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Phytochem Anal. 2011 Jul-Aug;22(4):367-73. doi: 10.1002/pca.1289. Epub 2011 Feb 19.

Validated high-performance thin-layer chromatographic method for the quantification of thymoquinone in Nigella Sativa extracts and formulations.

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Department of Pharmacology, Bombay College of Pharmacy, Kalina, Santacruz (E), Mumbai-400 098, India.



The surge of interest in naturally occurring phytochemicals with anticancer potential has led to the discovery of many molecules, one of them being thymoquinone (TQ) the bioactive constituent of the volatile oil of black seed, Nigella sativa L. (NS).


The aim of the present work was to develop and validate an HPTLC method for determination of TQ in NS extracts, commercially available marketed oils, polyherbal formulations and in lipid-based oral and parenteral formulations prepared in-house.


Analysis of TQ was performed on TLC aluminium plates pre-coated with silica gel 60F-254. Linear ascending development was carried out in twin trough glass chamber, saturated with mobile phase consisting of toluene-cyclohexane (8 : 2, v/v) at ambient temperature. Camag TLC scanner III was used for the spectrodensitometric scanning and analysis in absorbance mode at 254 nm.


The method was found to give compact spots for TQ (R(f) value of 0.28 ± 0.05) and was linear over the range 100-1400 ng/spot (r(2) = 0.9921 ± 0.0020). Accuracy, precision and repeatability were all within the required limits. The mean recoveries measured at three concentrations were higher than 95% with RSD ≤ 3%.


The HPTLC method developed was found to be relatively simple, rapid and accurate for the routine analysis of TQ in extracts, marketed oils, polyherbal and in-house formulations.

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