Quantitative Analysis of Salicylic Acid and its Derivatives in Primulae radix by High Performance Liquid Chromatography-Diode Array Detec- tion-Electrospray Ionization Mass Spectrometry (HPLC-DAD-ESI-MS) and Simultaneous Determination of Total Polyphenol Content (TPC)

摘要

Salicylic acid (SA) plays a key role against pathogens of plants and also induces resistance, hence it is an important signaling compound in plants. SA derivative, such as acetylsalicylic acid (Aspirin), is widely used and marketed today as a non steroidal anti-inflammatory drug (NSAIDs) which is one of the first "wonder drugs" of the 20th century. Here we report a method for quantification of SA and its derivatives i.e. 3,4-dihyroxybenzoic acid (3,4-DHBA), gallic acid (GA) and gallic acid methyl ester (GAME) obtained from Primulae radix. First, the total polyphenol content (TPC) of Primulae radix extracts was determined by Folin-Ciocalteu assay. In that context reflux and microwave assisted extracts (MAE) in different solvents, i.e. water, 100% ethanol, 100% methanol, 50% ethanol and 50% methanol were evaluated. 50% ethanol MAE was taken for the quantitative analysis of SA and its derivatives as the highest amounts of total polyphenol content were yielded. This extract was pre-concentrated by solid phase extraction (SPE) using Strata-X (surface modified copolymer of styrene-divinylbenzene) cartridges. Eluted sample was analyzed by a reversed phase liquid chromatography-electrospray ionization mass spectrometry (RPLC-ESI-MS) system in selected ion monitoring (SIM) mode. Lower limits of detection (LOD) of 0.74 ng/ml for SA, 1.39 ng/ml for 3,4-DHBA, 3.00 ng/ml for GA and 0.37 ng/ml for GAME and limits of quantification (LOQ) of 2.45 ng/ml for SA, 4.62 ng/ml for 3,4-DHBA, 10.00 ng/ml for GA and 1.23 ng/ml for GAME were determined. Correlation coefficient values > 0.99 for all standards were obtained. The determined mean concentrations were 28.80 and 29.34 μg/g for SA, 9.22 and 8.61 μg/g for 3,4-DHBA, 6.17 and 7.12 μg/g for GA and 0.14 and 0.03 μg/g for GAME using intra and inter-day analyses respectively. Finally, presented data prove that introduced method is highly sensitive and reproducible for the simultaneous quantification of SA, 3,4-DHBA, GA and GAME in Primulae radix extract.