María R. Alberto, Mario E. Arena, and María C. Manca de Nadra
Biogenic amines can be formed and degraded as a result of normal metabolic activity in animals, plants, and microorganisms and are usually produced by the decarboxylation of amino acids (1). Recent trends in food security are promoting an increasing search for trace compounds that can affect human health. Although they are present in fermented foods and beverages in low quantities, they exhibit interactions with normal human metabolism (e.g., having vasoactive or psychoactive properties) that justify the research on their presence in foods and the possible related toxicologi-cal effects that they may cause.
Estimation of the biogenic amines histamine, tyramine, agmatine, putrescine, and cadaverine is important not only from the point of view of their toxicity, but also because they can be used as indicators of the degree of freshness or spoilage of food. Until recently, because of the difficulty in detecting and quantifying amines reliably we have had insufficient information about their occurrence in different types of foods and beverages. These problems are related to matrix interference (e.g., the presence of free amino acids) and the low levels at which the amines are found.
Early techniques for the determination of biogenic amines in foods were based on thin-layer chromatography. More modern analytical techniques have since been developed that allow the acquisition of reliable quantitative data and better separation/resolution of various amines. The quantitative determination of biogenic amines is generally accomplished by overpressure-layer chromatography, high-performance liquid chromatography (HPLC), and gas chromatography (1).
From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ
The use of reverse-phase column and precolumn derivatization was more efficient and faster than the conventional ion-exchange techniques (2). This study was conducted to evaluate two HPLC derivatization methods for quantitative determination of biogenic amines: the method described by Gonzales de Llano et al. (3) for amino acid analysis and the method described by Eerola et al. (4).
The biogenic amine standards agmatine, cadaverine, histamine, putrescine, tyramine and 1,7-diaminoheptane were from Sigma. All solvents used in the derivatization process and in the chromatographic separation were HPLC quality.
2.2. OPA Method
1. Reverse-phase (RP)-HPLC.
a. An ISCO HPLC system (Lincoln, NE).
b. Model 121 fluorimeter (340 nm excitation filter and 425 nm emission filter).
c. A Waters Novapack C18 column, 3.9 x 150 mn, 4-^m particle size for the stationary phase, with a flowrate of 1.5 mL/min (see Note 1).
a. For derivatization: 200 mg o-phthaldialdehyde (OPA) in 9 mL methanol, 1 mL 0.4 M sodium borate, pH 10, and 160 ^L 2-mercaptoethanol (MCE; see Note 2).
b. For separation: A: methanol, 10 mM sodium phosphate buffer, pH 7.3, and tetrahy-drofuran (19:80:1); B: methanol and 10 mM sodium phosphate buffer, pH 7.3 (80:20).
2.3. Dansyl Method
1. Gilson system connected to a Gilson 118 UV detector at 254 nm.
2. A reversed-phase Phenomenex ODS2 column 4.6 x 300 mn id, 4 mm particle size, was used for the stationary phase, with a flowrate of 1.0 mL/min.
3. For deivatization dansyl chloride solution: 5 mg dansyl chloride in 0.5 mL acetone.
4. For separation: Solvent A: 0.1 M ammonium acetate; solvent B: acetonitrile.
1. A standard solution of biogenic amines was prepared by dissolving each amine into a 0.1 N HCl solution to reach a concentration of 2.5 ^mol/mL.
2. 50, 100, 200, and 500 ^L of these solutions were adjusted to 25 mL with 0.4 M borate buffer, pH 10, and filtered through a 0.45-^m filter.
3. Standards were derivatized prior to column injection as follows: 50 ^L of sample were reacted with 50 ^L OPA/MCE reagent for exactly 1 min, and 25 ^L of this solution were immediately injected (see Note 4).
4. Solvent gradient conditions were as follows: 8 min (20% B); 8 min (30% B); 12 min (40% B); 16 min (80% B); 6 min (100% B) and 12 min (20% B). See Note 5.
The external standard method was used (see Figs. 1 and 2 and Note 6).
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