Estimation of Protein in Anhydrous Milk Fat

A method for the estimation of protein in anhydrous milk fat is described. The protein concentration is estimated by quantifying the arginine and aspartic acid (ASX) released upon acid hydrolysis of the anhydrous milk fat. Arginine and aspartic acid are derivatized with the fluorescent tag 9-fluorenylmethoxycarbonyl (FMOC), the derivatives are quantified by reversed phase HPLC, and the protein concentration in the anhydrous milk fat is estimated as 8.77 × (Arg + ASX). Method suitability was defined by experimental assessments of FMOC-arginine and FMOC-aspartic acid linearity (R 2 averages > 0.999), protein estimate precision [day-to-day RSD values (n = 8 days) ranged from 7.3 to 14 % for protein concentrations of 9.42 to 40.0 mg/kg of anhydrous milk fat], protein estimate accuracy [spike recovery average = 95.6 % (9.3 %), n = 8, spiking level = 20 mg/kg and agreement of the experimental protein estimate for butter, 0.611 (0.044) g/100 g, n = 3 lots, with a published value 0.6/100 g], and analyte selectivity (baseline resolution of FMOC-arginine and FMOC-aspartic acid from the FMOC derivatives of other common amino acids). The method provides for a reliable estimation of the protein content of anhydrous milk fat, when present at concentrations >2 mg/kg.