Experimental design considerations in quantification experiments by using the internal standard technique: cholesterol determination by gas chromatography as a case study.

An overview on experimental design in quantification experiments using the internal standard technique is described. Particular emphasis is given to the modelling of the relationship analyte/internal standard and the estimation of the optimal amount of internal standard to be used throughout the analytical concentration range by using a uniform shell design with a minimum number of seven experiments. The described approach is demonstrated in the analysis of cholesterol in various reference materials by gas chromatography (GC), using alpha-5 cholestan as internal standard. The outlined approaches can be applied in the determination of any kind of analyte.