The RPC method is well suited to the analysis of aqueous samples lacking appreciable lipid and protein content. Human urine fits this description well, requiring only an optional dilution prior to direct analysis by RPC LC-MS. The analytical method can also be used for more complex sample types where the lipids and proteins present are first removed as to not compromise the analysis by accumulating or precipitating, respectively, on the column leading to system pressure increases, unpredictable hydrophobic metabolite elution and ionisation suppression effects. However, the complexity of the sample preparation and potential for introducing selectivity bias rapidly increases. For example, precipitation of protein (required in samples with appreciable protein to prevent on-column precipitation, increased LC system pressure and ultimately column blockage) by addition of organic solvent either requires drying and solvent exchange (i.e. reconstitution in water) or injection of the organic-solventcontaining sample in sub-optimal conditions for the analysis, given that retention of small molecules in biofluid samples generally requires loading and initial chromatographic conditions to be as close to 100% aqueous as possible given the composition and tolerance of the stationary phase. Similarly, depletion of lipids can be conducted by laborious methods such as liquid/liquid biphasic separation or consumables-driven approaches such as solidphase extraction (SPE). However, perturbation of the small molecule profile is a risk that must be minimised or avoided. Consequently, not all depletion methods are accepted as suitable for analysis of the small molecule content of a biological sample. Finally, note that this method would not be suitable for analysis of lipophilic species (e.g. by extending the gradient to higher strength elution) owing to their insolubility in the requisite aqueous sample diluent and chromatographic initial conditions.