Improved reagent for detection of allantoin in thin-layer chromatographic method for urine stains on foods and packaging

Ten detection reagents known to react with either primary amine groups or indole derivatives were tested to improve sensitivity for allantoin (ALN) and indican (IND) in the official AOAC thin-layer chromatographic method for urine metabolites (44.175-44.177). The lowest levels found using the official method were 500 ng ALN and 6 ng IND. The best reagent was p-dimethylaminocinnamaldehyde (pDMAC), which yielded intensely colored spots with both compounds. The lowest amounts consistently found were 125 ng ALN and 13 ng IND. pDMAC was also used as an overspray for ALN after the spray specified in the official method, p-dimethylaminobenzaldehyde (pDMAB), was applied. This resulted in a detection limit of 250 ng. The overspray procedure was incompatible with IND detection, but provided an easy way to gain slightly greater sensitivity for ALN when use of pDMAB gave negative or borderline results. The combined use of pDMAC for ALN, and the official sprays (pDMAB and sodium acetate) for IND, maximized sensitivity for both compounds.