Harnessing the Heme Soret Band: A Dual-Wavelength Ultra-High-Performance Liquid Chromatography Method for Selective Mass Quantification of Lactoperoxidase and Co-Purified Lactoferrin

A robust and selective reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) method was developed and validated for the simultaneous quantification of lactoperoxidase (LPO) and lactoferrin (LF) in commercial LPO raw materials. A key innovation of this method is the exploitation of the characteristic Soret band absorption of the LPO heme group. Separation was performed on an Excsep C₄ column (3 μm, 4.6 × 150 mm) at 60 °C, employing a gradient elution of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in acetonitrile. A novel dual-wavelength detection strategy was established: LPO was selectively monitored at 403 nm via its heme Soret band, while LF was quantified at 280 nm via aromatic amino acid absorption. The A₂₈₀/A₄₀₃ ratio serves as an intrinsic indicator of heme saturation and chromatographic peak purity, providing a basis for distinguishing heme-containing LPO from total LPO protein. This approach ensures high specificity, circumventing the interference common to single-wavelength or activity-based assays. Both analytes exhibited excellent linearity from 0.05 to 10 mg/mL (R² > 0.999). Limits of quantification (LOQs) were 0.49 g/100 g for LPO and 0.48 g/100 g for LF. Spike recoveries ranged from 93.0% to 102.4% with RSDs ≤ 2.4%. Application to eleven commercial LPO raw material samples revealed significant batch-to-batch compositional heterogeneity, consistently demonstrating that LF constitutes a major co-purified impurity. This method provides a critical analytical tool for the rigorous quality control and authentication of dairy-derived bioactive ingredients.