drugs

1. Determination of drugs of abuse in hair: evaluation of
external heroin contamination and risk of false positives
Guido Romano*
, Nunziata Barbera, Giorgio Spadaro,
Vincenzo Valenti
Dipartimento di Anatomia, Patologia Diagnostica, Medicina Legale, Igiene e Sanita` Pubblica,
Universita` di Catania, Via S. Sofia 87—Comparto 10, 95123 Catania, Italy
Received 31 August 2002; accepted 18 October 2002
Abstract
One of the most controversial point regarding the validity of hair testing is the risk of false positive due to external
contamination. The aim of our experience is to verify if a 5 consecutive days contamination with a small amount of a powdered
mixture of heroin hydrochloride and acetylcodeine hydrochloride (10:1 w/w) will last sufficiently long to make a contaminated
subject indistinguishable from active users, and if normal washing practices together with the decontamination procedure are
sufficient to completely remove the external contamination.
Our results suggest that decontamination procedures are not sufficient to remove drugs penetrated into hair from external
source. In fact, all contaminated subjects were positive for opiates (heroin, 6-MAM, morphine, acetylcodeine and codeine) for at
least 3 months.
Significant 6-MAM concentrations (>0.5 ng/mg) were found in each subject until 6th week. Further, 6-MAM/morphine ratio
were always above 1.3.
# 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Hair analysis; Contamination; Heroin; Opiates
1. Introduction
Hairtestingiswidelyemployedtoevaluatedruguse[1–23].
Accordingtothe most accepted theories,the presenceofdrugs
in hair is the result of one or some mechanisms of incor-
poration. Several models have been proposed to explain the
drug incorporation into hair matrix. The multi-compartment
theory suggests that drugs may be incorporated into hair from:
(a) blood; (b) sweat and sebaceous gland secretions; (c)
external environment [24]. The inaccessible interior domain
model supposes that interior of hair can be accessed with great
difficulty by exogenous drugs and aqueous solvents, so that,
under realistic contamination conditions, this domain is inac-
cessible to exogenous drugs [2]. On this basis, it has been
concluded that hair analysis should allow to evaluate the
actual or long-term abuse of drugs.
However,oneofthemostcontroversialpointsregardingthe
validity of hair testing is the risk of false positives due to
external contamination. The statement about the efficacy of
decontamination procedures was based on experimental stu-
dies not reproducing realistic contamination models [25–30].
Our study proposed a pattern of drug contamination
corresponding to a realistic scenario: in fact, the use of
drugs implies their handling and, therefore, the real possi-
bility to contaminate oneself or others.
The aim of our study was to verify the validity of hair
testing for heroin in order to discriminate active users from
false positives due to external contamination.
2. Materials and methods
2.1. Experimental groups
The study was conducted on six drug-free volunteers,
three males and three females (Table 1). Each subject
Forensic Science International 131 (2003) 98–102
*
Corresponding author. Tel.: þ39-95-256164;
fax: þ36-95-256165.
E-mail address: toxicologyct@hotmail.com (G. Romano).
0379-0738/02/$ – see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 7 9 - 0 7 3 8 ( 0 2 ) 0 0 4 1 3 - 9

2. gave informed consent. Before the start of the study, urine
and hair samples from each subject were analyzed to
exclude the presence of drugs or substances that could
interfere with hair test. Each week for the duration of the
study, urine samples from each subject were analyzed
to exclude the possibility of drug use during the study.
Each subject could wash their hair following their normal
routine.
2.2. Contamination procedure
Contaminationwascarried out asfollows:each subjectwas
asked to apply 5 mg of a powdered mixture of heroin hydro-
chloride (S.A.L.A.R.S. Como) and acetylcodeine hydrochlor-
ide (S.A.L.A.R.S. Como) (10:1 w/w) to their hands for 5 min
so that it was uniformly distributed. Each subject was then
instructedonhow tocontaminatetheirhair withtheir hands,as
uniformly as possible, from the roots to the ends for 10 min.
Contamination procedure was daily carried out for 5 con-
secutive days.
2.3. Hair samples
Each subject was asked to wash their hair before collec-
tion. Each hair sample (100 mg; 5 cm long) was cut as close
as possible to the scalp from the posterior vertex, having care
to exclude the new hair growth. The samples were collected
at the following times:
one sample before contamination;
one sample 24 h after the first contamination, and before
the second contamination (2nd day);
one sample 24 h after the last contamination (6th day);
one sample each week for 14 weeks.
Each sample was decontaminated and analyzed on the
day it was collected.
2.4. Decontamination procedure
Each sample underwent the decontamination procedure
proposed by Kintz [31]: hair sample was placed in a screw-
top glass tube and washed three times in 5 ml of dichlor-
omethane for 5 min at room temperature, by agitation. Each
wash solution was analyzed by gas chromatography-mass
spectrometry (GC-MS).
2.5. Hair extraction procedure
After decontamination procedure, each sample was finely
cut, spiked with 1 ng/mg of nalorphine and with 1 ng/mg of
scopolamine as internal standards, incubated at 45 8C for 24 h
in 1 ml of HCl 0.1 N. The aqueous solution was collected,
adjusted to pH 7.5–8 adding 1 M phosphate buffer (pH 7) and
some drops of 1 M phosphate buffer (pH 8.5) and then
extracted using 2 ml of chloroform:isopropanol (8:2 w/w).
2.6. Derivatization
The eluent was evaporated to dryness under nitrogen
flow. The 50 ml N,O-bis(trimethylsilyl)trifluoroacetamide
ðBSTFAÞ þ 1% trimethylchlorosilane (TMCS) was added
to the dry extract, which was sealed and heated at 70 8C for
30 min.
2.7. GC-MS analysis
A Hewlett-Packard (HP) 5890 gas chromatograph (Series
II) coupled to a 5989B mass selective detector (MSD) was
used. An HP Ultra-2 (Cross-linked 5%, phenyl, 95% methyl-
polysiloxane) capillary column (12 m  0:20 mm i.d.) with a
0.33 mmfilmthicknesswaslinkedtothe MSDthrougha direct
capillary interface. The injector temperature was 250 8C. The
oven temperaturewas maintained at 100 8C for2.25 min, then
programmed to 200 8C at 40 8C/min, 260 8C at 5 8C/min,
290 8C at 20 8C/min, and maintained at 290 8C for 2 min.
Interface temperature was 280 8C; source temperature was
230 8C; quadrupole temperature was 100 8C; the carrier gas
was helium with a flow rate of 1 ml/min. Each hair extract was
analyzed using two different acquisition methods (A and B),
both performed by select ion monitoring (SIM), in order to
resolve the substances with the same retention time (e.g.
morphine–TMS from acetylcodeine, heroin from nalorphine
TMS). Analysis of hair extracts was performed by monitoring
the following ions:
2.7.1. Method A
m/z 138; 154; 375 (scopolamine–TMS); m/z 371,3; 343,3;
234,3; 313 (codeine–TMS);
m/z 429,3; 414,3; 324,2 (morphine–TMS); m/z 399,3;
340,3; 287,2 (6-MAM–TMS);
m/z 455,3; 440,3; 414,3 (nalorphine–TMS).
Table 1
Characteristics of the subjects
ubject Sex Race Characteristics of hair
A F C Chestnut dyed hair, 32 cm long, medium thickness, wavy
B F C Black dyed hair, 20 cm long, thick, straight
C F C Meches dyed hair, 13 cm long, thin, straight
D M C Light chestnut virgin hair, 12 cm long, medium thickness, straight
E M C Grey hair, 5 cm long, medium thickness, straight
F M C Black virgin hair, 5 cm long, thick, wavy
F, female; M, male; C, Caucasian.
G. Romano et al. / Forensic Science International 131 (2003) 98–102 99

3. 2.7.2. Method B
m/z 138; 154; 375 (scopolamine–TMS); m/z 371,3; 343,3;
234,3; 313 (codeine–TMS);
m/z 341; 282; 298 (acetylcodeine); m/z 399,3; 340,3;
287,2 (6-MAM–TMS);
m/z 327; 369; 268; 310 (heroin).
Calibration curves were constructed by analyzing control
hair samples (100 mg) added with heroin, codeine, mor-
phine, acetylcodeine, 6-MAM (from 0.01 to 5 ng/mg), sco-
polamine (1 ng/mg) and nalorphine (1 ng/mg). Hair control
samples were extracted and analyzed using the same meth-
ods described above.
3. Results
All hair samples tested were positive for opiates; the
data are shown in Table 2. It has been found that heroin
concentrations were high during the first 2 weeks, while
decreased during the next 2 weeks; it was no longer detect-
able between 29th and 36th day. The 6-MAM concentrations
decreased during the first weeks, however significant levels
(0.5 ng/mg) were found in each subject until 43rd day.
Three months after contamination, every subject showed
detectable levels of morphine (0.05–0.18 ng/mg) and 6-
MAM (0.15–1.18 ng/mg).
Noteworthy, 6-MAM/morphine ratio were always above
1.3. Significant concentrations of acetylcodeinewere detected
until 50th–64th day. Also, all samples showed low levels of
codeine until the end of the study.
It is interesting to note that during the contamination
period, the first two wash solutions showed appreciable
heroin, 6-MAM and acetylcodeine concentrations, while
the GC-MS analysis of the third dichloromethane washing
did not reveal presence of opiates; during the next weeks, 6-
MAM traces were found only in the first wash solution.
Urinalyses of each experimental subject were negative for
the duration of the experience.
Table 2
Concentrations of opiates (ng/mg) found in hair samples after decontamination
Subject Analytes 2 6 8 15 22 29 36 43 50 57 64 71 78 85 92 99
A H 0.42 1.72 0.83 0.28 0.09 – – – – – – – – – – –
6-MAM 0.45 2.38 2.81 1.90 1.29 0.64 0.55 0.58 0.46 0.49 0.41 0.43 0.38 0.37 0.33 0.35
M 0.09 0.14 0.58 0.41 0.28 0.16 0.19 0.14 0.15 0.13 0.11 0.12 0.12 0.10 0.08 0.10
AC 0.07 0.38 0.35 0.21 0.18 0.04 0.02 0.01 – – – – – – – –
C – 0.06 0.16 0.11 0.09 0.03 0.02 0.02 0.01 0.02 0.01 0.02 0.02 0.02 0.01 0.01
B H 0.58 1.14 1.02 0.79 0.47 0.11 – – – – – – – – – –
6-MAM 0.73 1.60 1.47 1.38 1.52 0.74 0.61 0.63 0.58 0.59 0.55 0.37 0.36 0.38 0.32 0.31
M 0.22 0.42 0.84 1.12 0.78 0.32 0.29 0.27 0.24 0.2 0.16 0.12 0.12 0.13 0.11 0.1
AC 0.15 0.23 0.21 0.16 0.09 0.04 0.03 0.01 – – – – – – – –
C 0.02 0.06 0.12 0.09 0.07 0.05 0.04 0.06 0.05 0.04 0.05 0.03 0.02 0.01 0.02 0.01
C H 1.26 2.76 1.66 1.37 0.42 0.10 – – – – – – – – – –
6-MAM 1.56 2.93 2.83 2.70 1.99 0.86 0.72 0.51 0.35 0.31 0.28 0.29 0.25 0.26 0.21 0.19
M 0.31 0.41 0.77 1.07 0.51 0.17 0.15 0.11 0.10 0.11 0.09 0.08 0.07 0.08 0.06 0.05
AC 0.27 0.49 0.39 0.34 0.18 0.06 0.04 0.03 0.01 – – – – – – –
C 0.04 0.15 0.19 0.15 0.13 0.06 0.06 0.04 0.05 0.04 0.04 0.03 0.02 0.01 0.02 0.01
D H 0.84 2.80 0.99 0.49 0.33 0.17 – – – – – – – – – –
6-MAM 1.03 3.58 2.77 2.60 2.08 1.81 1.81 1.75 1.61 1.54 1.37 1.34 1.31 1.25 1.28 1.18
M 0.21 1.25 0.94 0.67 0.65 0.49 0.45 0.37 0.31 0.29 0.33 0.25 0.23 0.24 0.21 0.18
AC 0.25 0.86 0.45 0.38 0.26 0.19 0.12 0.10 0.06 0.03 – – – – – –
C 0.07 0.28 0.18 0.17 0.14 0.11 0.13 0.12 0.10 0.11 0.09 0.09 0.08 0.07 0.06 0.04
E H 0.18 1.34 0.76 0.40 0.31 0.15 – – – – – – – – – –
6-MAM 0.37 3.46 2.93 2.44 1.10 0.78 0.56 0.50 0.44 0.37 0.33 0.28 0.25 0.21 0.18 0.15
M 0.09 0.41 1.32 0.94 0.63 0.42 0.31 0.19 0.13 0.13 0.12 0.10 0.11 0.09 0.08 0.07
AC 0.08 0.25 0.21 0.19 0.15 0.08 0.05 0.01 – – – – – – – –
C 0.01 0.11 0.16 0.17 0.10 0.07 0.04 0.05 0.03 0.02 0.03 0.02 0.01 0.01 – –
F H 0.41 1.75 0.87 0.43 0.28 0.12 – – – – – – – – – –
6-MAM 0.63 3.38 2.75 2.37 1.73 1.57 1.43 1.31 1.38 1.22 1.18 1.03 0.79 0.83 0.72 0.67
M 0.14 0.71 1.07 0.81 0.68 0.50 0.53 0.36 0.29 0.21 0.23 0.18 0.19 0.15 0.11 0.12
AC 0.12 0.34 0.29 0.15 0.11 0.08 0.02 0.03 0.01 – – – – – – –
C 0.02 0.18 0.15 0.16 0.12 0.10 0.11 0.08 0.05 0.07 0.06 0.04 0.03 0.02 0.01 0.01
100 G. Romano et al. / Forensic Science International 131 (2003) 98–102

4. 4. Discussion and conclusions
Our study shows that a contamination with a rather
modest quantity of a mixture of heroin and acetylcodeine
(10:1 w/w) gives a positive hair test result despite the
adoption of the decontamination procedure. This finding
agrees with our previous studies demonstrating the possi-
bility of external contamination with cannabinoids [32]and
cocaine [33]. Positive hair test, resulting from external
contamination of surely not active consumers, makes its
validity questionable. Applying the evaluation criteria
suggested in literature [3,7,9,10,12,14–16,20,21,31,34,
35], the contaminated subjects resulted to be in the range
of values that defines the subjects as active users. The 6-
MAM concentrations higher than 0.5 ng/mg and 6-MAM/
morphine ratio above 1.3 are the criteria suggested to
establish a positive hair test result [9,37]. On this basis,
the subjects of our study should be considered active users.
In fact, in our experimental subjects 6-MAM concentra-
tions were higher than 0.5 ng/mg until 43rd day, and 6-
MAM/morphine ratio were always above 1.3. Our findings
suggest that the proposed criteria are not enough to dis-
criminate active users from a false positive due to external
contamination.
Several studies [25–30] exclude that passive contam-
ination could affect hair test results, assessing that the
drugs from external environment remain on the surface or
penetrate into the most superficial layers of hair, so that it
can be easily removed by hygienic washing, cosmetic
treatments or appropriate decontamination procedures.
However, our results, according to other studies reported
in literature [9,12,25,26,32,33,36], showed that neither
normal hygienic and cosmetic cares nor decontamination
procedures are sufficient to remove drug contamination.
Once penetrated into hair from external source, the
opiates persisted in the matrix for long time and were
detectable at least for 3 months following the contamina-
tion.
The finding of significant concentrations of 6-MAM,
morphine and codeine in hair matrix suggests that heroin
and acetylcodeine can enter into hair and can be trans-
formed into their metabolites. These data would seem to
exclude the existence of inaccessible domains hypotized by
other Authors [26,30,36]. As a result, cut-off values and
metabolite-to-drug ratio are not useful to evaluate passive
contamination. According to other studies [25,26,30,32,33,
38], hair testing does not allow either to discriminate the
external contamination share from the one due to active
consumption, or to distinguish active users from contami-
nated subjects.
In summary, while a negative hair testing result could be a
valid diagnostic tool only to rule out either active use or a
contact with drugs, a positive result cannot and must not be
interpreted as a sure sign of a state of drug use, but should be
confirmed by further toxicological tests (e.g. urinalysis and
clinical data).
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