A novel ultra-high performance liquid chromatography method for the rapid determination of ß-lactoglobulin as heat load indicator in commercial milk samples.

The level of undenatured acid-soluble ß-lactoglobulin can be used as an indicator to assess the heat load applied to liquid milk, thus further allowing the discrimination between milk originating from different thermal production processes. In this work, a new UHPLC method for the rapid determination of bovine ß-lactoglobulin in 1.8min only (total runtime 3min) is presented using simple UV detection at 205nm. Separation selectivity for possibly co-eluting other major whey proteins (bovine serum albumin, lactoferrin, α-lactalbumin, immunoglobulin G) was verified, and the method validated for the analysis of liquid milk samples regarding linearity (20-560µg/mL, R(2)>0.99), instrumentation precision (RSDs<2.8%), limits of detection and quantification (7 and 23mg/L milk), repeatability of sample work-up (RSDs≤2.6%) and method recovery (103%). In total, 71 commercial liquid milk samples produced using different preservation techniques (e.g., thermal or mechanical treatment), hence featuring different applied heat loads, were profiled for their intrinsic undenatured acid-soluble ß-lactoglobulin levels. As expected, pasteurized milk showed the highest concentrations clearly above 3000mg/L due to pasteurization being the mildest thermal treatment, while in contrast, ultra-high temperature heated milk featured the lowest amounts (<200mg/L). For extended shelf life (ESL) milk, quite diverse levels were determined ranging from ∼100 up to 4000mg/L, thus clearly illustrating variable applied heat loads and impacts on the "nativeness" of milk essentially due to the fact that the production technologies used for ESL milk may differ significantly, and are currently not regulated in the EU.

Application of UHPLC for the determination of free amino acids in different cheese varieties.

A rapid ultra-high performance liquid chromatography (UHPLC) protocol for the determination of amino acids as their respective 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives was successfully applied for assessing free amino acid levels in commercial cheese samples representing typical product groups (ripening protocols) in cheesemaking. Based on the Waters AccQ.Tag™ method as a high performance liquid chromatography (HPLC) amino acid solution designed for hydrolyzate analyses, method adaptation onto UHPLC was performed, and detection of AQC derivatives was changed from former fluorescence (λ(Ex) 250 nm/λ(Em) 395 nm) to UV (254 nm). Compared to the original HPLC method, UHPLC proved to be superior by facilitating excellent separations of 18 amino acids within 12 min only, thus demonstrating significantly shortened runtimes (>35 min for HPLC) while retaining the original separation chemistry and amino acid elution pattern. Free amino acid levels of the analyzed cheese samples showed a high extent of variability depending on the cheese type, with highest total amounts found for original Italian extra-hard cheeses (up to 9,000 mg/100 g) and lowest for surface mold- or bacterial smear-ripened soft cheeses (200-600 mg/100 g). Despite the intrinsic variability in both total and specific concentrations, the established UHPLC method enabled reliable and interference-free amino acid profiling throughout all cheese types, thus demonstrating a valuable tool to generate high quality data for the characterization of cheese ripening.

Application of UHPLC for the simultaneous analysis of free amino acids and biogenic amines in ripened acid-curd cheeses.

A new UHPLC method for the simultaneous determination of amino acids and biogenic amines in a single run, and its first application to profile ripened acid-curd cheeses was presented. After pre-column derivatization with 6-aminoquinolyl-N-hydroxy succinimidyl carbamate (AQC), 23 amino acids and 15 amines were separated in 9min only (12min total run time), and eluates monitored using their UV response at 249nm. Limits of detection (0.05-0.29mg/100g) and quantification (0.16-0.97mg/100g), repeatability for sample preparation (1.0-6.1% RSD) and method recoveries (83-120%) were found suitable for cheese analysis. In total, 47 acid-curd cheeses classified into sub-groups like cooked, Quargel-type or grey cheeses were analyzed for their free amino acid and amine (histamine, tyramine, putrescine, cadaverine, and tryptamine) contents, which (as expected) were highlighted by a great variability. Total free amino acid levels ranged between less than 100 and more than 4000mg/100g (median 567mg/100g), implying that for some cheeses less or not ripened/fresh quark was used for production or, in contrast, a higher degree of proteolysis had occurred. For the sum of biogenic amines, median concentration was determined at 7.0mg/100g, while only 5% of all cheeses had levels higher than 161.9mg/100g. Thus, the obtained results suggest quite acceptable biogenic amine levels for (mostly underrated) ripened acid-curd cheeses, although partly exceptional high concentrations (>250mg/100g) were indeed observed in individual samples.