51A-8

J. E. VALLS, M. Kodaira, and E. G. Tome. Instituto de Ciencia y Tecnología de Alimentos, Universidad Central de Venezuela, P.O. Box: 47.097. Los Chaguaramos., Caracas, 1041-A, Venezuela

JUSTIFICATION: Autolytic descomposition of fish has been mainly evaluated in terms of catabolites formation from the enzymatic breakdown of adenosine triphosphate (ATP). Liquid chromatography (LC), is one of the most commonly used technique for these studies, but there are only few reports on testing or validation LC procedure.

OBJECTIVES: The purpose of this study was to validate and improve a rapid isocratic LC method for simultaneously determining ATP-related compounds in sardine, tilapia and palometa.

METHODS: The method was validated by assesing the following aspects: specificity, linearity, recovery, limit of detection (LOD), limit of quantification (LOQ), UV detection wavelengths and stability of nucleotides under frozen storage. The methodology was applied to fresh fish samples.

RESULTS: LC procedure for simultaneously analyzing of ATP-related compounds in fish samples was found reproducible, specific, accurate and quick (less than 15 min. in chromatographic elapsed time). AMP, Hx, and INO peaks were well resolved while ATP, ADP, and IMP peaks showed some baseline fused, but responses to repeated injections of a mixed standard demostrated the reproducibility of this methodology. Data indicates that 254 was more suitable for measurement ATP-related compounds. The linearity shown ( r ) from 0.9949 to 0.9999. The ATP derivatives standard and samples extracts are highly stable at -40°C and not changes were observed during the storage of 20 weeks. The LOD and LOQ range from 0.10 to 1.86 mg/g and 0.45-7.35 mg/g respectively. Recoveries were from 92.7 to 104.8% and not significant statistical differences were found between the mean recovery of each nucleotide and theoretical value of 100%.

SIGNIFICANCE: LC methodology can be easily applied in fish samples and was found that good peak resolution and quantification were achieved. The use of a isocratic phase makes affordable for studies that involves large number of determinations.