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

Nizam      Uddin; Christof      Krieg; Wolfgang      Stoggl; Gunther      Stecher; Gudrun      Abel; Michael      Popp; Gunther   K.   Bonn; Christian   W.   Huck

Abstract

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 20^th 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.

Keywords: Primulae radix, salicylic acid, total polyphenol content (TPC), folin-Ciocalteu assay, solid phase extraction (SPE), RPLC-ESI-MS.

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