Improved Recovery of Milk Peptides from Digests and Calibration Standard Solutions for the Analysis of Allergenic Food Proteins

摘要

Poloxamer surfactant added to samples in simple solutions minimizes hydrophobic peptide losses to glass and polyethylene vial walls, improving sensitivity up to an order of magnitude. High sample organic solvent content somewhat reduces vial wall losses and is used to reconstitute peptide standards. Matrix components other than the analyte act as carriers, somewhat improving recovery without additives: (1) Peptide losses in milk digest require only 20percent the poloxamer concentration in simple solutions for maximum recovery. Maximum analyte recovery is used to standardize concentrations of "Heavy"-labeled and unlabeled peptides. Equal recovery using poloxamer is necessary to calibrate ~(15)N-labeled intact alpha-S1-casein stock concentration, equalizing observed response factors between simple solutions and different food matrices. Low analyte concentrations incurred in more complex cookie matrices do not require poloxamer additives for full recovery, but are comparable to standard response with poloxamer in simple solutions, as opposed to calibration in matched matrices. Equal relative losses for analyte and labeled peptide standards without poloxamer produce equal calculated results for alpha-S1-casein content, but some food matrices yield lower absolute sensitivity. Future work will assay contaminant milk concentrations incurred in other food matrices: (1) Of interest are cookies with higher fat content and/or different ingredients, chocolate, foods with different baking times; (2) Standardized ~(15)N-labeled intact alpha-S1-casein enables measurement of incurred milk protein extraction efficiency; (3) Benchmarked NIST NFDM can be used in standard additions of milk to "blank" food matrix samples.