Ανάπτυξη μεθόδων ταυτόχρονου προσδιορισμού βιογενών αμινών με υγρή χρωματογραφία υψηλής πίεσης και εφαρμογή σε αλκοολούχα ποτά

Abstract

In this study, eleven of the most oenologically important biogenic amines have been determined simultaneously in alcoholic beverages by Reversed-Phase High Performance Liquid Chromatography. The amines studied (methylamine, ethylamine, tryptamine, 2-phenylethylamine, isoamylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine) were pre-column derivatized with dansyl chloride, in the presence of 1,7-diaminoheptane (internal standard) and subsequently cleaned-up from derivatization by-products with solidphase extraction (SPE) through C18 cartridges. The methods developed involve the use of an ultraviolet (UV) and a fluorimetric detector used independently, as well as simultaneously, exploiting the unique feature of the dansyl derivatives of biogenic amines to absorb intensively in the UV region and at the same time to fluoresce. The separation of dansylamides was achieved on an Inertsil ODS-3 column (250x4 mm I.D., 5 μm) using a 35-min gradient elution with a binary system of acetonitrile - water, at a flow rate of 1 mL.min-1. UV detection was carried out at 254 nm, fluorescence detection at λex/λem = 320/523 nm and simultaneous detection was performed with the use of both detection systems connected in series. The derivatization and SPE procedures as well as the detection conditions were optimized, for each method. The applied postderivatization SPE procedures, increases the selectivity and sensitivity of the proposed methods. The identity and purity of the dansyl derivative peaks was confirmed by liquid chromatography- diode array/ atmospheric pressure chemical ionization mass spectrometry (HPLC-DAD-APCI/MS). Using UV detection, linearity of derivatization was obtained for concentrations ranging from 0.025 to 3.0 mg.L-1. The within- and between-day relative standard deviations (RSDs) ranged from 0.4 to 5.7% and 0.6 to 7.3% respectively. The fluorimetric detection of the dansylamides resulted in wider linear ranges, covering two orders of magnitude, and improved precision and recovery over other methods using derivatization with OPA and fluorimetric detection. Linearity of derivatization was obtained for concentrations ranging from 0.008 to 40.0 mg.L-1. Compared to UV detection, fluorimetric detection proved to be more sensitive and selective, offering at least one order of magnitude higher sensitivity for all amines (except histamine). The within- and between-day RSDs ranged from 0.2 to 7.6 % and 0.3 to 8.6 % respectively. The overall processes were successfully applied to identify and quantify biogenic amines in white, red and Retsina Greek wines and Greek beers, after treatment with polyvinylpyrrolidone (PVP), which removes the substances that interfere in the derivatization and subsequent quantification of the derivatives. Beer samples were degassed prior to PVP treatment. In all cases the results were satisfactory. The purpose of dual detection was to evaluate the simultaneous use of ultraviolet and fluorescence detection for quantitative determination of dansylated biogenic amines in alcoholic beverages and to show whether there are statistically significant differences between the results obtained by each detection method. The linearity of simultaneous detection was obtained for concentrations covering the common range of the respective linear plots of the independent detection modes. The analysis of alcoholic beverages by simultaneous detection was performed as usual, after PVP treatment, derivatization and subsequent SPE. The difference between results obtained by UV and fluorescence detection was calculated for each amine. Comparison of the detection methods was performed by means of significance tests (t-tests), at the 5% level, applied to within-day measurements of standard solutions, at a medium concentration level, recovery data and results from the analysis of wine and beer samples. On the basis of peak size, fluorescence detection showed higher sensitivity for all amines, except for histamine. On the other hand, both detection methods yielded similar accuracies, as indicated by the statistical comparison between their recovery data. UV and fluorescence detection yielded recoveries that were not statistically different for any of the amines. On the contrary, the results obtained from within-day measurements of standard mixtures were statistically different for all amines. The proposed methods offer several advantages over previously reported ones, including: The use of a simple gradient which effectively separates all the amines in a relatively short time, a simple, time- and cost- effective pretreatment of the samples, high precision and accuracy, satisfactory recoveries and an order of magnitude higher sensitivity when compared to other methods. Additionally, the proposed fluorimetric and simultaneous detection methods, the LC-DAD-MS identification and peak purity check of dansylamides, as well as the statistical comparison of the detection modes, by means of significance tests (t-tests) are applied here, for the first time in the literature, to alcoholic beverages.