An internal standard is necessary because of uncertainties introduced during the solid-phase extraction. For example, the volume of serum transferred to the solid-phase extraction cartridge, 0.5 mL, and the volume of solvent used to remove the analyte and internal standard, 0.25 mL, are very small. The precision and accuracy with which we can measure these volumes is not as good as when using larger volumes. In addition, the concentration of eluted analytes may vary from trial to trial due to variations in the amount of solution held up by the cartridge. Using an internal standard compensates for these variation. To be useful we must assume that the analyte and the internal standard are completely retained during the initial loading, that they are not lost when the cartridge is washed, and that they are completely extracted during the final elution.