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.

Results and discussion

Parameters for spectral library
search and quantification

The LC 3DChemStation evaluates
each set of raw data automatically.
This occurs immediately after the
chromatographic run. Once reten­tion time and peak area or height
have been determined by integra­tion, the library search begins on
these integrated peaks.

First, to compensate for baseline
drift caused by solvent composi­tion changes across the gradient
(especially at wavelengths shorter
than 220 nm), the spectral contri­bution of the baseline either
before or after the peak is sub­tracted from each peak's spec­trum.

Second, a check on peak purity
provides the analyst with a purity
level (maximum value 1,000) that
reflects how closely the spectra
match each other along the peak's
elution. Values above the pre­defined threshold of 990 indicate
peak purity; values below 990
indicate impurities, which are
then marked in the library search
report.

Finally, the actual spectra search
is contained within a search
window of ±4 percent of the
compound's retention time, to find
compounds with a retention time
close to the one of the unknown
peak. Peaks that match an entry in
the library can be considered iden­tified when the search results are
better than a significance limit of
990, as calculated by the LC

3D

ChemStation. If peak height is
very small and background
absorbance is relatively high, then
values of between
950 and 990 can possibly consti­tute an identification. Visual
control of the overlay spectra is
advised to avoid errors.

Detection limits will vary depend­ing on differences in analytes and
their sample matrices. In general,
identification of compounds with
absorption in the high wavelength
range is easier when sample matri­ces have fewer compounds, such
as with fresh serum. Ideal concen­trations for identification are
about 100 ng/ml.

Spectra sublibraries

The entire set of spectra is divided
into sublibraries to make peak
identification faster. Moreover,
through problem-oriented prese­lection of the matching sublibrary,
identification can occur with a
higher degree of certainty.

Table 2 shows the sublibraries of
the toxicology library. These subli­braries were created according to
the range of effects of individual
drugs. The Agilent ChemStation
makes it easy to modify these sub­libraries, customizing the library
to user needs.

Metabolites are in the same subli­brary as the parent drug.
However, because of possible mul­tiple effects, some compounds
reside in sublibraries that are
somewhat arbitrary.

HPLC analysis

Automated spectra library search

• Data acquisition of chromatogram(s) and spectra.

• Integration with determination of retention time
and peak area or height.

Compensation of baseline drift by
subtraction of baseline spectra
Peak purity analysis
Spectra comparison with spectra
library/sublibraries
Report with peak purity information and peak
confirmation with match factor.

1

2

3

4

Sublibrary Effect/Usage (listed in the database) #

TOX01

Agents with addiction potential, Analgetic (Btm), anesthetic, anoretic, antihypnotic, 122
illegal drugs, psychoactive barbiturate, central stimulant, hallucinogen, hypnotic,
agents, hypnotics illegal drug, narcotic, opiate, opiate antagonist, psycholeptic,
(see also TOX02) psychostimulating agent, psychotonic, sedative

TOX02

Psychopharmaceuticals, Analeptic, anticonvulsant, antidepressant, antiepileptic, 201
antiepileptics and similar anxiolytic, benzodiazepine, myotonolytic, neuroleptic,
effects (see also TOX01) spasmolytic, tranquilizer

TOX03

Analgetics, antirheumatics, Analgetics (except for Btm), antiarthritic, antigout agents, 138
antitussives and similar antimigraine, antineuralgetic, antipyretic, antirheumatic,
effects (see also TOX01) antiphlogistic, antitussive (except for opiate), bronchiolytic,

expectorant, local anesthetic, muscle relaxant, mucolytic
agents, secretolytic

TOX04

Antihistaminics, antiallergics, MAO-inhibitor, antiallergic, anticholinergic, antidot, 134
further CNS-effective agents antiemetic, antihistaminic, antiparkinsonian, cholinergic,
with different effects cholinesterase inhibitor, dopamine antagonist, mydriatic,

parasympatholytic, serotonin antagonist, a-sympathomimetic,
ß-sympathomimetic

TOX05

Heart-blood circulation- Antiarrhytmic, antihypercinetic, antihypertonic, 150
remedies (see also TOX04 antihypotonic, beta-blocker, calcium antagonist, cardiac
and TOX06) glycoside, cardiac, cardiotonic, vasoconstrictor, vaodilator,

venodynamic, venotonic

TOX06

Diuretics, remedies for blood ACE-inhibitor, ALDH-inhibitor, anticataractic, 137
coagulation, for the digestion anticoagulant (see also rodenticide antidiabetic),
system, other remedies with antidiarrhoeal, antifibribrinolytic, antihypoxemic, carbonic
different effects anhydrase inhibitor, carminative, choleretic, ecbolic, gastric

ulcer therapeutic, hepatotherapeutic, diuretic, hemostyptic
agent, hyperemisation agent, laxative, lipid lowering agent,
peristaltic stimulant, platelet aggregation inhibitor, remedy
against varicose veins, rubefacient, remedy against ulcus,
saluretic, uricostatic, vulnery, X-ray contrast agent

TOX07

Remedies with steroid Aldosterone antagonist, anabolic, androgenic, 146
structure, hormones, antiandrogenic, antiestrogenic, estrogenic, glucocortcoide,
endogenous agents, vitamins gonatotropine inhibitor, hormones, prostaglandines, vitamin,

thyreostatic, endogenous agents

TOX08

Cytostatics, antibiotics, Antibacterial, antibiotic, antimalarial, antiprotozoic, 179
other agents with chemotherapeutic, cytostatic, gramicinide,
anti-micro-organism effects immunostimulating agent, immunosuppressant, opthalmic,

tuberculostatic, virostatic

TOX09

Fungicides, disinfectants, Anthelminic, antimycotic, antiparasitic, antiscabatic, anti- 116
stabilizer for drug preparation septic, disinfectant, fungicide, stabilizer for drug preparation

TOX10

Insecticides, acaricides, Acaricide, acetylcholin esterase inhibitor, fungicide (see 158
nematicides, etc. also W09), insecticide, nematicide, pesticide, repellant,

rodenticide (see also W06)

TOX11

Herbicides Herbicide, growth regulator for plants, wood preserver 108

TOX12

Ecotoxicological agents Polychlorinated biphenyls, polycyclic aromatic 32
(see also TOX10 and TOX11) hydrocarbons, internal standards

Table 2
Sublibraries—divided according to the
agents' effect/usage

Validity of spectra

Below 200 nm, spectra are influ­enced by noise and are not very
reproducible. Therefore the wave­length range below 200 nm was
not included in the spectra library
search.

Acidic acetonitile/phosphate
buffer mixtures were used for the
mobile phase for three reasons:
because of their UV transparency
down to 195 nm, because of the
suitable retention times of the tox­icologically-relevant compounds,
and because of the high resistance
of RP-phases to these mobile

phases. The amount of acetonitrile
buffer, which is valid for both neu­tral and acidic compounds, can be
varied widely without significant
changes in spectra. The validity of
this buffer was proven through
many measurements in both
mobile phases A and B. However,
the acidic pH conditions must be
maintained for all compounds that
underlie acid-base-equilibria
because the dissociation of

-COOH, -OH or -SH groups or the
protonation of basidic (=alkaline)
N-atoms often leads to significant
changes in light absorption.

Minimal sample preparation

In many cases, analysts can use
the LC system with a simple sam­ple preparation. For a general
toxicological analysis with mini­mal equipment, the user can fol­low this typical procedure:

• 0.5 ml blood + 0.5 ml phosphate
buffer pH=2.3, or 0.5 ml blood +

0.5 ml carbonate buffer pH=9.4

• Extraction with 0.5 ml
methylenchloride

• Extraction with 0.5 ml
ethylacetate/ether

• Unify the organic phases and
evaporate to dryness and
dissolve with eluent.

Figure 1
ChemStation screen with the HPLC chromatogram and the peak identification of a urine sample.
1 mL of urine was enzymatically (glucuronidase/arylsulfatase) hydrolyzed and extracted with
ethylacetate at pH=9.4. Urine extracts contain a significantly higher number of matrix compounds
than blood or serum extracts. Therefore, under the chromatographic conditions given above, only
compounds of high concentration or high retention can be analyzed well. The sample displayed
in figure 1 was spiked with Amitriptyline and Lorazepam.

Conclusion

The spectral library developed by
the Institute of Forensic Medicine
at Humboldt University in Berlin
is a useful and efficient tool for
identifying toxic compounds. With
minimal sample preparation, ana­lysts can use this library to identi­fy poisons and their metabolites
quickly and confidently.

Moreover, the ability to link to a
database using Microsoft Access

®

or Microsoft Excel®makes the
library a valuable tool for
chromatographic method
development.

Figure 2
Chromatographic separation of a native
serum sample. 1 ml serum was extracted
with RP18-solid phase extraction at pH=7.4.
The extraction process results in very clean
extracts with peaks that can be well
characterized. The sample mainly contains
theophylline, caffeine, metochlopramide and
phenazone.

Figure 3
HPLC analysis of postmortal full blood sample.

0.5 ml were extracted with Dichloromethane

at pH=9.4. As can be seen from the small peak
at 6.178 min, the concentration of the com­pound of interest is very low and the spectra
are very noisy because of strong background.
Nevertheless this compound can be identified
as Zolpidem by comparison with the UV
spectra library.

Figure 4
Noisy spectrum of the chromatogram's
Zolpidem peak with only low UV absorption
and the pure library Zolpidem spectrum, both
normalized and overlayed.

Ordering Information

The spectral library is commercially
available. “UV Spectra of Toxic
Compound” Authors: F. Pragst, M.
Rothe, B.T. Erxleben and S. Herre.

FILT Forschungsgesellschaft mbH
Dr. Michael Rothe
Robert-Rössle-Str. 10, Building 79,
D-13125 Berlin-Buch, Germany.
Tel. [phone],
Fax [phone],
E-mail: [e-mail]

References

1.
Bogusz, M., and Wu, M., "Standardized
HPLC/DAD System, Based on
Retention Indices and Spectral
Library, Application for Systematic
Toxicological Screening," J.Anal.
Toxicol. 15 (1991) 188-197.

2.
Bogusz, M., and Erkens, M.,
"Reversed Phase High-Performance
Liquid Chromatographic Database of
Retention Indices and UV Spectra of
Toxicologically Relevant Substances
and Its Interlaboratory Use,"
J. Chromatogr. 674 (1994) 97-126.

3.
Ibid., "Influence of the Biological
Matrix on the Chromatographic
Behavior and Detection of Selected
Acidic, Neutral and Basic Drugs
Examined by Means of a Standardized
HPLC-DAD System," J. Anal. Toxicol.
19 (1995) 49-55.

4.
Hill, D.W., and Kind, A.J., "Reversed­Phase Solvent-Gradient HPLC
Retention Indexes of Drugs,"
J. Anal. Toxicol. 18 (1994) 233-242.

5.
Huber, L., and Zech, K., "Automated
Screening for Drug Metabolites with
High-Performance Liquid Chromatog­raphy, UV-Visible Diode Array Detec­tion and Spectra Library Search,"

J. Pharmaceutical & Medical
Analysis 6 (1988) 1039-1043.

6.
Koves, E.M., Wells, B. Sc., and
Wells, J., "Evaluation of a Photodiode
Array/HPLC-Based System for the
Detection and Quantification of Basic
Drugs in Postmortem Blood,"
J. Forensic Sci. 37 (1992) 42-60.

7.
Lambert, W.E., Meyer, E., Xue-Ping, Y.,
and DeLeenheer, A.P., "Screening,
Identification, and Quantification of
Benzodiazepines in Postmortem
Samples by HPLC with Photodiode
Array Detection," J. Anal. Toxicol. 19
(1995) 35-40.

8.
Lambert, W.E., Meyer, E., and
Deleenheer, A.P., "Systematic
Toxicological Analysis of Basic Drugs
by Gradient Elution of an Aluminia­Based HPLC Packing Material under
Alkaline Conditions," J. Anal. Toxicol.
19 (1995) 73-78.

9.
Logan, G.K., Stafford, D.T., Tebbett,
J.R., and Moore, C.M., "Rapid
Screening for 100 Basic Drugs and
Metabolites in Urine Using Cation
Exchange Solid-Phase Extraction and
High-Performance Liquid Chromatog­raphy with Diode Array Detection,"
J. Anal. Toxicol. 14 (1990) 154-164.

10.
Maier, R.D., and Bogusz, M., "Identifi­cation Power of a Standardized HPLC­DAD System for Systematic Toxico­logical Analysis," J. Anal. Toxicol. 19
(1995) 79-83.

11.
Mufhoff, F., and Daldrup, T., "A Rapid
Solid-Phase Extraction and HPLC­DAD Procedure for the Simultaneous
Determination and Quantification of
Different Benzodiazepines in Serum,
Blood and Postmortem Blood,"
Int. J. Leg. Med. 105 (1992) 105-109.

12.
Pragst, F., Erxleben, B.T., Herre, S.,
and Aberger, K., "HPLC in der system­atischen toxikologischen Analyse,"

GIT Spezial Chromatographie 2/94

(1994) 92-96.

13.

Turcant, A., Premel-Cabic, A.,
Cailleux, A., and Allain, P., "Toxico­logical Screening of Drugs by Micro­bore High-Performance Liquid
Chromatography with Photodiode­Array Detection and Ultraviolet
Spectral Library Searches,"
Clin. Chem. 37 (1991) 1210-1215.

14.

Turcant, A., and Kohn, A., "Confirming
Diagnosis of Poisoning by Automated
HPLC with UV Spectral Library,"
Agilent Technologies Publication
No.12-5091-5671E (1992).

Microsoft®, Microsoft Access®and Microsoft
Excel®are U.S. registered trademarks of
Microsoft Corporation.
Borland dBase®is a registered U.S. trademark

Authors:

Dr. Fritz Pragst
Institute of Forensic Medicine
Humboldt University Berlin,
Germany

Dr. Michael Rothe
Forschungsgesellschaft für Lungen­und Thoraxerkrankungen mbH
Berlin-Buch, Germany

Andrea Kohn
Agilent Technologies
Chemical Analysis Group
Hewlett-Packard Strasse

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Agilent Technologies

Innovating the HP Way

Copyright © 1998 Agilent Technologies
All Rights Reserved. Reproduction, adaptation
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Publication Number 5968-2596E