Development of a validated UPLC-qTOF-MS Method for the determination of curcuminoids and their pharmacokinetic study in mice

Background A specific and sensitive UPLC-qTOF-MS/MS method has been developed for the simultaneous determination of curcuminoids. These Curcuminoids comprises of curcumin, a principal curcuminoid and other two namely, demethoxycurcumin, and bisdemethoxycurcumin obtained from rhizomes of Curcuma longa an ancient Indian curry spice turmeric, family (Zingiberaceae). Methods These analytes were separated on a reverse phase C18 column by using a mobile phase of acetonitrile: 5% acetonitrile in water with 0.07% acetic acid (75:25 v/v), flow rate of 100 μL/min was maintained. The qTOF-MS was operated under multiple reaction monitoring (MRM) mode using electro-spray ionization (ESI) technique with positive ion polarity. The major product ions in the positive mode for curcuminoids were at m/z 369.1066, 339.1023 and 309.0214 respectively. The recovery of the analytes from mouse plasma was optimized using solid phase extraction technique. Results The total run time was 5 min and the peaks of the compounds, bisdemethoxycurcumin, demethoxycurcumin and curcumin occurred at 2.06, 2.23 and 2.40 min respectively. The calibration curves of bisdemethoxycurcumin, demethoxycurcumin and curcumin were linear over the concentration range of 2–1000 ng/mL (r2, 0.9951), 2–1000 ng/mL (r2, 0.9970) and 2-1000 ng/mL (r2, 0.9906) respectively. Intra-assay and inter-assay accuracy in terms of % bias for curcumin was in between −7.95to +6.21, and −7.03 to + 6.34; for demethoxycurcumin was −6.72 to +6.34, and −7.86 to +6.74 and for bisdesmetoxycurcumin was −8.23 to +6.37 and −8.47 to +7.81. The lower limit of quantitation for curcumin, demethoxycurcumin and bisdemethoxycurcumin was 2.0 ng/mL. Analytes were stable under various conditions (in autosampler, during freeze-thaw, at room temperature, and under deep-freeze conditions). This validated method was used during pharmacokinetic studies of curcumin in the mouse plasma. Conclusions A specific, accurate and precise UPLC-qTOF-MS/MS method for the determination of curcumin, demethoxycurcumin and bisdemethoxycurcumin both individually and simultaneously was optimized.


Background
Curcuma longa L. (Zingiberaceae) is a coloring agent, has been found to be a rich source of phenolic compounds, namely, curcuminoids (2-5%) [1]. C. longa consists of a mixture of three naturally occurring curcuminoids.
Curcuminoids are recognized for their broad spectrum biological activities and have been generally regarded as safe (GRAS) in foods or pharmaceuticals. Curcumin is widely used for coloring of foods like pickles and snacks.
Many pharmacological properties have been attributed to curcuminoids including anti-inflammatory and hepatoprotective activities [2], antioxidant and cholekinetic activities [3,4] and anti-protease activity [5,6]. In addition, apoptosis have been shown to induce in human cancer cells by the curcuminoids [7] and act as a chemopreventive agents for major types of cancer, including the stomach, lung, breast, prostate, colon and duodenal cancers, as well as leukemias [8][9][10][11][12] and display neuroprotective effects [13]. Curcumin has also been reported a more potent free radical scavenger than vitamin E [14].
It is also known for its potential use of curcumin in the treatment of infections such as human immunodeficiency virus (HIV) is also reported [15].
Quantification of the active metabolite, THC in plasma and urine by HPLC method has also been reported [16] and simultaneous quantification of diferuloylmethane and its metabolites in biological matrices has been reported by LC/MS/MS [17].
In recent times UPLC with qTof-MS is widely considered analytical technique for better quality data in terms of increased detection limits, and chromatographic resolution with greater sensitivity. This paper presents (i) a method for the simultaneous determination of curcuminoids by UPLC-qTOF-MS, and (ii) a pharmacokinetic study of curcumin in mice.

Material and methods
Curcumin, demethoxycurcumin and bis-demethoxycurcumin used as standards were isolated from the rhizomes of C. longa by the method already reported in literature. The isolated curcuminoids were identified on the basis of NMR and Mass spectral data. The purity of standards was >99%. All solvents/chemicals used were of HPLC grade and obtained from E-Merck, Mumbai, India. The HPLC grade water was obtained from a Water Purification System (Synergy UV, Millipore, USA).

Preparation of reference, standard and quality control solutions
Reference solutions of curcumin (C) (stock I), demethoxycurcumin (DMC), (stock II) and bisdemethoxycurcumin (BDMC) (stock III) were prepared by weighing 5 mg of each compound. The quantities were transferred to 5 mL volumetric flasks, dissolved and diluted suitably with HPLC grade methanol. All the reference solutions (1 mg/mL) were covered with aluminium foil and sealed with paraffin film to avoid photodegradation and loss due to evaporation. Stock I, stock II and stoke III were mixed together, and diluted suitably with methanol. A 50-uL of this solution was used to spike blank mouse plasma samples (450 uL) to achieve 8 calibration standards (CAL STD) containing curcuminoids combination. CAL STD-1: curcuminoids, 2 ng/mL; CAL STD-2: 5 ng/mL each; CAL STD-3: 10 ng/mL each; CAL STD-4: 50 ng/mL each; CAL STD-5: 100 ng/mL each; CAL STD-6: 200 ng/mL each; CAL STD-7: 500 ng/mL each; and CAL STD-8: 1000 ng/mL each. Three quality control (QC) standards (LQC: 2 ng/mL; MQC: 450 ng/mL; HQC 900 ng/mL each of curcuminoids) were prepared and used to spike blank mouse plasma.

Method validation procedures
The analytical method was validated to meet the acceptance criteria as per guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). The specificity of the method was established by comparing blank plasma samples with those spiked with the analytes to find out interference from endogenous components. The CAL STD solutions were utilized for establishment of linearity and range (linear least-squares regression with a weighting index of 1/x). The precision and accuracy parameters were ascertained in LLOQ, LQC, MQC, and HQC samples (7 replicates each in 3 sets) on the same day and on 3 consecutive days. The intra-assay and inter-assay accuracy (% bias) of the method was determined from mean measured concentrations and nominal concentrations as follows: % bias = [(mean measured conc.−nominal conc.)/ nominal conc.]×100. The intra-assay and inter-assay precision (% relative standard deviation or RSD) of the method was calculated from mean measured concentrations as follows: % RSD = (SD of mean measured conc./ mean measured conc.)×100. The stability of analytes in plasma was investigated under following conditions: (a) 1 month storage at deep freeze (−80°C); (b) 3 consecutive freeze-thaw cycles from −20°C to room temperature; (c) 24 h storage at room temperature; and (d) short-term stability (of processed samples) at 10°C for 24 h in autosampler. After specified storage conditions, samples were processed and analyzed. The matrix effect was investigated by post extraction spike method. Peak area (A) of the analyte in spiked blank plasma with a known concentration (MQC) was compared with the corresponding peak area (B) obtained by direct injection of standard in the mobile phase. The ratio (A/B×100) is defined as the matrix effect.

Sample preparation
The curcumin (C), demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) were recovered simultaneously from plasma using solid phase extraction (SPE) technique involving semi-automated vacuum chamber and vacuum pump (Supelco, USA). The various steps involved in the recovery procedure were: (a) conditioning of SPE cartridge (C18, 3 mL capacity, 100 mg bed, Samprep-Ranbaxy, Mumbai, India) with 1.0mLmethanol, followed by 1.0 mL water, (b) loading of diluted (1:4, v/v) plasma samples (1.0 mL) onto cartridge and drying under positive pressure, and (c) samples were washed with 2 mL of water followed by elution with 2 mL of methanol. The eluants were carefully collected in 2.0 mL capacity glass vials for direct analysis in UPLC-qTOF-MS system.

Experimental animals
Swiss mice (22-30 g) were obtained from the Animal House of this Institute, and kept in regulated environmental conditions (temperature: 25 ± 2°C, humidity: 60 ± 5%, 12 h dark/light cycle). Animals were fed on standard pelleted diet (Ashirwad Industries, Chandigarh, India) and water was provided ad libitum. Animal experiments were approved by Institutional Ethics Committee. Animals were fasted overnight before the experiment and segregated into different groups for the sample collections at different time intervals. All these animals were administered with curcumin (100 mg/kg, p.o.). Blood samples were collected in pre-heparinized glass tubes at different time intervals post dosing (0-24 hr). Blood samples were centrifuged (5000 rpm; 10 min at 20°C) to separate the plasma.

Pharmacokinetics
Concentration-time curves for Concentration-time curves were established for curcumin from the treated mice and used for the determination of pharmacokinetic parameters such as peak plasma concentration (Cmax), peak time (Tmax), extent of absorption (AUC), half-life (t1/2), clearance (Cl), and volume of distribution (Vd) by a non-

UPLC-qTOF-MS/MS analysis
Optimum chromatographic separation of curcuminoids was achieved by acetonitrile: 5% acetonitrile in water with 0.07% acetic acid (75:25 v/v). Flow rate of 100 μL/min was maintained. All the analytes were added simultaneously in the samples and the resulting chromatograms showed a retention time of 2.06, 2.23 and 2.40 min for bisdemethoxycurcumin, demethoxycurcumin and curcumin respectively ( Figure 1A Figure 2C).

Method validation Specificity
The method was found to be specific: Extracted blank plasma when compared with plasma samples spiked with curcuminoids did not show any interference at the respective retention times of each analyte.
Intra-assay and inter-assay precision (% RSD) were presented in Table 2.
The accuracy and precision of the method were within the acceptable limits of ±15%.

Stability
The stability of the analytes in plasma was investigated in LQC and HQC samples. The recoveries of the analytes after one month (storage stability), after 1, 2 and 3 cycles of freeze-thaw and after 24 h (stability at room temp.) relative to that at time zero are summarized in Table 3.

Pharmacokinetics
Concentration vs. time profile of Curcumin and pharmacokinetic parameters (Figure 3, Table 4). Each time point is mean±SE (n = 6). For details refer Section 1.6.

Discussion
Three curcuminoids were used as a standard in the present study showed separate peaks in the extracted ion chromatogram (EIC) at 2.06, 2.23 and 2.40 min for bisdemethoxycurcumin demethoxycurcumin and Curcumin respectively. The bisdemethoxycurcumin demethoxycurcumin and Curcumin, were also appeared in the  total ion chromatogram TIC. These three curcuminods were also shown together in the ESI spectra ( Figure 4). A method for the determination of curcumin, demethoxycurcumin and bisdemethoxycurcumin by UPLC-qTOF-MS/MS has not been reported, prior to this investigation, in which curcuminoids have been quantified on the basis of their major fragment. Previously reported HPLC-UV methods for the quantification and determination of curcuminoids have several disadvantages, such as unsatisfactory separation times (needs more analysis time), poor resolution and complicated solvent mixtures with gradient elution. These methods are not selective, rapid, so a timeconsuming pretreatment of a sample, or complicate gradient elution is required.
We have developed a simple, reliable and an isocratic UPLC-qTOF-MS/MS method which require only binary solvent system containing water and acetonitrile. This method has shown high degree of simplicity, accuracy, sensitivity, reproducibility and also provides short analysis time (5 min.). In the proposed method the linearity was in the range between 2 ng/mL to 1000 ng/mL which makes the method most suitable for the trace quantification of analytes. This method can also be used for the quantification of individual curcuminoids for routine analysis.
The method was validated in terms of specificity, accuracy, precision, sensitivity and stability of the analytes, and utilized for the determination of curcumin, demethoxycurcumin and bisdemethoxrcurcumin either individually or simultaneously in plasma (mice). After oral administration curcumin could be quantified only up to 24 h of sampling time. A pharmacokinetic parameters from plasma concentration-time data usually involves the maximum  Figure 4 The mass spectrum of curcuminoids in combination showing the molecular ion peaks, chemical structures and molecular weights.
(peak) plasma drug concentrations (C max ) and the area under the plasma concentration -time curve (AUC). The plasma drug concentration increases with the rate of absorption; therefore the most widely used general index of absorption is C max . AUC is another reliable measure for the extent of absorption. It is directly proportional to the total amount of unchanged drug that reaches systemic circulation.

Conclusions
A specific, accurate and precise UPLC-qTOF-MS/MS method for the determination of curcumin, demethoxycurcumin and bisdemethoxycurcumin both individually and simultaneously was optimized. Pharmacokinetic study of curcumin was carried out by using this validated method.