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 Organis ation for Standardisation, Guide to the Expression of Uncertainty and the E URACHEM/CITAC Guide "Quantifying Uncertainty in Analytical Measurement" wer e followed resulting in the expression of combined uncertainties as an expa nded uncertainty. The authors have identified the lack of a detailed descri ption on all possible sources of uncertainty on chromatographic milk protei n separations. Up to now, uncertainty has only been expressed as a random c hromatographic variability, being exclusively measured by regression equati ons. On the contrary, the establishment of a full uncertainty budget permit s a complete identification of all sources of uncertainty associated with t he measurement. Thus, a better comprehension of the method is obtained. Hav ing identified all sources of uncertainty, to the best of our knowledge, th ey are minimised (whenever possible) and combined. The importance of this i dentification is crucial, in particular, concerning previously neglected so urces, 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 unc ertainty statement, which should be based on a full uncertainty budget eval uation) is demonstrated. Moreover, due to the high precision of the propose d method, the total protein content, in addition to the group protein compo sition (i.e. total casein and whey protein content) is calculated with high reliability, which is an extremely useful factor regarding the establishme nt of milk provenance/adulteration. Combined relative standard uncertaintie s ranging from 3.3 up to 12.5% for individual protein concentration were fo und, whereas for total protein content, an overall combined relative standa rd uncertainty of 2.7% (liquid) and 3.3% (powdered samples) was achieved.