longer than 11 min. Liu et al. (2006) reported a LC-

MS/MS method for quantification of curcumin and its metabo-

lite tetrahydrocurcumin in rat plasma with a sample volume of

100 μL to achieve an LLOQ of 0.5 ng/mL and an mean extraction

recovery of 77.15%. In this method the plasma samples were

incubated to hydrolyze the crucumin and tetrahydrocurcumin

conjugates first, and then ethyl acetate was used to perform liquid–

liquid extraction. The sample pretreatment was complicated and

time-consuming. Yang et al. (2007) developed another LC-MS/

MS method to study oral bioavailability of curcumin in rat with

a LLOQ of 5 ng/mL and a run time of 5 min. Still another paper

described a method based on matrix-assisted laser desorption

ionization time-of-flight mass spectrometry for the study of cur-

cumin and its metabolites in mouse serum and mouse lung cell

cultures (May et al., 2005). Although LC-MS/MS method can pro-

vide excellent sensitivity and short run time, the apparatus is

expensive and the matrix effects are difficult to overcome. In this

study we successfully developed a fast, simple and sensitive

method based on protein precipitation in 96-well format and

HPLC-PDA for the determination of curcumin in rat plasma. After

full validation, the method was successfully applied to the study

of the pharmacokinetics of curcumin liposome in rats.

Experimental

Chemicals and Reagents

Curcumin and emodin (internal standard, IS, Fig. 1) were obtained

from the National Institute for the Control of Pharmaceutical and

Biological products (Beijing, PR China). The purities of curcumin

and emodin were >99.5%. Tetrahydrocurcumin with a purity

of >95.0% and a mixture containing 5% bisdemethoxycurcumin

(Fig. 1), 15% demethoxycurcumin (Fig. 1) and 80% curcumin were

supplied by Shineway Pharmaceutical Co. Ltd (Shijiazhuang, PR

China). Preparation of curcumin liposome was supplied by School

of Pharmacy, Second Military Medical University (Shanghai, PR

China). Acetonitrile (chromatographic grade) was purchased

from Baker (USA). Acetic acid was analytical grade and was from

Shanghai Chemical Reagent Company (Shanghai, PR China).

Deionized (18.2 MΩ/cm) water was generated in-house using a

Milli-Q System from Millipore (Bedford, MA, USA).

Apparatus

The analysis was carried out on a Varian HPLC system (Palo Alto,

CA, USA) consisting of a ProStar 430 autosampler and ProStar

210 pumps, The system also included a Photodiode Array (PDA)

detector and a computer running Varian workstation version

6.42 software for data acquisition and processing.

Liquid Chromatographic Conditions

The chromatographic separation was performed on a Diamonsil

C18 column (4.6 × 100 mm, 5 μm particle size, Dikma Technologies,

Beijing, China) with the mobile phase composed of acetonitrile–

5% acetic acid (75:25, v/v) at a flow rate of 1.0 mL/min. The mobile

phase was filtered through a 0.45 μm nylon membrane filter and

ultrasonically degassed prior to use. The wavelength of the detec-

tion was at 420 nm. The injection volume was 50 μL and the

analysis time was 3 min per sample.

Preparation of Stock Solutions, Calibration Samples and

Quality Control Samples

Stock solutions of curcumin and IS were prepared in acetonitrile

at concentrations of 0.5 mg/mL and 1.5 mg/mL, respectively. The

curcumin stock solution was diluted with acetonitrile to working

solutions ranging from 0.01 to 5 μg/mL. IS working solution

(0.15 μg/mL) was also prepared by diluting the stock solution

with acetonitrile. All described solutions were protected from

light and stored at 4°C. Calibration samples (1, 2, 5, 10, 20, 50, 100,

200 and 500 ng/mL) were prepared by spiking 10 μL of each work-

ing solution (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2 and 5 μg/mL) into

90 μL of rat blank plasma. For each validation and assay run, the

calibration curve standards were prepared fresh from the working

solutions. These standards were used to construct calibration

curves for the quantitation of curcumin at plasma concentrations

ranging from 1 to 500 ng/mL. Samples found to contain curcumin

at concentrations above 500 ng/mL were diluted appropriately

with rat blank plasma and re-assayed. Quality control samples

were independently prepared at three level concentrations of

2, 20 and 400 ng/mL. The QC samples were stored at −20°C and

brought to room temperature before use.

Extraction Procedure

Samples were prepared using protein precipitation in 96-well

format plate (1 mL, Varian, Inc., Palo Alto, CA, USA). An eight-channel

100 μL pipetting tool (Eppendorf Research®, Eppendorf AG, Ham-

burg, Germany) was used for liquid transfer steps. All the plasma

samples and standard working solutions were warmed to room

temperature before use. Aliquots of 90 μL rat blank plasma were

transferred to 96-well plates and 10 μL of standard working solu-

tions were added to 90 μL of rat blank plasma to make the fresh

Quantitation of curcumin in rat plasma

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calibration standards. A sum of 100 μL aliquots of plasma samples

and three levels of QC samples, respectively, were also pipetted

into 96-well plates. Using an eight-channel 100 μL pipetting tool,

100 μL aliquot of acetonitrile containing 0.15 μg/mL of IS was

added to each sample (standards, QCs and real samples) in order

to precipitate the plasma protein. Plates were capped and mixed

by vortex for 5 min and then subjected to centrifuge at 2500g

for 15 min to remove any precipitated material. A 100 μL aliquot of

the supernatant was transferred to another 96-well plate (350 μL),

this 96-well plate was covered to the autosampler and 50 μL of

the mixture was injected into the HPLC system.

Pharmacokinetic Study in Rats

Twelve male Sprague–Dawley rats (220–250 g) were obtained

from Shanghai SLAC Laboratory Animal Co. Ltd. They were kept

in an environmentally controlled breeding room (temperature:

22 ± 2°C, humidity: 60 ± 5%, 12 h dark–light cycle) with free

access to standard laboratory food and water for 3 days before

starting the experiment. They were fasted overnight before

dosing. Animal welfare and experimental procedures were

strictly in accordance with the guide for the care and use of

laboratory animals and the related ethical regulations of Second

Military Medical University.

Twelve rats were randomly separated into two groups. One

group of rats received curcumin liposome dissolved in normal

saline (5 mg/kg curcumin) intravenously via the tail vein, and the

other group was given curcumin liposome dissolved in normal

saline (100 mg/kg curcumin) orally. Animals had access to water

and food 4 h after drug administration. Through the catheters

which had been implanted into the right external jugular vein

of rats, blood samples (0.3 mL) were collected into heparinized

tubes at 0, 2, 5, 10, 20 min, 0.5, 0.75, 1, 2, 3, 4 and 6 h after intra-

venous injection or at 5, 10, 15 min, 0.5, 0.75, 1, 2, 3, 4, 6, 8 and 12 h

after oral administration. A 0.3 mL of normal saline was admin-

istered to compensate for the blood loss after every blood

sample. The plasma was separated from heparinized blood by

centrifugation and stored at −20°C prior to analysis.

Results and Discussion

Method Development

HPLC-UV conditions. An appropriate wavelength was impor-

tant for good sensitivity. It is shown in Fig. 1 that curcumin has a

special conjugation structure which leads to strong UV absorption

at the wavelength of 420 nm. Therefore the detection wavelength

was set at 420 nm. It was necessary to use an IS in extraction

techniques and HPLC method to compensate for extraction vari-

ation, efficiency and analytical errors. Emodin was adopted as

the IS in this study for the reasons that it is structurally similar

to curcumin and its behavioral characteristics and properties

conform to the chemical requirement for IS in HPLC. In addition,

emodin is commercially available in high purity, and it is stable

and nonreactive with sample or mobile phase. Meanwhile, it

also has good response at the detection wavelength of 420 nm.

The mobile phase was optimized through several trials to achieve

good resolution and symmetric peak shapes for both analyte and

IS, as well as a short run time. Acetonitrile has the least viscosity

compared with tetrahydrofuran and methanol, and it has strong

eluting power. Therefore, it was selected as organic phase. Accord-

ing to the chemical characteristics of curcumin and IS, appropriate

concentration of acetic acid added into mobile phase was

necessary for good peak shapes. It was found that a mixture of

acetonitrile–5% acetic acid (75:25, v/v) could achieve the above

purpose and was finally adopted as the mobile phase.

In our early study, several kinds of columns were compared,

such as Diamonsil C18 column (4.6 × 200 mm, 5 μm), Lichrospher

C18 column (4.6 × 150 mm, 5 μm), Agilent Zorbax SB-C18 column

(4.6 × 150 mm, 3.5 μm) and Waters Symmetry C18 column (4.6 ×150 mm, 5 μm). However, the retention times of curcumin and IS

were longer and the shapes of the peaks were not sharp enough.

After careful comparison, a Diamonsil C18 analytical column

(4.6 × 100 mm, 5 μm) was finally used with a flow rate of 1 mL/min

to produce good peak shapes and permit a run time of 3 min.

Sample Pre-treatment

Because of the complex nature of plasma, a pre-treatment pro-

cedure is often needed to remove protein and potential interferences

prior to HPLC analysis. The instrumentation is not the limiting

factor in high-throughput analysis, but the sample preparation.

Unlike liquid–liquid extraction and solid-phase extraction, protein

precipitation is commonly used for fast sample clean-up and

disrupting protein–drug binding. In order to increase sample

throughput, the protein precipitation in 96-well format plates

was used, which resulted in shorter sample preparation time.

Method Validation

The method was validated according to the guidelines of the

main regulatory agencies, such as those issued by the UnitedStates Pharmacopeia (United States Pharmacopeial Convention,

2005) and by the US Food and Drug Administration (US DHHS,

FDA, CDER, 2001). The validation experiments and results

obtained are described below.

Specificity. Specificity is described as the ability of a method

to discriminate the analyte from all potentially interfering sub-

stance. The specificity of the method was investigated by blank

plasma detection, peak purity and spiking experiments with

pure standard compounds. Figure 2(a) shows that blank rat

plasma had no interference when curcumin and the IS eluted.

Peak purity was evaluated by means of the Varian workstation

version 6.42 software. The total peak purity values of curcumin

and the IS were 1.0000 for both spiking and real samples. To avoid

the potential interference of other curcuminoids in the prepara-

tion, such as demethoxycurcumin and bisdemethoxycurcumin,

mixtures containing 5% bisdemethoxycurcumin, 15% demeth-

oxycurcumin and 80% curcumin were analyzed. Figure 2(b)

indicates that demethoxycurcumin and bisdemethoxycurcumin

could be separated from curcumin under the optimized chroma-

tography conditions. Previous reports (Pan et al., 1999; Liu et al.,2006) suggested that tetrahydrocurcumin may be the main

metabolite of curcumin in rat plasma. Tetrahydrocurcumin has

no such special conjugation structure as curcumin, thus it has

no UV absorption at the wavelength of 420 nm. That is to say,

tetrahydrocurcumin does not interfere with the determination

of curcumin under this chromatography condition [Fig. 2(c)].

Sensitivity and linearity. The LLOQ of the assay, defined as the

lowest concentration on the standard curve that can be quan-

titated with accuracy within 20% of nominal and precision not

exceeding 20% CV, was 1 ng/mL for curcumin. The reproducibility

J. Li et al.

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of LLOQ was determined by examining five LLOQ samples inde-

pendent of the standard curve, and the accuracy and precision

were 99.9 and 14.9%, respectively. A typical chromatogram of an

LLOQ sample is shown in Fig. 2(d).

The linearity of an analytical procedure is its ability to obtain

test results which are directly proportional to the concentration

of analyte in the sample. The linearity of the assay for the test

compounds was evaluated with a total of five calibration standards.

Calibration curves consisted of nine concentrations of curcumin

spiked in rat plasma: 1, 2, 5, 10, 20, 50, 100, 200 and 500 ng/mL.

The extraction procedure and HPLC analysis described above

were performed on the calibration samples.

Calibration curves were constructed by plotting the peak area

ratios (curcumin/IS) of plasma standards versus nominal concen-

tration. A weighted (1/x) linear regression was used to perform

standard calibration, giving a mean linear regression equation

for the calibration curve of: y = 0.02106 x + 0.04357, r 2 = 0.9997,

where y represents the peak area ratios of curcumin to that of IS,

and x represents the plasma concentration of curcumin in ng/mL.

Calibration curves of five different lots of plasma were linear in

the range of 1–500 ng/mL with r2 ≥ 0.999.

Accuracy and precision. The precision of an analytical procedure

expresses the closeness of agreement between a series of meas-

urements obtained from multiple sampling of the same homo-

geneous sample, while the accuracy of an analytical method

describes the closeness of the test results obtained by the

method to the normal value of the analyte. The intra-day accuracy

Figure 2. Representative chromatograms: (a) double blank plasma; (b) a mixture solution containing 13 ng/mL bisdemethoxycurcumin, 38 ng/mL

demethoxycurcumin and 200 ng/mL curcumin in methanol; (c) tetrahydrocurcumin in methanol at a concentration of 200 ng/mL; (d) blank plasma

spiked with 1 ng/mL curcumin and 150 ng/mL of IS; (e) blank plasma spiked with 10 ng/mL curcumin and 150 ng/mL of IS; (f ) plasma sample collected

from a rat 45 min after receiving a 100 mg/kg oral administration of curcumin liposome. The assayed concentration of curcumin in this sample was

11.8 ng/mL. (1) Curcumin; (2) emodin (IS); (3) bisdemethoxycurcumin; (4) demethoxycurcumin.

Quantitation of curcumin in rat plasma

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and precision of the assay were determined by analyzing replicates

(n = 5) containing curcumin at three different concentration levels.

The inter-day accuracy and precision were determined by analyz-

ing three concentrations of QC samples, five times at each con-

centration. Table 1 presents the intra- and inter-day accuracy

and precision for each of the QC samples. Our intra- and inter-

day accuracy and precision (%CV) acceptance criterion for each

QC was ≤15%. The assay successfully met the criterion.

Extraction recovery. To investigate extraction recovery, a set

of samples (n = 5 at each concentration in unique lots of plasma)

was prepared by spiking curcumin into plasma at 2, 20, and

400 ng/mL. Each of the samples (100 μL) was vortex-mixed with

100 μL IS at the working concentration of 150 ng/mL, then proc-

essed using the procedure described previously. A second set

of plasma samples was processed and spiked post-extraction

with the same concentrations of curcumin and IS that actually

existed in the pre-extraction spiked samples. Extraction recovery

for each analyte was determined by calculating the ratios of the

raw peak areas of the pre-extraction spiked samples to those of

the samples spiked after extraction. Mean extraction recoveries

of curcumin at concentrations of 2, 20 and 400 ng/mL were 95.7 ± 5.6,

97.7 ± 5.8 and 95.6 ± 5.0%, respectively, and the extraction

recovery of the IS was 96.6 ± 8.4%.

Stability. Bench-top stability was investigated to ensure that

curcumin was not degraded in plasma samples at room temper-

ature for a time period to cover the sample preparation, and was

assessed by exposing the QC samples to ambient laboratory

conditions for 4 h. Freeze–thaw stability was assessed over three

cycles. QC samples were thawed at room temperature and

refrozen at −20°C over three cycles and assayed. Because of the

need for occasional delayed injection or reinjection of extraction

samples, the stability of reconstituted samples in autosampler

vials was assessed at ambient temperature for 24 h. The freezer

storage stability of curcumin in rat plasma at −20°C was evalu-

ated by assaying QC samples at beginning and 2 weeks later. All

stability QC samples were analyzed in five replicates. The results

indicated that curcumin had an acceptable stability under those

conditions, as shown in Table 2.

Table 1. Intra- and inter-day accuracy and precision of curcumin (n = 5)

QC sample Nominal

concentration

(ng/mL)

Mean measured

concentration

(ng/mL)

Accuracy

(%)

Precision

(CV %)

Intra-dayLQC 2 2.12 106.23 7.51

MQC 20 18.83 94.15 4.26

HQC 400 402.81 100.70 1.74

Inter-dayLQC 2 2.14 107.05 9.24

MQC 20 21.13 105.65 5.77

HQC 400 403.62 100.92 3.37

Table 2. Stability of curcumin in rat plasma (n = 5)

Sample condition Nominal

concentration

(ng/mL)

Mean determined

concentration

(ng/mL)

CV

(%)

Accuracy

(%)

2 1.92 6.53 96.00

Bench-top stabilitya 20 20.80 2.41 104.00

400 405.65 1.95 101.41

2 2.22 3.82 111.00

Autosampler stabilityb 20 21.05 1.75 105.25

400 407.37 1.63 101.84

Freeze–thaw stabilityc 2 2.13 6.55 106.50

20 20.92 3.32 104.60

400 405.61 5.67 101.40

2 1.81 7.06 90.50

Two-week stabilityd 20 18.83 3.38 94.15

400 397.86 1.86 99.47

a Exposed at ambient temperature (25°C) for 4 h.b Kept at ambient temperature (25°C) for 24 h.c After three freeze–thaw cycles.d Stored at −20°C.

J. Li et al.

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Sample dilution. To investigate the ability to dilute and analyze

samples containing curcumin at concentrations above the assay

upper limit of quantitation, a set of plasma samples was pre-

pared containing curcumin at a concentration of 2000 ng/mL,

and placed in a −20°C freezer overnight prior to analysis. After

thawing, a 20 μL aliquot was withdrawn for analysis (n = 5),

diluted with 80 μL of control rat plasma, vortex for 30 s, then

treated as described above. The accuracy of the test was 99.7%

with a good precision (CV = 1.4%).

Application of the Assay

The method described above was applied to study pharmacoki-

netics of curcumin after a single intravenous (5 mg/kg) or oral

dose administration (100 mg/kg) of curcumin liposome in rats.

The representative chromatogram of a plasma sample, which

was collected from a Sprague–Dawley (SD) rat 45 min after receiv-

ing a 100 mg/kg oral administration of curcumin liposome, is

shown in Fig. 2(f ). The mean plasma concentration–time profiles

of curcumin after an intravenous or oral administration of curcumin

liposome are shown in Fig. 3. To determine the pharmacokinetic

parameters of curcumin liposome, the concentration–time data

were analyzed by non-compartmental methods using the Bioa-

vailability Program Package (BAPP, Version 2.0, Center of Drug

Metabolism and Pharmacokinetics, China Pharmaceutical Uni-

versity). The maximum plasma concentration (Cmax) and the time

to reach Cmax (Tmax) were obtained directly from the concentration–

time data. The area under the plasma concentration–time curve

from time zero to the last sampling time (AUC0−t) was calculated

by the trapezoidal rule. The terminal phase was determined by

visual inspection of the log-transformed concentration–time

data. The elimination rate constant, kel, was obtained from linear

regression analysis of the terminal log–linear phase of the con-

centration vs time curve. Plasma AUC0−∞ values were estimated

by the combination of AUC0−t and AUCt−∞, where AUCt−∞ repre-

sents the residual area of drug from time t to infinity and were

calculated by dividing the last plasma concentration value

measured by the elimination rate constant. The elimination half-

life (t1/2) was calculated as 0.693 divided by kel. The mean resi-

dence time (MRT) was estimated from AUMC/AUC, where AUMC

is area under the first moment curve. The main pharmacokinetic

data after curcumin liposome administration (5 mg/kg, i.v. and

100 mg/kg, p.o.) in rats are shown in Table 3. Compared with

the reported data, it took less time to reach Cmax and the t1/2 was

much longer in our experiments (Yang et al., 2007). In this study

the absolute bioavailability of curcumin liposome in a freely

moving rat was about 2.7% while the data in the reported paper

was about 1% (Yang et al., 2007). The special preparation of

Figure 3. (a) Mean plasma concentration–time curve in six rats when administered 5 mg/kg curcumin lipo-

some i.v. (with log-transform scale inset). (b) Mean plasma concentration–time curve in 6 rats when adminis-

tered 100 mg/kg curcumin liposome p.o. (with log-transform scale inset).

Quantitation of curcumin in rat plasma

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curcumin liposome enhanced water solubility and oral absorp-

tion of curcumin and this may be the main reason for the higher

bioavailability (Lin et al., 2007; Chen et al., 2009; Meng et al.,2008).

Conclusion

A rapid, sensitive and specific HPLC-UV method has been developed

for the determination of curcumin in rat plasma. The adequate

selectivity, sensitivity, precision, accuracy and appropriate reten-

tion time make it suitable for high-throughput pharmacokinetic

study. Because of the simple HPLC conditions and straightfor-

ward sample pre-treatment procedure, the method is easy and

fast to perform. Finally, the method has been successfully applied

to the pharmacokinetic study of curcumin liposome in rats and

the absolute bioavailability of curcumin liposome in rats was

about 2.7%.

Acknowledgements

This work was supported by Platform on Research of Metabolism

Technology of Traditional Chinese Medicine founded by Science

and Technology Department of Shanghai, P. R. China (04DZ19216).

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Table 3. Pharmacokinetic parameters of curcumin in male

SD rats generated by non-compartmental analysis

Paramenters Curcumin

i.v. (n = 6) Oral (n = 6)

Dose (mg/kg) 5 100

C0 (ng/mL) 3527.91 ± 1145.01 —

Cmax (ng/mL) — 42.67 ± 13.23

Tmax (h) — 0.30 ± 0.10

t1/2 (h) 1.57 ± 0.16 4.83 ± 0.57

MRT (h) 0.81 ± 0.31 5.58 ± 0.95

AUC0–τ (h ng/mL) 168.69 ± 50.79 84.58 ± 11.58

AUC0–∞ (h ng/mL) 172.86 ± 50.99 98.59 ± 15.59

Data are expressed as mean ± SD.