Chromatography–Mass Spectrometry at Hong Kong Baptist University
The major research of this group focuses on method development and applications of chromatography coupled with mass spectrometry
for trace analysis in complex systems, such as the environment, traditional Chinese medicines (TCM), food and biological matrices.
The group is equipped with a wide range of state-of-the-art instrumentation, including GC, GC–MS (quadrupole, ion-trap and
magnetic sector), HPLC, LC–MS (ion-trap and quadrupole-time of flight) and MALDI-TOFMS. Current research programmes include:
HRGC–HRMS for the trace analysis of dioxins and dioxin-like PCBs; PBDEs in environmental and biota samples related to e-waste
recycling; drug metabolism and pharmacokinetics of active components from TCM; metabolomics; and proteomics.
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Chromatography plays an important role in successful analytical method development for trace analysis in complex matrices.
To provide a specific example, they have focused on LC–MS method development for fast, specific and sensitive analysis of
endogenous nucleotides and the phosphorylated metabolites of anti-HIV drugs, such as the nucleoside reverse transcriptase
inhibitors (NRTIs). Nucleoside drugs that can block the production of new mitochondria have been developed against human HIV.
Consequently, the determination of generated nucleotides in cells and tissues at trace levels has become more and more important
for understanding the mechanism of action of these analogues in drug discovery and development. Reversed-phase HPLC–MS is
an attractive approach to accurately qualify and quantify phosphorylated metabolites because of its separation efficiency
and detection sensitivity. However, most nucleotides are not effectively retained on reversed-phase columns using conventional mobile phases because
of their extremely high polarity. The difficulty of separating analytes from the interference background has caused problems
for sensitive detection and identification. Development of a suitable method to improve the retention times of nucleotides
is therefore critical for the trace analysis of nucleotides in biological samples. Although several chromatographic conditions
have been reported for the retention and separation of nucleotides, they were mostly "unfriendly" to ESI-MS. For the application
of ion-paring LC–MS, the group found that dimethylhexylamine (DMHA) caused minimal interference with the ESI-MS analysis and
allowed the simultaneous detection of nucleoside drugs and their corresponding nucleotides under the positive ion mode. Efforts
have been made to apply the minimal levels of ion-paring agent; for example, by using smaller diameter columns to minimize
ion suppression and ESI-MS source contamination from the ion-paring agent. The developed methods have been successfully applied
to support several research programmes, including the trace analysis of phosphorylated metabolites in human liver cells treated
with Ziagen (abacavir — a carbocyclic nucleoside analogue with activity against HIV).
To avoid using ion-pairing agents to improve ESI-MS sensitivity, the group is now exploring new approaches with various chromatographic
conditions, particularly the selection of modified reversed-phase LC columns. Recent results have shown that porous graphitic
carbon (PGC) columns might be promising. It has been found PGC columns can provide much better retention and selectivity for
polar compounds compared with traditional and modified reversed-phase columns — allowing the direct sensitive detection of
nucleotides with negative ion ESI-MS. Another advantage of PGC columns is their tolerance to a wide pH range, especially at
pH > 8, under which the detection sensitivity of nucleotides might be greatly improved with the negative ion mode of ESI-MS.
The LC–MS method with PGC columns has been developed and successfully applied for supporting biochemical research projects.
For more information, contact Professor Zongwei Cai on e-mail: zwcai@hkbu.edu.hk
, or visit http://www.hkbu.edu.hk/~dioxin/ and http://www.hkbu.edu.hk/~caigroup/
