Figure 6: LC–MS-MS postcolumn infusion studies: (a) protein precipitation (Captiva, Agilent), (b) solid-phase extraction using
a neutral polymeric cartridge, (c) liquid–liquid extraction with methyl-tert-butyl-ether and (d) lipid-stripped protein precipitation
(Captiva NDLipids, Agilent).
A second example is also a protein precipitation but considering an area where ion suppression comes into play. The presence
of phospholipids in plasma can cause ion suppression if analytes of interest coelute in the portion of a chromatogram where
these phospholipids appear. Phospholipid MS-MS selectivity can be achieved by considering the m/e 184 → m/e 184 transition indicative of phospholipid and lysophosphatidylcholine elution. Figure 6(a) shows a typical infusion experiment
result that is obtained from protein-precipitated plasma, followed by Captiva filtration (Agilent Technologies, Delaware,
USA). If the drug or its metabolites were to coelute with these compounds, ion suppression may occur and the analytical results
would be jeopardized. Thus, in this case the simple protein precipitation sample preparation procedure may not be enough to
provide reliable data. By performing the more complex SPE [Figure 6(b)] or liquidliquid extraction [Figure 6(c)], the extract
is now cleaner and many of the phosphorus-containing lipids are greatly reduced. To get the best overall performance, an even
more sophisticated phospholipid reduction may be achieved with a selective SPE phase that removes the last traces of phosphorylated
compounds [Figure 6(d)]. Luckily, a product called Captiva NDLipids , which is a combined membrane filtration and phospholipid removal 96-well plate, performs both operations at once and thus
is a simple just enough solution to this problem.
Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Seven Common Faux Pas in Modern HPLC