The third alternative to sample cleanup is solid-phase extraction (SPE). SPE methods consist of loading samples onto pre-conditioned
sorbent-filled cartridges, often arranged in a 96-well plate format. Loaded samples are then washed with an appropriate solvent,
and the analyte(s) is subsequently eluted. Method selection and extract cleanliness are determined by the retention mechanism
and the ability of the wash stage to effectively remove endogenous components. For example, ionic interactions, as represented
by cation and anion exchange, offer a greater degree of analyte selectivity and, therefore, extract cleanliness when compared
to C18-based sorbents. In general, extracts from SPE are cleaner than those from PPT.
SPE gained in popularity because of its compatibility with automation, especially with sorbent material packed into a 96-well
format plate (17–18). Technological improvements include the development of polymeric SPE sorbents that no longer suffer from
sorbent drying problems while enjoying extended working pH ranges. Taking advantage of the full pH range of the sorbent, a
specific pH and organic modulated SPE (i.e., an optimized SPE) method can be developed to provide clean sample extracts. The
hydrophilic-hydrophobic nature of these polymeric supports is amenable to generic extraction techniques.
A generic protocol should achieve high recovery; however, high recoveries do not necessarily correlate with high sensitivity
in LC–MS/MS. Achieving high sensitivity is usually a trade-off between recovery and chemical interference or ion suppression.
Nevertheless, high sensitivity and high recovery are achievable by selective retention of a basic drug using strong cation
exchange SPE. Fortunately, the majority of drug candidates have a basic functionality that can be leveraged for selective
retention on an appropriate strong cation exchange SPE material.
As described, SPE methods often involve evaporation and subsequent reconstitution of the eluent before LC–MS/MS analysis.
These steps not only take time and effort, but can also lead to the loss of valuable sample. Therefore, the ability to elute
in very small volumes of solvent is desirable to minimize sample preparation time and reduce sample loss. Low sorbent mass
and novel 96-well plate designs, including SPE pipette tips and discs, have alleviated some of these concerns (19).
Another approach to consider is the direct coupling of SPE to the LC–MS/MS system (i.e., on-line SPE) (20). Differences in
flow rates during load and elution steps afford additional opportunities to enhance the extraction process. For example, sufficiently
high flow rates can induce turbulent flow chromatography that actually involves a combination of size-exclusion and adsorption
phenomena (21–24). If the analyte fraction has a high enough affinity for the stationary phase inside the pores, then it will
remain there until a solvent with the appropriate strength desorbs it.
Online SPE methods have the potential to significantly enhance sensitivity because no dilution of sample occurs. Eliminating
analyte collection, evaporation, reconstitution, and injection not only improves reproducibility, but also saves time, labor,
The general idea of sample cleanup is that all elements of a method should contribute to its required sensitivity and selectivity.
Issues to consider when selecting a bioanalytical method for sample cleanup should include what matrix the analyte is in,
the detection limit and dynamic range required, the number of samples to be analyzed, analyte stability to extraction, and
the amount of matrix available.
The final method should be orthogonal to maximize selectivity and reduce ion suppression. If C18 SPE is used for sample extraction,
then the analyst should consider cation exchange or phenyl column chromatography. Alternatively, if strong cation exchange
SPE was selected for sample extraction, then any reverse-phase LC method can be used. The sample cleanup method should be
assessed for recovery, selectivity, precision, accuracy, and ruggedness. Formal validation may also be required (25–27).
Roger N. Hayes is vice-president and general manager of laboratory sciences at MPI Research, 54943 North Main Street, Mattawan, MI 49071.