A chromatographic survey of methods for extacting long-chain grass fructans

Authors: Kagan, Isabelle; Kirch, Brett; Strickland, James

Submitted to: Grass and Forage Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/24/2011
Publication Date: 4/1/2011
Citation: Kagan, I., Kirch, B.H., Strickland, J.R. 2011. A chromatographic survey of methods for extacting long-chain grass fructans. Grass and Forage Science. 66:434-448.

Interpretive Summary: Fructans are chains of fructose molecules, with variable lengths, that are produced by some monocots and dicots. The amounts of fructans produced by these plants can increase dramatically in response to some environmental stresses, including low temperatures and intense light. Methods were developed to aid in analyzing the fructan content of forage grasses (monocots) grown under various controlled environmental conditions. Protocols were developed for analyzing fructans by high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD). Different fructan extraction methods were evaluated by comparing the total fructan content of each extract to that of a sample extracted by the method expected to yield the least fructan. These comparisons were made within each of four species: bluegrass, tall fescue, orchardgrass, and timothy. No fructans were detected in bluegrass or tall fescue at the concentrations used. In orchardgrass, most extraction methods yielded similar amounts of fructans, except that yields nearly doubled when macerated, frozen tissue was extracted twice at boiling temperature. In timothy, most extraction methods also yielded similar amounts of fructans, except that yields decreased 24-40% when ground, frozen tissue was extracted twice at ambient or 3 times at boiling temperature. The information gained from this study will help in detecting a potentially wide range of fructans in samples from future studies, as well as in extracting and purifying fructans of different chain lengths to use as standards.

Technical Abstract: Fructans were extracted under different conditions from four cool-season forages in order to develop extraction, cleanup, and chromatographic separation protocols permitting optimal analysis of long-chain fructans. Adequate sample cleanup was achieved by passage through a C18 solid-phase extraction (SPE) column, followed by diluting 2.5-fold. Fructans were separated by anion-exchange high-performance liquid chromatography (HPLC), using a sodium acetate gradient in sodium hydroxide, and detected by pulsed amperometric detection (PAD). Within each species, all extraction methods yielded similar chromatographic profiles. Fructan yields in orchardgrass were increased about 2-fold when macerated, frozen tissue was extracted at boiling instead of ambient temperature. In timothy, most extraction methods yielded similar amounts of fructans, but yields decreased 30-50% when ground, frozen tissue was extracted twice at ambient or 3 times at boiling temperature. Tall fescue and bluegrass did not contain fructans similar to those in the standard mix or in the other two species studied. Two HPLC protocols were developed: one for profiling short- and long-chain sugars, and one for profiling only long-chain sugars. The latter protocol sped up the analysis time but left some questions about which peaks corresponded with those in the former protocol.