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Figure 7.8
UPLC-MS spectra of pooled extracted plasma reported by Bruce et al.
(2008).
weight compounds. Therefore, caffeine and its metabolites are only a few of
many other compounds investigated when using such methodologies.
Bruce et al (2008) described a protocol designed to perform high-throughput
metabolic profiling analysis on human blood plasma by UPLC-MS. They
investigated different protocols of extraction, as well as UPLC columns with
three different stationary phases (C8, C18, and phenyl). Gradient elution with
a mixture of 0.1% formic acid and acetonitrile was used for chromatographic
separation. A time-of-flight (ToF) MS equipped with an ESI source operating
in positive ion mode was employed for detection and all mass spectral data
from m/z 50 to 850 were acquired. Caffeine was observed as a positive ion with
m/z 195.09 and retention time of 2.74 min (Figure 7.8).
Caffeine analysis in the background of doping has also been the subject of
novel LC-MS methodologies. Even though it was removed from the World
Anti-Doping Agency (WADA) prohibited list in 2004, caffeine is currently
part of the WADA monitoring program in order to detect patterns of misuse
of caffeine in sport. Badoud et al (2009) developed a multi-analyte high-
throughput screening method based on UPLC coupled to hybrid quadrupole
time-of-flight (Q-ToF) MS to identify 103 forbidden substances from various
classes (such as stimulants, diuretics, narcotics, anti-estrogens) and WADA-
monitored compounds, such as caffeine in urine. Due to the high sensitivity
and selectivity of the method, urine samples could be simply diluted 2-fold
prior to injection. Caffeine was observed as a positive ion with m/z 195.0882,
retention time of 2.03 min and showed an adequate limit of detection, of
25 ng mL
-1
.
Human pharmacokinetic studies that involve the simultaneous quantifica-
tion of multiple agents require specific analytical methods such as LC-MS.
When complicating factors such as the widely different polarities of the drugs
and their wide range of concentrations found in plasma are taken into account,
LC-MS-MS methods are the most adequate. Moreover, LC-MS-MS allows
for simpler sample preparation protocols and more rapid run times. Cold
medications usually combine
an analgesic like paracetamol and an anti-
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