Urine and blood samples from 1730 drivers involved in road accidents (July 2012- December 2015) were analyzed for the evaluation of driving under influence of drug of abuse according to the Lombardia Region guideline. The 22.5 % of urine screenings tested positive for at least one class of drugs. 10.6% of the 1730 drivers were under the influence of drug, being blood concentration above the cut-off limit for at least one active substance; the prevalence of illicit drugs in blood was cocaine (5.7 %), cannabinoids (3.7 %), opiates (1.4 %), methadone (1.4 %), amphetamines (0.2 %). Trend in prevalence showed similar percentage (about 5 %) of cocaine and cannabinoids consumption in the last two years. Poly-drug of abuse consumption emerged in the 10.4 % of the positive blood and alcohol was above the legal limit in 47 % of the subjects driving under the influence of drugs

Biffi, Marco

Di Tuccio, Marcello

Papa, Pietro

Rocchi, Loretta

Rolandi, Laura Maria

Valli, Antonella

Annali dell'Istituto Superiore di Sanità

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Key words
• drugs of abuse
• blood
• urine 
•  driving under the 
influence of drugs  
of abuse
Illicit drugs in Emergency Department 
patients injured in road traffic accidents
Pietro Papa1, Loretta Rocchi1, Laura Maria Rolandi1, Marcello Di Tuccio1, Marco Biffi2 
and Antonella Valli1
1Laboratorio di Tossicologia Analitica, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy 
2Laboratorio di Chimica Clinica, Azienda Ospedaliera di Treviglio, Treviglio (BG), Italy
Ann Ist Super Sanità 2017 | Vol. 53, No. 1: 35-39
DOI: 10.4415/ANN_17_01_08
Abstract
Urine and blood samples from 1730 drivers involved in road accidents (July 2012 − De-
cember 2015) were analyzed for the evaluation of driving under influence of drug of abuse 
according to the Lombardia Region guideline. The 22.5% (95% CI 20.5 to 24.5) of urine 
screenings tested positive for at least one class of drugs. 10.6% (95% CI 9.2 to 12.1) of the 
1730 drivers were under the influence of drug, being blood concentration above the cut-off 
limit for at least one active substance; the proportion of illicit drugs in blood was cocaine 
5.7 % (95% CI 4.7 to 6.9), cannabinoids 3.7 % (95% CI 2.9 to 4.7), opiates 1.4% (95% CI 
0.9 to 2.1), methadone 1.4% (95% CI 0.9 to 2.1), amphetamines 0.2% (95% CI 0.04 to 
0.5). Trend in proportion showed similar percentage (about 5%) of cocaine and cannabi-
noids consumption in the last two years. Poly-drug of abuse consumption emerged in the 
10.4% (95% CI 6.4 to 15.7) of the positive blood and alcohol was above the legal limit in 
47% (95% CI 39.6 to 54.5) of the subjects driving under the influence of drugs.
INTRODUCTION
In Italy, driving under the influence of drugs of abuse 
(DUID) is illegal, but the law does not establish either 
the list of substances to be controlled or the punish-
able concentration limits for drugs of abuse. In order 
to regulate the analytical procedure in DUID cases, 
Lombardia Region issued, in 2012, a guideline to sug-
gest a list of illicit drugs to be controlled and the blood 
concentration limits of the active substances (parent 
drug and/or metabolite) above which the driver can be 
prosecuted for DUID [1]. This procedure includes also 
the detection of inactive metabolites.
The aim of this study is to present the results of toxi-
cological investigations carried out on blood and urine 
samples collected from drivers involved in road traffic ac-
cidents. The proportion of illicit drugs after the introduc-
tion of the Lombardia Region procedures is analyzed.
MATERIALS AND METHODS
Selection of cases 
The population consisted of 1730 drivers involved in 
non-fatal traffic accidents and taken to seven Emergen-
cy Departments (ED) of Pavia and province and four 
hospitals of the Azienda Ospedaliera di Treviglio (BG), 
from July 2012 to December 2015. In case of road acci-
dents, according to the Italian law, Police systematically 
requested the analysis for drugs of abuse, regardless of 
a clear suspect of DUID; a chain of custody for the 
collected samples was put into effect and the patients 
were asked for consent. Only in case of positive consent 
or when the patient was not able to provide a valid con-
sent, biological samples were collected and sent to our 
lab. The laboratory proceeded to analyze biological ma-
trices only in the presence of patient’s consent or, if not 
available, of the magistrate’s request. 17 patients were 
not included in this study, because they were not able 
to provide valid consent and the magistrate’s request 
never arrived.
The mean age of population was 41.1 years + SD 
15.7; males were heavily predominant, 80.5% vs female 
19.5 %. The circumstances (i.e. type of the vehicle, sus-
pected cause or victim of the accident etc.) of the road 
accidents were not available.
For this study, population data were anonymized and 
a unique, sequential number was assigned to each case.
Biological samples and storage
Blood was collected into glass tubes containing po-
tassium fluoride as preservative and EDTA as antico-
agulant. Urine was collected into polyethylene tubes. 
Samples were stored at 4 °C for periods not exceeding 
72 h, otherwise were frozen at -20 °C. Analysis was rou-
tinely completed within 10 days, later only in case of 
absence of timely consensus.
Analytical procedures
The analytical strategy consisted of the following 
steps:
Address for correspondence: Pietro Papa, Laboratorio di Tossicologia Analitica, Fondazione IRCCS Policlinico San Matteo, Piazzale Golgi 19, 27100 
Pavia, Italy. E-mail: p.papa@smatteo.pv.it.
Pietro Papa, Loretta Rocchi, Laura Maria Rolandi et al.
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A) urine screening for six classes of drugs of abuse/
metabolites (e.g. opiates, cocaine, cannabinoids, am-
phetamines, methadone and buprenorphine – Table 1) 
by immunological tests (Siemens, EMIT II Plus) and 
quantification of creatinine by enzymatic assay (Sie-
mens) as index of urine suitability. Creatinine concen-
tration < 20 mg/dl is consistent with over dilution of the 
sample and in this case, blood analyses is mandatory 
even if urine screening is negative;
B) blood identification and quantification of the par-
ent drugs/metabolites tested positive in urine: drugs 
were isolated by solid phase extraction (SPE). Cocaine/
benzoylecgonine blood analysis was performed by Gas 
Chromatography-Mass Spectrometry (GC-MS) in 
SIM mode after pentafluoropropionic anhydride de-
rivatization, while the other drugs were analysed by 
Liquid Chromatography-Tandem-Mass Spectrometry 
(LC-MS/MS).
C) urine analysis in case of blood negativity for active 
and inactive compounds: an aliquot of urine, different 
from that used for the screening test, was processed to 
confirm the positive screening result. Urine confirma-
tion test was performed by GC-MS analysis in SIM 
mode after SPE extraction for all drugs except for can-
nabinoids that were  isolated by liquid/liquid extraction.
Cut-off values for the different classes and analytes 
are summarized in Table 1. According to the regional 
procedures, a sample is positive if the measured con-
centration for each analyte is above the cut-off level 
considering the confidence range of the methods (10% 
for urine screening and confirmation tests, 20% for 
blood quantification).
RESULTS AND DISCUSSION
The 22.5% (95% CI 20.5 to 24.5) of the screening 
tests in urine resulted positive for at least one class of 
drugs and in 10.6 % (95% CI 9.2 to 12.1) of the 1730 
cases, subjects were driving under influence of drug, be-
ing blood concentration above the adopted cut-off limit 
for at least one active substance (parent drug and/or 
active metabolite).
Table 2 summarizes the positive results of the screen-
ing urine tests along with the positivities for active sub-
stances in blood and for inactive substances in blood 
and urine. 
Cocaine and cannabinoids were the most frequently 
detected illegal drugs. In particular, cocaine/metabo-
lites resulted positive in urine in the 11.4% (95% CI 
Table 1
Cut-off of drugs of abuse/metabolites in urine and blood
Class Analyte Urine Blood
    Screening cut-off Confirmation cut-off Cut-off
Opiates  150 ng/ml   
  Morphine 100 ng/ml 10 ng/ml
  6-acetylmorphine 10 ng/ml 10 ng/ml
  Codeine 100 ng/ml 10 ng/ml
Methadone 150 ng/ml
  Methadone 100 ng/ml 10 ng/ml
  EDDP1 100 ng/ml 10 ng/ml
Buprenorphine 5 ng/ml
  Buprenorphine 5 ng/ml 5 ng/ml
  Nor-Buprenorphine 5 ng/ml 5 ng/ml
Cocaine 150 ng/ml
  Cocaine 50 ng/ml 10 ng/ml
  Cocaethylene 50 ng/ml 10 ng/ml
  Benzoylecgonine 50 ng/ml 50 ng/ml
Amphetamines 500 ng/ml
  Amphetamine 200 ng/ml 50 ng/ml
  Methylamphetamine 200 ng/ml 50 ng/ml
  MDMA2 200 ng/ml 50 ng/ml
  MDA3 200 ng/ml 50 ng/ml
  MDE4 200 ng/ml 50 ng/ml
Cannabinoids 25 ng/ml
  THC5 2 ng/ml
  THC-COOH6 15 ng/ml 5 ng/ml
Creatinine  > 20 mg/dl    
1EDDP: 2-Ethylidene-1,5-DimethylDiphenyl-3,3-Pyrrolidine; 2MDMA: 3,4-Methylendioxymethamphetamine; 
3MDA: 3,4-Methylendioxyamphetamine; 4MDE:3,4- methylendioxy-N-ethylamphetamine;
5THC: Delta9-Tetrahydrocannabinol; 6THCCOOH = 11nor-9-carboxy -delta9- THC.
IllIcIt drugs In road accIdents 
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9.9 to 13.0) and 5.7% (95% CI 4.7 to 6.9) of cocaine 
blood concentrations exceeded the adopted cut-off; co-
caethylene was positive in the 26.3% (95% CI 18-36) 
of cocaine blood positivities; one case tested positive 
for cocaethylene with cocaine concentration below the 
cut off. Blood cocaine positivities were associated with 
almost another different active drug in the 16.2% (95% 
CI 9.5 to 25).
The proportion of cocaine presented in this work dif-
fers from the data found in two previous Italian popula-
tion studies [2, 3], where cocaine proportion in blood 
was, respectively, 2.7 and 7.1%, even if  Legrand et al. 
[2] considered seriously injured drivers only, while our 
population is not selected on the basis of severity of in-
jury.
Cannabinoids tested positive in 14% (95% CI 12.4 to 
15.8) of urine samples and THC in blood was detected 
above the adopted limit in 3.7% (95% CI 2.9 to 4.7) of 
the total cases; six of the 64 positive cases for THC in 
blood (9.4%, 95% CI 3.5 to 19.3) were associated to 
cocaine positivity.
Low percentages of cocaine and THC blood positivity 
compared to proportion in urine can be due to the rapid 
decrease of these active compounds in blood and slow 
excretion of metabolites in urine [4-6]. In this study 
(Table 3), the concentration ratios of THC/THCOOH 
and cocaine/benzoylecgonine measured in blood were 
low (0.21 + 0.11 and 0.16 + 0.13 respectively). The 25th 
percentile of THC blood concentration (3.1 ng/ml) re-
sulted near to the cut off adopted in the guideline (2.0 
ng/ml with 20% of analytical confidence). The same 
situation applied to cocaine, where the 10th percentile 
of blood concentrations was 16 ng/ml versus an adopted 
limit of 10 ng/ml with 20% of analytical confidence. It 
is possible that the time elapsed between the road ac-
cident and the specimen collection played an important 
role in the decrement of the active substances, but, un-
fortunately, this information was not available. 
Table 2
Percentage and 95% confidence interval (CI) of positive cases in urine and blood vs drug/class of drugs
Drug/class Positive urine samples and 
percentage vs total cases 
(1730)
Positive  blood samples for active
substances vs positive urine samples
and vs total cases
Positive blood samples for 
inactive metabolites only*
Cocaine 197 (11.4%; CI 9.9 to 13.0) 99/197 (50.2%;  CI 43.1 to 57.4)
99/1730 (5.7%; CI 4.7 to 6.9)
27/197 (13.7%; CI 9.2 to 19.3)
27/1730 (1.6%; CI 1 to 2.3)
Cannabinoids 243 (14.0%; CI 12.4 to 15.8) 64/243 (26.3%; CI 20.9 to 32.3)
64/1730 (3.7%;   CI 2.9 to 4.7)
67/243 (27.6%; CI 22 to 33.6)
67/1730 (3.9%; CI 3 to 4.9)
Opiates 56 (3.2%; CI 2.4 to 4.2) 24/56 (42.9%; CI 29.7 to 56.8)
24/1730 (1.4%;  CI 0.9 to 2.1)
-
-
Methadone 24 (1.4%; CI 0.9 to 0.21) 24/24 (100%; CI 85.7 to 100)
24/1730 (1.4%; CI 0.9 to 2.1)
-
-
Amphetamines/
ecstasy
5 (0.3%; CI 0.09 to 0.7) 3/5 (60%; CI 14.7 to 94.7)
3/1730 (0.2%;  CI 0.04 to 0.5)
-
-
Buprenorphine 3 (0.2%; CI 0.04 to 0.5) 0/3
0/1730
-
-
*Benzoylecgonine, THCCOOH, EDDP.
Table 3
Number of positive cases, mean concentrations, ranges and percentiles for the drugs, metabolites and their ratio detected in blood of 
drivers involved in nonfatal road accidents
Substance LOQ1
(ng/ml)
N
(positive)
Mean + SD
(ng/ml)
Range
(ng/ml)
p10
(ng/ml)
p25
(ng/ml)
p50 
(ng/ml)
p75
(ng/ml)
p90 
(ng/ml)
Cocaine 10 99 70.9 +  71.6 12.6-378 16.0 26 45.5 85.3 159.8
Cocaethylene 10 27 33.6 + 16.5 12.9-90 14.2 20 31.6 44.5 53.1
Benzoylecgonine 50 126 561.7 + 687.4 62-3595 89.0 161 312 636 1209
Cocaine/benzoylecgonine 99 0.16 + 0.13 0.02-0.60 0.04 0.07 0.10 0.22 0.37
Cocaine/cocaethylene 43 2.14 + 1.39 0.52-5.65 0.88 1.15 1.71 3.02 4.58
THC 2 64 5.6 + 3.6 2.6-22 2.7 3.1 4.5 6.2 11.7
THCCOOH 5 131 24.3 + 22.4 6.2-120 7.8 10 15.8 30 54.4
THC/THCCOOH 64 0.21 + 0.11 0.03-0.57 0.1 0.12 0.20 0.27 0.36
Morphine 10 24 54.2 + 115.4 13.2-470 13.3 16.9 25 31.5 216
Methadone 10 24 187.8 + 152.4 16.9-582 32.3 93.2 122.5 279.5 448.7
MDMA 50 3 848 +  998 235-2000 235 235 309 2000 2000
1Analytical confidence: 20%
Pietro Papa, Loretta Rocchi, Laura Maria Rolandi et al.
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As regard the positive cases for opiates in blood (n 
= 24), the analyses revealed morphine in 23 subjects 
(presence of 6-monoacetylmorphine in 1 case) and only 
codeine in 1 case.
24 subjects resulted positive for methadone in blood 
and no information about patient’s methadone therapy 
was available. 
Amphetamines/ecstasy were positive in blood in 3 
cases: two cases were positive for MDMA in associa-
tion with cocaine, 1 case was positive for amphetamine 
in association with THC. 
Figure 1 shows the distribution of the proportion of 
drugs in blood over the considered period: one notable 
finding is the higher proportion of cocaine compared to 
cannabinoids in the period 2012-2013, while in the last 
two years the data is similar.
Poly-drug consumption emerged in the 17.5% (95% 
CI 13.8 to 21.6) of positive urine screenings and in 
the 10.4% (95% CI 6.4 to 15.7) of the cases positive 
in blood for active substances. Moreover, alcohol was 
above the legal limit (0.5 g/L) in 47 % (95% CI 39.6 
to 54.5) of the subjects driving under the influence of 
drugs, and the proportion of the population positive for 
ethanol only was 28.2% (95% CI 26 to 30.5).
Table 4 and Figure 2 show the distribution of the drugs 
tested positive in blood samples versus different age 
groups: THC was mostly encountered in the age group 
18-30, while cocaine was frequently detected in a wider 
age range (18-50 years). The 86% (95% CI 65 to 97) of 
poly-drug abuse cases was found in the population 18-40 
year old. As regard to gender, the 91.9% (95% CI 84.7 to 
96.4) and 84.4% (95% CI 73.1 to 92.2) of the population 
positive for cocaine and THC respectively, are males.
CONCLUSIONS
We analyzed urine and blood samples of 1730 drivers 
(July 2012-December 2015) involved in nonfatal road 
accidents in order to verify the driving under influence 
of drugs of abuse according to Lombardia Region proce-
dure: urine immunoassay screening for illicit drugs and 
quantitative analysis in blood by chromatography cou-
pled to MS detector methods. In the considered period, 
the most common drug of abuse quantified in blood 
above legal limits was cocaine (5.7%), followed by THC 
(3.7%); however, in the last two years the proportion of 
cocaine and cannabinoids resulted similar (about 5%). 
These data mostly differ from those collected in other 
countries were the most common drug found in blood 
was THC [2, 6-13]. However, in all these reports, the 
cut-off for THC in blood is lower (from 0.4 to 2.2 ng/ml) 
then ours (2 ng/ml with 20% of analytical confidence). 
Methadone, opiates and amphetamines resulted less fre-
quently involved (1.4%, 1.4% and 0.2% respectively). 
The percentage of urine samples positive for cocaine, 
cannabinoids and opiates was significantly higher than 
the blood one (see Table 2). This difference may arise, as 
0
1
2
3
4
5
6
7
8
2012 2013 2014 2015
Cocaine
THC
Opiates
Methadone
Amphetamines
Year
Pr
ev
al
en
ce
 %
Figure 1
Prevalence in percentage of active drugs/metabolites posi-
tive in blood: distribution by year in the sampling period, July 
2012- December 2015.
Table 4
Age and sex distribution of the patients and the results of detected proportions of drug use by substance in the specific age-sex groups
Analytes Age groups (years) and sex vs number of positivities in blood 
16-18 19-30 31-40 41-50 50
M F M F M F M F M F
Cocaine 1 /99
(0.1%)
0/99 33/99
(33.3%)
3/99
(3%)
34/99
(34.3%)
3/99
(3%)
23/99
(23.2%)
2/99
(2%)
0/99 0/99
THC 3/64
(4.7%)
1/64
(1.6%)
32/64
(50%)
5/64
(7.8%)
7/64
(11%)
2/64
(3.1%)
8/64
(12.5%)
2/64
(3.1%)
4/64
(6.2%)
0/64
Opiates 0/24 0/24 10/24
(41.7%)
0/24 6/24
(25%)
0/24 6/24
(25%)
0/24 2/24
(8.3%)
0/24
Methadone 0/24 0/24 8/24
(33.3%)
0/24 8/24
(33.3%)
1/24
(4.2%)
5/24
(20.8%)
1/24
(4.2%)
1/24
(4.2%)
0/24
Amphetamines 0/3 0/3 2/3
(66.6%)
0/3 1/3
(33.3%)
0/3 0/3 0/3 0/3 0/3
Poly-drugs 0/22 0/22 11/22
(50%)
1/22
(4.5%)
6/22
(27.3 %)
1/22
(4.5%)
3/22
(13.6%)
0/22 0/22 0/22
IllIcIt drugs In road accIdents 
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well as from a previous drug consumption, also due to 
the time elapsed between the road accident and sample 
collection that plays a decisive role in the outcome of 
the analysis.
The presented data confirm the low value of urine 
results for DUID aim, even if immunoassay urinary 
screening remains a simple and reliable tool to select the 
cases for the more complex quantitative blood analysis.
Conflict of interest statement
There are no potential conflicts of interest or any fi-
nancial or personal relationships with other people or 
organizations that could inappropriately bias conduct 
and findings of this study.
Received on 12 May 2016.
Accepted on 25 November 2016.
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5
10
15
20
25
30
35
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Poly- drugs
Codeine
Amphetamines
Methadone
Morphine
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Illicit drugs in Emergency Department patients injured in road traffic accidents

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Illicit drugs in Emergency Department patients injured in road traffic accidents

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