SPSFAM ERP

1. Abstract The Fructan Assay Kit is an enzymatic test kit developed by Megazyme for the determination of fructan [inulin, FOS, agave (branched) fructan and levan] in a variety of animal feed and food samples and in ingredients. For the purpose of this submission, the kit has been validated in a single laboratory for analysis of pure inulin, FOS (non-reducing), agave fructan and levan and a range of fructan containing samples. The commercially available Fructan Assay Kit (Megazyme cat no. K- FRUC) contains all components required for the analysis. Quantification is based on complete hydrolysis of fructan to fructose and glucose by a mixture of exo -inulinase, endo -inulinase and endo -levanase and measurement of these sugars using the PAHBAH reducing sugar method, which gives the same color response with fructose and glucose. Before hydrolysis of fructan, interfering sucrose and starch in the sample are hydrolyzed to glucose and fructose and these sugars and any other reducing sugars in the sample extract are “removed” by borohydride reduction. The Single Lab Validation (SLV) outlined in this document was performed on commercially available inulin (Raftiline) and agave fructan (Frutafit ® agave fructan), levan purified from Timothy grass, two grass samples, a sample of legume hay, two animal feeds and two barley flours, one of which (Barley MAX ® ) was genetically enriched in fructan through plant breeding. Parameters examined during the validation included Working range, Selectivity, Recovery, Limit of Detection (LOD), Limit of Quantification (LOQ), Trueness (bias), Precision (reproducibility and repeatability), Robustness and Stability. The method outlined within allows accurate enzymic measurement of fructan in a wide range of animal feeds, pet foods and ingredients. It is a robust, quick and easy method for analysis of fructan, based on the commercial Fructan Assay Kit (K- FRUC) as developed by Megazyme.

2