A liquid chromatography–tandem mass spectrometric method for quantification of curcuminoids in cell medium and mouse plasma
Introduction
Curcumin, a major constituent of turmeric (Curcuma longa L.), has been valued worldwide as a potential food additive, coloring agent and spice, and more importantly showing pharmacological activities such as anti-inflammatory, antimicrobial, antioxidant, anti-parasitic, anti-mutagenic and anti-cancer activities [1], [2], [3], [4]. Due to its potential inhibition of mutagenesis and chemically induced carcinogenesis via perturbation of multiple molecular targets during these processes, it has become a vital lead molecule for the development of various potential anti-cancer agents that can be used for the prevention and/or treatment of various cancers [5], [6], [7], [8].
In the course of discovery and development of novel DNA methylation inhibitors from natural sources, curcumin, its two natural analogs: demethoxycurcumin and bisdemethoxycurcumin, and one of its reduced metabolites tetrahydrocurcumin (THC) have been found as potent DNMT1 inhibitors and a pilot structure–activity relationship study demonstrated that the beta-diketone is an essential pharmacophore of curcuminoids for inhibition of M. SssI [9]. However, they suffer from low oral bioavailability and rapid in vivo metabolism. To circumvent these problems, two curcumin analogs: 1,7-bis (3,4-dimethoxyphenyl)-4,4-dimethyl-1,6-heptadiene-3,5-dione (TMC) and 1,7-bis(3,4-dimethoxyphenyl)-4-cyclohexyl-1,6-heptadiene-3,5-dione (DMCHC) have been synthesized to enhance their stability by blocking the two metabolic sites, the phenolic and C4 methylene moieties. Both compounds have shown inhibitory activity on M. SssI similar to that of curcumin and THC (Poster, M1114, AAPS, 2009). Preclinical pharmacokinetics is yet to be performed.
Several methods based on high performance liquid chromatography (HPLC) [10], [11], [12], capillary electrophoresis [13], liquid chromatography–tandem mass spectrometry (LC–MS/MS) [14] and matrix assisted laser desorption/ionization (MALDI) [15] were reported for quantification of curcumin and its analogs from turmeric powder, food products, rat plasma, and other biological matrices. Recently, a simple and useful method for the analysis of curcumin analogs from rat plasma samples using solvent extraction and negative mode LC–MS/MS was reported [16], and this method can be efficiently used for the rapid screening of curcumin and THC [16]. However, due to conversion of 4-OH group to 4-methoxy group in TMC and DMCHC and the introduction of the hydrophobic moieties in TMC and DMCHC, it is expected that their mass signal in negative mode will be attenuated and their interaction with the surface of a reverse phase C18 column is stronger and results in a rather long retention time, therefore, in this paper, we report a sensitive and rapid LC–MS/MS method for simultaneous determination of these four curcuminoids in cell medium and mouse plasma on a C8 column under a positive mode, and its application in their stability in these matrices and a pilot plasma pharmacokinetic study of curcuminoids in mice.
Section snippets
Reagents and chemicals
Curcumin (95%) was purchased from Acros Chemicals (USA) and is used as such without any further purification. THC, TMC, DMCHC were synthesized and purified as per the respective reported methodologies [17]. Their structures and purities were determined by HPLC, NMR and mass spectrometric methods. All the chemicals were more than 95% pure. Analytical HPLC grade methanol, acetonitrile, ethyl acetate and formic acid were obtained from Fisher Scientific (Waltham, MA, USA). The internal standard
Results and discussion
Curcumin has shown significant anti-cancer activities in various rodent models [1], [3], [4], [6], [7], [18], [19], [20]. It can form complexes with metal ions, e.g. cupuric [21] ion due to the presence of the ortho methoxy, phenolic and diketo groups in the molecule, which has been proposed to be one of the possible mechanisms for its instability when it is added to the solutions containing metal ions like PBS, cell medium, and plasma. Also, curcumin contains two active double bonds, which are
Conclusion
A sensitive LC–MS/MS method for simultaneous quantification of curcumin, THC, TMC and DMCHC with an LLOQ of 5.0 ng/mL for curcumin and 1.0 ng/mL for other three curcuminoid was developed in cell medium and mouse plasma, respectively. The stability study in these matrices at 37 °C using this method demonstrated an increase in stability in the order of curcumin, THC, TMC and DMCHC, which is consistent with their structural optimization to block the three metabolic sites of curcumin: conversion of
Acknowledgements
This work was supported by National Institute of Health (NIH) grants [R21 CA135478] (ZL), grant # IRG-67-003-44 from the American Cancer Society (JRF) and Biomedical Mass Spectrometric Laboratory (KKC and ZL) at The Ohio State University.
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