This document provides an overview of analyzing drugs from hair samples for forensic analysis. It discusses how drugs become incorporated into hair, specimen collection procedures, stability of drugs in hair, effects of cosmetic treatments, hair digestion procedures, drug analysis methods including immunoassays, chromatography, and sectional analysis. Applications of hair analysis include drug facilitated crimes, verifying drug history, determining gestational drug exposure, and post-mortem toxicology. Two case studies are presented where hair analysis aided investigations.

1. FORENSICANALYSISOFDRUG FROM
HAIRSAMPLE

2. CONTENTS
 Introduction
 Mechanisms of drug incorporation into hair
 Specimen collection and procedures
 Stability of drugs in hair
 Decontamination procedures
 Effects of cosmetic treatments
 Hair digestion procedures
 Drug analysis
 Sectional analysis
 Applications
 Case study
 Conclusion
References

3. 1. INTRODUCTION
 Hair is recognized as an attractive and commonly used biological
specimen for drug testing.
 The major practical advantages of hair testing are:
1) larger detection windows (from 3days to years), depending on the
length of the hair shaft, compared to those of urine/blood
2) evaluation of long term history to short term history
3) the sample collection is non-invasive.
 There is also no valid reason for making hair specimens unlikely to
be tampered.
 Drugs in hair can be detected and determined at the levels of pico-
mole/mg.
 Hair analysis has found even broader applications in evaluating
environmental exposure to toxicants, in doping controls and in drug
abuse studies in the fields of forensic toxicology, clinical toxicology
and clinical chemistry.

4. MECHANISMS OF DRUG INCORPORATION INTO
HAIR
3 Modes of
Drug
Incorporation
(3)
Incorporation
from External
Contamination
(2) Incorporation
from Sweat and
Other Secretions
(1) Incorporation
from the
Bloodstream

5. • Drugs circulating in the blood delivered to the hair
follicle and are entrapped in inaccessible regions of
the hair.
• Distinct bands of drug are present in hair related to
hair growth.
• This model is termed the “entrapment model”.
(1)
Incorporation
from the
Bloodstream
• Drugs and metabolites are present in sweat and
sebum and may be transferred easily to hair.
• Lipophilic substances more accumulate in hair.
• This model is called the “sweat model”.
(2)
Incorporation
from Sweat
and Other
Secretions
• Passive exposure of the hair to the drug, either from
vapour phase or solid-phase contact.
• Drugs in hair are bind through ionic and van der
Waals interactions with the protein chain.
• Diffusion of drug is proportional to a number of
factors (concentration, nature of the object, pore size,
area, etc.).
(3)
Incorporation
from External
Contamination

6. Figure Showing Drug Incorporation Into Hair

7.  Vertex posterior (back of the head) is the best area for sample
collection due to the following:
 Most of the hairs exist in the same growth phase.
 The growth rate of most of the hairs is also the same in this
region.
 Less influence of age and sex.
SPECIMEN COLLECTION AND PROCEDURES
Figure showing Vertex posterior and Cutting of hair sample

8. COLLECTION PROCEDURE
 Hair strands are cut as close as possible to the scalp, and the
location of the root-tip must be indicated.
 Hair samples must be collect before the autopsy has started.
 In case of curly hair, pluck hair and cut every hair strand
separately at defined intervals from the root.
 Plucked hair may also be preferred in suspected fatal poisonings.
 Hair samples are stored in an envelope, aluminium foil, or a
plastic zip lock bag and stored at ambient temperature.
 Tape should be avoided.
 Hair should be carefully aligned for segmental hair analysis.

9. Collection and packing of hair samples in aluminium foil

10. STABILITY OF DRUGS IN HAIR
 Drug incorporated in the
hair is very much stable
in favourable conditions,
e.g., ambient temperature
and dry atmosphere.

11. EFFECTS OF COSMETIC TREATMENTS
 Drug concentration is change in hair by the cosmetic
treatment of hair.
 The products used for cosmetic treatments, such as
bleaching, permanent waving, dyeing or relaxing; cause
hair damage and affect drug content (by loss) or directly
affect drug stability.
 Hair cosmetic treatments affect and damage the cuticle
and hence drug concentration.

12. DECONTAMINATION PROCEDURES
 Contaminants can be care products (hair gel), sweat, and
drug contaminants from the environment, or anything else.
 The goal of washing is to remove only the external
contaminants, dirt and grease from the surface of the hair
and avoidance of false positive.
 Decontamination agents used in washing are detergents
such as Prell shampoo, surgical scrubbing solutions,
surfactants such as 0.1% sodium dodecyl sulfate, phosphate
buffer, or organic solvents such as acetone, diethyl ether,
methanol, ethanol, dichloromethane, hexane or pentane.
 In hair analysis, the metabolites of drugs are analyzed,
which would not exist in external contamination.

13. HAIR DIGESTION PROCEDURES
Hair
Digestion
Procedures
Acidic
Extraction
Method
Enzymatic
Digestion
Method
Organic
Solvent
Method
Alkaline
Digestion
Method

14. Alkaline Digestion Method
• Incubate hair sample in 0.1∼2.5 M NaOH, at 37˚ C
overnight, at pH 9.
• Aqueous solution is extracted with solid phase extraction.
• Alkaline stable compounds, such as morphine,
amphetamines and cannabinoids have to be analyzed.
Acidic Extraction Method
• Incubate hair sample at 37˚ C overnight in 0.1 M HCl
solution or at 120 °C for 30 min in 0.6 M HCl solution.
• Solution is neutralized and extracted with solid phase
extraction (SPE).
• 6-AM and cocaine can be extracted.

15. Organic Solvent
Method
• Hairs are digested in an ultrasound bath for several hours
at 45 °C using ethanol or methanol as solvent.
• Evaporating the organic solvent, the sample can be
analyzed directly by GC-MS.
• Unstable drugs like 6-AM can be analyzed.
Enzymatic Digestion Method
• Hairs are treated with a solution of Tris buffer, dithiothreitol,
proteinase K, and sodiumdodecyl sulfate.
• Enzymes digest hair by acting on hair protein (keratin) without
altering or destroying the concentration of drug and its
metabolites.
• Analyze unstable compounds like heroin/6-AM and cocaine.

16. DRUG ANALYSIS
 Immunological methods
• Immunoassays are used as screening tests because of their
sensitivity, speed and convenience.
• It provides either a positive or negative result, indicating the
presence or absence of detectable drug.
• Quantification by immunoassay is difficult to achieve.
 Radioimmunoassay
• RIA is the most common screening test for drug testing.
 ELISA
• ELISA is used for methadone, benzodiazepines, opiates,
methamphetamine and for amphetamine.
• ELISA test is not useful for tetrahydrocannabinol.
 Positive and negative controls should be also made up for
immunological hair analysis.
 A second analytical method based on a different property of the
analyte must be always performed, like HPLC, or GC/MS for
confirmation.

17. CHROMATOGRAPHIC METHODS
 Chromatographic methods have been used as screening
and confirming tests.
 Quantification of drugs
achieved.
 Gas Chromatography
and drug metabolites can be
• Gas chromatography (GC) separates a mixture of
compounds into individual components and the detector
coupled with GC identifies each component.
 Gas Chromatography–Mass Spectrometry (GC-MS)
• GC-MS is by far the most widely used analytical tool for
drug determination in hair specimens.
• Analysed the opiates, cocaine and related drugs,
amphetamines, cannabinoids, benzodiazepines,
therapeutical drugs and pesticides.

18.  HPLC coupled to MS
• LC–MS is an analytical chemistry technique that
combines the physical separation capabilities of liquid
chromatography (or HPLC) with the mass analysis
capabilities of mass spectrometry (MS).
• Use of LC-MS(-MS) owing to its excellent specificity and
its extraordinary sensitivity, which allow, in some special
cases, the detection of a single exposure to a specific drug.
 In recent years, tandem mass (MS–MS) have been used
for hair analysis in order to increase sensitivity and detect
GC-unstable compounds.

19. SECTIONAL ANALYSIS
 Multi-sectional analysis
involves taking a length of hair
and cutting it into sections to
measure drug use during
shorter periods of time.
 Segmental hair analysis is used
to verify both their previous
drug history and their recent
enforced abstinence.
 The switch from one drug (e.g.
heroin) to another drug
(codeine, ethylmorphine,
dihydrocodeine) can be
established with accuracy.

20. The effect of disarray of hair strands on the analytical results. Striped bars represent
drug concentrations in segments containing a drug (circular areas in the hair).

21. APPLICATIONS OF HAIR ANALYSIS
1) Drug-Facilitated Crime
• Drug-facilitated crimes (sexual assault, robbery,
incapacity, etc.) increase recently.
• Some drugs possess amnesic properties, as a result of
which a delay in reporting the crime, natural processes
have eliminated the drug from typical biological specimens.
In these situations, blood or even urine can be of little
interest.
• Hair is suggested as a valuable specimen in such situations.
2) Verification of drug history through sectional
analysis of hair
• To measure a person’s drug abuse history for a relatively
short period of time, sectional analysis of hair is performed
by cutting hair into short fragments.

22. 3) Determination of gestational drug exposure
• Analysis of new-born hair provide history of maternal drug
abuse during gestational period.
4) Dope testing
• Hair follicle tests provide a much large window of detection
of drugs used over a period of time by athletes.
5) Drug screening for pre-employment and driving
license fitness testing
• Level of drug abuse, single, multiple, or chronic users can
also be confirmed. Hair analysis in combination with
psychological tests can help to trace out the drug abuse
history, which can be used for re-granting or cancelling the
license.
• Studies showed that hair analysis is more effective than
urinalysis for “fitness to drive assessment”.

23. 6) Post-mortem toxicology
• For forensic investigation, it is helpful to gain maximum
possible information regarding the victim’s regular use of
drugs before death.
• The presence of a drug or its metabolite in the hair can
confirm whether the person was a regular user of a drug or
not.
7) Comparison with urine testing
• There is no tampering dispute with hair sample as for
other biological samples.
• Urine does not indicate the frequency of drug intake.
• Urine specimens cannot distinguish between chronic use
or single exposure, hair analysis can make this distinction.

24. CASE STUDY
 Case-1
In July of 1990, a Miami man became extremely ill after drinking
an imported malted beverage from Colombia. After drinking the
contents of the bottle, the subject thought he may have been
poisoned; he stated the beverage tasted bad, and his mouth and
tongue were numb. The man went into a coma immediately
after making the statement and was rushed to the hospital. At
the hospital he was diagnosed as suffering from acute cocaine
intoxication after a urinalysis test.
Cocaine was detected in the residue of the bottle consumed by the
victim. The subject was maintained alive for 24 days. After the
victim died, hair samples were collected to determine whether
he was a regular cocaine user who had overdosed or the victim
of a product tampering (during the period after the incident but
before his death, the victim’s hair grew approximately 1 to 1.5
cm).

25. Historical information on his drug usage was gathered by
conducting segmental analysis on the victim's 2.5 cm length hair.
The hair was cut into half-centimeter segments and analyzed.
The hair segments contained a peak concentration of almost 100
nanograms (ng) per mg at a time period that corresponds to the
ingestion of the suspect beverage (segment 1-1.5 cm). Witness
interviews substantiated results of the segmental hair analysis
during the investigation, which revealed the victim was a chronic
cocaine user.

26.  Case-2
A rape investigation was aided by hair analysis for cocaine. A
request was made for hair analysis by a small town's police
department to contest the alibi of a suspect after a woman
reported an acquaintance had raped her in her own home.
The suspect stated he and the victim were dating, engaging
in sex, and had used crack cocaine together on numerous
occasions. She denied his allegations and proof was
needed to refute or confirm his alibi. Since the suspect was
positive for cocaine and the victim was negative for use of
cocaine over the previous several months, hair testing was
effective in contradicting the alibi.

27. CONCLUSION
 The testing of hair for drugs has been an invaluable aid and
often a necessary tool for law enforcement. It has given the
forensic investigator a glimpse into the past. In conjunction
with the use of urinalysis, hair testing can give a more
detailed drug history on a test subject. The two tests should
be considered complementary.
the confirmation of chronic environmental
 However,
exposure
represents
to substances, by performing hair analysis,
a challenge for the toxicologist and careful
evaluation of the restrictions in the analysis of every class of
substances is further required.

28. REFERENCES
Boumba, V., Ziaavrou, K., Vougiouklais. T. 2006. Hair as a Biological
Indicator of Drug Use, Drug Abuse or Chronic Exposure to
Environmental Toxicants. International Journal of Toxicology.
25:143–163.
Kintz, P. 2007. Analytical and Practical Aspects of Drug Testing in Hair.
First Edition. CRC Press Taylor & Francis Group 6000 Broken Sound
Parkway NW, Suite 300 Boca Raton. PP- 5-25.
Moffat, A.C., Osselton, M., Widdop, B. 2011. Clarke’s Analysis of Drugs
and Poisons. Fourth Edition. Pharmaceutical Press 1 Lambeth High
Street, London SE1 7JN, UK. PP- 323-334.
Nakahara, Y. 1999. Hair analysis for abused and therapeutic drugs.
Journal of Chromatography B. 733: 161–180.
Slanger, S. K. 1999. Detection of multiple drugs in human hair from
seven drug classes using one digestion/extraction protocol. Graduate
Student Theses, Dissertations, & Professional Papers. 9252: 20-36.