Accepted Manuscript

Title: Proactive drugs in DFSA cases: toxicological findings in

an eight-years study

Authors: Elisabetta Bertol, Maria Grazia Di Milia, Alessia

Fioravanti, Francesco Mari, Diego Palumbo, Jennifer P.
Pascali, Fabio Vaiano

PII: S0379-0738(18)30665-0
DOI: https://doi.org/10.1016/j.forsciint.2018.08.032
Reference: FSI 9456

To appear in: FSI

Received date: 25-5-2018

Revised date: 2-8-2018
Accepted date: 25-8-2018

Please cite this article as: Elisabetta Bertol, Maria Grazia Di Milia, Alessia Fioravanti,
Francesco Mari, Diego Palumbo, Jennifer P.Pascali, Fabio Vaiano, Proactive drugs
in DFSA cases: toxicological findings in an eight-years study, Forensic Science
International https://doi.org/10.1016/j.forsciint.2018.08.032

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Proactive drugs in DFSA cases: toxicological findings in an eight-years study

Elisabetta Bertol, Maria Grazia Di Milia, Alessia Fioravanti, Francesco Mari, Diego Palumbo,

Jennifer P. Pascali*, Fabio Vaiano

Forensic Toxicology Unit, Department of Health Sciences, University of Florence, Largo

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Brambilla 3, Florence, Italy; U.R.I.To.N- Unit of Research of University of Florence, Florence,

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Italy

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Corresponding author:

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jenniferpaola.pascali@unifi.it

+30 055 2751787

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ORCID: 0000-0002-1363-3400
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Highlights
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 Drug facilitated sexual assault are the most common type of Drug Facilitated

Crimes.
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 Some countries have no jurisprudence in DFSA area, others consider it an

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aggravating circumstance
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 Substances causing disinhibition and amnesia are preferred, particularly if not

detected by the victim.

 Most of results concerned ethanol alone and co-assumption with

benzodiazepine.

 There is no a single substance that is uniquely associated with DFSA.

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Abstract

In case of drug-facilitated sexual assault (DFSA), the evidence is frequently anecdotal, with few

investigations based on scientific evidences being carried out and thus most cases are diagnosed as an acute

drug or alcohol intoxication. The reason may lay in the lack of specific knowledge by the victim on the

possibility to retrospectively study the allegedly events and to the absence of standardized and shared

protocols among health, forensic and police subjects. On this basis, in 2015 the Unit of Forensic Toxicology

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of University of Florence and the Sexual Assaults Centre in Hospital Careggi have fixed a common protocol

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to be applied in case of DFSA. The purpose of the study was to describe the results of the application of the

shared protocol for toxicological findings among women seeking health care after sexual assault, and to

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assess the relationship with so-called proactive DFSA drugs. We conducted a study on female patients above

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18 years of age consulting the Sexual Assault Centre between 2010 and July 2018. Among the 256 patients

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included, 37.1 % was positive at least for a substance. Alcohol was the most detected substance (57 cases),

followed by Cannabis (19 cases), cocaine (15 cases) and opiates/methadone (heroine: 5; morphine:1;
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methadone: 6); benzodiazepines and amphetamine were found in 13 and in 2 cases, respectively. Only case
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of gamma-hydroxybutyrate (GHB) consumption was observed while new psychoactive substances were not
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detected. Among the patients suspecting proactive DFSA, sedative drug findings, not explained by voluntary
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intake, were encountered.

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Keywords: DFSA, drug- facilitated crimes, Sexual Assaults Centre, GHB, benzodiazepine, ethanol
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Introduction

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The type of abuse that can be committed against a woman, or generically a subject, can be physical,

psychological, stalking, economical (inheritance, money transfer etc.) and in particular, sexual assault. The

victim of an abuse can incur into a variety of problems concerning the psychological aspects deriving from

the traumatic subjugation event/events, which include depression, anxiety, fear, grieve and, in case of sexual

abuse, physical outcomes (physical damage, infections, pregnancy) are also possible [1-3]. All these negative

aspects do not only affect the single individual but can also relapse to the entire society, with public health

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consequences when pathological behaviours such as drug dependence and/or alcoholism arise. In general, the

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rapidity in reporting is of detrimental importance for the medicolegal or forensic examination, however,

especially in case of sexual assault, a frequent delay between the fact and the reporting to the authorities is

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observed; this negatively reflects a promptly identification of physical and psychological evidences with the

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result of a loss of important information that may lead to an incorrect strategy both in health care actions and

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judicial measures (i.e. judicial report of the crime). Special attention should then be paid in case of sexual

assault committed on a victim who is under the influence of a psychoactive substance, which is defined as a
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drug facilitated crime (DFC) particularly where drug(s) have been given surreptitiously. Rape or other types
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of sexual assault are referred to as drug facilitated sexual assault (DFSA) and are usually the most common
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type of DFC. In DFC substances that cause sedation, disinhibition and amnesia are preferred, particularly if
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they can be dissolved readily in drinks without being detected by the victim. In these cases, the potential

victim has impaired consciousness and reduced ability to resist unwanted sexual advances. Psychoactive
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substances most often detected include the sedating benzodiazepines (BDZ), related hypnotics and sedatives

such as zopiclone and zolpidem, anaesthetics, gamma-hydroxybutyrate (GHB) and drugs of abuse such as
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cannabis and even the amphetamine-type stimulants that also can facilitate an altered state of mind [4]. A
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Canadian study showed that DFSA was most frequently detected with the most common drugs in urine being

cannabinoids (40%), cocaine (32%), amphetamines (14%), 3,4-methylenedioxymethamphetamine (MDMA,

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9.2%), ketamine (2.3%) and GHB (1.1%)[5]. Ethanol was included in the Norwegian study, where 40% of

DFSA cases were positive to ethanol as only detected substance and 5% of positive results related to

benzodiazepine only [6]. In addition, following the introduction of new psychoactive substances into the

market, findings related to their use as proactive DFSA have been reported [7]. While from the medical

viewpoint the used drugs are well-known, and their presence can be clinically and analytical verified, the

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legal consequences of their use in subsequent criminal prosecution is less clear-cut. Some European

countries have no jurisprudence in this area, while others consider the use of drugs as an aggravating

circumstance [8]. In Italy, DFSA is considered an aggravating circumstance per sè of sexual abuse since

1996, punished with a jail sentence of 6-12 years, irrespective of the victim’s voluntary consumption (artt.

609 bis and ter). If the criminal law is clear in matter of DFSA, the collection of suitable and unquestionable

forensic evidence to support the crime under the influence of psychoactive substances in judicial field

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remains challenging and some of these critical aspects will be considered in this manuscript.

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In our paper we conducted a study on female patients consulting the Sexual Assault Centre at Careggi

University Hospital, Florence, Italy after an allegedly case of sexual abuse. They were examined between

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2010 and July 2018 for a total of more than 250 cases and in agreement with the protocol adopted by our

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research Unit in collaboration with the Hospital Sexual Assaults Centre, urine and/or blood were analysed

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for ethanol and selected medicinal/recreational drugs. A shared routine protocol had been defined in 2015 to

make sure every step of the examination was properly done and all samplings were performed in the correct
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order by all physicians. We describe step-by-step management procedures for victims of sexual assault,
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taking into consideration the different samplings to help the physicians, which are responsible for both the
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health care of sexually assaulted persons and for conducting forensic medical examinations on them, to
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collect corroborative evidence to aid in legal proceedings.

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Materials and methods

Study design and settings

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A total of 256 of female patients who accessed (voluntary or accompanied by their parents in case of minors)
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to the University Hospital Sexual Assaults Centre in the period between January 2010 and July 2018 were

examined. The Sexual Assaults Centre is inside the University Hospital Careggi in Florence, Italy and
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collects victims from the “Area Vasta Firenze”, including 4 cities for a total of more than 1.500.000

inhabitants. The procedure for identification and for data and sample collection at the victim arrival was

clearly described in an agreed protocol between the Centre and the Unit of Forensic Toxicology of the

University of Florence, approved by the Medical Ethical Committee. The list of detected substances is

reported in table 1.

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Circumstantial data and sample collection

To support forensic toxicological investigation, information on the aggression modality, self-reported alcohol

or medicinal/recreational drugs assumption, time elapsed between the fact and the report were acquired from

the victim. Urine (10-20 ml) and blood (10 ml) were directly collected at the Sexual Assaults Centre for

toxicological analysis by medical personnel. Subjects were properly informed of the aim and modality of the

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tests, before samples collection, by informative material specifically delivered. Subjects were also asked to

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sign the privacy law statement. Materials were kept refrigerated at 2-8 °C until delivery to the laboratory for

toxicological analyses under proper chain of custody. Victims were proposed also to provide hair sample 3

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months after the event to perform segmental hair analysis for retrospective information on the history of drug

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exposure in the victim. All statistics were developed on the basis of self-declarations by the victims and

toxicological findings.
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Toxicological analysis
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Chemical and reagents

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Hydrochloric acid (HCl), methanol (MeOH), acetonitrile (ACN), ethyl acetate (AcOEt) ammonium
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hydroxide (NH4OH), potassium hydroxide (KOH) glacial acetic acid, hexane and formic acid were acquired

from J.T. Baker (Deventer, Holland). Dichloromethane (DCM), toluene, N-methyl-N-(trimethylsilyl)

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trifluoroacetaminde (MSTFA), N,O-Bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane

(BSTFA 1% TMCS), LC-MS CHROMASOLV® MeOH, LC-MS CHROMASOLV® ACN and LC-MS
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CHROMASOLV® water were purchased by Sigma–Aldrich (Merck KGaA, Darmstadt, Germany). Sodium
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hydroxide (NaOH), ammonium acetate and isopropanol were provided by Panreac Quimica S.L.U. (Castellar

del Vallès, Spain). All standards were provided by LGC Standard (Teddington, Middlesex, UK) and diluted
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with the appropriate solvent to the working concentrations. Bond Elut LCR-certify 130 mg solid-phase

extraction (SPE) cartridges were obtained from Agilent Technologies (Palo Alto, CA, USA). Stepbio Isolute

HAX SPE cartridges were purchased from Biotage (Uppsala, Sweden)

EMIT® Immunoassay Screening Test

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Urine samples were firstly analysed with an EMIT1 Siemens VIVA-E drug testing system (Siemens, Newark

DE) in order to investigate the common drugs of abuse: cocaine, opiates, cannabinoids, amphetamines,

barbiturates, methadone and benzodiazepines according to the manufacturer’s instructions.

Blood alcohol content (BAC)

BAC was determined by means of our Laboratory standard procedure [9]. Briefly, 1 mL of blood was added

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with 2-butanol (internal standard, IS) in a vial for headspace analysis and loaded in a headspace sampler

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Agilent 7697A Headspace (Agilent Technologies, Palo Alto, CA, USA); the oven was set at 60 °C. After 30

min of incubation, the gas was injected in an Agilent 7890B GC system (Agilent Technologies) equipped

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with a flame ionization detector. The column was an Alltech Superox II, 10 m length, 0.54 mm i.d. and 1.2

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mm film thickness (Alltech Associates INC., Deerfield, IL, USA). Chromatographic run was carried out at

60 °C for 4.5 min.

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Amphetamines, cocaine, methadone and opiates
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Urine samples required hydrolyzation before to be extracted. This phase was achieved by adding 1 mL of
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HCl 36% at 110 °C for 1 h.

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Blood and hydrolysed urine samples (1 mL) were added with 1 mL of phosphate buffer (0.1 M, pH 6), 10 µL

of nalorphine (50 ng/µL, IS) and few millilitres of NaOH 5 M (up to pH 6–7). Then the mixture was
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centrifugated at 4000 rpm for 5 min and added to the Bond Elut LCR-certify SPE cartridge preconditioned

with 2 mL of MeOH and 2 mL of phosphate buffer (0.1 M, pH 6). Washing phase was carried out with 2 mL
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of water, 1 mL of acetic acid 1 M and 2 mL of MeOH. Analytes were eluted twice with 1 mL of a
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DCM/isopropanol 8:2 (NH4OH 2%) mixture and dried under nitrogen (N2) stream at 75 °C. The residue was

incubated at 75 °C for 15 min with 25 mL of MSTFA and then added with 25 mL of toluene.
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Cannabinoids

A first hydrolyzation step was achieved on 500 µL of sample with 1 mL of KOH 10 M at 60 °C for 15 min.

After cooling, the solution was added with 10 µL of α-cholestane (2.5 ng/µL, IS) 100 µL of ammonium

acetate buffer (pH 4.5) and then loaded to a Stepbio Isolute HAX SPE cartridge. The latter was washed with

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6 mL of a 1:1 MeOH/water solution. Elution was performed with 5 mL of a 75:25 hexane/AcOEt (+1% of

glacial acetic acid) mixture. The extract was dried under N2 stream at 45 °C and then derivatized with 25 µL

of BSTFA 1% TMCS at 60 °C for 20 min.

GHB

GHB determination was performed following the standard procedure in our Laboratory [10]. Briefly, 100 µL

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of biological fluid were added by 50 µL of GHB-D6 (10 ng/µL, IS) and liquid-liquid extracted by 500 µL of

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AcOEt. The supernatant was dried under N2 stream at 45 °C and the residue was derivatized with 75 µL of

BSTFA 1% TMCS at 90 °C for 5 min.

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BDZ

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Protein precipitation for blood sample was performed adding 1 mL of specimen with 2 mL of MeOH. The

sample was agitated for 1 min and then centrifuged at 4500 rpm for 5 min. Urinary detection for BDZs was
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performed as routinely in our laboratory [11,12].
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New psychoactive substances (NPS)

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Since 2016, a new screening method for 64 NPS has been adopted in our Forensic Toxicology Unit [13]. The

procedure, as previously published, consisted in deproteination of 200 µL of blood with 600 µL of ACN.
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The supernatant was dried under N2 stream and reconstituted in 100 µL of MeOH.
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Gas chromatography-mass spectrometry (GC-MS) instrumentation

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The GC-MS system consisted of an Agilent 7890A GC system equipped with an Agilent 7683B series

autosampler and interfaced via electronic impact source to a single quadrupole Agilent 5975C mass
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spectrometer. Helium was used as gas carrier at constant flow of 1 mL/min. The columns were: Agilent HP-5

(30 m 0.32 mm, 0.25mm) for GHB, cocaine, methadone and opiates; Alltech H46 (10 m 0.53 mm, 1.20mm,

Alltech Associates Inc., Columbia, MD) for amphetamine and cannabis. The temperature programs were: i)

for cannabis, starting temperature was 100 °C for 1 min, raised to 325 °C (increase rate: 30 °C/min; hold

time: 3 min); ii) for cocaine, methadone and opiates, starting temperature was 100 °C (hold time: 1 min),

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raised to 300 °C (increase rate: 20 °C/min; hold time: 1 min); iii) for amphetamines, starting temperature was

100 °C (hold time: 1 min), raised to 125 °C (increase rate: 25 °C/min) and then 180 °C (increase rate: 15

°C/min); iiii) for GHB temperature was set initially at 60 °C for 0.5 min and programmed to raise up to 130

°C (increase rate: 10 °C/min) and up to 300 °C (increasing rate: 8 °C/min). Injection volumes were 1 µL.

Acquisition was in selected-ion monitoring: 6-monoacetyl-morphine (6-MAM), 399, 340, 287 m/z; α-

cholestane: 217, 357, 372 m/z; amphetamine, 91, 116, 192 m/z; benzoylecgonine, 240, 82, 361 m/z; cocaine,

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182, 82, 303 m/z; 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 277, 276, 220 m/z; GHB,

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233, 234, 147; GHB-d6, 239, 240 and 147; morphine, 429, 414, 401 m/z; methylenedioxyamphetamine

(MDA), 116, 135, 236 m/z; MDMA, 130, 250, 91 m/z; methadone, 72, 294, 223 m/z; methamphetamine,

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130, 91, 206 m/z; nalorphine, 455, 414, 440 m/z; Δ9-tetrahydrocannabinol (THC), 386, 371, 315 m/z; 11-

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Nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), 371, 473, 488 m/z.

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Liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrumentation
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Analysis was conducted using an HPLC Agilent 1290 Infinity system coupled via electrospray ion (ESI)
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source to an Agilent 6460 Triple Quad LC/MS. The source parameters were: gas temperature 325 °C; gas
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flow rate 10 L/min; nebulizer 20 psi; capillary 4000 V. Chromatographic separations were carried out on a
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Zorbax Eclipse Plus C18 column (2.1 mm x 50 mm, 1.8 m, Agilent Technologies). Elution gradients and

multiple reaction monitoring transitions for BDZ and NPS are described in previous papers [11,13].
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Results
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The statistics concerning rape or sexual assault and DFSA are usually accepted as under reported in many
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countries. In most of the times, in case of DFSA the evidence is frequently anecdotal, with few investigations

based on scientific evidences being carried out and most cases are diagnosed as an acute drug or alcohol
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intoxication, thereby bypassing sexual abuse diagnosis and appropriate care [14]. The reason may lay in the

lack of specific knowledge by the victim on the possibility to retrospectively study the allegedly events and

to the absence of standardized and shared protocols among health, forensic and police subjects that may

cause a loss of important information that cannot be retrieved later. On this basis, in 2015 the Unit of

Forensic Toxicology of University of Florence and the Sexual Assaults Center in Hospital Careggi have

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fixed a common protocol to be applied in case of DFSA, where clinical symptoms, modality, self-assumption

and time of the event are reported. Blood and urine samples are usually collected immediately at the arrival,

as well as any material retrieved on the crime scene, while the victim is invited to produce a hair sample after

at least 3 months. The chain of custody and a correct samples storage are assured throughout the procedure.

The panel of “rape drugs” investigated by the forensic laboratory include hypnotic BDZ such as

flunitrazepam, or Z-drugs such as zolpidem and zopiclone, GHB and hallucinogenic-type drugs such as

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ketamine or ecstasy, many NPS, beside the most common psychotropic drugs (cannabinoids, opioids,

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cocaine, amphetamines, methadone) and ethanol.

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Overall statistics

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From 2010 to July 2018, Laboratory of Forensic Toxicology Division analysed a total number of 256 cases

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of allegedly DFSA. More than 70% (73.0%, n: 187) of women arriving at the Centre was from foreign

countries, suggesting either a poor confidence of Italian women to report or instead, due to the high presence
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of tourists and young students in Florence. The age of presumed victims ranged from 7 to 54 years (median:
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22 years); 69.2 % was 18-30 years old (fig. 1). We analysed about 28 cases per year, with the highest number
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in 2012 (43, table 2). No hair samples were provided. 37.1 % of cases was positive at least for a substance,
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with peaks in 2013 (43%), in 2017 (46%) and in the first seven months of 2018 (40%, table 2 and fig. 2). In

66.0% of all cases, the victim arrived at the Sexual Assaults Center up to 24 hrs after the event, 12.1 %
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between 24-48 hrs and 14.5% later than 48 hrs (up to 3 months after); in 7.4 %, it was not possible to

estimate the time frame from the sexual assault and the hospitalization (fig. 3). The percentage of positives
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results gradually decreased at greater time lapse, confirming the importance of a prompt report and rapid
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sample collection. Indeed, 81.1 % of all the positivity were concentrated in the time frame ≤ 24 h (fig. 3).

Alcohol was the most detected substance (57 cases), followed by Cannabis (19 cases), cocaine (15 cases) and
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opiates/methadone (heroine: 5; morphine:1; methadone: 6); BDZ and amphetamine were found in 13 and in

2 cases, respectively. Regarding GHB, only one case has been reported in Florence in the time range so far

studied. None of detectable NPS has been found since the application of the new screening method in 2016.

Our findings were consistent with the European statistics, where alcohol, Cannabis and Cocaine are the most

found drugs of abuse [15]. Poly-assumption was registered in 18 cases, with the associations of cocaine-

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Cannabis (n: 4), alcohol-Cannabis (n: 3) and alcohol-BDZ (n: 3) as the most frequent. This finding supports

the evidence that there is no single substance that is uniquely associated with DFSA [16].

Alcohol

Many surveys reported that alcohol is the most common drug detected in case of alleged sexual assault, with

a prevalence ranging from 40 to 70% [16-21]. Young et al. stated that alcohol was involved in 12 to 20 % of

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sexual assaults, with higher rates occurring in female adolescents 16 years of age or older [22]. These

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observations are in line with our outcome, where alcohol alone was present in 50 over 95 cases of drug

detection (52.6 %) and 7 in association with other substances (mainly BDZ and Cannabis). Alcohol is

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consumed to increase confidence in interacting with potential sexual partners and to arouse sexual desire.

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Moreover, ethanol induces amnesia and loss of inhibitions, effects usually sought by the perpetrators. In

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DFSA, the victim is encouraged to drink more than him/her had planned or to drink beverages added with

alcohol in order to enter into a state of chemical submission. However, it is difficult to distinguish what
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quantity has been covertly administered and what has been voluntarily consumed [23].
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In our study, BAC was adopted as marker of alcohol exposure and its values ranged from 0.12 to 3.43 g/L,
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with a median of 1.33 g/L. Hurley et al. [24] reported that the average BAC in suspected DFSA victims was
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1.10 g/L. Time sampling plays a key role in estimation of the alcoholic intoxication state at the time of the

sexual assault. Alcohol half-life is approximately of 4 h and elimination rates are between 0.1 and 0.25 g/L/h
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[20]. Even if the metabolism rate can vary with gender, tolerance (social versus chronic drinkers), and

phenotypic differences in alcohol dehydrogenase [25,26], for this reason back calculations are not always
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reliable [20]. In our survey, 84.2 % (n: 48) of alcohol positive cases was detected in a time frame, from the
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assault and the sampling, of ≤ 24 h; in particular, 32 out of 48 cases (66.7 %) were collected within 5 h (fig.

4). Only 3 cases of alcohol positivity were detected above 24 h (24-48 h, 1; < 48 h, 2). In 6 cases was not
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possible to determine the time lapse. These findings well underline the importance of the time lapse for the

detection of alcohol intoxication.

Cannabis

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Cannabis products are the most widely used recreational drugs and have been often linked to sexual

behaviour for a long time. Many studies reported high presence of THC in urine samples from suspected

DFSA [18-20]. Beside THC, biological specimens were also investigated for the presence of THC-COOH,

because plasma THC concentrations rapidly decrease after exposure [27, 28]. Small quantities are however

detectable in blood for longer time in frequent users due to its redistribution in fat tissues and highly

vascularized tissues [29, 30]. About 30 % of THC dose is excreted in the urine and THC-COOH is the

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primary glucuronide conjugate with a urinary excretion half-life of 30 h after seven days and 44-60 h after

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twelve days of monitoring [31,32]. Peak concentration in urine was observed after 4 h [33,34]. For all these

reasons, it appears clearly necessary to include the urinary matrix and THC-COOH in the panel of the

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detectable compounds. This is further highlighted by our findings: THC-COOH was found in all positive

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samples (n: 19), while THC was not; blood specimens were positive in only 5 cases (fig. 5). It must be

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underlined that 17 out 19 positive cases were related to samples collected within 24 h (fig. 5). However, the

long-time detectability of THC-COOH in urine entails interpretative issues as the consumption may even be
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dated days before the suspected assault. Cannabis is used in DFSA as it can cause the impairment of short-
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term memory and the enhancement of the sexual pleasure by stimulating relaxation and sense of touch [35].
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Cannabis was found in association with other drugs in 9 cases (+cocaine, n: 4; +alcohol, n: 3; +BDZ, n: 2).
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Cocaine
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Cocaine is a central nervous system stimulant producing tachycardia, hyperactivity, restlessness, euphoria,

increased energy, appetite suppression and increased self-confidence and libido [36]. It is also well-
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considered as a substance that delays the orgasm and, therefore, prolongs coitus [37], although the extension
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of the intercourse can result in genital abrasions increasing the risk of transmitting infections. Cocaine half-

time is estimated in 0.7-1.5 h [38] and it is eliminated in the urine primarily as unchanged drug (1-9%),
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benzoylecgonine (35-54%) and ecgonine methyl ester (32-49%) [39]. The prevalence of cocaine in samples

submitted for analysis for DFSA ranges from 8% to 11% [18-20]. In our experience, we obtained a lower

value: 15 out of 256 cases (5.6 %) were positive for cocaine and its main metabolite, benzoylecgonine. In 7

cases, cocaine was in association with other drugs (+Cannabis, 4; +alcohol, 1; +opiates, 2). Fourteen of the

15 positive samples were collected within 24 h (fig. 6).

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BDZ

These substances are ideal agents for DFSA, especially in combination with alcohol [40]. They induce

dizziness, disorientation, lack of coordination, slurred speech, loss of consciousness, flaccid muscle tone,

nystagmus, and anterograde amnesia [41]. In a large study, 58 % of DFSA victims were positive for BDZ,

even if they are found as a single drug less frequently than alcohol, Cannabis or cocaine [19]. The most

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detected molecules are oxazepam, diazepam, lorazepam and clonazepam. In our studied cases, BDZ were

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detected alone in 5 cases over the 13 samples positive for this class. The most identified compounds were

diazepam (and its metabolites: nordiazepam, oxazepam and temazepam), alprazolam, lorazepam, midazolam

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and clonazepam. All BDZ were detectable both in blood and urine specimens. In figure 7 is reported the

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distribution of positive cases among the range of time sampling.

Opiates and methadone U

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Heroin, morphine and methadone are not so commonly associated to DFSA. In a study, El-Bassel et al. [42]
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examined sexual experience in 38 women recruited from methadone maintenance programs.

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The subjects believed that drugs, particularly heroin, increased their and their partners’ sexual performance,
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libido and pleasure. Many participants deemed that heroin, methadone and other drugs were responsible for

their partners’ abusive and coercive behaviour. On the base of the pharmacokinetics of heroin, evaluation of
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its consumption was performed on morphine and 6-MAM concentrations in blood and urine. Indeed, heroin

(t½: 2-6 min) is rapidly deacetylated in whole blood to 6-MAM (t½: 6-25 min), which in turn is further
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hydrolysed to morphine (t½: 2-3 h) at a somewhat slower rate. Heroin was detected in 5 samples, 4 of which
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collected within 24 h (for the other case, the time frame was not determinable, fig. 8). Morphine was

detected in only 1 case. Regarding methadone, it was always detected together with its main metabolite,
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EDDP, in 6 samples (time frame, ≤ 24 h: 5; nd: 1; fig. 9). In 3 cases, MT and heroine were associated to

other substances: once with BDZ; twice with cocaine.

Amphetamines

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This class of substances is highly valued for its ability to induce euphoria, empathy, loss of shyness, sexual

desire and to intensify and prolong the orgasm [35]. These compounds can be easily and secretly added to

drinks by perpetrators in DFSA. Studies report that amphetamine and methamphetamine were found in 4%

to 7% of samples from DFSA cases [18], often in combination with other drugs such as alcohol, cocaine,

benzodiazepines and cannabis. We observed only 2 cases of positivity for amphetamine. The first one was

registered in 2014 and was related to a 25 years-old female. She accessed to the hospital about 7 h after the

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presumed sexual assault. Amphetamine was the only substance detected in her samples. The second case

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occurred in 2017 when the victim was 22 years old. She was hospitalized 3.5 h after the violence with a BAC

of 2.38 g/L. Amphetamine was detected both in blood and urine specimens.

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GHB

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The drug most likelihood connected to proactive DFSA, at least in theory, showing the perfect characteristics

of amnesic effects, availability, unlikely to be detected due to a short half-life or a lack of routine screening,
N
is GHB [43]. Indeed, related with its use as DFSA, one of the biggest difficulties the substance presents is
A

the rapid elimination from the body, what implies that it will not able to be detected in blood after 2-8
M

hours and in urine after 8-12 hours. Effects are easily confused with those of alcohol ingestion and are syn-
ED

ergistic with alcohol. Victims often recover spontaneously in as little as 1 to 2 hours [25,44]. Only one case

has been reported in Florence in the time range so far studied, concerning a young woman of 22 years,
PT

approaching the Sexual Assaults Center in the morning. She referred to have been at a party the night before,

where she had some alcohol before leaving the party with a young man, recovering the morning after in her
E

car without her underwear. She reported no memories of any fact. Toxicological findings revealed the
CC

presence of GHB at the concentration of 2.85 µg/ml and ethanol at the concentration of 0.68 g/l in blood, in

the 7-8 hrs following the event. Urine sample was also positive to GHB, at the concentration of 18.5 µg/ml.
A

It is widely accepted that GHB administration is tricky to prove for forensic toxicologists, mostly for its

variable endogenous presence and to the short half-life the molecule down to endogenous levels [45].

However, in this case exogenous GHB persisted detectable in blood and urine for 8 hrs after the

administration. Unfortunately, although invited, the woman did not present herself for hair sample collection

after the 3-months period.

13
Conclusion

Italy is the only no English-speaking European country considering drug and/or ethanol an aggravating

circumstance in case of DFSA and thus proper sample collection as soon as possible to sustain forensic

medical evidence is of utmost importance for legal aspects. We believe that victims of sexual assault should

have easy and fast access to emergency health care with a trained staff and should be encouraged to seek

T
immediate help. Toxicological screening should also be routinely offered to achieve a comprehensive

IP
assessment in each individual case. This analytical investigation must be performed by specialized personnel

who adopts the most sensible and advanced technologies (such as GC-MS and LC-MS/MS). Moreover, the

R
correct choice of the detected compounds (drugs and their metabolites) and of matrices (blood, urine, hair

SC
and so on), beside very short time lapse for sampling, play a key role for reliable estimation of drug intake in

U
DFSA. For this reason, since 2015 the Unit of Forensic Toxicology and the Sexual Assaults Center of

University Hospital of Careggi in Florence collaborate in a unique and shared protocol to be offered to the
N
victims in case of allegedly DFSA.
A
M

Maria Grazia Di Milia and Diego Palumbo carried out the experiments. Fabio Vaiano and Alessia
ED

Fioravanti wrote the manuscript with the support and suggestion by Jennifer Pascali. Francesco
Mari helped supervise the project. Elisabetta Bertol conceived the original idea. All authors
discussed the results and contributed to the final manuscript.
E PT

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Figure captions
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Figure 1. Distribution of cases among the age ranges.

Figure 2. Trend of analysed cases and positivity per year (2018: from January to July).
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Figure 3. Number of cases and positivity versus the time frames from suspected sexual assault and sampling

(nd: not determinable).

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Figure 4. Distribution of alcohol positive cases among the time frames from suspected sexual assault and
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sampling (nd: not determinable).

Figure 5. Distribution of Cannabis positive cases among the analysed matrices and the time frames from
A

suspected sexual assault and sampling (nd: not determinable).

Figure 6. Distribution of cocaine positive cases among the analysed matrices and the time frames from

suspected sexual assault and sampling (nd: not determinable).

Figure 7. Distribution of BDZ positive cases among the analysed matrices and the time frames from suspected

sexual assault and sampling (nd: not determinable).

18
Figure 8. Distribution of heroin positive cases among the analysed matrices and the time frames from

suspected sexual assault and sampling (nd: not determinable).

Figure 9. Distribution of methadone positive cases among the analysed matrices and the time frames from

suspected sexual assault and sampling (nd: not determinable).

T
R IP
SC
U
N
A
M
ED
E PT
CC
A

19
 A
CC
EPT
ED
M
A
N
U
SC
RIP
T

20
 A
CC
EPT
ED
M
A
N
U
SC
RIP
T

21
 A
CC
EPT
ED
M
A
N
U
SC
RIP
T

22
 A
CC
EPT
ED
M
A
N
U
SC
RIP
T

23
 A
CC
EPT
ED
M
A
N
U
SC
RIP
T

24
Table 1. List of detected substances and used instrumentations in case of suspected DFSA. Metabolites are
shown in italic.

Class Substance Instrumentation

Cannabinoids THC, THC-COOH GC-MS

Aa, MDA, MDMA,
Amphetamines GC-MS, LC-MS/MS
MDE, MA
BDZ [ref. 12] LC-MS/MS
Cocaine,
Cocaine GC-MS

T
Benzoylecgonine
Ethanol BAC HSGC-FID

IP
GHB GHB GC-MS

R
Methadone Methadone, EDDP GC-MS

SC
NSP [ref. 13] LC-MS/MS

Opiates Morphine, 6-MAM GC-MS

a
Amp: amphetamine; Meth: methamphetamine. U
N
A
M
ED
E PT
CC
A

25
Table 2. Number of analysed cases and positivity to the main illicit drugs per year.
Opiates
Year Cases Positivity BACa Coc MT Can BDZ A GHB
Her Mor
2010 19 7 (36.8%) 5 - - - 1 - 1 - -
2011 34 9 (26.5%) 5 1 1 - 1 1 - - -
2012 43 15 (34.9%) 12 3 2 - 1 1 2 - -
2013 32 15 (46.9%) 11 2 - 1 - 4 - - -
2014 34 13 (38.2%) 6 2 1 - 2 1 3 1 -
2015 21 7 (33.3%) 4 1 - - - 2 2 - -
2016 22 8 (36.4%) 2 2 - - - 4 2 - 1
2017 26 11 (42.3%) 8 2 - - - 2 1 1 -

T
2018* 25 10 (40.0%) 4 2 1 - 1 4 2 - -

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total 256 95 (37.1%) 57 15 5 1 6 19 13 2 1
*January-July
a
A: amphetamine; Can: Cannabis; Coc: cocaine; Her: heroin; MT: methadone; Mor: Morphine.

R
SC
U
N
A
M
ED
E PT
CC
A

26