analytical toxicology in forensic toxicology

1. Toxicological Analysis
Maysam nameer
Higher diploma in toxicology
Supervised by
Dr. Ammar ali hussein
Baghdad university college of pharmacy
2020

2. introduction
Analytic toxicology involves the application of the tools of analytic
chemistry to the qualitative and/or quantitative estimation of chemicals
that may exert adverse effects on living organisms.
analytical toxicology involves the application of the tools of analytical
chemistry to the qualitative and/or quantitative estimation of chemicals
that may exert effects on living organisms.

3. advantage
Analytical toxicology can assist in the diagnosis, management,
prognosis, and prevention of poisoning. In addition analytical
toxicology laboratories may be involved in a range of other
activities such as the assessment of exposure following chemical
incidents, therapeutic drug monitoring, forensic analyses, and
monitoring for drugs of abuse. They may also be involved in
research, for example in determining the pharmacokinetic and
toxicokinetic properties of substances or the efficacy of new
treatment regimens.

4. Before the analysis begins, several factors must be considered, including
the amount of specimen available,
the nature of the poison sought, and
the possible biotransformation of the poison.
 oral administration of the poison, the gastrointestinal (GI) contents are analyzed
first because large amounts of unabsorbed poison may be present.
 The urine may be analyzed next, as the kidney is the major organ of excretion for
most poisons and high concentrations of toxicants and/or their metabolites often
are present in urine.
 After absorption from the GI tract, drugs or poisons are carried to the liver before
entering the general systemic circulation; therefore, the first analysis of an internal
organ is conducted on the liver.
 If a specific poison is suspected to have caused or contributed to a death, the
toxicologist may first analyze the tissues and fluids in which the poison
concentrates.

5. *the knowledge of drug biotransformation is often essential
before an analysis is performed. The parent compound and any
major pharmacologically active metabolites should be isolated
and identified.
In some cases , the metabolites provide the only evidence that
a drug or poison has been administered. Many screening tests,
such as immunoassays, are specifically designed to detect not
the parent drug but its major urinary metabolite.

6. To determine a profile of each analyte present in a
specimen, chromatographic procedures such as
gas chromatography(GC) and
liquid chromatography (LC) coupled with mass spectrometry(MS)
or tandem MS are commonly used, which facilitates the
simultaneous separation and quantification of each compound.

7. when the nature of a suspected poison is unknown, a systematic, standardized approach
must be used to identify the presence of most common toxic substances.:
1. Gases—Gases are most simply measured by means of gas chromatography.
2. Volatile substances—These are generally liquids of various chemical types that vaporize
at ambient temperatures. Gas chromatography is the simplest approach for separation
and quantitation.
3. Corrosive agents—These include mineral acids and bases. Many corrosives consist of
ions that are normal tissue constituents. Chemical techniques can be applied to detect
these ions when they are in great excess over normal concentrations.
4. Metals—Metals are encountered frequently as occupational and environmental hazards.
Separation involves destruction of the organic matrix by chemical or thermal oxidation.
5. Anions and nonmetals—These present an analytical challenge as they are rarely
encountered in an uncombined form.

8. *The analysis may be complicated
by the normal chemical changes
that occur during the decomposition
of a cadaver.
The autopsy and toxicological analysis
should be started as soon after death as
possible.
*The natural enzymatic and nonenzymatic
processes of decomposition and microbial
metabolism may destroy a poison that was
present at death or produce compounds with
chemical and physical properties similar to
those of commonly encountered poisons.

9. For examples':
*The concentration of cyanide and
ethyl alcohol and the carbon monoxide
saturation of the blood may be
decreased or increased, depending on
the degree of putrefaction and
microbial activity.
*However, many poisons—such as
arsenic, barbiturates, mercury, and
strychnine—are extremely stable and
may be detectable many years after
death.

10. Before analysis, the purity of all chemicals used in laboratory
procedures should be established.
* All reagents and solvents should be of the highest grade
possible and should be free of contaminants
* Specimen containers, lids, and stoppers should be free of
contaminants such as plasticizers,
*clean laboratory environment. This is of particular concern in the
analysis of metals, as aluminum, arsenic, lead, and mercury are
environmental and reagent contaminants.

11. Forensic toxicology laboratories analyze
specimens by using variety of analytical
procedures
. Initially, nonspecific tests designed to determine the
presence or absence of a class or group of analytes on the
specimens.Positive results obtained with these tests must
be confirmed by a second analytical procedure that
identifies the particular drug identify and confirm the
presence of the analyte.
Today, GC-MS and LC-MS are the most widely applied
methodology in toxicology and are generally accepted to
identification for all drugs.

12. most cases require reliable estimates of poison concentrations
for forensic interpretation. For quantitative analysis, the
accuracy, precision, linearity, and specificity of the procedure
must also be established.
Precision, is determined by replicate analyses of a specimen of
a
known concentration.
quantitative result will deviate spuriously from the true value.
Therefore,
replicate quantitative determinations are highly recommended

13. Applications of analytical toxicology
1. Clinical toxicology
2. Therapeutic drug monitoring (TDM):aminoglycoside..lithium ,
antipsychotics
3. Occupational and environmental toxicology
4. Drug abuse screening , alcohol , laxative and diuretics to weight loss
5. Forensic toxicology .. to identify substance and determine apattern
of use or influence there death.

14. Case study
• A 97-years old woman was hospitalized because of severe symptoms,
including drowsiness, convulsions, pallor and hematoma. The
laboratory tests showed abnormal values for coagulation parameters
(prothrombin time-international normalized ratio, PT-INR = 12.46;
activated partial thromboplastin time, aPTT = 60 s; aPTT ratio = 1.82).
After intravenous (IV) administration of 10 mg vitamin K and one day
monitoring, the patient recovered from the hemorrhagic syndrome
(PT-INR = 1.45; aPTT = 40.6 s; aPTT ratio = 1.23) and was dismissed.
After 1 week, the woman was taken again to the Emergency
Department (ED) because she reported the same symptoms. Her
coagulations parameters were the following: PT-INR = 13.31; aPTT =
68 s and aPTT ratio = 2.06. After IV administration of vitamin K and
three blood transfusions, the patient recovered once more.

15. • During the treatment of the second episode, a blood sample was
collected and screened for anticoagulants, in order to find possible
explanations. Screening for further substances, which may account
for convulsive symptoms, were not performed. Because the blood
sample resulted positive to difenacoum, the case was reported to the
Public Prosecutor's office, which took jurisdiction of the case. A fruit
mousse allegedly used to poison the victim was seized by the Police.
Our laboratory was asked by the Prosecutor to determine the content
of the mousse and to estimate for how long the poisoning occurred.
In order to respond to the latter query, the victim was asked to give a
hair sample on which to perform the inherent toxicological analyses.
The patient's hair sample was taken 2.5 months after her first
hospitalization.

16. Application to a real case
• The extraction of the fruit mousse was performed by the QuEChERS
method, which is a streamlined and effective extraction and cleanup
approach for the analysis of a variety of analyte residues in food
matrices .The fruit mousse sample tested positive for difenacoum and
α-chloralose, at 2 and 50 µg/g concentrations, respectively.
• To perform segmental analysis, proximal and distal extremities of the
hair sample were identified. Assuming that the hair growth rate
generally ranges from 1.0 to 1.3 cm/month, a relationship between
hair length and investigation chronology was obtained..

17. • Difenacoum was detected in the first (proximal) 3-cm hair segment at
the concentration of 2.9 pg/mg. To our knowledge, this is the first
study to report that exposure to difenacoum is detectable in real hair
samples . For most drugs, concentrations in the low picogram per
milligram range are expected in the circumstances of single intake,
such as in drug-facilitated crimes and drug offences Differently, long-
term intoxication usually leads to nanogram of drug per milligram of
hair levels .Therefore, we concluded that in the present case the
victim was administered difenacoum in either a single or few isolated
occurrences, possibly immediately before the two admissions into the
ED.

18. • The other target analyte found in the hair sample was α-chloralose,
which was detected in the proximal (0–3 cm) segment at the
concentration of 85 pg/mg. The two subsequent and consecutive
segments (3–6 cm and 6–9 cm) showed only traces of difenacoum
(below LOQ) and low but quantifiable concentrations of α-chloralose
(29 and 6 pg/mg, respectively). Sporkert et al. reported a case of
segmental hair analysis which yielded α-chloralose concentrations in
the range from 75 to 338 ng/mg for each segment, suggesting
repetitive exposure of the victim to this substance.

19. • On the other hand, numerous factors may account for an observed
longitudinal migration of drugs along the hair shaft , suggesting that the
detection of a drug in two or three hair segments does not necessarily
implies multiple exposures. For example, drugs released in the sweat are
prevalently incorporated into the proximal hair segment, but partly also in
distal segments, especially when the entire hair length is kept in contact
with the skull by a pillow, a foulard, a hat or similar clothes. This is even
more likely in elderly people spending most part of the day in armchairs
and bed, as in the present case. In order to interpret apparently
contradictory segmental hair analysis data after single drug exposure, Kintz
proposed to consider that the highest concentration must be detected in
the segment corresponding to the period of the alleged event, and this
measured concentration should be at least three times higher than those
measured in the preceding or following segments. In the case presented
hereby, we concluded that the victim was repeatedly exposed to α-
chloralose in the period corresponding to the first segment of hair. Possible
contamination of the remaining hair segments may be accounted for by
the fact that the victim used to spend most of her time in bed or on
armchairs.

20. • Several harmful substances are easily available on the market in large
quantities. Therefore, these compounds are often involved in
intoxication cases and detected in biological specimens, including
hair, in circumstances of attempted or accomplished poisonings.
Cumulative exposure to organophosphorus pesticides was
demonstrated by Kavvalakis et al who reported results on hair
samples from both the general population and exposed populations.
Concentrations of nonspecific metabolites of organophosphorus
pesticides, dialkylphosphates, ranged from 40 to 165 ppb for the
general population and from 181.7 and 812.9 ppb for the exposed
population. Similar hair concentrations were reported also by
Tsatsakis et al.

21. • Kavvalakis et al. 2013 demonstrated a dose-dependent accumulation
of Imidacloprid, a relatively new neuro-active neonicotinoid
insecticide, in rabbit hair, after a chronic subacute long-term exposure
to the insecticide , while Schummer et al. measured 50 pesticides
including 39 molecules from different chemical families currently
used in agriculture and 11 organochlorines in hair of farm workers in
order to evaluate the exposure to pesticides . These results
demonstrate that hair analysis can provide extensive information on
human exposure to pesticides and harmful substances in general.

22. Conclusion
• An UHPLC–MS-MS method for the simultaneous determination of ten
anticoagulant rodenticides and α-chloralose in human hair was
developed and validated. The method proved to be simple, accurate,
rapid and highly sensitive, allowing the simultaneous detection of all
compounds. The method was successfully applied to a real case of
difenacoum and α-chloralose poisoning and proved sensitive enough
to detect occasional exposure of the victim to the two analytes by
segmental analysis.

23. references
• klaassen c, eaton d. the basic science of poisons , forensic toxicology .
9th ed. 2019. 1639 p.
• Leporati M, Salomone A, Golè G, Vincenti M. Determination of
Anticoagulant Rodenticides and α-Chloralose in Human Hair.
Application to a Real Case. J Anal Toxicol. 2016;40(4):277–85.

24. Thank you