Evaluation of the uncertainty associated with the off line HPLC–GC(FID) determination of 4-desmethyl sterols in vegetable oils

This paper discusses the estimation of the uncertainty of the chromatographic determination of 4-desmethyl sterols in vegetable oils, combining the off line HPLC fractionation of the analytes, from the unsaponifiable fraction of the samples, with their determination as TMS derivatives by GC(FID), using the data obtained from a single internal calibration (one surrogate) at one level and “bottom up” approach. The methodology used, makes possible to identify the main uncertainty contributions, find their origins, and reduce them. The final results show that the main contributions to the relative overall uncertainty are those closely related with the chemical aspects of the method, i.e. those related to derivatization reaction and quantification of the analytes, although others aspects, such as the addition of a mass of surrogate, are not negligible.