Year : 2009 | Volume
: 5 | Issue : 17 | Page : 71--74
A Sensitive Reversed Phase HPLC Method for the Determination of Curcumin
Yadav Vivek Ramshankar, Sarasija Suresh
Department of Pharmaceutics, Al-Ameen college of Pharmacy, Hosur Road, Bangalore - 560027, India
Department of Pharmaceutics, Al-Ameen college of Pharmacy, Hosur Road, Bangalore - 560027
A simple, precise, rapid and accurate, binary reverse phase high performance liquid chromatographic method has been developed for the determination of curcumin a natural drugs with short run time. Chromatographic separation was achieved by using Merck C15 (250 cm X 4.6 mm) Column with mobile phase acetonitril: tetrahydrofuran: 2% acetic acid 50:30:20 (2%) was used. The flow rate was 0.7 ml/min. The retention time was 4.587 minutes. The limit of detection and limit of quantification of curcumin were between 3.68 to 8.125 ng/ml for 50-μL injection volumes. The percentage recovery of curcumin was found to be 97.2 to 98.4 and Relative standard deviation was 0.345 % and 1.160 %. The developed HPLC method can therefore be applied to both in vitro studies of curcumin formulations as well as drug estimation in biological samples.
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Ramshankar YV, Suresh S. A Sensitive Reversed Phase HPLC Method for the Determination of Curcumin.Phcog Mag 2009;5:71-74
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Ramshankar YV, Suresh S. A Sensitive Reversed Phase HPLC Method for the Determination of Curcumin. Phcog Mag [serial online] 2009 [cited 2021 Apr 12 ];5:71-74
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Curcumin [1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3, 5-dione] is the major yellow pigment extracted from turmeric, a commonly used spice, derived from the rhizome of the herb Curcuma longa Linn  . It is a naturallyoccurring polyphenolic phytochemical currently being examined in preclinical trials for cancer chemoprotective drug development, with pharmacological actions that including antioxidant , , anti-inflammatory , , and cancer chemopreventive actions ,, . Curcumin, the major yellow-orange pigment extracted from turmeric, may be responsible for much of the bioactive effects. In a recent study, products of curcumin reduction and conjugation had a reduced ability to inhibit cyclooxYgenase-2 (COX-2) expression, which correlated to a decrease in the inhibition of prostaglandin biosynthesis when compared to intact curcumin, indicating that the metabolic conversion of curcumin results in pharmacologic deactivation  . Curcumin is also a potent scavenger of various reactive oxygen species (ROS) including superoxide anions  and hydroxyl radicals , . In addition, there have been indications that curcumin may help prevent and treat patients with Alzheimer's disease by reducing oxidative damage, plaque burden, and suppressing specific inflammatory factors  .
A limitation to the studies cited above was the inability to quantitate low curcumin concentrations. Quantitation of curcumin concentrations below 10 ng/ml would allow better characterization and understanding of the disposition and absorption kinetics of this compound. Although several methods of detection for curcumin have been published, only one has reported a limit of quantitation below 10 ng/ml  . Of these methods, several involve spectrophotometric  , liquid chromatography- mass spectrophotometric , , and radiolabeled determination of curcumin  . HPLC methods have also been developed in order to quantitate curcumin in biological samples ,,, . Ireson et al.  utilized a HPLC gradient system that produced reasonable separation and sensitivity. The retention time for curcumin, however, was greater than 35 min. We therefore focused on developing a rapid and more sensitive HPLC binary method for the estimation of Curcumin.
Chemicals and reagents
Curcumin was obtained from Natural remedies, Bangalore. Acetonitrile, tetrahydrofuran and acetic acid was obtained from Thomas Baker, India. Water was deionised by the Milli-Q Plus system (Millipore).
The LC system consists of a Shimadzu SPD-10TVP, Binary pump equipped with a normal sample injector with a 50-microliter loop, SPD-10AVP variable wavelength UV detector and Spincotech station for data analysis.
The stock solutions were prepared by dissolving 5.0 mg of Curcumin was dissolved in 50 ml mobile phase to get a concentration of 1,00,000 ng/ml. Analytical standard solutions for linearity were prepared by diluting the stock solution with 50% acetonitrile, 30% tetrahydrofuran and 20% acetic acid (2%v/v) immediately prior to use. All the preparations were made in borosilicate glass tubes.
Chromatographic separations were achieved using a Shimadzu ODS C15 , 1 cm long Guard column (4.6 X 250 mm, 5 μm). The mobile phase consisting of acetonitril: tetrahydrofuran: 2% acetic acid 50:30:20 (2%) V/V was passed through a 0.22 μm membrane filter and degassed by ultrasonication under vacuum before use. The flow rate was maintained at 1.0 ml/min and the effluent was monitored for UV absorption at 425 nm. The injection volume was 50 μL. All separations were performed at ambient temperature.
The objective of this study to develop method for the determination of Curcumin with short run time, which can also be used for its formulations and biological samples. The column chosen for this study was 250 mm length, 4.6 mm internal diameter and 5-micron particle size. Good sample separation was observed on silica based C 15 Mark column using mobile phase acetonitril: tetrahydrofuran: 2% acetic acid 50:30:20 (2%) v/v. The retention time of Curcumin was found to be 4.59 min. The system suitability results were given in [Table 1].
Precision of the procedure was determined by repeatability method. A solution of Curcumin containing 10Ong and 5000ng/ml respectively was injected into the system repeatedly six times. The percentage RSD of injection repeatability and analysis repeatability for Curcumin was found to be 0.812 % and 0. 856% respectively. The results obtained confirm good precision of the method developed.
The linearity of the method for Curcumin was checked at ten concentration levels over the concentration range of 50-100000 ng/ml. The typical equation describing the calibration curve is y=0.830x where y is the peak area of Curcumin and x is the concentration of Curcumin, with a mean correlation coefficient (R2) of 0.9997.
Quantification of Curcumin in natural samples
The standard addition and recovery experiments were conducted to determine the accuracy of the present method for the quantification of Curcumin samples. The recovery of Curcumin was calculated from the slope and the intercept of the calibration curve drawn in the concentration range of 50-100000ng/ml. The percentage recovery of Curcumin was ranged from 98.6 % to 99.4 % in samples of Curcumin. The results were shown in [Table 2]. A HPLC chromatograph of Curcumin in samples was shown in [Figure 1].
Limit of detection
The limit of detection represents the concentration of analyte that would yield a signal to noise ratio equal to 3σ(DL=3σ/S) 5 . The limit of detection for Curcumin was found to be 3.68 to 8.125 ng/ml for 50μL injection Volume. The limit of quantification represents the concentration of analyte that would yield a signal to noise ratio equal to 10σ ((DQ=10σ/S) 5 . Limit of quantification for Curcumin was 8.125 ng/ml for 50μL injection Volume.
Solution stability of Curcumin was studied by leaving the solution (10 and 5000ng/ml prepared in diluent) in tightly capped ambered colour volumetric flasks at room temperature for three days. Content of Curcumin was checked for 12 hours interval and compared with freshly prepared solutions. No variation was observed in the content of Curcumin for the study period, which indicates that the Curcumin sample solutions prepared in the said diluents are stable for at least 3 days.
A simple and sensitive HPLC method was developed for Curcumin. This assay method provided excellent sensitivity, accuracy and precision, with relatively short retention time for Curcumin. This HPLC method can therefore be applied to both in vitro studies of Curcumin formulations as well as drug estimation in biological samples.
The authors would like to thank Prof. B.G. Shivananda, Principal, Al-Ameen College of Pharmacy for his kind support and encouragement. This study was supported by SRF grant to Vivek Yadav from Indian Council of Medical Research, India.
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