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

• The application of UHPLC for the determination of β-lactoglobulin in milk is proposed.
• The level of acid-soluble β-lactoglobulin was used to assess the applied heat load.
• Commercial liquid milk samples produced with different technologies were analyzed.
• β-Lactoglobulin amounts varied greatly within extended shelf life (ESL) milk samples.

The level of undenatured acid-soluble β-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 β-lactoglobulin in 1.8 min only (total runtime 3 min) is presented using simple UV detection at 205 nm. 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 (20–560 μg/mL, R2 > 0.99), instrumentation precision (RSDs < 2.8%), limits of detection and quantification (7 and 23 mg/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 β-lactoglobulin levels. As expected, pasteurized milk showed the highest concentrations clearly above 3000 mg/L due to pasteurization being the mildest thermal treatment, while in contrast, ultra-high temperature heated milk featured the lowest amounts (<200 mg/L). For extended shelf life (ESL) milk, quite diverse levels were determined ranging from ∼100 up to 4000 mg/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.