Development of a microplate FAN method—Not always as straightforward as expected

Analytical Session
Mark R Schmitt, USDA Agricultural Research Service, Madison WI USA
Co-author(s): Allen Budde, USDA Agricultural Research Service (retired), USA

ABSTRACT: Many malting quality analysis methods have several versions available, often an original manual method and a newer automated version. For determination of wort free amino nitrogen (FAN) concentration, a long-established manual method (ASBC Wort-12) is available but infrequently used, and an automated segmented flow analysis (SFA) version has recently been recommended for acceptance as an approved method (MacLeod et al, JASBC 69(4):295, 2011). A third format, a 96-well microtiter plate-based assay, is attractive in certain situations. In this study, we compared the manual Wort-12 method for FAN analysis, a standard SFA method (plus several variations), and two microplate versions, using chemistry derived from either the manual or the SFA version. We examined a number of variables, including reagent (ninhydrin) source and grade, incubation time and temperature, and reductant concentration, for the two microplate versions. In general, the adaptation of the SFA reagent yielded a more robust assay with results that matched manual and SFA assays. In contrast, direct adaptation of the chemistry from Wort-12 to a microplate format generated results that did not consistently match those from the other methods. In addition, the Wort-12 process in a microplate format required longer incubations (up to 15 min) at higher temperatures (up to 99°C) for full color development, indicating the assay could be more susceptible to variations in color development. The assay derived from the SFA reagents worked well, generated results that matched those from existing manual and automated methods, and is recommended for FAN analysis in a microplate format.

Mark Schmitt received a Ph.D. degree in plant physiology/plant biochemistry from the University of Wisconsin, Madison. He joined USDA’s Agricultural Research Service in Madison in 2003. His research emphasis is on malting quality.