Identifying Identifying Toxins Using an Extensive User Manual

The Institute of Legal Medicine at Humboldt University in Berlin,

Germany, initiated development of a spectra library to help clinical

and forensic toxicologists identify poisons as quickly as possible. The

library contains more than 1,600 UV spectra of compounds relevant to

pharmacology and toxicology. The spectra were measured at the

Forschungsgesellschaft für Lungen- und Thoraxerkrankungen (FILT) in

Berlin and tested with extracts of human serum, spiked full blood or

urine samples and samples taken during the investigation of many real

cases of poisoning. The library is divided into sublibraries that enable

faster peak identification. Problem-oriented preselection of the match-

ing sublibrary allows identification of an analyte with a higher degree of

accuracy. Using this library and a diode-array detector software, ana-

lysts can compare the spectrum of an unknown peak with those in the

library in a very short time. With relatively simple sample preparation

methods, analysts can use this library to identify poisons and their

metabolites fast and confidently. The library also offers a link to a data-

base that gives each compound's nonproprietary name, CAS number,

effect or use, retention time and spectroscopic maxima, minima and

shoulders. An easy-to-use manual provides the same data, a printout of

each spectrum and the structural formula of each compound. All of this

makes the library a valuable tool in forensic, clinical and toxicological

analysis.

Identifying Toxins Using an Extensive,
Fast and Automated HPLC Spectral Library

Dr. Michael Rothe,
Prof. Dr. Fritz Pragst,
Andrea Kohn

Pharmaceutical and
Toxicological Analysis

Agilent Technologies

Innovating the HP Way

Application Note

Introduction

During the past decade, High
Performance Liquid Chromato­graphy (HPLC) has become one
of the most important analytical
techniques in the chemical, bio­chemical and medical practice.
HPLC is particularly effective for
separating and identifying com­pounds used in pharmacology and
toxicology. Applications include
controlling environmental and
food contamination and determin­ing the levels of active agents in
medicinal drug candidates.

Almost all compounds of interest
in the chemical and medical fields
absorb light in the UV spectral
region. Therefore, most HPLC
analyses use UV-visible detectors.
The introduction of the diode­array detector and the similarly­operating rapid-scan UV-visible
detector opened another dimen­sion in UV detection. These detec­tors allow each chromatographic
peak to be identified not only by
retention time but also by its UV
spectrum. The UV spectrum of a
compound is very reproducible
and its full shape is much more
compound-specific than generally
assumed.

A prerequisite for identifying com­pounds with diode-array detection
is a library containing the UV
spectra of as many compounds of
interest as possible. Such a library
is laborious to generate. And
because many compounds are not
available, creating a comprehen­sive library is often expensive and
time-consuming. Most such
libraries, therefore, remain very
limited.

The Institute of Forensic Medicine
at Humboldt University in Berlin,
Germany, initiated development
of a spectra library that contains
more than 1,600 UV spectra rele­vant to pharmacology and toxicol­ogy, including a large variety of
metabolites, which are particularly
challenging to identify. The library
was created because clinical and
forensic toxicologists need to
diagnose poisonings as quickly as
possible. The large number and
variety of potential poisons make
diagnosis challenging, particularly
when there is little information
about the type and amount of poi­son ingested.

Spectra were measured and tested
at the Forschungsgesellschaft für
Lungen- und Thoraxerkrankungen
(FILT) in Berlin and tested with
human serum, spiked full blood or
urine samples or samples taken
during the investigation of real
poisonings. The identification and
correctness of the spectra were
thoroughly controlled by multiple
measurements and comparisons
with the literature.

In addition to peak identification,
this library is a valuable tool for
designing optimal detection and
quantification conditions for new
analysis methods.

In addition to the spectra files, the
library offers a link through each
compound’s CAS number to a
database that contains each com­pound’s nonproprietary name and
CAS number, pharmacological or
toxicological use or effect, relative
retention time and UV spectro­scopic maxima, minima and shoul­ders. These data can be read by
Microsoft Excel®, Microsoft
Access®or Borland dBase®. The
library also comes with an easy-to­use manual that includes a print­out of each spectrum and the
structural formula together with
instructions for the proper use of
the library.

Furthermore, the user can quickly
generate a new library extracted
from the entire library with only
the user’s spectra of interest.
Relative retention times, based on
internal standards, indicate the
expected elution order, helping to
overcome the well known difficul­ties of reproducibility even with
the same type of column and the
same mobile phase.

This application note summarizes
the development of the toxicologi­cal library and provides examples
of its use.

Experimental

Selected compounds

The Institute's HPLC spectra
library contains approximately
1,600 spectra that match a broad
user range in pharmacological and
toxicological analysis. All kinds of
drugs were considered, with drugs
that are most used included with­out question. A large number of
metabolites were included, as
available. Illegal drugs, such as
opiates, cannabinoids, cocaine,
amphetamins, commonly used hal­lucinogenes and other so-called
designer drugs were also incorpo­rated into the library. Various drug
manufacturers generously provid­ed the pure compounds.

Most herbicides, wood preserva­tives and pesticides became part
of the library. Environmental tox­ins were restricted to a series of
polycyclic aromatic hydrocarbons,
polychlorinated biphenyls and
some phenols. Alcaloids such as
strychnine, brucine and nicotine
were included.

Nomenclature of the compounds
applies international names, when
available, or IUPAC nomenclature.
Many of the compounds were
used in their salt form for spectral
analysis, that is, as hydrochloride
or sodium salt. The identification
code was generated with the first
letter of the compound's name
and a three-digit number.
Caffeine, for example, became
C042. Compounds analyzed using
a different mobile phase have an
additional "b" added to their
names, such as A018b for
Amiodarone.

Instrumentation and analytical
conditions

Table 1 lists the instrumentation
and conditions used in the devel­opment of the pharmaceutical
and toxicology library. Certainly
these analyses can be done on a
Agilent 1100 Series HPLC system
as well.

The library contains corrected rel­ative retention times calculated
according to the general formula:

RRT = (RT

compound

- dead time) /

(RT

standard

- dead time)

Dead time (the time of an unre­tained peak) was determined by
injection of histamin hydrochlorid,
which showed no retention in the
acidic mobile phase. The absolute
retention time is stored under
Acquisition in the data file.

Table 1
HPLC system configuration and conditions

HPLC system HP 1090 Series Liquid Chromatograph with

Agilent ChemStation

HPLC column Lichrosorb RP-8, 5.0 µm, 240 x 4.6 mm

Mobile phase A • 156 g (=200 ml) acetonitrile (UV grade)

• 340 g phosphate buffer, pH=2.3, made with 4.8 g H3PO4and

6.6 g KH2PO4in 1 L water, with pH control by glaselectrode.
Retention times were calculated as 5- (4-Methylphenyl)-5-phenyl
hydantoine (MPPH, code M000, RRT=1.000), as standard

Mobile phase B • 625 ml acetonitrile
for a smaller number of • 375 ml phosphate buffer, pH 2.3
compounds having Retention times were calculated based upon
retention times that p-Phenylbenzophenone (Code P191b, RRT=1.000) as standard
exceeded 30 minutes
(RRT>3 in mobile phase A)

Degassing Helium or vacuum

Column temperature 40 °C

Flow rate 1 ml/min

Injection volume 1 µg of each compound (10 µl of a solution of 0.1 mg/ml in the

mobile phase or acetonitrile)

Detector Diode-array detector

UV spectra Taken at the peak apex between 195 and 380 nm after background

correction.