Validated LC/MS/MS assay for curcumin and tetrahydrocurcumin in rat plasma and application to pharmacokinetic study of phospholipid complex of curcumin

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Abstract

To study pharmacokinetic properties of curcumin, a fast sensitive assay method was developed to determine curcumin and its metabolite tetrahydrocurcumin in rat plasma. The assay was based on tandem mass spectrometry detection (LC/MS/MS). Salbutamol was used as the internal standard (IS). The method had the lower limit of quantitation (LLOQ) of 0.5 ng/ml in rat plasma, which corresponds to 2.5 pg for the 5 μl injection volume. Good linearity was got to 500 ng/ml. The precision, accuracy, recovery and applicability were found to be adequate for pharmacokinetic studies.

Phospholipid complex of the natural compound curcumin was prepared in order to improve its bioavailability. Complex formation resulted in an obvious increase in bioavailability of curcumin in rat in vivo according to the assay by above LC/MS/MS method.

Introduction

Curcumin, the yellow pigment in Turmeric, is a naturally occurring polyphenolic phytochemical isolated from the powdered rhizome of the plant Curcuma Longa that possesses anti-inflammatory properties [1] and inhibits cancer formation in mice [2]. The National Cancer Institute is currently developing curcumin as an anticancer agent [3].

The absorption, metabolism, and tissue distribution of curcumin after oral administration of 400, 80 and 10 mg of [3H] curcumin in rats [4], [5], [6] has been studied. Chuang et al. [7] showed that tetrahydrocurcumin, β-glucuronidic conjugates of curcumin and tetrahydrocurcumin are the major metabolites of curcumin in rat in vivo, and curcumin has low bioavailability in rat. The possible application of this compound in therapy is hampered by its poor absorption. Since 1980s, phospholipid complex began to be a common try to increase drugs’ bioavailability since it can improve the gastrointestinal absorption to achieve higher drug concentration in plasma and lower kinetic elimination (Ke), and the technique is easy to practice by using suitable solvent treatment [8]. Several natural drugs, such as silymarin [9], dolichol [10], sapinins from Centella asiatica [11], has been found obvious bioavailability improvement through phospholipid complex formulation. In this paper, we attempted to improve the bioavailability of curcumin in vivo by preparation of its phospholipid complex and a simple method was developed to simultaneously determine curcumin and tetrahydrocurcumin in plasma after oral administration of curcumin phospholipid complex. The method requires an HPLC with mass spectrometric detection. Mass spectrometric detection with ESI offers a detection technique capable of analyzing both curcumin and tetrahydrocurcumin with high sensitivity. The combination with liquid chromatography allows the simultaneous analysis of both analytes within a 5 min run time. The precision, accuracy, recovery and applicability were proved to be good enough for pharmacokinetic studies.

According to the pharmacokinetic study data acquired, we can believe that the preparation of phospholipid complex improved curcumin's bioavailability obviously.

Section snippets

Materials

Curcumin (purity > 99%, by HPLC) and tetrahydrocurcumin (purity > 99%, by HPLC) were prepared in our laboratory and identified by NMR techniques, and salbutamol (purity > 99%) as internal standard were purchased from National Institute for the Control of Pharmaceutical and Biological Products (China). Fig. 1 represents the structures of curcumin, tetrahydrocurcumin and salbutamol. A 95% soya phospholipids were purchased from Panjin pharmaceutical Co., Ltd. (Liaoning Province, China). Sulfatase-free

Mass spectrum

Mass spectrum of curcumin [M + H]+ m/z 369, tetrahydrocurcumin [M + H]+ m/z 373 and the internal standard salbutamol [M + H]+ m/z 240 are shown in Fig. 2.

Chromatography and specificity

Under optimized HPLC and MS conditions, curcumin, tetrahydrocurcumin and the internal standard were detected respectively (Fig. 3), and MS revealed a double peak with tetrahydrocurcumin, such phenomenon reflected rapid transition between the keto–enol β-diketone molecular species of tetrahydrocurcumin as published before [13], the single tautomer of

Discussion

The method is generally highly sensitive and specific, and it simplify the sample cleanup. In this method, the elution time can be kept short that makes the sample analysis very efficient, no appreciable ion suppression was found in MRM transitions of m/z 373.2  137.1 and 240.2  148.1 for curcumin and tetrahydrocurcumin, respectively, when the ethyl acetate extraction residue of rat plasma was spiked with the analytes.

Later, we were able to shorten the retention time to about 4.5 min with some

Conclusion

A highly sensitive and specific LC/MS/MS method for the quantitation of curcumin has been developed. The method has been validated with a routine sensitivity limit of 0.5 ng/ml in 0.1 ml rat plasma. Using this method, pharmacokinetics of curcumin and curcumin phospholipid complex at 100 and 300 mg/kg respectively following the oral route in the rat was investigated. The plasma levels of the two drugs have been detected at lower level and shorter duration than previously documented.

According to our

Acknowledgement

Authors thank Dr. Y.H. Gao for samples analyses and Prof. G.H. Lee for collection of rat plasma.

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