Liquid-liquid extraction (LLE) using organic solvents offers sample cleanup with analyte enrichment steps, and is a rugged
off-line sample preparation process that is well suited for routine high-throughput LC–MS/MS analysis. The basic concept of
LLE is to partition an analyte into a volatile organic solvent away from polar proteins and lipids that remain in an aqueous
phase. The organic phase is removed, evaporated, and the sample reconstituted for injection onto an LC–MS/MS system. By careful
choice of organic solvent, LLE is amenable to automation in a 96-well format (12–15). In order, the acceptability of solvents
for automated LLE is methyl t-butyl ether > 95/5 hexane/ethanol >;>t; ethyl acetate. LLE does, on occasion, suffer from emulsion
formation, which may be resolved by extended centrifugation.
LLE can also be performed in the solid state by using diatomaceous earth (Hydromatrix or Celite). Several such products are
available commercially for performing supported liquid extractions (SLE) in a 96-well plate format.
Plasma sample preparation by protein precipitation (PPT) is the most widely used technique for LC–MS/MS analysis because of
its simplicity, low cost, and universality. PPT is amenable to automation in a 96-well format (16). Precipitation of plasma
proteins is most commonly performed by using organic solvents like acetonitrile or methanol. Following denaturation, the sample
is centrifuged and the supernatant is directly injected. However, organic solvents are inefficient in precipitating proteins
and often require significant dilution of the plasma sample, typically by two- to three-fold. Overcoming the impact of dilution
by injecting larger volumes of the plasma extract may be precluded when using reversed-phase HPLC because of the high organic
content. Evaporation of the extracted samples to near dryness followed by reconstitution in an appropriate solvent is usually
required. In order, the preference of solvent for automated PPT is 95/5 (v/v) acetonitrile/acetone >; acetonitrile >;>; methanol.
Alternative choices for precipitation of plasma proteins include trichloroacetic acid (TCA) or zinc sulfate. Reagents like
TCA and zinc sulfate, however, are unable to remove small proteins, polypeptides, and salts, thereby contributing to high
ionic strength of the supernatant that can subsequently suppress ionization and attenuate LC–MS/MS response.
Despite the attractiveness of PPT as a rapid approach to sample preparation, potential drawbacks exist. For example, because
only proteins are removed, ion suppression from co-eluting matrix components can significantly reduce sensitivity, especially
when using electrospray ionization. Therefore, assessing the potential for ion suppression over the elution time is an important
step in developing a rugged method. Although using stable isotope-labeled internal standards can compensate for response variability,
ultimately such a loss in sensitivity limits the achievable limit of quantification. 96-well filter plates are available commercially
and they provide the analyst the ability to conduct, in an automated fashion, the protein crash procedure, the mix step, and
the filtration of protein precipitants in the same 96-well flow-through plate.