Accepted Manuscript

Title: Detection of drugs in 275 alcohol-positive blood

samples of Korean drivers

Author: Eunmi Kim Sanggil Choe Juseon Lee Moonhee Jang

Hyeyoung Choi Heesun Chung

PII: S0379-0738(16)30052-4
DOI: http://dx.doi.org/doi:10.1016/j.forsciint.2016.02.030
Reference: FSI 8337

To appear in: FSI

Received date: 7-10-2015

Revised date: 15-2-2016
Accepted date: 16-2-2016

Please cite this article as: E. Kim, S. Choe, J. Lee, M. Jang, H. Choi, H. Chung, Detection
of drugs in 275 alcohol-positive blood samples of Korean drivers, Forensic Science
International (2016), http://dx.doi.org/10.1016/j.forsciint.2016.02.030

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Detection of drugs in 275 alcohol-positive blood samples of Korean
drivers

Eunmi Kim†1, Sanggil Choe1, Juseon Lee2, Moonhee Jang2, Hyeyoung Choi3 and Heesun
Chung4

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†
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Corresponding author : Eunmi Kim (emkim4725@korea.kr)
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Co author : Sanggil Choe (csk91@korea.kr)
Juseon Lee (sunnyljs@korea.kr)
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Moonhee Jang (jmh1229@korea.kr)

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Hyeyoung Choi (hychoi22@korea.kr)

Heesun Chung (hschung1024@gmail.com)
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1
Busan Institute, National Forensic Service, 50 Geumoh-ro, Mulgeum-eup, Yangsan, Korea
2
Seoul Institute, National Forensic Service, 139 Jiyang-ro, Yangcheon-gu, Seoul, Korea
3
Daejeon Institute, National Forensic Service, 1524 Yuseong-ro, Yuseong-gu, Daejeon, Korea
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4
Graduate School of Analytical Science and Technology, Chungnam National University,
Daejeon, Korea

Abstract
Since driving under the influence of drugs (DUID) is as dangerous as drink-driving, many
countries regulate DUID by law. However, laws against the use of drugs while driving are not
yet established in Korea. In order to investigate the type and frequency of drugs used by drivers
in Korea, we analyzed controlled and non-controlled drugs in alcohol-positive blood samples.

Page 1 of 21
Total 275 blood samples were taken from Korean drivers, which were positive in roadside
alcohol testing. The following analyses were performed: blood alcohol concentrations by GC;
screening for controlled drugs by immunoassay and confirmation for positive samples by GC-
MS. For the detection of DUID related drugs in blood samples, a total of 49 drugs were selected
and were examined by GC-MS. For a rapid detection of these drugs, an automated identification
software called "DrugMan" was used. Concentrations of alcohol in 275 blood samples ranged

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from 0.011 to 0.249% (average 0.119%). Six specimens showed positive results by

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immunoassay: one methamphetamine and five benzodiazepines I. By GC-MS confirmation,
only benzodiazepines in four cases were identified, while methamphetamine and

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benzodiazepine in two cases were not detected from the presumptive positive blood samples.
Using DrugMan, four drugs were detected; chlorpheniramine (5)*, diazepam (4),

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dextromethorphan (1) and doxylamine (1). In addition, ibuprofen (1), lidocaine (1) and
topiramate (1) were also detected as general drugs in blood samples (* indicates frequency). The

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frequency of drug abuse by Korean drivers was relatively low and a total 14 cases were positive
in 275 blood samples with a ratio of 5%. However it is necessary to analyze more samples
including alcohol negative blood, and to expand the range of drug lists to get the detailed
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information.

Keywords : DUID (Driving under the influence of drugs) · Korean driver · Drug abuse ·
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Blood
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1. Introduction

According to a report by the National Police Agency of Korea [1], there were 223,656 cases
of traffic accidents reported in 2012. Among them, drink-driving cases were 29,093, taking up
13% of the total traffic accidents. Since driving under the influence of drug (DUID) is as

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dangerous as drink-driving, many countries regulate DUID by law. However laws against the

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use of drugs while driving are not yet established in Korea. Furthermore, there are little reported
cases related to DUID.

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DUID cases have been reported in many countries. Augsburger and Rivier [2] reported that
the most abused drugs were cannabinoids (57%), followed by opiates (36%), ethanol (36%) and

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benzodiazepines (15%) in drivers suspected of DUID from Canton de Vaud area of Switzerland
during a 13 years period (1982-1994). Jones et al [3] reported unusually high concentrations of

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diazepam and nordiazepam in blood of impaired drivers from Sweden during 2001 and 2002.
By the roadside test using saliva in Germany, Toennes et al [4] reported that cannabis was most
prevalent (78%) in 177 cases of drivers suspected of DUID. Wylie et al [5] analyzed oral fluid
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samples from drivers in the United Kingdom and reported that 16.8% were positive for at least
one drug, and the most detected drug was 3,4-methylenedioxymethamphetamine (MDMA).
Drummer et al [6] also performed 13,176 roadside drug tests from randomly selected drivers in
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Australia, and reported that 313 cases were positive to drugs, in which the most abused drug
was methamphetamine (269 cases), followed by MDMA and cannabis. However there have
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been no reports about DUID cases in Asian country with an exception of one report from
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Turkey. Acar et al [7] evaluated the type of psychoactive drugs (with or without alcohol)
detected in blood samples in Istanbul during one year. And reported that alcohol was present in
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44% of suspected DUID cases (n=4274), and psychoactive drugs were 15.4% of cases. Among
them, cannabis was found to be 17.4%, 8.7% for benzodiazepines, 4.3% for barbiturates, and
4.3% for antidepressants.
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In this study, in order to investigate the type and frequency of drugs used by Korean drivers,
controlled and non-controlled drugs were searched in alcohol-positive blood samples. A total of
49 drugs related to DUID were selected and were detected using gas chromatography-mass
spectrometry (GC-MS) in whole blood samples. For the rapid detection of drugs, an automated
identification software called "DrugMan", a newly developed macro modules with modified
MSD Chemstation® was used.

2. Materials and methods

Page 3 of 21
2.1. Specimens

A total of 275 blood samples were taken from Korean drivers positive to roadside alcohol test
from Nov to Dec of 2011. At first, blood-alcohol analysis was performed in the collected
samples, and then the residues were stored at -20℃ until drug analysis.

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2.2. Experimental flowchart

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The analytical strategy was composed of following three steps: First, ethyl alcohol (alcohol)

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in blood samples was confirmed and quantified by head-space gas chromatography (GC).
Second, for a preliminary test, blood samples were screened for controlled drugs by Evidence

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investigator™ (Randox, U.K.) which is a biochip array analyzer based on immunoassay. Final
confirmation of positive samples by immunoassay was performed by GC-MS. Third, drugs

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related to DUID were examined in 275 blood samples by GC-MS, and the detection of 49 drugs
was performed using a newly developed automated identification software, DrugMan. In
addition, general drugs were also examined from the extracts of blood samples.
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2.3. Ethyl alcohol (alcohol) analysis
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Six standard alcohol solutions for calibration curve were purchased from Accu Standard Inc.
(USA), in which the concentrations were 0.02, 0.05, 0.10, 0.20, 0.30 and 0.40% w/v,
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respectively. Tertiary butanol (0.05% w/v) was used as internal standard (IS). Chromatographic
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separations were performed on 0.2% Carbowax 1500, 60/80 Carbopak C column (1.8 m × 1/8
inch × 2.1 mm, Stainless Steel) using an Agilent 6890 GC with a 7694 autosampler. The
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detector type was flame ionization detector (FID), and the oven temperature was set at 110℃
isothermally. The injector temperature was 210℃ and the detector temperature was 250℃.
Helium was used as the carrier gas at flow rate of 20 mL/min. Sample preparation was as
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previously reported [8], and the aliquot volume of upper layer (head-space) from the vial was
injected into the GC. Alcohol concentration of sample was calculated as the peak area ratio of
alcohol to internal standard in sample using the standard calibration curve.

2.4. Immunoassay

Controlled drugs in blood samples were screened by Evidence investigator™ (Randox, U.K.),
which is a biochip array analyzer based on immunoassay. In total nine groups of controlled
drugs were analyzed. The list of drugs, calibration curve ranges (or detection range) and cutoff

Page 4 of 21
levels of each group are shown in Table 1.
Reagents and materials were provided by Randox, and employed according to the
manufacturer instructions. At first 50 µL of blood sample was diluted with 150 µL of sample
diluent, and 60 µL of diluted sample was used for the assay. Diluted sample, 120 µL of assay
diluent, 120 µL of conjugate solution were added to the biochip well, and then mixed together.
The biochip was transported to incubator and agitated for 30 min at 33℃ at 330 rpm. After

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washing biochip surface with wash buffer, the well was placed into to the Evidence analyser for

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the assay.

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2.5. Drug detection by GC-MS

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2.5.1. Selection of drugs related to DUID

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A total of 49 drugs related to DUID were selected in present study. Selection was based on
the report of Chung et al [9] in 2005, and included antidepressants, 1st generation antihistamines,
and controlled drugs. Unfortunately cannabinoids were excluded from the lists, because of their
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low extraction recoveries. The list of drugs for DUID is shown in Table 2. Methamphetamine
(MA) was not counted in the lists, because MA was extracted with a separate sample
preparation procedure and confirmed by GC-MS, in case of presumptive positive results for
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methamphetamines in immunoassay.
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2.5.2. Chemicals and reagents

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The following analytical standards (1.0 mg/mL methanolic solutions) were purchased from
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Cerilliant (Round Rock, TX, USA); desalkylflurazepam, nordiazepam, oxazepam, diazepam,

lorazepam, nitrazepam, temazepam, flunitrazepam, 7-aminoflunitrazepam, 7-aminoclonazepam,
clonazepam, prazepam, alprazolam, α-hydroxyalprazolam, triazolam, α-hydroxytriazolam,
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estazolam, morphine, codeine, 6-monoaceylmorphine, nalbuphine, cocaine, benzoylecgonine,

ketamine and norketamine. Among them, morphine, codeine, 6-monoacetylmorphine,
nalbuphine, cocaine, benzoylecgonine, ketamine and norketamine were diluted to 20 µg/mL,
and other standards were diluted to 10 µg/mL with methanol.
Powder type of analytical standards were obtained from Sigma-Aldrich Co. (St. Louis, MO,
USA): zolpidem, amitriptyline, nortriptyline, clomipramine, chlorpromazine, citalopram,
doxepine, fluoxetine, imipramine, paroxetine, sertraline, pheniramine, chlorpheniramine,
brompheniramine, doxylamine, diphenhydramine, hydroxyzine, triprolidine, dextromethorphan,
dextrorphan, phentermine, phenmetrazine, phendimetrazine and fenfluramine. Firstly these

Page 5 of 21
standards were prepared to 1 mg/mL solutions with methanol, and finally were diluted to 20
µg/mL.
The deuterated IS, Cocaine-D3 (Cerilliant, 100 µg/mL methanolic solutions) was diluted to 50
µg/mL with methanol. The derivatization reagent, N,O-bis-(trimethylsilyl) trifluoroacetamide
(BSTFA) with 1% trimethylchlorosilane (TMCS) was obtained from Sigma-Aldrich Co. All
standard solutions were stored at -25℃ until analysis.

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Blood sample extraction was performed on automatic solid-phase extraction (SPE) equipment,

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RapidTraceTM (Zymark Co., USA). The SPE column, Drug-Clean™ C (200 mg), was purchased
from Alltech Co., USA. 2-Propanol, methylene chloride, methanol, hexane were high

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performance liquid chromatography (HPLC) grade, the eluent used was as mixture of 2-
propanol and methylene chloride (75:25, V:V). 0.15 M carbonate buffer (pH 9.5) and 0.1 M

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KH2PO4 (pH 4.5) were prepared for SPE extraction.

2.5.3. Sample preparation and SPE extraction

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1 mL of whole blood was taken to test tube, followed by 30 µL of IS and 3 mL of 0.1 M
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KH2PO4 (pH 4.5), and then mixed well. The mixed blood sample was centrifuged for 10 min at
3000 rpm, and finally 3 mL of upper layer was transferred to test tube (borosilicate, 16 x 100
mm) for loading into the SPE column. SPE extraction in present study was modified from a
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previous report [10]. Finally the eluent was evaporated under N2 gas and the residue was
derivatized (trimethylsilylation, TMS) with 30 µL of BSTFA and 30 µL of ethyl acetate at 90℃
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for 15 min (Scheme 2).

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2.5.4. GC-MS analysis

An Agilent GC-MS system consisting of HP 6890 GC, HP 7673 autosampler injector and
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5975 mass selective detector (MSD) was used in this study. For separation, HP-5MS capillary
column (30 m x 0.25 mm x 0.25 µm) was used. The injector was operated in the splitless mode
and the injection volume was 2 µL. The injector temperature was 250℃. The ionization energy
was 70 eV; the transfer line temperature was 320℃. Oven temperature was programmed as
follows; initial temperature was 100℃ held for 1 min, then increased to 300℃ by 20℃/min
and held for 15 min. Full scan mode was used as an acquisition mode, and the mass range was
from m/z 40 to 550.

2.5.5. Methamphetamine (MA) analysis

Page 6 of 21
 Methamphetamine (MA, 1.0 mg/mL in methanol) and amphetamine (AM, 1.0 mg/mL in
methanol) were obtained from Cerillant, and were diluted to 10 µg/mL with methanol. The
deuterated ISs, MA-D5 and AM-D5, (Cerilliant, 100 µg/mL in methanol) were diluted to 10
µg/mL. The derivatization reagent, pentafluoropropionic anhydride (PFPA, 99%) was obtained
from Sigma-Aldrich Co.
Sample preparation and GC-MS analysis were based on a previous report [11]. The same GC-

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MS system and column described in section 2.5.4 were used. Identification and quantitation of

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MA and AM in blood samples were performed by selective ion monitoring (SIM) mode with
the following ions; MA-PFP, m/z 204, AM-PFP, m/z 190, MA-D5-PFP, m/z 208 and AM-D5-

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PFP, m/z 194.

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2.6. Automated drug identification software, DrugMan

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Choe et al [12] developed an automated drug identification software, DrugMan. In summary,
it is an application program developed with Visual Basic (VB) and its function is to manage
target drugs information. This application manages lists, parameters and generates text files.
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Utilizing these text files, text reports and graphic reports are generated from the modified
Enhanced Data Analysis program of MSD Chemstation®. Parameters for data management
include 49 drug names (Table 2), three major fragment ions, retention times (RT) by GC-MS,
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relative retention times (RRT), which are calculated from target drug RT divided by internal
standard RT, and RT margins. The RT and major fragment ions for 49 drugs are shown in Table
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3. Text report offers two different types of report modes; Brief mode (Fig 1, left) and Full mode
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(Fig 1, right). Brief mode includes RT, RRT, peak height and area of three fragment ions for 49
drugs. Full mode includes extracted ion chromatograms (EIC) of target drugs.
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3. Results
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3.1. Blood alcohol concentrations

Concentrations of alcohol in 275 blood samples ranged from 0.011 to 0.249% (average
0.119%), and the distribution is shown in Fig 2. The concentration range with the highest
frequency was 0.05 - 0.074% (54 cases), followed by 0.075 - 0.099% (51 cases) and 0.125 -
0.149% (44 cases). The frequency of concentration range less than 0.050%, not punishable by
law, was 11 cases, taking up 4% of total. Especially, the frequency of concentration higher than
0.100% was relatively high (159 cases), taking up 58% of total. Since samplings were taken
from Nov to Dec 2011, it is assumed that the results reflected Korea’s heavy drinking culture in

Page 7 of 21
year-end holidays.

3.2. Immunoassay and drug confirmation by GC-MS

Screening of controlled drugs was performed by immunoassay and final confirmation was by
GC-MS. Nine groups of controlled drugs were screened in 275 blood samples, and only six

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samples showed positive results in immunoassay, i.e., one methamphetamine and five

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benzodiazepines I, taking up 2.1% of total (Table 4).
Concentrations of controlled drugs were calculated by calibration curves and positive (+) or

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negative (-) was decided by cutoff levels of controlled groups (Table 1).
As shown by Table 4, the numbers in parenthesis beside + or – mean the concentration

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(ng/mL) of the sample. For example, ‘+(139)’ in sample 85063, benzodiazepines I was detected
with the concentration of 139 ng/mL. Negative (-) means that samples are below cutoff level or

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they are not detected (zero, 0). In sample 85063, MAMP and AMPH were detected with the
concentration of 52 and 2.4 ng/mL, respectively. However these concentrations were below
cutoff levels for MAMP and AMPH, thus the results were negative to both MAMP and AMPH.
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For positive case such as ‘+(>219)’, benzodiazepines I in four samples (84178, 84285, 84573
and 84787) were detected over the calibration range (0 - 219 ng/mL). Meanwhile, sample 84768
was positive to methamphetamine with the result of ‘(>392)’, however, it was negative to
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AMPH as indicated ‘–’. It was assumed that the sample was false positive to MAMP, because
methamphetamine and its metabolite amphetamine may be detected together in the sample.
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By GC-MS confirmation, only benzodiazepines (diazepam, nordiazepam and temazepam) in

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four cases were confirmed, while methamphetamine (#84768) and benzodiazepine (#85063) in
two cases were not detected from the positive blood samples by immunoassay (Table 5).
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3.3. Drug detection by DrugMan

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Four target drugs were screened with good matching results by DrugMan, they were
chlorpheniramine, diazepam, dextromethorphan and doxylamine. All were within the RT margin
and verifiable by graphic report that showed the EIC of target drugs. Chlorpheniramine was
detected in five samples and diazepam in four samples. Dextromethorphan and doxylamine
were both detected in one sample. In addition, ibuprofen, lidocaine and topiramate were also
detected in one sample as general drugs.
A total of 14 cases of drugs were detected in 275 blood samples with a positive ratio of 5%.
The drug with the highest frequency was chlorpheniramine followed by benzodiazepines. Total
identified drugs are summarized in Table 6.

Page 8 of 21
4. Discussion

In forensic laboratory, drug testing is the most process for DUID cases. Thus it is necessary
to test with appropriate scope to identify the drugs in the specimen [13]. Farrell et al [14], based
on the survey data and author’s experience, proposed 44 drugs for DUID investigation and

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recommended LODs in blood and urine, for both screening and confirmation. These drugs

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included cannabis, central nervous system (CNS) stimulants, CNS depressants, narcotic
analgesics and dissociative drugs. For a comprehensive approach to screening in DUID, authors

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suggested that some of the listed drugs that are not commercially available for immunoassay,
such as gamma-hydroxybutyrate (GHB), zolpidem, amitriptyline, diphenhydramine,

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carisoprodol, phenytoin, carbamazepine, topiramate, dextromethorphan and so on, needed some
additional chromatographic analysis such as mass spectrometry.

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In this study 49 drugs were selected for DUID investigation and the confirmation of these
drugs was performed by GC-MS (Table 2). Among them only three controlled groups
(benzodiazepines, opiates and coca alkaloids) were included in the lists. Even though cannabis
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is ranked at the second abused drug in Korea following methamphetamine,
tetrahydrocannabinol (THC) and carboxytetrahydrocannabinol (THC-COOH) were not included
in the lists. In our extraction conditions, recoveries of THC and THC-COOH in blood were
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lower less than 50%. Cutoffs both of them for GC-MS confirmation were 50 ng/mL in our
conditions, which were 10-25 times higher than the recommended cutoffs for cannabinoids in
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blood in DUID investigations [13,14]. The method for cannabinoids is under development and
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further data will be soon available.

In the analytical process, Evidence investigator™ was used as the preliminary test for the
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screening of controlled drugs in blood samples. It was developed based on the biochip array
technology, which it used the chemiluminescence of substrate with a horseradish peroxidase
label for the detection of antibodies or analytes bound to the biochip surface. A charge-coupled
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device camera recorded the light emission from all substrates on each of the nine biochips, and
according to the intensity of signal output the quantities of antibody or antigen are measured
[15]. The advantage of using biochip technology is that simultaneous screening of multiple
controlled groups with small volumes of sample can be done. In fact, only 15 μL of blood was
used in this study to screen nine groups of controlled drugs simultaneously. However the cost of
biochip was quite expensive compared to multiple single assay testing, and in order to be fully
equipped for the automation of the entire process, the cost may be an issue.
Meanwhile, Ishida et al [16] introduced a novel automatic drug screening system, the
NAGANATA software, which enables the detection and quantification of drugs simultaneously

Page 9 of 21
by GC-MS. The software is similar to ours, DrugMan, in terms of using the Chemstation
software package with Agilent GC-MSD instruments. Using DrugMan software, both
identification of target drugs and reporting the results were performed within one minute. If the
semi-quantitation function of target drugs are added and more drugs are registered to the
DrugMan database, it will be a powerful tool to screen target drugs in forensic toxicology.
According to the annual domestic report published in 1998 by National Institute of Scientific

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Investigation (NISI, former name of NFS), 22 Korean drivers’ urine samples related to traffic

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accidents were submitted to NISI from the Police for drug testing [17]. Two controlled drugs,
cannabinoids and methamphetamine were detected. In addition uncontrolled drugs,

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dextromethorphan, nalbupine, carisoprodol and imipramine were detected from urine samples as
well. Currently, dextromethorphan, nalbuphine and carisoprodol are regulated as controlled

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drugs in Korea.
As shown in Table 6, the frequency of drug abuse by Korean drivers is relatively low. Even

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though these results are limited to alcohol positive blood samples, to our knowledge, this is the
first international report on DUID cases in Korea. However it is necessary to analyze more
samples including alcohol negative blood, and to expand the range of drug lists to get the
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detailed information.

5. Conclusion
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This study examined the type and frequency of drugs used by Korean drivers. For the drug
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screen, 49 drugs related to DUID were selected and DrugMan, a newly developed automated
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identification software, enabled the detection of these drugs within one minute. The frequency
of drug abuse by Korean drivers was relatively low and total 14 cases were positive in 275
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blood samples with a ratio of 5 %. The highest frequency of drug was chlorpheniramine
followed by benzodiazepines. However, these results were limited to alcohol positive blood
samples, thus, it is necessary to analyze more samples including alcohol negative blood to get
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further detailed information.

References

1) http://www.police.go.kr/portal/main/contents.do?menuNo=200375 (last access date :

02/Nov/2015)
2) M. Augsburger and L. Rivier, Drugs and alcohol among suspected impaired drivers in
Canton de Vaud (Switzerland), Forensic Sci. Int., 85 (1997) 95-104.
3) A.W. Jones, A. Holmgren, P. Holmgren, High concentrations of diazepam and

Page 10 of 21
 nordiazepam in blood of impaired drivers: association with age, gender and spectrum
of other drugs present, Forensic Sci. Int., 146 (2004) 1-7.
4) S.W. Toennes, S. Steinmeyer, H-J Maurer, M.R. Moeller, G.F. Kauert, Screening for
drugs of abuse in oral fluid-correlation of analysis results with serum in forensic cases,
J. Anal. Toxicol., 29 (2005) 22-27.
5) F.M. Wylie, H. Torrance, A. Seymour, S. Buttress, J.S. Oliver, Drugs in oral fluid Part

t
II. Investigation of drugs in drivers), Forensic Sci. Int., 150 (2005) 199-204.

ip
6) O.H. Drummer, D. Gerostamoulos, M. Chu, P. Swann, M. Boorman, I. Cairns, Drugs
in oral fluid in randomly selected drivers, ), Forensic Sci. Int., 170 (2007) 105-110.

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7) F Acar, M Asirdizer, RG Aker, EE Kucukibrahimoglu, I Ates, Y Erol, A Sahin, A
review of suspected cases of driving under the influence of drugs (DUID) involved in

us
traffic accidents in Istanbul (Turkey), J Forensic Leg Med, 20 (2013) 626-31.
8) Y. Kim, J. Kim, J. Yang, S. Park, E. Kim, K. Yang, A reciprocal relationship of ethyl

Annual Report of NISI, 39 (2007) 335-341.

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alcohol concentration between brain tissue and blood in postmortem specimens,

9) H. Chung, N. Kim, C. Chung, The research on the regulation of DUID, by Police

M
science institute, 2005, pp. 15-21.
10) E. Kim, J. Lee, H. Choi, M. Lim, H. Chung, Simultaneous analysis of fifteen
benzodiazepines in urine by solid-phase extraction and negative chemical ionization
d

gas chromatography-mass spectrometry (NCI-GC-MS), Korean Journal of Forensic

Science, 8 (2007) 24-32.
e

11) E. Kim, J. Lee, H. Choi, E. Han, Y. Park, H. Choi, H. Chung, Comparison of

pt

methamphetamine concentrations in oral fluid, urine and hair of twelve drug abusers
using solid-phase extraction and GC-MS, Annales de Toxicologie Analytique, 20
ce

(2008) 145-153.
12) S. Choe, S. Kim, H. Choi, H. Choi, H. Chung, Automated toxicological screening
reports of modified Agilent MSD Chemstation® combined with Microsoft Visual
Ac

Basic® application programs, ), Forensic Sci. Int., 199 (2010) 50-57.

13) B.K. Logan and M.D. Osselton : Chapter 5. Driving under the influence of drugs,
Clarke's analysis of drugs and poisons in pharmaceuticals, body fluids and postmortem
material, 4rd edi., by Moffat AC, Osselton MD, Widdop B, Pharmaceutical Press, 2011,
pp. 115-126.
14) L.J. Farrell, S. Kerrigan, B.K. Logan, Recommendations for toxicological investigation
of drug impaired driving, J. Forensic Sci., 52 (2007) 1214-1218.
15) R.M. Molly, R.I. McConnell. J.V. Lamont, S.P. FitzGerald, Automation of biochip
array technology for quality results, Clin. Chem. Lab. Med., 43(2005) 1303-1313

Page 11 of 21
 16) T. Ishida, K. Kudo, S. Naka, K. Toubou, T. Noguchi, N. Ikeda, Rapid diagnosis of drug
intoxication using novel NAGANATATM gas chromatography/mass spectrometry
software, Rapid Commun. Mass Spectrom., 21 (2007) 3129-3138
17) H. Choi, W. Jin, W. Yang, E. Han, C. Kim, H. Chung, Y. Yoo, Pharmacokinetics of
Carisoprodol in rat, Annual Report of NISI, 31 (1998) 585-602.

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Fig legend

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Fig 1. Report generation (left; Brief mode text report. Right; Full mode text report)

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Fig. 2. Distribution of alcohol concentration in 275 blood samples

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Scheme legend
Scheme 1. Total experimental flow
Scheme 2. Extraction procedure for 49 drugs related to DUID in whole blood.

Table 1
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Lists of the screened controlled drugs, their calibration ranges and cutoff levels by Immuno
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assay (Evidence investigator™ ) in whole blood
Calibration range Cutoff level
Group Target drug
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(ng/mL) (ng/mL)
Amphetamines d-Amphetamine 0.00 - 159.00 50
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Methamphetamines d-Methamphetamine 0.00 - 392.00 100

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Cannabis THCCOOH 0.00 - 42.00 10

Coca alkaloids Benzoylecgonine 0.00 - 248.00 50
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Opiates Morphine 0.00 - 95.00 25

Barbiturates Phenobarbital 0.00 - 251.00 50
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Benzodiazepines I1) Oxazepam 0.00 - 219.00 50

Benzodiazepines II2) Lorazepam 0.00 - 261.00 50
Methadone Methadone 0.00 - 131.00 25
1)
Benzodiazepines I ; a broad spectrum of benzodiazepines
2)
Benzodiazepines II ; lorazepam, lorazepam glucuronide, clonazepam, desalkylflunitrazepam.

Page 12 of 21
Table 2
Lists of 49 Drugs for DUID by GC-MS
Groups
Drugs
(the number of drugs)
Antidepressants Amitriptyline, Nortriptyline, Citalopram, Clomipramine,

t
ip
(10) Chlorpromazine, Doxepine, Fluoxetine, Imipramine,
Paroxetine, Sertraline

cr
1st Generation Pheniramine, Chlorpheniramine, Brompheniramine,
Antihistamines Diphenhydramine, Triprolidine, Hydroxyzine, Doxylamine

us
(7)
Controlled drugs -Benzodiazepines (17) ;
(32) Desalkylflurazepam, Nordiazepam, Oxazepam, Diazepam,
an
Lorazepam, Temazepam, Clonazepam, 7-Aminoclonazepam,
Flunitrazepam, 7-Aminoflunitrazepam, Alprazolam, α-
Hydroxyalprazolam, Triazolam, α-Hydroxytriazolam,
M
Prazepam, Nitrazepam, Estazolam
d

-Anorectics (4) ;
Phentermine, Phendimetrazine, Phenmetrazine, Fenfluramine
e
pt

-Controlled drugs (11) ;

Morphine, Codeine, 6-Monoacetylmorphine, Cocaine,
ce

Benzoylecgonine, Dextromethorphan, Dextrorphan,

Ketamine, Norketamine, Nalbuphine, Zolpidem,
(Methamphetamine)
Ac

Page 13 of 21
Table 3
Retention time and selected three major mass fragment ions of drugs
Retention Time Mass Fragment Ions (m/z)
Drug name
(min) Q1 Q2 Q3

t
ip
Cocaine-D3* 10.01 185 306 275

6-Acetylmorphine-TMS* 11.60 399 340 287

cr
7-Aminoclonazepam-diTMS 11.93 429 394 414

7-Aminoflunitrazepam-TMS 12.21 355 327 326

us
Alprazolam 14.00 279 308 204

Amitriptyline 9.97 58 202 215

Benzoylecgonine-TMS

Brompheniramine
10.29

9.54
82

247
an 240

249
361

58

Chlorpheniramine 9.04 203 205 167

M
Chlorpromazine 11.33 58 318 272

Citalopram 10.86 58 238 324

d

Clomipramine 10.92 268 58 314

e

Clonazepam-TMS 12.03 387 352 306

Codeine-TMS 11.09 371 178 196

pt

Desalkylflurazepam-TMS 10.24 359 360 341

ce

Dextromethorphan 9.73 271 214 150

Dextrorphan-TMS 9.96 329 272 150

Diazepam 11.09 256 283 221

Ac

Diphenhydramine 8.31 58 73 165

Doxepine 10.13 58 165 178

Doxylamine 8.58 58 71 167

Estazolam 13.61 259 294 239

Fenfluramine 4.30 72 159 216

Flunitrazepam 11.89 312 285 286

Fluoxetine 8.22 44 104 309

Hydroxyalprazolam-TMS 14.49 381 383 396

Page 14 of 21
Hydroxytriazolam-TMS 15.65 415 417 430

Hydroxyzine-TMS 13.66 201 203 165

Imipramine 10.10 234 235 193

Ketamine 8.31 180 182 209

Lorazepam-diTMS 11.16 429 431 347

t
Morphine-diTMS 11.29 429 236 196

ip
Nalbuphine-diTMS 13.65 446 447 501

cr
Nalbuphine-triTMS 13.79 518 428 519

Nitrazepam-TMS 11.41 352 353 306

us
Nordiazepam-TMS 10.30 341 342 327

Norketamine-TMS 8.60 238 225 73

Nortriptyline 10.06 44 202 215

Oxazepam-diTMS 10.68 429

an
430 313

Paroxetine 11.66 44 192 329

M
Phendimetrazine 5.93 85 57 191

Pheniramine 8.03 169 168 58

Phenmetrazine 5.81 71 105 177

d

Phentermine 3.82 58 91 134

e

Prazepam 12.06 269 295 324

pt

Sertraline-TMS 11.44 274 276 73

Temazepam-TMS 11.71 343 257 283

ce

Triazolam 14.86 313 238 342

Triprolidine 10.24 208 209 278

Ac

Zolpidem 12.92 235 236 307

*; internal standard, TMS; trimethylsilylation

Page 15 of 21
Table 4
Lists of positive samples by Immunoassay
Sample MAMP BARB BENZ1 BENZ2 MDONE OPIAT BZG THC AMPH
ID

84178 - - +(>219) - - - - - -

t
84285 - - +(>219) -(5.29) - - - - -

ip
84573 - - +(>219) -(0.84) - - - - -

cr
84768 +(>392) - - - -(0.1) -(0.01) - - -

84787 - - +(>219) - - - - - -

85063 -(52) - +(139) - -(0.58) -(2.06) - - -(2.4)

us
MAMP: methamphetamine, BARB: barbiturates, BENZ1: benzodiazepines I, BENZ2: benzodiazepines II, MDONE:

methadone, OPIAT: opiates, BZG: benzoylecgonine, THC: cannabinoids, AMPH: amphetamine

an
M
e d
pt
ce
Ac

Page 16 of 21
Table 5
Comparisons of Immunoassay and GC-MS results
IA (ng/mL)
Sample Confirmation
Target Drug Calibration range
ID Results by GC-MS

t
(cutoff)

ip
0.00 - 392.00
Methamphetamine 84768 Pos.: + (>392) Neg.
(100)

cr
84178 Pos.: + (>219) Pos.: DZ

84285 Pos.: + (>219) Pos.: DZ, NDZ, TZ

us
0.00 - 219.00
Benzodiazepines I 84573 Pos.: + (>219) Pos.: DZ, NDZ
(50)
84787 Pos.: + (>219) Pos.: DZ, NDZ, TZ

85063 Pos.: + (139) an Neg.

IA; Immunoassay, DZ; Diazepam, NDZ; Nordiazepam, TZ; Temazepam Pos.; positive, Neg.; negative
M
e d
pt
ce
Ac

Page 17 of 21
Table 6
Identified drugs in 275 whole bloods, according to the drugs present in each sample.

Drugs Number of samples

Chlorpheniramine 5

t
Benzodiazepines 4

ip
Diazepam (1)

cr
Diazepam / nordiazepam* (1)

Diazepam / nordiazepam* / temazepam** (2)

us
Dextromethorphan 1

Doxylamine 1

Ibuprofen
an 1

Lidocaine 1
M
Topiramate 1

Total 14
d

*, ** Metabolites of Diazepam
e
pt

Highlights
ce

The type and frequency of drugs used by drivers in Korea were investigated.
A total of 49 DUID related drugs selected and examined by GC-MS in blood
samples.
Ac

The identification software, DrugMan, enabled to detect drugs in one minute.

Page 18 of 21
 t
ip
cr
us
an
Fig 1
M
de
pt
ce
Ac

Page 19 of 21
 t
ip
cr
us
an
M
Fig. 2.
de
pt
ce
Ac

Scheme 1

Page 20 of 21
 t
ip
cr
us
an
M
d

Scheme 2
e
pt
ce
Ac

Page 21 of 21