Purpose: The objective of this study was to evaluate several promising modifications to the official AOAC fluorometric method with the intention to reduce assay time and increase sample throughput while maintaining the performance of the original method.
Methods: The following modifications to the AOAC method were investigated: replacement of the ion-exchange resin with solid phase extraction (SPE) cartridges; use of pre-treated AG 1-X8 resin; reduction of resin column length from 8 to 4 cm; reduction of sample size from 10 to 5 g; omission of the heating step in the extraction; and replacement of the cuvette style fluorometer with a microplate reader. Yellowfin tuna (Thunnus albacares), mahi-mahi (Coryphaena hippurus), and Spanish mackerel (Scomberomorus maculatus) samples spiked with histamine in concentrations ranging from 5-250 ppm were used to test the modified method. Recovery, precision, and detection limits were evaluated by applying FDA guidelines for single laboratory validation of chemical methods.
Results: Replacing the ion-exchange resin with a SPE cartridge or pre-treated ion-exchange resin, reducing column length of resin from 8 to 4 cm, and reducing sample amount from 10 to 5 g had an adverse effect on the performance characteristics of the method. However, omitting the heating step in the extraction and replacing the cuvette style fluorometer with a microplate reader retained method performance while increasing sample throughput. The recovery, precision (relative standard deviation), and limit of detection of the modified method assessed by the single laboratory validation ranged from 92 to 105%, 1 to 3%, and 0.2 to 0.5 ppm, respectively, in tuna, mahi-mahi, and Spanish mackerel samples.
Significance: We conclude that the AOAC 977.13 fluorometric method, modified as described, will improve assay time and sample throughput efficiency cumulatively, as the number of sample units analyzed increases. This modified method could likely be used by regulatory agency and other laboratories following successful multi-laboratory validation.