Uncertainty estimation on the quantification of major milk proteins by liquid chromatography

A detailed uncertainty budget is evaluated for the quantification of major milk proteins separated by reversed phase ion-pair high performance liquid chromatography (RP-IP HPLC). Recommendations from the International Organisation for Standardisation, Guide to the Expression of Uncertainty and the EURACHEM/CITAC Guide "Quantifying Uncertainty in Analytical Measurement" were followed resulting in the expression of combined uncertainties as an expanded uncertainty. The authors have identified the lack of a detailed description on all possible sources of uncertainty on chromatographic milk protein separations. Up to now, uncertainty has only been expressed as a random chromatographic variability, being exclusively measured by regression equations. On the contrary, the establishment of a full uncertainty budget permits a complete identification of all sources of uncertainty associated with the measurement. Thus, a better comprehension of the method is obtained. Having identified all sources of uncertainty, to the best of our knowledge, they are minimised (whenever possible) and combined. The importance of this identification is crucial, in particular, concerning previously neglected sources, in this example the combination of the interpolated injected mass of protein and its purity, having a dominant influence on the total combined uncertainty. The quality of the measurement (measured by its associated uncertainty statement, which should be based on a full uncertainty budget evaluation) is demonstrated. Moreover, due to the high precision of the proposed method, the total protein content, in addition to the group protein composition (i.e. total casein and whey protein content) is calculated with high reliability, which is an extremely useful factor regarding the establishment of milk provenance/adulteration. Combined relative standard uncertainties ranging from 3.3 up to 12.5% for individual protein concentration were found, whereas for total protein content, an overall combined relative standard uncertainty of 2.7% (liquid) and 3.3% (powdered samples) was achieved.