Determination of Curcumin in Rat Plasma by Liquid–liquid Extraction using LC–MS/MS with Electrospray IonizationAssay Development, Validation and Application to a Pharmacokinetic Study

Curcuma spp. contain turmerin, essential oils, and curcuminoids, including curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-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.


Introduction
Curcuma spp. contain turmerin, essential oils, and curcuminoids, including curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-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

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 L aliquots of the processed samples on a Supelco Discovery C18 column (4.6 × 50 mm, 5.0 m). 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 m 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 (

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 g/mL. The IS primary stock solution of 1000 g/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 g/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 g/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 g/mL) was prepared in methanol from primary stock solution of 1000 g/mL in methanol. Calibration standards and QC samples were prepared by spiking 90 L of control pooled rat plasma with the appropriate working solution of curcumin (10 L) and IS (10 L) 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 L of plasma in a tube, 10 L of IS solution (biochanin at 1 g/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 L of the mobile phase and 10 L 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  [13][14][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/X 2 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 (100L) 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.

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 m).

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 plasma calibration curve was constructed using eight calibration standards (viz., 10-2000 ng/mL). The calibration standard curve had a reliable reproducibility over the standard concentrations across the calibration range. 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/X 2 ). 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.  Table 2: Intra-day assay precision and accuracy for curcumin in rat plasma (n = 6).