Method Development, Validation, and Sample Analysis for Regulated Quantitative Bioanalysis Using LC-MS/MS - LC-MS in Drug Bioanalysis - page 47

Table 2 Common phospho-
lipid SRM transitions

Phospholipid SRM transitions

Lyso-phosphatidylcholine; 16:0 496 → 184

Lyso-phosphatidylcholine; 18:0 524 → 184

Phosphatidylcholine; 30:1 704 → 184

Phosphatidylcholine; 34:2 758 → 184

Phosphatidylcholine; 38:6 806 → 184

phenomenon of “matrix effects,” even though the sources causing the matrix effects
were unknown at that time.
The term “matrix effect” can be broadly applied to any change in the behavior of
a compound (analyte or internal standard) due to the presence of any other coeluting
compound(s). Specifically for LC-MS/MS application, the impact could be either
ionization enhancement or suppression. Coeluting compounds could be salts;
endogenous compounds such as fatty acids and triglycerides; exogenous compounds
such as drugs and their metabolites, including their corresponding internal stan-
dards; dosing vehicles such as carboxy-methyl-cellulose, DMSO, phosphate buff-
ered saline, polyethylene glycol, etc.; anticoagulants such as EDTA or heparin; or
constituents from the laboratory analysis process such as polymers, surfactants, and
mobile phase additives. Although the reasons behind the matrix effects remained
unclear for several years, the infusion experiment was well known and popularly
used to showcase the matrix effect in a qualitative manner [ 13 ] . In this experiment,
an extracted control blank sample is injected while the analyte of interest was
infused into the HPLC effluent postcolumn using a syringe pump. Any ion suppres-
sion or enhancement would be observed as a decrease or increase of the MS signal
at the time of analyte elution, without any real information as to the source of the
effect. In 2003, as one of the pioneer research groups, our laboratory reported that
endogenous phospholipids (PLs) were the primary source of the matrix effects
observed when using ESI in the positive ion mode [ 14, 15 ] . Since then, numerous
additional publications addressing matrix effects in great detail have been published
as the phenomenon becomes better understood each year [ 16- 20 ] .
To ensure that appropriate resolution and lack of interference is obtained during
method development, it is highly recommended actively monitoring PLs and aware
of the retention/elution times and intensity of the PLs relative to the retention/elu-
tion times for the analytes of the interest. Table 2 summarizes several representative
Selective Reaction Monitoring (SRM) transitions for PLs monitoring purpose dur-
ing method development.
In general, phospholipids can be managed either via extraction or chromato-
graphic separation. The best approach is to selectively remove the phospholipids
from the sample during extraction. However, if nonselective extraction has to be
used, significant amounts of phospholipids remaining in the sample will be injected
onto LC column along with the analytes. In general, if the chromatography requires
a higher organic composition, the PLs will either elute in the same injection or sub-
sequent injection(s) and cause ion suppression (or enhancement) and may result in
various analytical problems including, but not limited to: decreased or increased