Forensic Science International 186 (2009) e9–e11

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Case report

Identification of GHB and morphine in hair in a case of drug-facilitatedsexual assault

Riccardo Rossi a,*, Massimo Lancia b, Cristiana Gambelunghe b, Antonio Oliva a, Nadia Fucci a

a Institute of Legal Medicine, Catholic University of the Sacred Heart, L.go F. Vito, 1 00 168 - Rome, Italyb Institute of Legal Medicine, University of Perugia, Via del Giochetto snc, 06100 - Perugia, Italy

A R T I C L E I N F O

Article history:

Received 6 May 2008

Received in revised form 20 January 2009

Accepted 25 January 2009

Available online 3 March 2009

Keywords:

GHB

Hair analysis

GC/MS

Drug-facilitated sexual assault

A B S T R A C T

The authors present the case of a 24-year-old girl who was sexually assaulted after administration of

gamma-hydroxybutyrate (GHB) and morphine. She had been living in an international college for foreign

students for about 1 year and often complained of a general unhealthy feeling in the morning. At the end

of the college period she returned to Italy and received at home some video clips shot by a mobile phone

camera. In these videos she was having sex with a boy she met when she was studying abroad.

Toxicological analysis of her hair was done: the hair was 20-cm long. A 2/3-cm segmentation of all the

length of the hair was performed. Morphine and GHB were detected in hair segments related to the

period of time she was abroad. The analyses of hair segments were performed by gas chromatography/

mass spectrometry (GC/MS) and the concentration of morphine and GHB were calculated. A higher value

of GHB was found in the period associated with the possible criminal activity and was also associated

with the presence of morphine in the same period.

� 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Gamma-hydroxybutyrate (GHB) is a substance naturallypresent in mammalian species. It has been used clinically fromthe 1960s as an intravenous anaesthetic, in the management ofalcohol and opiate withdrawal syndromes and for a variety ofpurposes including the treatment of sleep disorders, depressionand anxiety [1–5,14].

In 1980 this substance began to be used for illegal purposes, atfirst in the field of sport because of its assumed stimulatingfunctions on the growth hormone (there are still some doubtsabout this) and afterwards in ‘‘rave’’ parties and in clubs due to itshallucinating effects.

For 10 years there has been an increase in the use of GHB forsexual abuse [5–7,12,13]. Together with ketamine and flunitraze-pam, GHB is used in drug-facilitated crimes. Doses of 10 mg/kgcause amnesia, 20–30 mg/kg induce sleep, and doses of 50 mg/kgor higher produce anaesthesia [3,6,8]. It is attractive for rapistsbecause it can be found easily (on the street, fitness centres, andInternet) and moreover because it can be delivered mainly as anodourless, colourless liquid and so it is often consumed unwit-tingly, mixed in spiked drinks.

* Corresponding author. Tel.: +39 06 35507031; fax: +39 06 35507033.

E-mail address: riccardo.rossi@rm.unicatt.it (R. Rossi).

0379-0738/$ – see front matter � 2009 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.forsciint.2009.01.017

These drug-facilitated sexual assaults are extremely difficult todocument from an analytical point of view because of theendogenous presence of gamma-hydroxybutyrate in biologicalhuman fluid, its narrow detection window and the powerfulsedative effects of the molecule [9,10]. In fact, 24 h after the allegedassault, the victim’s urine has generally a normal physiologicallevel of GHB [9]. To address a response to this caveat, hair wassuggested as a valuable specimen. Several studies have proved theefficacy of hair analysis, in highlighting both single doses of GHBand multiple exposures over time [3,6,9].

In this study the authors present the case of a 24-year-old womanwho was sexually assaulted after unintentional intake of GHB andmorphine. The analytic findings obtained by gas chromatography/mass spectrometry analysis of hair samples demonstrated exposureto GHB and morphine. Based on the hair analysis and the girl’shistory authors’ conclusions are that it is very likely she wasassaulted after deceptive exposure to GHB and morphine.

1.1. Case history

A 24-year-old girl had been studying abroad for about 1 year.During this period she met a young man who became her boyfriend.During the whole year abroad sometime she woke up with a strongheadache and nausea and noticed some small abrasions on one arm,similar to pricks (punctures). Despite all this she was not tooworried. She thought her confused state of mind was due to the fact

Fig. 1. GHB and morphine concentration obtained from the hair samples.

Fig. 2. Selected ion chromatograms of GHB.

R. Rossi et al. / Forensic Science International 186 (2009) e9–e11e10

of being abroad, far from home, stressed and a hangover. One monthafter she had returned to Italy, she received at home some video clipsshot by a mobile phone camera. In these videos she was having sexwith a boy she met when she was studying abroad, but she noticedshe was in a confused state of mind with strange behaviour that shecould not recall at all. She was convinced that she had been rapedunder the effect of drugs. On the basis of this assessment she decidedto have her hair analysed. She admitted a moderate consumption ofalcohol and no drug intake during the period she was abroad.

2. Materials and methods

2.1. Specimen

The hair was not cosmetically altered or treated and the cut was performed as

close as possible to the scalp. Two strands, 20 cm long, with a total weight less than

500 mg were collected and stored in plastic tubes at room temperature.

Investigations were performed on four segments corresponding approximately

to the period before and after the sexual assault. In detail we used four different 3-

cm sections for the proximal part, and four 2-cm segments for the distal part.

2.2. Chemicals and reagents

Ethyl acetate, methylene chloride and chloroform were purchased from Carlo

Erba Milan, Italy. The derivatization agent 1% N,O-bis(trimethylsilyl) trifluoroace-

tamide (BSTFA) + 1% trimethylchlorosilane (TMCS), and pentafluoropropionic

anhydride (PFPA) and 2,2,3,3,3-pentafluoropropanol 97% was purchased from

Sigma–Aldrich srl (Via Gallarate 154-20151, Milan Italy). Standards of GHB,

gamma-hydroxybutyric acid hexadeuterated (GHB-d6) morphine and morphine-

d3 from Promochem (via Venezia, 23-20090 Sesto San Giovanni, Italy)

2.3. Toxicological analysis

2.3.1. GHB analysis

Hair collected by the vertex posterior was decontaminated twice using 5 ml of

methylene chloride for 2 min at room temperature, and then cut into four 3-cm

proximal segments and four 2-cm distal segments. About 20 mg of decontaminated

hair was incubated in 0.5-ml NaOH (0.01N), 16 h at 56 8C overnight, adding 25 ml of

GHB-d6 (20 ng/ml) used as internal standard. After cooling, the homogenate was

neutralized with 50 ml 0.1N HCl, and 2 ml ethyl acetate was added together with

50 ml of 0.01 M H2SO4. After agitation and centrifugation, the organic phase was

evaporated to dryness under nitrogen flow. The residue was derivatized by adding

20 ml of the BSTFA (1% TMCS) and 20 ml ethyl acetate for 20 min at 60 8C. The

aqueous washing was analysed under the same analysis condition.

2.3.2. Morphine analysis

The biological sample was examined for the presence of morphine using GC/MS.

About 20 mg of the washed hair sample (twice with dichloromethane) was analysed

as follows: the sample was added with deuterated internal standard analogs of

morphine-d3 and then incubated with KOH 0.1 M overnight at 40 8C. The sample was

submitted to liquid/liquid extraction with a mixture of chloroform and isopropanol.

After evaporation of the solvent the dry extract was derivatized adding PFPA (50 ml)

and pentafluoropropanol (30 ml) at 100 8C for 30 min. The residue was evaporated

and reconstituted with 25 ml ethyl acetate and directly analysed by electron impact

GC/MS operated in SIM mode with ions chosen for morphine and deuterated.

2.4. GC/MS procedure

One-microliter aliquot of the derivative extract was submitted to GC/MS analysis

on a model 5890 gas chromatography fitted with a split-splitless injector (270 8C)

equipped with a HP 1 capillary column (12 m to 0.2 mm i.d.) 0.3 mm film thickness

methylsilicone. A linear program from 70 to 280 8C at 10 8/min increments with a 5-

min final isotherm was chosen. Helium was employed as the carrier gas at a column

head pressure of 10 psi. The capillary column was connected to a mass analyzer (HP

5971A) operated by electron impact (70 eV) electronic impact in total ion

monitoring (40–500 amu) to identify GHB in the hair sample. Data were recorded

in full scan and ions monitored were: m/z 233–204–147 and 239 for GHB and GHB-

d6, respectively (ions m/z 233 and 239 were used for quantification). The same

temperature program was employed for morphine; the mass analyzer operated in

selected ion monitoring mode with the choice of ions: m/z 414–577–558 and

relatively deuterated ions (417–580–561). Limit of detection (LOD) and limit of

quantitation (LOQ) were, respectively, 0.01 and 0.1 ng/mg.

3. Results and discussion

The aqueous washings were analysed to exclude if the hair wasexternally contaminated with morphine or GHB and no drugs were

detected. The analysis of the segmented hair strand revealed a highconcentration of GHB in the proximal segment while the value ofGHB was lower in the distal segment. Potential conversion of GHBto GBL during the preparation process did not occur as verified bypH measurements [15]. The concentration of GHB in distalsegments was 1 ng/mg. On the contrary, a concentration of 5–4–3–4 ng/mg was detected in the proximal segments. Besides thepresence of morphine was revealed at a concentration of 1 ng/mgin the proximal segment whereas the presence of morphine (cut-off 0.5 ng/mg) in the distal section was excluded. Fig. 1 shows theGHB and morphine concentration obtained from the hair samples.Fig. 2 shows ion chromatogram of GHB obtained from hair samples.GHB was positive in the range 3–5 ng/mg, which is above thephysiological concentrations (<2 ng/mg, n = 20) referred in litera-ture [1]. Moreover, according to these data, the authors underlinethat the quantitative value from different hair segments is notimportant for an exact evaluation. On the contrary, a comparisonbetween the values obtained for the segment not related to thecriminal event (distal segment) is necessary to determine thelevels incorporated in the hair matrix before and after theexposure. It is well known that to demonstrate the intake ofGHB with sufficient confidence, it is necessary to determine thebasic levels by segmented hair analyses for each victim to compareGHB levels at the suspected period [3,9,11]. Since GHB is present inthe hair of the general population at a physiological concentration,

R. Rossi et al. / Forensic Science International 186 (2009) e9–e11 e11

toxicologists must be able to discriminate between endogenouslevels and a concentration resulting from exposure [11].

Comparison between different patients is not justified because itis well known that the individual GHB basal levels are verychangeable. Some authors assume that a high concentration of GHBin a hair segment is not significant and should always be comparedwith individual basic levels [3,9]. Hair analysis is the only methodused to prove repetitive exposure to a toxic substance. Thisobservation reveals that a high concentration of GHB in hair reflectsan acute overexposure to GHB and can be documented severalmonths after the sexual assault. In general it must be specified thatthe possibility given by hair analysis should not prevent the victimand the medical examiner from taking urine, blood, and sweatsamples as soon as possible after the event. Hair analysis may be auseful adjunct to conventional drug testing in sexual assault, and itshould not be considered an alternative to urinalysis, but acomplement [3]. We believe that hair analysis could be a usefuladdition to conventional drug testing in sexual assault, but we dobelieve that further studies may confirm the usefulness of thistechnique and establish the definition of legally defensible cut-offvalues. We think other studies regarding individual basic levels ofGHB are very necessary especially to obtain a reliable demonstrationin cases of single exposures through hair analysis.

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