Biomedical Engineering Reference
In-Depth Information
the ionization yield observed for an analyte in a complex matrix (e.g. supernatant of
a serum specimen after protein precipitation) is lower than observed for the target
analyte dissolved in a seemingly pure solvent, the term “ion suppression” is used;
while “ion enhancement” describes the increase in ionization yield due to matrix
constituents when compared to a “pure,” “neutral” or “inert” matrix. It should be
noted, however, that “HPLC-grade” solvents such as methanol, acetonitrile or water
also interact with analytes during ionization—on the one hand due to their inherent
specific chemical properties or on the other hand by containing impurities [ 44, 45 ] .
Thus, there exist no “matrix free” analyses in LC-MS/MS at all.
Factors that cause ion suppression or enhancement can include salts and hydro-
philic small molecules which occupy or provide ions (i.e. hydrogen, sodium or
ammonium ions) or compounds affecting the droplet formation as surface active
compounds. Also late eluting sample constituents as phospholipids may cause ion
suppression, in particular becoming more serious if gradient elution is applied.
Matrix effects depend on the mode of ionization used as well as on the selected ion
polarity. Generally, positive ion electrospray ionization (positive ESI) exhibits more
pronounced effects, while more selective API techniques as negative ESI (which is
applicable only for a minority of target analytes), atmospheric pressure chemical
ionization (APCI), and atmospheric pressure photo ionization (APPI) methods are
affected.
Ion suppression effects are investigated by most analysts during method develop-
ment and validation by applying post-column infusion of dissolved target analyte
into the eluate of the HPLC column via a T-piece (Fig. 1 ). This infusion generates a
sustained background signal in the selected reaction monitoring (SRM—also known
as multiple reaction monitoring—MRM) trace of the target analyte. Typically, in
this set-up the injection of analyte free biological matrix (e.g. deproteinized plasma)
by HPLC leads to a drop in the baseline SRM-signal generated by an infused ana-
lyte for seconds or up to several minutes. If the target analyte elutes during this
period of baseline depression, a specific analyte is said to be subject to ion suppres-
sion. It shall not be overlooked, that this type of experiment is strictly qualitative
and does not allow evaluating the impact of the observed ionization attenuation onto
the final readout of the assay—the quantitative analysis result. Hence quantitative
approaches as spiking experiments or the internal standard balanced matrix factor
evaluation procedure should be added to any validation protocol.
Regarding the HPLC settings, it should be generally desired to develop chro-
matographic conditions which avoid elution of the target analyte during the period
of ion suppression within a chromatographic run [ 46 ]. However, this approach is
typically associated with increased run-times leading to diminished sample through-
put and increased instrument costs per analyzed sample. The sample preparation
protocol applied in a method in part determines the duration of ion suppression dur-
ing a chromatographic run: typically laborious methods as solid phase extraction
(SPE) or liquid-liquid extraction (LLE) achieve short periods of ion suppression
associated with the sample solution solvent front, while simple protein precipitation
protocols (only removing one dominant macromolecule class from the specimen) is
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