Mass Spectrometry Determination of Fining-Related Allergen Proteins in Chilean Wines

Casein (milk powder) and ovalbumin (egg white powder) are often used as wine fining agents with the objective to promote interactions with undesirable compounds, e.g., polymeric phenols. This kind of proteins may trigger allergic reactions in susceptible individuals; therefore, their occurrence in wines could become a human health risk, moreover when their presence is not reported. The objective of this work was to establish a mass spectrometry method to determine casein and ovalbumin in Chilean wines. To the best of our knowledge, the present work reports for the first time the presence of these proteins in Chilean wines. Proteins were extracted combining the use of ultrafiltration membranes and protein precipitation with organic solvents. Thereafter, proteins were digested with trypsin during 7h with a 1:10 enzyme protein ratio. Chromatographic separation was carried out on a Kinetex XB C-18 (100x4.6mm, 2.6m) column set at 35 degrees C using an acidified (0.1% v/v formic acid) mobile phase composed of ultrapure water (A) and acetonitrile (B). For a highly selective evaluation, quantification was carried out by mass spectrometry applying multiple reaction monitoring (MRM). Validation was established according to the International Conference Harmonization guidelines. Calibration data (n=6) fitted a linear regression model with determination coefficients (R-2) higher than 0.99. Repeatability (n=6) and intermediate precision (n=3) showed RSD values 1.36% and 1.53%, respectively. Recovery (n=3) at three levels ranged from 86.40 to 106.98%. Detection and quantification limits ranged from 4.70 to 8.50gL(-1) and 10 to 20gL(-1), respectively. Twenty Chilean wine samples were analyzed; 18 samples showed quantifiable levels, from which three samples presented a total casein and ovalbumin concentration (0.24-0.26mgL(-1)) closer or higher to the European limit for mandatory labeling (0.25mgL(-1)).