Determination of Putrescine and Tyramine in Fish by Micellar Liquid Chromatography with UV Detection Using Direct Injection

ABSTRACT

This paper shows how fish samples could be directly injected into a chromatograph, without any previous extraction, using micellar liquid chromatography, taking as an example the determination of two biogenic amines: putrescine and tyramine, derivatized with 3,5-dinitrobenzoyl chloride. First, the method has been optimized, using an interpretive model, and the best conditions for analysis were: mobile phase containing 0.15 M SDS-6% (v/v) butanol-pH 7, C18 column (125 x 0.46 mm, 5 µm particle size), UV detection of derivatives set at 260 nm. Second, the method has been validated using spiked samples following the guidelines proposed by the US Food and Drug Administration: linear in the range from 0.5 to 1000 ppm (r2>0.999), detection limits of 30 and 40 ppb for putrescine and tyramine, respectively, a 500 ppb limit of quantification in both cases, intra- and inter-day precision (RSD) below 2%, and accuracy between 99.5% and 102.7%. The method is adequate for routine analysis in the determination of these compounds in fish sauces.