There are several sample preparation options that havebeen used for many years with HPLC and studies haveevaluated the degree of matrix effects for these differenttechniques [24,27,29,30,32]. It is considered that proteinprecipitation using an organic solvent or dilute and shoot arethe bdirtiestQ sample preparation techniques and thusproduce the most matrix effects compared to solid phaseor liquid–liquid extraction [24,30]. More recently, on-linetwo-dimensional chromatography, using a column-switchingdevice, has been shown to provide an alternate samplepreparation that gives minimal matrix effects [35]. A clearadvantage of this technique is the ability to automate theanalytical process but column breakthrough will limit howmuch crude sample can be loaded. The selection of whichextraction technique to use for an application is not justlimited to providing the cleanest sample but linked also tothe chromatographic separation when optimizing a method.