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  • br Experimental br Results and discussion First of

    2021-09-22


    Experimental
    Results and discussion First of all we had to choose suitable electrolyte´s system. We utilized advantageously the composition of the background electrolyte for biogenic amines separation published in our work [13] to which we adapted the leading and terminating electrolytes. With respect to an excess of sodium in food samples we chose it as a leading cation which was buffered with adipic TNF-alpha, recombinant human protein to pH 5.5 (close to the pH of the background electrolyte). By the addition of EDTA into the leading electrolyte calcium and magnesium were removed from the cationic separation as negatively charged complexes, thereby reducing the analytical column load. Histidine buffered with adipic acid served as the terminating electrolyte for cITP step. Methanol addition into the electrolytes had several functions: The background electrolyte was used in nearly unchanged composition (the only EDTA was not added because of it is in the leading electrolyte) as that used in our CZE method [13]. Electropherograms (CZE step) of model mixture of some biogenic amines including HA and a sample extract of sprat meal are shown in Fig. 1. It is clear from these records that HA is fully separated from other biogenic amines and from sample matrix. Relative step height of HA is 0.17 (cITP step is not shown). Table 1 summarizes measured method characteristics, i.e., linearity, intra-assay, accuracy (recovery), limit of quantification, and detection limit. Relatively high deviation of migration time of HA is caused by variable sodium content in samples, the higher sodium content the longer ITP step resulting in longer migration time of HA. So this is not a fluctuation of MT at the CZE step, but time-shift of the start of the CZE step. On the contrary, thanks to the use of a hydrodynamically closed separation system (suppression of electroosmotic flow), the reproducibility of MT at the CZE step is excellent in the order of seconds, i.e., deep below 1% rel. To illustrate the sensitivity of the developed cITP-CZE method with conductometric detection (cITP-CZE-COND), a comparison with the previously published methods for histamine determination is given in Table 2. The developed cITP-CZE method provides up to two orders of magnitude higher sensitivity in comparison with published electrophoretic methods. The sensitivity of this method is the same or even higher than that of HPLC with fluorimetric and/or MS detection. This high sensitivity is achieved by the on-line isotachophoretic pre-concentration and the application of very sensitive conductivity detection in the CZE step. On a representative series of 37 food and feed samples the developed cITP-CZE-COND method was compared with accredited HPLC method with post-COlumn derivatization and fluorimetric detection. The obtained results are summarized in Table 3 and Fig. 2. The correlation between cITP-CZE and HPLC results is excellent (r = 0.996). Detailed statistical analysis of the obtained data revealed that the electrophoretic and chromatographic methods give comparable results. Because of the slope ± standard deviation of the regression equation includes unity (0.983 ± 0.031) and the intercept on the Y axis ± standard deviation includes zero (-0.2 ± 1.2) it can be stated that the difference of results between the electrophoretic and the chromatographic method is not statistically significant, i.e., both methods give the same results.
    Conclusions The presented results showed that the developed cITP-CZE method with conductometric detection for the determination of HA in food and feed samples is reliable and reproducible. On representative series of food/feed samples was proved that cITP-CZE gives comparable results as routine accredited HPLC method with fluorimetric detection, and thus be easily an alternative method to HPLC. Low laboriousness (no derivatization step or sample cleaning), sufficient sensitivity (˜ ng/mL), and low running cost (no separation column, small volume of aqueous diluted electrolyte) are the advantages of the presented cITP-CZE-COND method. Unlike the published electrophoretic methods [[16], [17], [18]] the developed cITP-CZE-COND can be used for the determination of HA in salty samples. This method can be potentially applied for the determination of another biogenic amines in foodstuffs with comparable sensitivity as for histamine.