|Harnly, James - Jim|
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/9/2003
Publication Date: 1/10/2004
Citation: Rybak, M.E., Calver, E.M., Harnly, J.M. 2004. Quantitative determination of allicin in garlic: supercritical fluid extraction and standard addition of alliin. Journal of Agricultural and Food Chemistry. 52:682-687. Interpretive Summary: Allicin, a thiosulfinate and the major sulfur-containing compound found in garlic, has been reported to have health-promoting properties. Allicin is responsible for the aroma and taste that we associate with this pungent bulb of the Allium family. Allicin is produced when the garlic bulb is physically altered (sliced, crushed, bruised, etc.) - alliin (a non-coded amino acid) reacts with alliinase (an enzyme) to form allicin. Allicin is unstable (half decays in 4 days) and reacts to form a wide variety of other sulfur-containing compounds. Because of its instability, allicin cannot be used as a primary standard and must be handled at low temperatures. We have developed a method that uses alliin as the primary standard and uses supercritical fluid extraction (high pressure and low temperature), separation by liquid chromatography, and spectrophotometric detection. This method provides a much more robust method for the analysis of allicin and other thiosulfinates. This method will be useful to researchers and for producing data suitable for the USDA National Nutrient Database for Standard Reference.
Technical 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 (HPLC) analysis with UV-visible 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 phenylsulfone) 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 126.96.36.199). 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 thus more amenable for use as a standard for routine analysis.