Liquid-liquid extraction (LLE) is a technique used to separate analytes from interferences in the sample matrix by partitioning between two immiscible liquids. A given volume of aqueous sample solution (e.g., plasma) containing analytes is mixed with an internal standard in solution. A volume of buffer at a known pH (or a strongly acidic or basic solution that adjusts pH) is then added to maintain the analytes in their unionized (uncharged) state. The resulting solution is then vigorously mixed with several ratio volumes of a water-immiscible organic solvent or mixtures of two or more solvents such as hexane, diethyl ether, methyl teri-butyl ether (MTBE), or ethyl acetate. Substances in the sample matrix distribute between these two liquid phases (aqueous and organic) and partition preferentially into the organic phase when analytes are unionized (uncharged) and demonstrate solubility in that organic solvent. Isolation of the organic phase, followed by evaporation and reconstitution in a mobile-phase-compatible solvent, yields a sample ready for injection (Figure 17.2). The method provides efficient sample cleanup, as well as sample enrichment.
Plasma (100 uL) Internal Standard (25 uL) Buffer (100 jiL)
Organic Solvent (1 mL) j, Seal
^ Mix thoroughly x 10 min ^ Centrifuge x 5 min
Isolate Organic Layer (Discard Aqueous Layer)
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