Quantitative Determination of Casein in Facial Masks Labeled as Containing Milk by UHPLC‐ESI‐QqQ‐MS

Casein-based ingredients are widely used and labeled in cosmetic products, especially facial masks. However, a reliable determination method has not been established, and label authenticity cannot be readily verified. Therefore, in the present study, we selected four previously verified marker peptides corresponding to the four milk casein subtypes (αs1, αs2, β, and κ) and developed a quantitative UHPLC-ESI-QqQ-MS method for determining caseins in facial mask samples. Compared with organic-solvent precipitation and isoelectric precipitation, an optimized in-gel enzymatic digestion using 8% polyacrylamide, followed by three cycles of ultrasound-assisted extraction with 0.1% formic acid-acetonitrile, afforded the highest recovery. αs1-, αs2-, and κ-casein showed good linearity from 0.1 to 4 µmol/L, while β-casein was linear from 0.2 to 8 µmol/L (all R² > 0.990). For β-casein, the detection limit and quantitation limit were 10 and 30 nmol/L, respectively. Matrix effects (ME) were calculated as (response in matrix/response in neat solution) - 1. The ME values were 0.5, 0.4, 0.3, and 0.4 for the selected peptides of αs1-, αs2-, β-, and κ-casein, respectively, indicating that the corresponding peak areas in the milk matrix were 50%, 40%, 30%, and 40% higher than those obtained in neat solution. Therefore, all four peptides exhibited moderate-to-strong signal enhancement rather than ion suppression. Method accuracy and precision were satisfactory, with recoveries of 87.0%-108.0% and relative standard deviations of 2.8%-8.2%. Applied to 20 batches of facial masks labeled as containing milk protein extract, casein was detected in 11 batches. In conclusion, the method is suitable for authenticity verification of casein in cosmetic products.