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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).