DETERMINATION OF CINNAMIC ACID AND 4-COUMARIC ACID IN ALFALFA (MEDICAGO-SATIVA L) CELL-SUSPENSION CULTURES BY GAS-CHROMATOGRAPHY

A capillary gas chromatographic (GC) method for the assay of cinnamic acid and 4-coumaric acid in alfalfa (Medicago sativa L.) cell extracts is described. These compounds were assayed as their trimethylsilyl de rivatives; hydrocinnamic acid was used as the internal standard for qu antifications. This method was qualitatively validated by GC/mass spec tral analysis and quantitatively validated by measuring the extraction recovery from alfalfa cells, the recovery from derivatization, the st ability of silylated derivatives, the limits of detection, the lineari ty of the detector response to increasing amounts of analytes, and the intra- and inter-assay precisions. Cinnamic acid and 4-coumaric acid were present in alfalfa cell extracts and were resolved from all other components by this method. As little as 4 pmol of each acid could be quantified, and the detector response was linear over a range covering five orders of magnitude. The cellular concentrations of free cinnami c acid and 4-coumaric acid ranged from ca. 5-100 mumol/kg fresh weight tissue.