Thin-Layer Chromatographic Separation and Validated HPTLC Method for Quantification of Ursolic Acid in Various Ocimum Species
KEDAR KUMAR ROUT1, RAJESH KUMAR SINGH1, DURGA PRASAD BARIK2 AND
SAGAR KUMAR MISHRA3
1. Post Graduate Department of Chemistry, NorthOrissaUniversity, Orissa, India
2. Department of Botany, School of Life Sciences, RavenshawUniversity, Cuttack-753003, Orissa, India
3. Pharmacognosy and Phytochemistry Division, University Department of Pharmaceutical Sciences, Utkal University, Vani Vihar, Bhubaneswar-751004, Orissa, India
(Received: October 13, 2011; Accepted: June 13, 2012)
ABSTRACT
An improved thin-layer chromatographic separation and quantitative estimation of ursolic acid in different plant parts of various Ocimum species have been developed and validated. Excellent separation of the components was achieved on high-performance precoated TLC plates by using optimized ternary mobile phase consisting of toluene : acetone : formic acid (7.8 : 2.2 : 0.15, v/v/v). Quantification and densitometric determination were performed after derivatization of the plate with methanol-sulphuric acid reagent in reflection/absorption mode at 540 nm. The limit of detection and limit of quantification were found to be 20 and 35 ng/spot, respectively. The response of ursolic acid was linear over the range of 40 to 440 ng/spot with a correlation coefficient of r2=0.9995, indicating good relationship between peak area and concentration. Recovery values from 98.36 to 100.06% showed excellent accuracy of the method. The method was validated according to ICH protocol for precision, repeatability and accuracy. Ursolic acid was efficiently separated from the other components by the proposed method which was very simple, rapid, precise, sensitive and accurate for the quantification of ursolic acid in different plant parts of Ocimum species. The maximum ursolic acid content was found in the stems.
Key words: HPTLC, Ocimum basilicum, Ocimum sanctum, TLC, ursolic acid