A modification of the established protocol was required, as the
original standard curve had a broad protein percentage range
beyond that normally encountered in lean beef and the results
were not similar to the Dumas obtained results. Therefore, a new
standard curve using homogenized lean beef samples (n = 10) with
known protein content (wet matter basis) of 18.5–24.5%, as
determined using the Dumas method, was employed. The new
calibration curve was develop using the internal software installed
in the equipment (Sprint). For each reference point, five replicates
were obtained and two outliers (>3 times SD from mean) were
removed to reduce the variability at each point and strengthen
linearity of the standard curve. Slope, intercept and coefficient of
determination (R2) were provided by the software based on the
standard values from Leco (provided previous to the analyses) and
the internal readings for variation in the iTAGTM dye absorbance.