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Conclusions and Future Perspectives
The scope of this thesis was the development of a chromatographic screening method for the
determination of compounds in urine with main focus on those substances that are difficult to
detect with common STA methods in plasma, such as alkaloids and other polar basic compounds in
order to replace time-consuming and/or specialised methods formerly used for this purpose at the
Institute of Toxicology-Clinical Toxicology and Poison Control Centre Berlin. This method was
supplemented with two further methods: a toxicological screening method for plasma analysis and
a method for the determination of neutral, weakly acidic and weakly basic compounds in urine. The
following conclusions for the development can be drawn:
Firstly, a fully automated method for the qualitative determination of basic drugs from urine was
established and validated. The use of on-line extraction replaced tedious and time-consuming
purification steps and led to a system that could work unattended. The elution under isocratic
conditions as well as the use of common HPLC solvents and equipment simplified the method and
the method set-up. The analysis of authentic toxicological samples proved the utility for
toxicological applications, such as the analysis of alkaloids and other basic compounds in general
and demonstrated its applicability for STA and DOA confirmation analysis for amphetamine,
cocaine/BEC, opiate and methadone/EDDP above the given LOD. A comparison with an existing
urine screening method, the RemediTM-HS showed that the developed system presents an adequate
alternative to the latter system, but offers higher versatility, modern equipment and sample analysis
at low costs. The method has been successfully introduced to toxicological routine use and will
eventually be commercially marketed as an alternative to the RemediTM-HS, which will be taken
out of service in 2008. All obtained validation data met the criteria for the investigated parameters
set in international guidelines for bioanalytical methods and confirmed the reliability of the
method.
In conclusion, the developed on-line extraction HPLC-DAD method for basic compounds allowed
simple and reliable determination of basic drugs in urine at low costs and is suitable for the routine
use (STA and DOA confirmation screening) as results of authentic sample analyses showed.
Secondly, in addition to the on-line extraction HPLC-DAD method for basic compounds, a
toxicological screening method for plasma was established in the analytical system. The plasma
method covers most basic and neutral compounds that can be separated on the coupled NucleosilTM
100 C8 columns following LLE and allows quantitation of therapeutic and toxic concentrations.
The set-up of the method was successfully controlled by a performance test and proven with the
parallel analysis with a reference system.
Thirdly, an on-line extraction method for the determination of neutral, weakly acidic and weakly
basic compounds in urine was developed and integrated in the analytical system. This method was
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especially useful for the determination of the important group of benzodiazepines as could be
concluded from the achieved validation results and as was illustrated with the analysis of clinical
cases. Barbiturates could be identified qualitatively in high concentrations (> 1.0 µg/mL cyclo-,
crotyl- and phenobarbital, > 0.1 µg/mL methohexital and pentobarbital).
Sample extraction as well as the switching between the different methods is fully automated and
therefore easy to perform.
For all established methods, the use of the DAD system gave access to the spectra library of
approximately 2600 spectra and additional spectra of the method-specific libraries and allowed the
detection of metabolites by comparing the spectrum of the proposed metabolite and the parent
compound. Thus, the spectra library will be continuously expanded with toxicological relevant
compounds and metabolites which are not available as reference standards.
The developed system showed to be very useful and efficient for toxicological screening, as it
covers a broad range of relevant compounds and particularly in its ability to screen either plasma or
urine with complementary methods. In comparison to other chromatographic methods, no sample
derivatisation (GC-MS) is required and direct injection of urine without carry-over problems (LCMS) is possible.
The developed screening method for basic compounds in urine has been patented and will be
distributed by Shimadzu under the name Prominence TOX.I.S.. Prominence describes the HPLC
equipment series, TOX.I.S. stands for TOXicological Identification System. Therefore, it will be
used in different fields of research resulting in inter-laboratory exchange concerning its advantages
and limitations. Moreover, besides the continuous enlargement of the method-specific library
through analysis of clinical routine samples at the Institute of Toxicology-Clinical Toxicology and
Poison Control Centre Berlin, contributions from other laboratories are expected to lead to an
increase of accessible spectra and RRT data.
Detailed characterisation of the substance spectrum for each method will be gained by continuous
analysis of samples by all established methods, detecting gaps in either extraction or analytical
separation for certain compounds in accordance to the used method.
Optimisation of the LEC method might be of interest for laboratories that previously performed
analysis of LEC in urine by the RemediTM-HS. For this aim investigation of appropriate analytical
columns will be necessary.
A further research objective will include the structure elucidation of appearing metabolites by MS
in order to specify the library data. The expansion of the library with MS data will also give the
opportunity of MS coupling. Thus, with the combination of UV and MS detection, higher
sensitivity will be gained. However, a suitable volatile mobile phase component will have to be
found for this purpose.
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Replacement of LLE by automated on-line extraction for the toxicological screening method for
plasma would be another interesting task, which would lead to a fully automated analytical system.
To accomplish this aim, the use of RAM for protein removal may be an option.
Finally, more data about the screening method for neutral, weakly acidic and weakly basic
compounds for toxicological routine use will be gained. Optimisation of the latter method
concerning cleaner sample extracts will be subject to further investigations dealing with the testing
of new on-line extraction materials.