UPLC analysis of free amino acids in wines: profiling of on-lees aged wines.

The evolution of free amino acid (FAA) profiles intrinsic to on-lees aged white wines was determined by ultra performance liquid chromatography (UPLC™). On basis of the AccQ.Tag™ method as a commercialized amino acid analysis solution for HPLC, a new protocol for dedicated amino acid analysis using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) for pre-column derivatization was established by method transfer onto UPLC™ conditions. Since AQC derivatives enable both fluorescence (AccQ.Tag™ method) and UV detection, the performed method transfer additionally included changing to a more versatile UV detection. Emphasizing enhanced performance of UPLC™, the newly established protocol facilitated rapid and reliable separations of 24 amino acids within 23 min, hence proved to be superior compared to the original HPLC protocol due to significant improvements in resolution and reduced runtime. Applying UV detection enabled adequate quantifications of AQC amino acid derivatives at µM level (LOQs from 0.12 to 1.10 µM), thus proved sufficient sensitivity for amino acid profiling in wine samples. Moreover, this compiled methodology was successfully applied to monitor the changes of FAA concentrations in four distinct sets of on-lees aged white wines (fermented with different yeasts) at three progressing ripening periods, each (control, 3 and 6 months aging). For the control wines, the applied winery yeast significantly affected total FAA amounts (1450-1740 mg L(-1)). During maturation, the proceeding yeast autolysis implied a rather complex impact on FAAs, yielding total FAA excretions up to 360 mg L(-1). However, the magnitude for increases of specific FAAs (up to +200%) highly depended on the individual amino acids as well as on the applied fermenting yeast. Given the overall complexity of yeast autolysis in winemaking, the application of efficient LC techniques such as UPLC™ may indeed contribute as a valuable tool in wine research for product monitoring and characterization of intrinsic developments during wine maturation.

Characterization of amino acid profiles of culture media via pre-column 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization and ultra performance liquid chromatography.

The AccQ.Tag™ method, as a well-established protocol for amino acid analysis combining derivatization procedure, dedicated HPLC separation and fluorescence detection, was properly transferred and accordingly optimized for the application on ultra performance liquid chromatography (UPLC™) and UV detection. Capitalizing on sub-2 µm particles, this newly established UV-UPLC™ technique facilitated efficient chromatographic separation of 21 amino acid derivatives within 12 min. In addition, UPLC™ demonstrated significant improvements due to superior performance and reduced run times compared with the former 35 min of the original HPLC protocol. Using UV instead of fluorescence detection, amino acid quantification after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) yielded appropriate sensitivities within the low pmol range with corresponding detection limits varying from 0.11 to 0.57 pmol per injection. Moreover, the UPLC™ method was applied to characterize changes in the free (FAA) as well as total amino acid (TAA) profiles specific to culture media at three distinctive stages of fermentation: fresh medium, fermentation broth after cell mass production prior to induction and after product expression at the end of fermentation. Amino acid profiles intrinsic to the fresh, sterilized medium indicated free, hence more bioavailable, amino acids at a FAA/TAA ratio of 40%, whereas ongoing fermentation implied a rather specific, successive decline in selective FAA concentrations. Thereby, the most distinctive variations in FAAs were highlighted by aspartic acid, serine and threonine, each exhibiting an almost complete uptake from the culture media (-96% to -99%), with remaining FAA/TAA ratios of 1%, 8%, and 1%, respectively. This indeed may indicate limitations and shortages within the nutrient broth. Thus, amino acid monitoring utilizing high-throughput chromatography, such as UPLC™, can be considered as a valuable tool to facilitate rapid adjustments of fermentation broths and to optimize culture media to specific requirements.

Characterization of isoflavone composition in soy-based nutritional supplements via ultra performance liquid chromatography.

The specific isoflavone composition of nutritional supplements is commonly not-labeled, although the stated amounts are strongly dependent on the present isoflavone conjugates. Hence, 11 soy-based dietary supplements were characterized via a newly established ultra performance liquid chromatography (UPLC) method, on both their native conjugated isoflavone spectra, as well as on quantitative amounts derived as total aglycones after enzymatic hydrolysis utilizing Helix pomatia juice. Capitalizing on sub-2 microm particles, the established RP-UPLC technique facilitated efficient chromatographic separation of all 12 soy intrinsic isoflavone forms within 10 min. Derived native isoflavone profiles implied a certain variability, comprising conjugated forms, especially glycosides, as the predominant isoflavonic constituents throughout the majority of supplements, whereas only two samples indicated the more bioavailable free aglycones as prevailing compounds. Moreover, the robust quantification as total aglycones subsequent to enzymatic hydrolysis, unexceptionally yielded negative deviations referring to the labeled specifications, thus implying that stated amounts were typically calculated on basis of the high molecular isoflavone conjugates. Thus, especially in regard to better comparability, regulations concerning an uniform labeling basis are needed.

A new ultra-pressure liquid chromatography method for the determination of biogenic amines in cheese.

A fast and reliable ultra-performance liquid chromatography (UPLC) method for the determination of biogenic amines (ethanolamine, methylamine, agmatine, histamine, dimethylamine, ethylamine, octopamine, pyrrolidine, dopamine, isopropylamine, propylamine, tyramine, putrescine, butylamine, cadaverine, tryptamine, 2-phenylethylamine, 3-methylbutylamine, spermidine, spermine) in cheese was established. After pre-column derivatization with 6-aminoquinolyl-N-hydroxy-succinimidyl carbamate (AQC), 20 primary and secondary biogenic amines were separated on an Acquity UPLC column (BEH C(18), 1.7 microm; 2.1 mm x 50 mm) within 9 min. Limits of detection (mg/100g cheese) ranged from 0.04 (ethanolamine) to 1.62 (spermine), and limits of quantification were between 0.16 (ethanolamine) and 6.09 (spermine). The UPLC method was applied to the analysis of 58 cheese samples as retailed in Austria. About 13.8% of samples had a histamine content above 10mg/100g, and 22.4% had a tyramine content above 10mg/100g. Moreover, 8.6% of samples had a putrescine or cadaverine content higher than 10mg/100g. The total concentration of biogenic amines in two cheese samples was about 194 mg/100g. Thus, obligatory monitoring of biogenic amines should be considered to ensure quality of cheese in future.