Quantitative Determination of Allicin in Garlic: Supercritical Fluid Extraction and Standard Addition of Alliin

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Abstract
A quantitative method is described for the determination of allicin (2-propene-1-sulfinothioic acid S-2-propenyl ester) in garlic, using standard additions of alliin (l-(+)-S-allylcysteine sulfoxide) in conjunction with supercritical fluid extraction (SFE) and high performance liquid chromatography analysis with UV−vis absorbance detection. Optimum CO2-SFE conditions provided 96% recovery for allicin with precision of 3% (RSD) for repeat samples. The incorporation of an internal standard (allyl phenyl sulfone) in the SFE step resulted in a modest improvement in recovery (99%) and precision (2% RSD). Standard additions of alliin were converted to allicin in situ by endogenous alliinase (l-(+)-S-alk(en)ylcysteine sulfoxide lyase, EC 4.4.1.4). Complete conversion of the spiked alliin to allicin was achieved by making additions after homogenization-induced conversion of the naturally occurring cysteine sulfoxides to thiosulfinates had taken place, thus eliminating the likelihood of competing reactions. Concentration values for allicin determined in samples of fresh garlic (Allium sativum L. and Allium ampeloprasum) and commercially available garlic powders (Allium sativum L.) by standard addition of alliin were found in all cases to be in statistical agreement (95% confidence interval) with values determined using a secondary allicin standard (concentration determined using published extinction coefficients). This method provides a convenient alternative for assessing the amount of allicin present in fresh and powdered garlic, as alliin is a far more stable and commercially prevalent compound than allicin and is thus more amenable for use as a standard for routine analysis. Keywords: Alliin; allicin; alliinase; cysteine sulfoxides; thiosulfinates; standard addition; supercritical fluid extraction; high performance liquid chromatography