LIQUID-LIQUID EXTRACTION

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.