Quantitative Determination of Seven Flavonoids in Bark and Seeds of Oroxylum indicum by HPLC-UV, UPLC-UV Methods and Confirmation by Mass Spectrometry

Oroxylum indicum Vent., family Bignoniaceae is a rich source of flavonoid compounds [1]. The seeds and bark are used medicinally for alleviating pain and as an antiphlogistic medicine. Chrysin, baicalein and the other flavonoids in Oroxylum have anti-inflammatory and anti-allergy effects [1,2]. A simple and specific analytical method for the quantitative determination of flavonoids in bark and seeds of the Oroxylum indicum was developed using HPLC-UV and UPLC-UV. By LC-UV, the separation was achieved by reversed-phase chromatography on a C18 column with water and acetonitrile (both containing 0.1% acetic acid) as the mobile phase. In UPLC-UV system, acetic acid was replaced by formic acid. Detection was performed at 280 nm. The methods were validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). The limits of detection and limits of quantification by HPLC-UV was found to be 0.2 µg/mL and 1 µg/mL for baicalein (1), OA-6 (2), OA-7 (3), chrysin (4), oroxylin A (5), 5,7 dihydroxy-6-methoxy-2-phenylchroman-4-one (DHMF, 6) and acetyl chrysin (7), respectively and 0.02 µg/mL and 0.1 µg/mL by UPLC-UV method. An LC-MS coupled with electrospray ionization (ESI) interface method is described for the identification and quantification of flavonoids in various plant samples. This method involved the use of the [M+H]⁺ ions of flavonoids in the positive ion mode with extractive ion monitoring (EIM). The developed methods were applied for the determination of seven flavonoids in bark and seeds of Oroxylum indicum. All seven flavonoids were detected in bark and only chrysin was found in the seeds of Oroxylum indicum. Acknowledgements: This research is funded in part by “Science Based Authentication of Dietary Supplements“ Funded by the Food and Drug Administration grant number 2 U01 FD 002071-07 and the United States Department of Agriculture, Agricultural Research Service Specific Cooperative Agreement Number 58-6408-06-067. Also this research is supported jointly by CSIR and M/S Zandu pharmaceuticals and NCNPR. The CSIR budget code for this project is ”Fellowship abroad p-80104”. References: [1] Suresh BK, et al. (2006) Bioorganic and Medicinal Chemistry Letters 16(1): 221–224. [2] Mao AA (2002) Indian Journal of Traditional Knowledge 1(1): 17–21.

Fig. 1 UPLC and HPLC Chromatograms of a mixture of standard (A), Seeds (B) and Bark (C) of Oroxylum indicum at 280 nm