Recently, several novel technologies have emerged with substantial benefits in
toxicological analysis. These include the development of beadbased
multiplex
immunoassay (Suspension Bead Array, SBA), the use of reduced-volume
centrifugal ion-exchange
extraction (SpinSPE),
and Ultra-Performance
(TM) liquid chromatographic
separation coupled to mass spectrometry (UPLC(TM)/MS n ). This work sought to investigate
the efficacy and practicality of these innovative approaches against a benchmark of
established methods and instrumentation for the screening and confirmation of
amphetaminetype
substances.
This study begins with a statistical survey of amphetaminetype
substances encountered in
an accredited forensic laboratory supporting the Australian Capital Territory and regional
New South Wales. Over the 5year
period 2001-2005, it was determined that 6683 case
submissions required presumptive screening for amphetamines. Of these cases, 1269
(19.0%) required confirmative analysis of amphetaminetype
substances, including
amphetamine, methamphetamine, MDA, MDMA, MDEA, ephedrine, pseudoephedrine,
and phentermine. Such analytical needs were then used in comparative assessment of the
novel and established methodologies, including examination of immunoassay specificity,
extraction efficiency, chromatographic resolution, general resource efficiency, and total
analysis time.
Development of a beadbased
immunoassay platform (SBA) for multiplex amphetamines
analysis proved to be a complex task. Efforts to multiplex the amphetamine and
methamphetamine immunoassay models into a single assay exhibited a significant degree
of non-specific
antibody cross-reactivity.
However, the merits of the individual bead
assays were demonstrated. Upon comparison with commercially available enzyme-linked
immunosorbent assays for amphetamine or methamphetamine (ELISA), it was observed
that the SBA models exhibited specificity comparable to that of the ELISA assays and
linearity over a concentration range of toxicological relevance (0-1000 ng/mL
amphetamine or methamphetamine). In addition, the results indicated the practical
applicability of the individual SBA assays for an oral fluid matrix, and demonstrated
significant reductions in the volumes of reagents required and length of time of analysis.
Additionally, in an optimised multiplex system, the amount of sample required for
screening could be reduced as the SBA technology theoretically permits analysis of up to
100 different drugs or metabolites from one volume of sample.
The aspect of forensic sample conservation was further explored with investigation of
reduced-volume
extraction techniques, such as the application of centrifugal ionexchange
extraction columns (SpinSPE).
Following initial development, the SpinSPE
technique
was applied to the isolation of amphetaminetype
substances from oral fluid and compared
with a mixedmode
SPE method for both extraction and resource efficiency. From the
observed results, both extraction methods were demonstrated to be effective in the
isolation of amphetamine, methamphetamine, ephedrine, pseudoephedrine, PMA, MDA,
MDMA, MDEA, MBDB, and 2CB from an oral fluid matrix with detection by
heptafluorobutyric acid derivatisation (HFBTA) and GC/MS. The SpinSPE
model
demonstrated comparable efficacy with reduced sample volume (200 쌩, as well as
significant reductions in the volumes of reagents required for column conditioning,
washing, and elution. In addition, the linear working range (0-2000 ng/mL) and
sensitivity of the method indicated the potential to further reduce sample volume.
In the confirmative separation and identification of drug compounds, the technological
advancement of UltraPerformance
(TM) liquid chromatography (UPLC(TM)) has recently
evolved from efforts to improve LC resolution, sensitivity, and time of analysis. In this
research, UPLC(TM) coupled to mass spectrometry was demonstrated to be capable of
rapidly identifying several amphetaminetype
substances (phenylethylamine,
amphetamine, phentermine, methamphetamine, ephedrine, pseudoephedrine, PMA, 4MTA,
MDA, MDMA, MDEA, MBDB) and ketamine in an analysis time of less than five
minutes. In addition, UPLC(TM)/MS demonstrated a resolving power comparable to GC/MS
with significantly reduced instrumental analysis time.
This research reveals the promise of these new applications in advancing towards a more
efficient and modernised systematic toxicological approach. The continued development
and optimisation of SBA multiplex immunoassays will permit customisable systems
capable of simultaneously detecting numerous compounds with antibodybased
sensitivity
and selectivity. In circumstances where low sample volumes are required for confirmation
of drug use, such as in roadside saliva drug testing for driving under the influence
offences, reducedvolume
SpinSPE
has been demonstrated to be a practical and effective
alternative for sample preparation. In addition, a more streamlined procedure is further
enhanced with the use of UPLC(TM) coupled to mass spectrometry for analyte separation
and molecular identification.
It is expected that illicit drug use will remain a significant public concern. With the
continued desire for more rapid and comprehensive methodologies, further study of these
and other innovative technologies will be of considerable future benefit to laboratories
such as that serving the Australian Capital Territory region.
| Identifer | oai:union.ndltd.org:ADTP/202607 |
| Date | January 2007 |
| Creators | Apollonio, Luigino Giuseppe, n/a |
| Publisher | University of Canberra. Applied Science |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | ), Copyright Luigino Giuseppe Apollonio |
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