Automated kinetics-enhanced flow-injection method for histamine in regulatory laboratories: rapid screening and suitability requirements Original Research Article

Scombroid poisoning continues to be a leading cause of finfish-borne illness. Histamine found in implicated fish has been traditionally used as a marker, if not the cause, of scombrotoxicity. The most accepted method for detecting histamine in fish meat requires extensive manual manipulations and sample cleanup steps. To address the need for more rapid methodology, an automated, kinetically-enhanced flow injection method for histamine has been used to screen thousands of sub-samples of various fish and seafood products. Selectivity (expressed as molar fluorescence ratios) for histamine versus histidine in the sample matrix is a function of reaction kinetics. Fine-tuning this effect is sufficient to remove the need for ion-exchange sample conditioning. This fine-tuning of reaction kinetics is easily accomplished via control of flow rates, reaction temperature, etc. Method performance is excellent, with detection and quantitation limits near 0.8 and 2.4 mg/kg, and linearity to approximately 340 mg/kg. Each sample produces a response in <1 min. The method is more rapid and more practical than other proposed screening methods including alternative chemistries, HPLC methods, and immunoassays. For widespread use in the regulatory environment, an analytical method must produce similar results in other laboratories. Success requires simplified rugged procedures, a knowledge of which specific variables are important in determining method ruggedness, and demonstration of system suitability. Consistent with the methods’ reliance on chemical kinetics, the most important factors are reaction time and temperature, and the condensation reagent concentration. These factors are not so sensitive that they limit ruggedness and practicality. A temperature change of 3°C results in a 4% change in peak height, and a 1 cm change in reactor length results in a 0.4% difference. These effects are then easily controlled using commercial instrumentation. With no sample cleanup other than extraction and crude filtration, tuna can be screened for histamine with R.S.D. of <3% (N=5 extracts, five injections/extract). Recoveries at 50 and 100 mg/kg were 97 and 118% (each spike extracted to five replicates, five injections/extract). Agreement with histamine levels using the alternate (official) method is also very good.