Download Hair analysis for drug testing

Hair analysis for drug testing
After the entering of drug (generally nox) into the human organism its absorption, distribution and
metabolism occurs. Traditional biological material to be analyzed in order to detect the presence of
drugs or their metabolites, can establish that a person is under the influence of drugs. The
intoxication of an organism is impossible to re-establish for distance longer period by analysis of
blood or urine because of a gradual elimination of drugs and their metabolites from the human body
(primarily in the urine and feces).
Blood and urine may be removed also post mortem (after death) to determine whether the dead was
dead at the time of his death under the influence of this substance and that the level of the substance
found in the blood is not related to the death. For these purposes, hairs are not suitable material for
analysis. By hair analysis for drug testing is not possible to demonstrate the influence of drug into
the human organism. But hair can be used to determine the long-term abuse of drugs. Hair can be a
very good biological material for toxicological analysis even in the case when no other biological
material obtained post-mortem to be analyzed, due for example advanced decay of bodies after
death. The investigation of hair significantly enhances the detection time after dose consumption.
Hair has as a biological material to the analyze specific characteristics which need to be taken into
account when sampling, pretreatment to analysis, actual analysis and also the interpretation of the
obtained results.
Hair is composed of proteins (65-95%), lipids, trace minerals, polysaccharides and water. It has a
slightly alkaline pH. The pH of the hair significantly influences the type of substances to be stored
in the hair. About the deposition of a particular substance in hair decides affinity agent to melanin,
lipofility of the substance and its acid-base properties. Hair matrix has a lower pH than blood and
therefore the resulting pH gradient preferable the transition of bases rather than neutral or acidic
substances. Even the hair color affects the storage of chemicals in hair. The concentration of drug is
usually larger in dark hair than in blond hair. The penetration of chemicals from the blood into the
hair is possible by three mechanisms:
(i) passive diffusion from blood capillaries (by this way the hair receives the greatest proportion of
substances and their metabolites),
(ii) exclusion on the surface of hair from sweat and sebum, and partial diffusion in the internal
structures of hair and other eventual diffusion of the deep skin compartments,
(iii) capture on the surface from the external environment, ie. external contamination.
External contamination (e.g. dyes from colored hair, depending on the surrounding body in the case
of substances found in the ground) must be removed because it cancels determination. One of the
important modifications prior to the extraction of drugs from hair and noxes is thus hair washing.
The hair sampling
It is best to remove hair from rear area of the head. Unlike other areas of the head, the rear region
has a lowest variability in the rate of hair growth.
The amount of hair in the growth phase is more stable and less affected by age-dependent gander
factors. Hair strands are taken as close to the scalp and place of collection must be registered.
Hair sample can be stored at room temperature in the aluminum foil, envelope or a plastic tube. The
size of samples differs depending on the desired analysis, the used analytical methods. The amount
of sample to be withdrawn, most often ranges from one hair to 200 mg of sample. For purposes of
analysis it is ideal subscribe strand of hair with a thickness of a pencil.
The general procedure for processing of hair sample
First, it is necessary to decontaminate the hair to remove interfering chemicals from the hair
surface. Decontamination is performed by washing the hair by surfactant solution. Next, the
degreasing of hair samples is carried out mostly by acetone. Subsequently it is necessary to do the
homogenization, usually shearing is sufficient. While the performing of sequence analysis is
required (i.e. the detection of noxes or drugs in influence of hair growth along its length), the hair is
cut into segments about 1 cm, which are then processed and analyzed separately. Further
homogenization into millimeter pieces by scissors is followed.
The next step is the disintegration of hair, which is performed to release noxs or drugs from hair in
the greatest quantity. Disintegration of the hair is carried out with methanol or hydrolyzed with a
mineral acid or an alkali hydroxide. For disintegration the temperature and time for which the
disintegration takes place are important parameters. After the disintegration the extraction and preconcentration steps follow. As the extraction techniques, L-L extraction, solid phase extraction SPE,
SPME and below are used.
The concentration of noxs in the hair ranges at very low levels; therefore it is necessary the
extraction and pre-concentration step with subsequent analysis of extracted and concentrated
analyte using highly sensitive analytical techniques. The most common techniques are: gas or liquid
chromatography coupled with tandem mass spectrometry (GC-MS/MS and LC-MS/MS).
Hairs are not used as biological material for the detection of acute drug intoxication, but for the
detection of chronic drug abuse. It can only establish whether the examinee enjoyed a long period
of time a substance in significant doses, because all materials have in hair certain limit of detection,
i.e. an amount which can be determined by the analytical methods. Sequence analysis serves for the
survey during the usage of certain substances to examinee. This allows us to determine when and in
what doses the substance was submitted.
Task: Perform the identification and quantification of amphetamines in submitted hair
samples.
Tools: ultrasound, beakers, graduated cylinder, test tubes, SPE extraction thimble (hydrophilic
polymer phase), automatic pipettes and tips
Chemicals: methanol, acetonitrile, ammonium hydroxide, acetic acid, trifluoroacetic acid,
amphetamine, methamphetamine and ephedrine standards in methanol 1 mg/mL
LC conditions
Column: Ascentis C18, 15 cm x 2.1 um, 5 um particlesize
Mobile phase:
A: deionized water + 0.05% trifluoroacetic acid
B: acetonitrile + 0.05% trifluoroacetic acid
Rate: 200 uL/min
Gradient
0 min
7 min
10-11 min
90% A
70% A
10% A
10% B
30% B
90% B
MS conditions:
ESI ionization: positive mode, + 5,5 kV,
temperature of ion source 300 C,
collision gas 6 psi
Workflow:
1. Cut the present sample into 3 mm pieces and weigh accurately 1 g of sample into the glass
vessel on an analytical balance.
2. Perform the disintegration of weighed hair sample in 5 mL of methanol using sonication for 20
minutes.
3. Filtered the disintegrated sample and apply the filtrate onto SPE column for amphetamine
capturing according to the following procedure:
a) Condition the SPE phase by 1 mL of methanol, 1 mL of water and again by 1 mL of methanol.
b) Apply 3 mL sample of extracted hairs.
c) Perform the washing (removing ballast) of SPE column using 1 mL of 5 % methanol containing
2% ammonium hydroxide and subsequently 1 mL of 20% methanol containing 2% ammonium
hydroxide.
d) Perform the elution of captured amphetamines by 0,5 mL of 20% methanol containing 2 %
acetic acid.
e) Reconstitute the eluate in 150 uL of mobile phase.
4. Prepare the set of standard solutions containing amphetamine, methamphetamine and ephedrine
in concentration range (0.05 g/L to 5 g/L) in mobile phase and measure them with tutors help.
Evaluate the content of amphetamine, methamphetamine and ephedrine in submitted hair sample.
Questions:
1. Can the results of the hair analysis for drugs to make inferences about the acute intoxication
organism?
Literature:
1. B. Levine, Principles of Forensic Toxicology, AACC Press; Fourth edition, 2013.
2. M.J. Bogusz, R. Smith, Forensic Science, Volume 6, Second Edition (Handbook of
Analytical Separations), Elsevier Science; 2 edition (2007).
3. S.H.Y. Wong, I. Sunshine, Handbook of Analytical Therapeutic Drug Monitoring and
Toxicology, CRC Press; 1 edition (1996).