A rapid and sensitive analytical method for quantification of polyacetylenes in carrot roots was developed. The traditional extraction method (stirring) was compared to a new ultrasonic liquid processor (ULP)-based methodology using high-performance liquid chromatography-ultraviolet (HPLC-UV) and mass spectrometry (MS) for identification and quantification of three polyacetylenes. ULP was superior because a significant reduction in extraction time and improved extraction efficiencies were obtained. After optimization, the ULP method showed good selectivity, precision [relative standard deviations (RSDs) of 2.3-3.6%], and recovery (93% of falcarindiol) of the polyacetylenes. The applicability of the method was documented by comparative analyses of carrots grown organically or conventionally in a 2 year field trial study. The average concentrations of falcarindiol, falcarindiol-3-acetate, and falcarinol in year 1 were 222, 30, and 94 mug of falcarindiol equiv/g of dry weight, respectively, and 3-15% lower in year 2. The concentrations were not significantly influenced by the growth system, but a significant year-year variation was observed for falcarindiol-3-acetate.