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HPLC SPECTRAL LIBRARY FOR TOXICOLOGICAL SCREENING
Identifying Poisons Quickly and Confidently
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Final graphical presentation of a urine analysis, with all identified peaks labeled. Urine extracts
contain a significantly higher number of matrix compounds than blood or serum extracts. This sample was
spiked with Amitriptyline and Lorazepam.
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By Michael Rothe, Forschungsgesellschaft für Lungen- und Thoraxerkrankungen mbH, Berlin-Buch; Fritz Pragst,
Institute of Forensic Medicine, Humboldt University, Berlin; Andrea Kohn, Hewlett-Packard Co., Waldbronn
(all Germany)
The large number and variety of toxic compounds often complicate the diagnosis in poisonings, especially when
little is known about the type and amount ingested.
The Institute of Legal Medicine at Humboldt University in Berlin developed a spectral library to help clinical and
forensic toxicologists identify poisons as quickly as possible. The library contains more than 1,600 UV spectra
relevant to pharmacology and toxicology.
The spectra were measured at the Forschungsgesellschaft für Lungen- und Thoraxer-krankungen (FILT) in Berlin and
tested with extracts of human serum, spiked full blood or urine samples taken during the investigation of many
real cases of poisoning. Analysts can use the library to identify poisons and their metabolites quickly and
confidently with very little sample preparation.
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HPLC analysis of postmortal full blood sample. A sample of 0.5 mL was extracted with dichloromethane at
pH 9.4. The small peak at 6.178 min shows very low concentration of the compound of interest. Despite the high
noise level of the spectrum, attributable to strong background, the compound can be identified as Zolpidem by
comparison with the UV spectra library.
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Evolving UV Detection
During the past decade, HPLC has become an important analytical technique in chemical, biochemical, and medical
practice. Because many compounds of interest in the chemical and medical fields absorb light in the UV spectral
range, most HPLC analyses use UV-visible detectors. The introduction of diode-array detectors opened yet another
dimension in UV detection. Each chromatographic peak could now be identified not only by retention time but also
by its UV spectrum, allowing even more accurate qualitative analyses.
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The noisy spectrum of the Zolpidem peak (with only low UV absorption) is shown here with the pure
library Zolpidem spectrum (both normalized).
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The Prerequisite: A UV Library
A prerequisite for identifying compounds with diode-array detection is a library containing UV spectra of
potential compounds of interest. Such libraries have been very limited, because they are laborious and
often expensive to generate.
The library developed by the Institute contains more than 1,600 UV spectra, including a large variety of
metabolites (which are particularly challenging to identify).
Organized for Fast Peak Identification
To enhance fast peak identification, this extensive library is divided into sublibraries. Preselection of
the matching sublibrary is problem-oriented and allows the identification of sample compounds with a high
degree of accuracy. Analysts can compare the peaks in a sample with those in the library automatically and
quickly and confirm whether the peak is a known drug or toxin.
The library contains the CAS number and provides access to compound data from other software. By extraction from
the entire library, the user can quickly compile a new library containing only the spectra of interest. Relative
retention times, based on internal standards, indicate the expected elution order
and thereby help to overcome the well-known difficulties of reproducibility even when the same type of column
and mobile phase are used.
Choosing and Verifying Content
In the process of compilation, all kinds of drugs were considered, with those most often encountered included without
question. A large number of metabolites, illegal drugs, commonly used hallucinogens and other so-called designer
drugs were incorporated. So were most herbicides, wood preservatives, and pesticides. Alcaloids such as strychnine,
brucine, and nicotine were also included. Environmental toxins were restricted to a series of polycyclic aromatic
hydrocarbons, polychlorinated biphenyls, and some phenols.
Spectra were measured and tested at the FILT in Berlin. The identification and correctness of the spectra were
thoroughly controlled by multiple measurements and comparisons with the literature.
Click for a description of the HPLC spectral library and its development.
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