Biomedical Engineering Reference
In-Depth Information
Liquid-liquid and SPE extractions tend to produce much
cleaner samples. Liquid-liquid extractions involve the addition of
an immiscible organic solvent such as methyl-t-butyl ether (MTBE)
or ethyl acetate to an aqueous matrix sample, typically at a ratio of
3:1 (organic solvent:aqueous sample) or higher to achieve suitable
recovery in a single extraction step. After mixing, the organic and
aqueous layers are allowed to separate. The organic solvent layer
containing the extracted drug is evaporated and the sample is recon-
stituted in a compatible solvent for analysis. For ionizable drugs, the
pH can be adjusted to provide for additional cleanup steps. For
instance, a sample can be made basic to keep an acidic drug in an
ionized state and therefore not extractable. The organic solvent can
then be used to remove interfering matrix components with an
initial extraction, leaving the drug behind in the aqueous layer.
After pH adjustment to an acidic condition, the organic solvent
can be added again and the now non-ionized acid drug extracted.
Liquid-liquid extraction is suitable for ocular fluids, tissue homo-
genates, and even intact tissues (liquid-solid extraction using vigor-
ous conditions) and can yield very clean extracts for analysis. When
using liquid-liquid extraction for intact ocular tissues and homo-
genates, very long mixing times or multiple extraction steps may be
required for acceptable recovery of drug. Lastly, SPE has been used
successfully in assays of plasma and other biological fluids, but it is
less suitable for tissue homogenates since homogenates contain
solid particles which can clog the SPE columns. For SPE to have
utility for ocular tissue matrices, the extraction procedure must be
coupled to one or more additional sample cleanup procedures to
produce a suitable solution for loading onto the SPE column. For
instance, organic supernatant of a precipitated tissue homogenate
may be diluted with aqueous buffer and then loaded onto a SPE
column for additional sample cleanup. Care must be taken to ensure
that a high percentage of drug is released into the homogenization
buffer or solvent prior to loading the SPE column and to ensure that
drug bound to soluble tissue components does not simply flow
through the SPE column to waste.
Regardless of the extraction procedure used, it is important to
include an internal standard within the assay. The internal standard
is a chemical which is structurally similar to the drug to be analyzed
and is added in equal amounts to all samples in an analytical run. The
internal standard is used to correct for sample-to-sample inconsis-
tencies in sample processing and chromatographic analysis. An
internal standard can be added at one of several steps of the extrac-
tion procedure depending on assay design and need for reanalysis of
the sample. The internal standard should be added as early as possi-
ble in the procedure to compensate for losses through processing.
Use of a poor internal standard can result in difficulties in method
development, restrictions in sample processing methodology,
and an assay, which is subject to high failure rates, especially with
