June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Research Paper

Evaluation of four drug screening devices for
detection of psychoactive drugs in pericardial ﬂuid
1,2
1,2
1,2
Masae Iwai *, Tadashi Ogawa , Tomohito Matsuo ,
1,3

1,4

1,2

Katsutoshi Kubo , Fumio Kondo , Hiroshi Seno
1

Department of Legal Medicine, Aichi Medical University School of Medicine,1‒1 Yazakokarimata, Nagakute, Aichi 480‒1195, Japan
2
Poison Analysis Center, Aichi Medical University School of Medicine,1‒1 Yazakokarimata, Nagakute, Aichi 480‒1195, Japan3
Department of Oral Pathology/Forensic Odontology, Aichi Gakuin University School of Dentistry,
1‒100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464‒8650, Japan
4
Department of Biomedical Sciences, Chubu University College of Life and Health Sciences,
1200 Matsumoto-cho, Kasugai, Aichi 487‒8501, Japan
Abstract
SIGNIFY

We evaluated the applicability of four immunoassay-based drug screening devices, Triage TOX Drug Screen,®

TM

ER, IVeX-screen M-1, and DRIVEN-FLOW M8-Z, in the detection of psychoactive drugs in pericardial ﬂuid

as an alternative to urine. A total of 38 pericardial ﬂuid samples from forensic autopsies were analyzed with the four drug
screening devices. To conﬁrm the results, the concentrations of psychoactive drugs in pericardial ﬂuid samples were measured together with those in urine and blood samples by liquid chromatography-tandem mass spectrometry. Only IVeXscreen M-1 precisely detected psychoactive drugs without false positive results, whereas Triage TOX Drug Screen and®

SIGNIFYTM

ER showed several false positive results, and DRIVEN-FLOW M8-Z led to many false positive results. These

results suggest that IVeX-screen M-1 is more useful than other screening devices for psychoactive drugs in pericardial ﬂuid
and that pericardial ﬂuid is a valid alternative material when urine is not available.
Key words: drug screening devices, psychoactive drugs, pericardial ﬂuid, liquid chromatography-tandem mass spectrometry

3‒5)

Introduction

detection of amphetamines. These false positive results
are particularly important in the context of forensic autop-

The detection of possible drug consumption is a central

sies, as they can be exacerbated by the production of putrefactive amines, such as 2-phenethylamine, by saprogenic

1)

aspect of forensic toxicological examinations . Devices used
for urine drug screening can provide a rapid indication of the2)

presence of analytes of interest during autopsy , but thesebacteria in moderately or heavily decomposed bodies.

devices are associated with several drawbacks. For example,

In our previous report, we evaluated the performance of®

®

Triage DOA frequently returns false positive results in theﬁve drug screening devices, Triage TOX Drug Screen,TM
ER, IVeX-screen M-1, Status DS10, andSIGNIFY* Corresponding author

DRIVEN-FLOW M8-Z, in the detection of amphetamines

Masae Iwai

and methamphetamines in urine containing putrefactive
amines. The results suggested that DRIVEN-FLOW M8-Z

Department of Legal Medicine, Aichi Medical University
School of Medicine, 1‒1 Yazakokarimata, Nagakute, Aichi

was more useful than other screening devices for screening

480‒1195, Japan
E-mail: masaoshi@aichi-med-u.ac.jp

Received: November 30, 2023. Accepted: March 31, 2024.

of methamphetamines in the presence or absence of
2-phenethylamine, while none of the tested devices

Epub May 20, 2024.

detected amphetamines precisely .

6)Another issue with the forensic detection of drug use is

DOI: 10.24508/mms.2024.06.003
19

June 2024

Medical Mass Spectrometry Vol. 8 No. 1that collection of urine is not always feasible. In these

zepam and ﬂunitrazepam (Eisai, Tokyo, Japan); brotizolam

cases, blood, pericardial ﬂuid, cerebrospinal ﬂuid, vitreous
humor, or other alternative ﬂuids must be analyzed. In par-(Sumitomo Pharma, Tokyo, Japan); estazolam (Takeda
Pharmaceuticals, Tokyo, Japan); phenobarbital, amitripty-

ticular, pericardial ﬂuid often can be readily obtained fromline, nortriptyline, diazepam and triazolam (Fujiﬁlm Wako
Pure Chemical, Osaka, Japan); temazepam, zolpidem, diaz-the pericardial cavity. Typical volumes of pericardial ﬂuid
amount is suﬃcient for forensic drug testing. Because of

epam-d5, and phenobarbital-d5 (Sigma-Aldrich, St. Louis,
MO, USA). High-performance liquid chromatography

the potential utility of this material in forensic analyses, in(HPLC)-grade methanol was obtained from Fujiﬁlm Wako

the present study we evaluated the applicability of four®
drug screening devices, Triage TOX Drug Screen, SIGNI-

Pure Chemical (Osaka, Japan). Other common chemicals

7)

taken at the time of autopsy range from 5 to 35 mL ; thisFY

TMused were of the highest purity commercially available.
Laboratory distilled water was puriﬁed using a Direct-Q

ER, IVeX-screen M-1, and DRIVEN-FLOW M8-Z, tothe detection of psychoactive drugs in pericardial ﬂuid from
autopsy samples. In this analysis, liquid chromatogra-

UV 3 system (Millipore, Molsheim, France).

phy-tandem

Biological samples

mass

spectrometry(LC-MS/MS)

was

Pericardial ﬂuid, urine and blood samples were obtained

employed as a sensitive and quantitative tool to compare
the performance of these immunoassay-based devices.

from autopsy cadavers at Aichi Medical University from2021 to 2023. Samples were collected in 5- or 15-mL tubes

Materials and Methods

and stored at −80°
C until analysis. Additionally, pericardial

Chemicals and materials

ﬂuid samples were assessed without any pretreatment.

Amphetamine was kindly provided by Dr. Kenji Hara
(Fukuoka University). The following standard drugs were

Devices and their principles of detection

used: methamphetamine (Dainihonseiyaku, Osaka, Japan);amobarbital (NIPPON SHINYAKU, Kyoto, Japan); broma-

Triage TOX Drug Screen (Alere San Diego, CA, USA),TM
SIGNIFY ER (Innovacon, CA, USA), IVeX-screen M-1

Table 1.

®

Cutoﬀ values of psychoactive drugs for four drug screening devices
Cutoﬀ values (ng/mL)

Drug classiﬁcation

Barbiturates
Sedative-hypnotics

Tricyclic antidepressantsStimulants

Drug name

Abbreviation*

1®

Triage TOX
Drug Screen

SIGNIFYER

TM

IVeX-screen
M-1

DRIVENFLOW M8-Z

Phenobarbital

BAR

230

100

150

2200

Amobarbital

BAR

250

300

200

2000

Bromazepam

BZO/BZDBZO/BZD

750
̶*2

1562
̶*2

600

Brotizolam＞10000Estazolam

BZO/BZD

400

2500

400

500
̶*2
̶*2

Flunitrazepam

BZO/BZD

200

390

400

̶*2

Lorazepam

BZO/BZD

200

1562

2000

Midazolam

BZO/BZD

200

12500

Temazepam

BZO/BZD

200

98

10000
̶*2

550
̶*2
̶*2

Triazolam

BZO/BZD

300

ZOL

2500
̶*2

600

Zolpidem

100
̶*2

50

50

Amitriptyline

TCA

600

1500

300

1000

Nortriptyline

TCA

900

1000

1000

1000

Amphetamine

AMP

500

10000085000

MethamphetaminemAMP/METH/MET

500

1000
̶*2

500

500

*1BAR; barbiturates, BZO/BZD; benzodiazepines, ZOL; zolpidem, TCA; tricyclic antidepressants, AMP; amphetamines, mAMP/METH/
MET; methamphetamines.
*2Not available.

20June 2024

Medical Mass Spectrometry Vol. 8 No. 1(Biodesign, Tokyo, Japan) and DRIVEN-FLOW M8-Z

m/z 225 → 42 for amobarbital, m/z 136 → 91 for amphet-(Alfa Scientiﬁc Designs, CA, USA) were assessed in thisstudy. All four devices are based on a competitive immuno-

for amitriptyline, m/z 264 → 233 for nortriptyline, m/zamine, m/z 150 → 91 for methamphetamine, m/z 278 → 91

316 → 209 for bromazepam, m/z 393 → 314 for brotizolam,assay and give qualitative responses to the presence orm/z 285 → 193 for diazepam, m/z 295 → 267 for estazolam,m/z 314 → 268 for ﬂunitrazepam, m/z 301 → 255 for temaze-absence of drugs, and they are recommended to be used forurine only by their manufacturers. Cutoﬀ values that arepam, m/z 343 → 308 for triazolam, m/z 308 → 235 for zolpidem, m/z 290 → 154 for diazepam-d5 and m/z 236 → 42 forused by the devices to determine positive results are shownin Table 1.

phenobarbital-d5.

LC-MS/MS analysis

Ethics approval

Samples of human pericardial ﬂuid, urine, or blood(100 μL) were mixed with 100 μL methanol and 200 μL ace-

All experiments were approved by the Ethics Committeeof Aichi Medical University (approval no. 2020-172).

tonitrile after addition of 40 μL internal standard solution(0.5 μg/mL diazepam-d5 and 25 μg/mL phenobarbital-d5).

Results

The mixture was vortexed for 60 s and centrifuged at

A total of 38 pericardial ﬂuid samples with matched

15,000 g for 10 min, and the supernatant was transferred toanother tube, followed by addition of 100 μL of 0.1% triﬂu-

urine and blood samples were obtained during forensic

oroacetic acid in acetonitrile. The solvent was removed
with a centrifugal evaporator (CVE-200D; Tokyo Rikaki-

autopsies. The presence of psychoactive drugs was ﬁrst®
evaluated in urine by Triage TOX Drug Screen or SIGNI-

kai, Tokyo, Japan). The residue was reconstituted in 100 μL
methanol and centrifuged at 15,000 g for 1 min. A 5 μL ali-

FY

quot of supernatant was subjected to analysis by LC-MS/
MS. Samples containing high concentrations of target com-

The concentrations of the drugs in urine and blood samples
were also determined by LC-MS/MS. The concentrations ofpounds were analyzed after dilution as needed.
LC-MS/MS analyses were performed using a Nexera X2

drugs that were present in these samples were determined
to be within the ranges of 2.0 to 20,300 ng/mL in urine sam-liquid chromatograph coupled to an LCMS-8040 massples (Table 2) and 0.50 to 3230 ng/mL in blood samples

spectrometer (Shimadzu, Kyoto, Japan). For separation, a(Table 3). Because of low sample volumes, four urine andthree blood samples were not analyzed by LC-MS/MS.

TM

ER. Nine urine samples (cases 1‒9) tested positive

for the presence of at least one psychoactive drug (Table 2).

Kinetex column (2.1 mm I.D. × 100 mm, particle size
2.6 μm; Phenomenex, Cheshire, UK) was used. The column

Pericardial ﬂuid samples were then subjected to analysis

temperature was maintained at 40°
C. The gradient systemwith four drug screening devices: Triage TOX Drug
TM
ER, IVeX-screen M-1 and DRIVScreen, SIGNIFY®

used for separation included mobile phase A (a solution ofand mobile phase B (a solution of 0.1% formic acid in

EN-FLOW M8-Z; these samples were also analyzed by
LC-MS/MS. LC-MS/MS analysis revealed that 9 samples

10 mM ammonium formate in methanol). The ﬂow rate was(cases 1, 2, 3, 6, 7, 8, 9, 11, 14 and 16) contained at least

0.5 mL/min. The elution gradient involved a linear increase

one type of psychoactive drug at a concentration between
9.8 and 3270 ng/mL (Table 4). A representative LC-MS/MS

0.1% formic acid in 10 mM ammonium formate in water)from 5% B to 95% B over 3.0 min, followed by constant
5% B over 0.01 min and maintained at 5% B for 3.0 min to

analysis of a pericardial ﬂuid sample (case 7) shows peaks
with the same retention times as those of standard amitrip-

equilibrate the column for the next sample. The desolvation

tyline (6.25 min) and nortriptyline (6.30 min) (Fig. 1a).

line temperature and heat block temperature were 250°
C

Diagnostic fragment ions and their ion abundance ratios
were also fully consistent with those of standard amitripty-

95% B for 1.5 min. The mobile phase was then returned toand 400°
C, respectively.

line and nortriptyline, thus conﬁrming the presence of these

Electrospray ionization was applied in the negative mode

compounds in this pericardial ﬂuid sample (Fig. 1b).

for phenobarbital and amobarbital and positive mode forother compounds. Quantiﬁcation was performed by

The results from the application of the four drug screen-

selected reaction monitoring (SRM) using the peak area.

ing devices to pericardial ﬂuid samples are summarized in

The SRM transitions were m/z 231 → 42 for phenobarbital,®

Table 4. The Triage TOX Drug Screen device returned
21June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Table 2.
Case

Summary of results for drug screening devices and LC-MS/MS in urine samples

drug screening device®

mAMP, BZO, BAR

Phenobarbital (4630), Methamphetamine (1140),
Amphetamine (247), Estazolam (37.3)®

AMP, mAMP, BZO

Methamphetamine (1520), Amphetamine (317),
Bromazepam (150),

1

Triage TOX Drug Screen Drug Screen

2

Triage TOX Drug Screen

3

SIGNIFYTM

BAR

Phenobarbital (6250)®

ER

mAMP, BZO

Methamphetamine (13260), Amphetamine (970),
Temazepam (55.3), Diazepam (7.0)®

BZO

Lorazepam (4.0)®

BAR

Phenobarbital (1030)®

BZO, TCA

Amitriptyline (513), Nortriptyline (200),
Triazolam (1.2), Flunitrazepam (0.89)®

BAR

Amobarbital (380)®

AMP, mAMP, BZO

Methamphetamine (20300), Amphetamine (2680),
Brotizolam (7.4), Flunitrazepam (5.2)
̶
NT*

4

Triage TOX Drug Screen

5

Triage TOX Drug Screen

6

Triage TOX Drug Screen

7

Triage TOX Drug Screen

8

Triage TOX Drug Screen

9

Triage TOX Drug Screen

10

Triage TOX Drug Screen

11

SIGNIFY

TM

ER

̶

12

SIGNIFY

TM

̶

®

13
14

ER

̶

®

Triage TOX Drug Screen

̶

®

̶

®

̶

NT*
̶

®

̶

Zolpidem (0.20)
̶

®

̶

̶

Triage TOX Drug Screen

15

Triage TOX Drug Screen

16
17‒28

Triage TOX Drug Screen

29‒38

LC-MS/MS (concentration, ng/mL)

Positive test result

NT*
NT*

®

Triage TOX Drug Screen or
IVeX-screen M-1
Triage TOX Drug Screen or
SIGNIFYTM ER*Not analyzed due to insuﬃcient sample quantity.

Table 3.

Quantiﬁcation of psychoactive drugs in blood samples by LC-MS/MS

Case

Psychoactive drug (concentration, ng/mL)

1

Phenobarbital (3230), Estazolam (117), Methamphetamine (28.0), Amphetamine (5.1), Flunitrazepam (13.3)

2

Bromazepam (280), Methamphetamine (40.4), Nitrazepam (23.5), Amphetamine (17.5)

3

Phenobarbital (2610)

4

Methamphetamine (23.0), Temazepam (3.1), Amphetamine (3.0)

5Lorazepam (3.7)

6

Phenobarbital (1750)

7

Amitriptyline (986), Nortriptyline (221), Flunitrazepam (6.9), Clonazepam (4.8), Triazolam (2.9)

8Amobarbital (670)9Methamphetamine (190), Amphetamine (38.3), Flunitrazepam (19.1)
̶

10
11
12

Midazolam (3.4)
NT*

14

NT*
NT*

15

̶

16

Zolpidem (0.50)
̶

13

17‒28
29‒38

̶

*Not analyzed due to insuﬃcient sample quantity.

22

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Summary of results for four drug screening devices and LC-MS/MS in pericardial ﬂuid samples

Table 4.
®

Triage TOX
Drug Screen

SIGNIFY

1

BAR

BAR

2

̶

3

BAR

4

AMP*
̶

Case

5
6

TM

ER

Phenobarbital (744), Methamphetamine (7.0),
Amphetamine (0.95) Estazolam (35.8)

BZO* , TCA*̶

TCA* , ZOL*

2

Bromazepam (24.1), Methamphetamine (8.9),
Amphetamine (2.5)

BAR
̶

BAR
̶

2
BAR, ZOL*
2ZOL*

Phenobarbital (3270)

̶

̶

ZOL*
2
ZOL*

2

BAR
3
TCA*

BAR
3
TCA*

BAR

8

3
BAR*

̶

9

BZO*

3
BAR*
3
METH*

3

11

̶

12

̶

13
14

BZO*
3
BZO*

TCA*
̶
̶

̶

15

*1

̶

̶

16
17‒28

̶

̶

̶

29‒38

̶

1

2

̶
̶

2

̶

̶

Methamphetamine (6.3), Amphetamine (0.60)
̶
Phenobarbital (505)

3

̶

3
BZO*

2

2

TCA

AMP*
2
TCA*

10

LC-MS/MS (concentration, ng/mL)

2
2
TCA* , ZOL*

7

1

DRIVENFLOW M8-Z

BAR
3

1

IVeX-screen
M-1

TCA*
2
ZOL*

Amitriptyline (488), Nortriptyline (33.1)

MET
2
2
TCA* , ZOL*

Methamphetamine (73.2), Amphetamine (9.8)
̶

2
ZOL*
2
2
TCA* , ZOL*

Midazolam (1.6)
̶

2
ZOL*
2
2
TCA* , ZOL*̶

Amobarbital (116)

2
ZOL*
3
ZOL*

̶

2
ZOL*
2
2
TCA* , ZOL*

Midazolam (38.7)
̶Zolpidem (0.928)
̶̶

2* Undeterminable. * False positive.
*3Positive even though the sample drug concentrations determined by LC-MS/MS analyses were under the cutoﬀ values.

tive result using DRIVEN-FLOW M8-Z. Other samples

three ambiguous results (cases 4, 9 and 15) for unknown
reasons, and it returned one false positive result for benzodiazepines (case 13). The SIGNIFY

TM

returned negative results for the spiked psychoactive drugs.

ER device produced

three false positive results for tricyclic antidepressants. The
IVeX-screen M-1 device returned no false positive results.

Discussion

The DRIVEN-FLOW M8-Z device returned false positive

devices with respect to their performance in detecting psy-

results for zolpidem or tricyclic antidepressants in 35 of thechoactive drugs in forensic pericardial ﬂuid samples. Oneof these devices, IVeX-screen M-1, was found to be moreIn the present study, we investigated four drug testing

38 cases.

useful than the other three screening devices. This device
accurately detected phenobarbital (cases 1, 3 and 6), ami-

Eleven samples (cases 2, 7, 8, 9, 11, 14 and 16) tested
positive for psychoactive drugs according to drug screening
devices, even though the sample drug concentrations deter-

triptyline (case 7), amobarbital (case 8) and methamphet-mined by LC-MS/MS were under the immunoassay cutoﬀ
values that are considered to indicate positive results (Tableamine (case 9), and returned no false positive results. On®
the contrary, the Triage TOX Drug Screen and SIGNI-

4). Therefore, to investigate this potential discrepancy,

FY

blank pericardial ﬂuid samples spiked with psychoactive
drugs at the concentrations found by LC-MS/MS analyses

positive results. To our knowledge, the only informationTM

ER devices returned several false positive results,
and the DRIVEN-FLOW M8-Z device returned many false

were subjected to analyses with the drug screening devices.

concerning the applicability of drug testing device to theUsing a blank sample spiked with the concentration of TCA

detection of psychoactive drugs in pericardial ﬂuid has®

been reported by Tominaga et al . This report showed thatTOX Drug Screen; the concentration of BZO from case 14

Triage TOX Drug Screen returned a few false positive

found in case 7 returned a positive result using Triage

5)

®

®

returned a positive result using Triage TOX Drug Screen;
and the concentration of ZOL from case 16 returned a posi-

results as ours and recommended the combined usage ofanother device to minimize misinterpretation prior to instru23

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Fig. 1. Representative LC-MS/MS analysis of a pericardial ﬂuid sample (case 7).
(a) Selective ion monitoring liquid chromatograms show the same retention times (6.25 min and 6.30 min) as those of standard
amitriptyline and nortriptyline. (b) Diagnostic fragment ions with ion abundance ratios of the noted fractions from the pericardial
ﬂuid sample were fully consistent with those of standard amitriptyline and nortriptyline, thus conﬁrming the presence of these compounds in this pericardial ﬂuid sample.

the pericardial ﬂuid is found in a closed compartment, con-mental analysis.
The principal advantage of the use of pericardial ﬂuid istamination by microorganisms is less likely as compared tothat pericardial ﬂuid is easily obtained from the pericardial

blood and cerebrospinal ﬂuid, and pericardial ﬂuid is thus

cavity, and the usual volume of pericardial ﬂuid taken at therelatively stable in the postmortem period . In the present

8)

7)time of the autopsy is enough for forensic drug testing . As

study, however, we found that drug concentrations were24

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

lower in the pericardial ﬂuid than in the urine. Speciﬁcally,

the immunoassay-based devices for the detection of psycho-

the concentrations of methamphetamine and amphetamine

active drugs. Thus, we recommend that positive resultsin pericardial ﬂuid were considerably lower than those in

obtained using the screening devices should be conﬁrmed
by LC-MS/MS.

urine samples. We conclude, then, that pericardial ﬂuid is
an important alternative material when urine is not avail-It should be noted that the concentrations of the drugs inand amphetamine because of low concentrations compared

the blood samples seem to be higher than those in the pericardial ﬂuid, except for a couple of drugs such as phenobar-

to urine.

bital in case 3. Because drug concentrations in pericardialable, but it is unsuitable for detecting methamphetamine

ﬂuid can be thought to be inﬂuenced from, or almost thesame with those of blood from anatomical perspective, peri-

One of the disadvantage of immunoassays, including
those investigated in the present study, is that antibodies
tend to be variable in terms of their potentials for cross-re-cardial ﬂuid thus has the potential to be very useful alterna-

activity . This cross-reactivity can lead to inconsistent
4)

tive when blood is not available.

results or false positive signals. In our previous study, we
found that the DRIVEN-FLOW M8-Z device was particu-

In conclusion, pericardial ﬂuid is an important alternative
material for forensic analyses when urine is not available.
Among the four drug screening devices tested for this appli-

larly useful for the detection of methamphetamines inforensic urine samples . One advantage of this device

cation, we found IVeX-screen M-1 to be the most useful, as

when screening urine samples was the lack of false positive

it accurately detected the highest number of psychoactive

signals even in the presence of the putrefactive amine
2-phenethylamine. However, in the present study, thisdrugs without returning false positive results. Despite the6)accuracy of this device, it is strongly recommended
LC-MS/MS methods should be used to conﬁrm any posi-device led to many false positive results for zolpidem andtricyclic antidepressants. False positive results are rela-tive results achieved with immunoassay-based screening

tively common with immunoassays, as antibodies tend tobe variable in terms of their potentials for cross-reactivity.

devices.

Conﬂict of Interest

In this case, we hypothesize that antibodies employed by
the DRIVEN-FLOW M8-Z device may cross-react with

The authors declare no conﬂicts of interest.

substances commonly found in the pericardial ﬂuid, thus
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