June 2018

Medical Mass Spectrometry Vol. 2 No. 1

Short Communication

In vivo metabolic proﬁling for acetamiprid in rat plasma byliquid chromatography quadrupole time-of-ﬂight mass spectrometry
Tadashi Ogawa *, Jun Ueyama , Takayoshi Suzuki , Masae Iwai , Xiao-Pen Lee ,
1

2

3,4

1

3

1

1

Takeshi Kumazawa , Keizo Sato , Fumio Kondo , Hiroshi Seno

3

1

1

Department of Legal Medicine, Aichi Medical University School of Medicine,1‒1 Yazakokarimata, Nagakute, Aichi 480‒1195, Japan2
Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine,1‒1‒20 Daiko-Minami, Higashi-ku, Nagoya, Aichi 451‒8673, Japan3
Department of Legal Medicine, Showa University School of Medicine,1‒5‒8 Hatanodai, Shinagawa-ku, Tokyo 142‒8555, Japan
4
Seirei Christopher University School of Nursing,3453 Mikatagahara-cho, Kita-ku, Hamamatsu, Shizuoka 433‒8558, Japan
Abstract The metabolism of acetamiprid, a neonicotinoid insecticide, in rats was investigated by monitoring time-dependent changes in plasma levels of acetamiprid and its metabolites using liquid chromatography quadrupole time-of-ﬂight
mass spectrometry (LC-QTOF-MS). Acetamiprid was administrated to rats intravenously at 7.1‒21.7 mg/kg for analysis ofplasma at 0‒96 h. Acetamiprid and its four metabolites could be detected and tentatively identiﬁed in rat plasma samples.
Plasma levels only estimated from peak areas of acetamiprid decreased, with a half-life of about 4 h, and were completely-cyano-acetamidine was most strongly detected at 4 h and was misseliminated by 24 h. N-[(6-Chloro-3-pyridyl)methyl]-N′
ing at 48 h. The peak areas of N-[(6-chloropyridin-3-yl)methyl]-acetamide increased over time, reached the maximum at-methylacetamidine and 6-(methylsulfanyl)
12‒24 h, and the metabolite was then completely eliminated at 72 h. N-Cyano-N′-cyano-acetamidine. These ﬁndings
nicotinic acid showed almost the same behaviors as N-[(6-chloro-3-pyridyl)methyl]-N′
suggested that LC-QTOF-MS enabled us to tentatively estimate acetamiprid metabolism in rats and that these four metabolites may also be useful biomarkers for acetamiprid exposure in humans.
Key words: acetamiprid, neonicotinoid, metabolite, liquid chromatography quadrupole time-of-ﬂight mass spectrometry

Introduction

insecticides are being phased out due to the emergence ofSince the late 1990s, the agricultural use of neonicotinoid

insects resistant to conventional pesticides. Acetamiprid is a

insecticides, including acetamiprid, has been increasingneonicotinoid insecticide with selective toxicity and is used
for the control of sucking-type insects on vegetable, fruits,

1‒4)

worldwide

because organophosphorus and pyrethroid

1‒4)

cotton, and ornamental plants and ﬂowers* Corresponding author. One of the

toxic characteristics of acetamiprid is high mammalian tox-

Tadashi Ogawa

icity of neonicotinoids; the oral median lethal dose (LD50)
5,6)
was in the range of 146‒217 mg/kg body weight , similar

Department of Legal Medicine, Aichi Medical University
School of Medicine, 1‒1 Yazakokarimata, Nagakute, Aichi
480‒1195, Japan
Tel: ＋81‒561‒62‒3311 (Ext. 12266), Fax: ＋81‒561‒63‒8120

to those of organophosphorus and pyrethroid insecticides.

Received December 5, 2017. Accepted February 21, 2018.

to monitor the levels of acetamiprid and its metabolites in

Epub April 20, 2018.

body ﬂuids, such as blood and urine.

Homicide or suicide cases using acetamiprid are also
expected to increase in the future. Therefore, it is importantE-mail: ogawatd@aichi-med-u.ac.jp

In several reports, the detection of acetamiprid has been

DOI: 10.24508/mms.2018.06.003
34

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Medical Mass Spectrometry Vol. 2 No. 1

8‒15), plants ,15)

ment. All procedures involving animals and their care con-21)

, animals , and humans. Taira et al.
-cyano-acetreported that N-[(6-chloro-3-pyridyl)methyl]-N′

formed to international guidelines, Principles of Laboratory
Animal Care (NIH publication no. 85-23, revised 1985),

amidine (acetamiprid-N-desmethyl) is the most dominanturinary metabolite of acetamiprid and that 6-chloronicotinicand Guiding Principles for the Care and Use of Laboratory

7)

reported on environmental water , soil
16‒18)

19)

insects

20‒25)

Animals of Nagoya University.

acid was found in the urine of patients suspected of sub20)

Animal experiments

acute exposure to neonicotinoid pesticides . Yeter and22)reported the results of autopsy samples (blood,
liver, stomach contents, and urine) in cases of fatal intoxi-

anaesthetized with an intraperitoneal administration of

cation. However, available information related to chronological changes in the levels of acetamiprid and its metabo-sodium pentobarbital (25 mg/kg body weight), and the rightjugular vein was cannulated with polyethylene tubes (Nat-19)

sume, Tokyo, Japan) for blood collection and drug adminis-

reported the levels of acetamiprid in mouse plasma, buttration. The doses of acetamiprid were set at a nonacute
symptomatic level (7.1 mg/kg body weight) and one-tenth

Aydın

lites in body ﬂuids is very limited. Ford and Casida

One day before the start of the experiments, rats were

they did not provide detailed results on metabolites.
Recently, liquid chromatography-mass spectrometry (LC-the LD50 (21.7 mg/kg body weight). Blood samples werecollected at designated intervals (0.25, 0.5, 1, 2, 4, 6, 12,
24, 48, 72, and 96 h after intravenous injection of each acet-MS) has been used for analysis of complex samples; thispowerful analytical technique combines the resolving
power of LC with the accuracy of mass separation.
LC-quadrupole time-of-ﬂight MS (LC-QTOF-MS) has been

amiprid). Plasma samples were immediately obtained byused for proﬁling drug metabolites in various biologicalstored at −80°
C until analyses.

centrifugation at 6000×g for 10 min at 4°
C. Samples were26‒30)

samples. In this study, we investigated the metabolite

proﬁling for acetamiprid in rats by monitoring time-dependent changes in peak areas of acetamiprid and its metabo-

Solid-phase extraction procedurelites in plasma using LC-QTOF-MS.

metabolites was performed according to our previous
31)
report with minor modiﬁcations. Brieﬂy, a 100-μL vol-Materials and Methods

ume of plasma was mixed with 50 ng IS and 4.0 mL of
250 mM sodium bicarbonate solution, vortex-mixed for

Solid-phase extraction (SPE) of acetamiprid and its

Materials
Acetamiprid was purchased from Wako (Tokyo, Japan);acetamiprid-N-desmethyl(N-[(6-chloro-3-pyridyl)methyl]-N′

1 min, and centrifuged at 1600×g for 5 min. For SPE, thecyano-acetamidine; M1) from Sigma-Aldrich Japan (Tokyo,
Japan); isotope-labeled acetamiprid (acetamiprid-d6) to be(60 mg, 3 cc; Waters, Milford, MA, USA) preconditioned
with 3 mL methanol and 3 mL distilled water. Each car-used as an internal standard from Hayashi Pure Chemical

tridge was washed with 3 mL distilled water and 3 mL ofInd. (Osaka, Japan). Laboratory distilled water was puriﬁed

5% methanol aqueous solution. The cartridges were driedusing a Synergy UV (Millipore, Molsheim, France). Other

under vacuum for 10 s. The analytes were eluted with 3 mL

common chemicals used in this study were of the highest

methanol. The elute was transferred to another tube andevaporated with a centrifugal evaporator (CVE-2000;

supernatant fraction was applied to an Oasis HLB cartridge

purity commercially available.

Tokyo Rikakikai, Tokyo, Japan). The residue was reconstituted in 90μL of 0.1% formic acid and 10μL acetonitrile

Animals

Thirteen-week-old male Wistar rats (approximately

and mixed by vortexing for 1 min.

300 g) were purchased from Japan SLC (Hamamatsu,
Japan). The rats were housed under controlled environmen-

Instrumental analysis
The LC instrument used in combination with a
QTOF-MS detector was a Waters Xevo G2 QTof system,

tal conditions (temperature of 23±1°
C and humidity of 55%±5%) with a commercial food diet (CLEA Rodent Diet
CE-2; CLEA Japan, Tokyo, Japan) and water freely avail-including an Acquity UPLC binary pump and a sample

able to animals under a 12-h light/dark cycle (lights on
from 08:00 to 20:00 h) for at least 3 days before the experi-

manager (Waters). The column used for chromatographic
separation was an Acquity UPLC HSS C18 (100×2.1 mm
35

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Medical Mass Spectrometry Vol. 2 No. 1

i.d., 1.8μm; Waters). The column temperature was main-on substrate elemental compositions using structure-based
C-heteroatom dealkylation to construct appropriate masstained at 40°
C, and a gradient system was used with mobile32)

defect ﬁlters for substrate . Filtered sample and control(zero-substrate) data were compared to identify changes

phase A (0.1% formic acid aqueous solution) and mobile
phase B (0.1% formic acid in acetonitrile) delivered at

potentially associated with drug metabolism and electrophilic trapping using the speciﬁc acetamiprid trapping fea-

0.4 mL/min. The linear gradient program was as follows:
95% A/5% B (0 min) to 5% A/95% B (until 8 min). The
ﬁnal mobile phase was held for 2 min to avoid carry-overtures in the MetaboLynx XS program, and lists of expectedfrom the previous injections. After the elution program, theand unexpected potential trapped reactive metabolites were

mobile phase composition was returned to 95% A/5% B

populated.

within 2 min for the next run. The total run time for eachsample analysis was 12 min. The autosampler was main-

Results and Discussion

tained at 4°
C, and the injection volume was 5 μL.

Metabolic proﬁle of acetamiprid

The LC system was interfaced by electrospray ionization

We searched for candidate metabolites of acetamiprid(ESI) to a Waters Xevo G2 QTOF-MS operated in full-scan
EE
MS mode with positive ionization. The MS mode com-using the MetaboLynx XS program and selected the target
32)

metabolites in reference to previous studies . We used
LC-QTOF-MS to tentatively identify the metabolites ofprised two interleaved full-scan functions, one acquired
under low-energy conditions to obtain accurate mass dataacetamiprid in rat plasma samples collected after intrave-for intact precursor ions, and one acquired under high-en-

nous administration of acetamiprid. The metabolites were
-cyadetermined to be N-[(6-chloro-3-pyridyl)methyl]-N′

ergy conditions to obtain fragment ions and corresponding
accurate mass data. No compound-speciﬁc tuning of ioniza-

no-acetamidine (M1), N-[(6-chloropyridin-3-yl)methyl]-ac-methylacetamidine (M3), andetamide (M2), N-cyano-N′tion parameters was employed. Data were acquired over them/z range of 50‒700 using a capillary voltage of 3.0 kV,

6-(methylsulfanyl)nicotinic acid (M4) (Table 1). Extracted

sampling cone voltage of 30 V, source temperature of 150°
C, and desolvation temperature of 500°
C. For the high-en-

ion chromatograms of acetamiprid (m/z 223.0750), M1 (m/zergy scan function, a collision energy ramp of 10‒30 eV

209.0594), M2 (m/z 185.0481), M3 (m/z 98.0718), M4 (m/z170.0275), and acetamiprid-d6 (m/z 229.1127) obtained

was applied. A continuous lockspray reference compound(leucine enkephalin, m/z 555.2693) was sampled at 10-sfrom plasma samples 12 h after intravenous administrationof acetamiprid by LC-QTOF-MS are shown in Fig. 1. Theintervals for centroid data mass correction.

peaks of ﬁve analytes and the IS were clearly observable.

Data processing

times of 6 and 24 h after administration according to peak

We also listed the ranks of the four metabolites at theData acquisition was achieved with MassLynx version

area intensities obtained. The M1 and M3 ranked the top

4.1 (Waters). Data were processed with the MetaboLynx

and second at both times, respectively.

XS program, a component of MassLynx. Data were initially
ﬁltered (±0.03 u) according to accurate mass defects based
Table 1.

Identiﬁcation of acetamiprid and its metabolites in rat plasma using LC-QTOF-MS, together with the ranks of metabolite peak area intensities at 6 and 24 hCompounds

FormulaRetention time(min)

Measured mass(m/z)

Acetamiprid
N-[(6-Chloro-3-pyridyl)methyl]-cyano-acetamidine (M1)N′
N-[(6-Chloropyridin-3-yl)methyl]acetamide (M2)-methylacetamidine (M3)
N-Cyano-N′

C10H11ClN4

3.19

223.0752

223.0750

0.90

̶

̶

C9H9ClN4

2.88

209.0597

209.0594

1.43

1

1

C8H9ClN2O

2.22

185.0478

185.0481

−2.16

4

3

C4H7N31.28

98.0716

98.0718

−2.04

2

2

C7H7NO2S

3.21

170.0274

170.0275−0.59

C10H5D6ClN4

3.18

229.1124

229.1127

−1.31

3
̶

4
̶

6-(Methylsulfanyl)nicotinic acid (M4)
Acetamiprid-d6 (IS)36

Theoretical mass Mass error Rank Rank
(m/z)
(ppm)
(6 h) (24 h)June 2018

Medical Mass Spectrometry Vol. 2 No. 1

2.88 min. The low-energy mass spectrum showed an [M＋＋H] ion at m/z 209.0597. The mass error between measured
and theoretical mass values was 1.43 ppm. The high-energy
mass spectrum provided a fragment ion at m/z 126.0113,
which was formed by loss of C4H6N3. Acetamiprid wasmetabolized to M1 by N-demethylation. The mass error
between measured and theoretical mass values was
1.59 ppm.
Three metabolites, i.e., M2, M3, and M4, were also
detected at retention times of 2.22, 1.28, and 3.21 min,respectively, although the intensities were lower than that of＋

M1. The [M＋H] ions and mass errors were m/z 185.0478
and −2.16 ppm for M2, m/z 98.0716 and −2.04 ppm for
M3, and m/z 170.0274 and −0.59 ppm for M4, respectively.
＋

The [M＋H] ion of M2 was 24 Da lower than that of M1;
therefore, M2 was identiﬁed as N-[(6-chloropyridin-3-yl)＋
methyl]-acetamide. The [M＋H] ion of M3 was 125 Da
lower than that of acetamiprid. The Metabolynx XS pro--methylacetamidine
gram suggested that M3 was N-cyano-N′by mass defect ﬁltering. The metabolite M4 was 6-(methyl19)

sulfanyl)nicotinic acid in reference to a previous study

and mass defect ﬁltering. Additionally, the mass spectra ofthese three metabolites by high-energy collision did not pro-

Fig. 1. Extracted ion chromatograms of (a) acetamiprid(m/z 223.0750), (b) M1 (m/z 209.0594), (c) M2 (m/z185.0481), (d) M3 (m/z 98.0718), (e) M4 (m/z170.0275), and (f) acetamiprid-d6 (m/z 229.1127) obtained from plasma samples 12 h after intravenous
administration of acetamiprid recorded by liquid
chromatography quadrupole time-of-ﬂight mass
spectrometry. M1: N-[(6-chloro-3-pyridyl)methyl]-cyano-acetamidine, M2: N-[(6-chloropyridin-3N′-methylacetyl)methyl]-acetamide, M3: N-cyano-N′
amidine, M4: 6-(methylsulfanyl)nicotinic acid.

Time-dependent changes in peak areas of acetamiprid
and its metabolites in rat plasma

Identiﬁcation of acetamiprid and its metabolites in rat
plasma samples

amiprid was detected in plasma samples taken between

vide structurally informative fragment ions because of low＋
intensities of their [M＋H] ions. Therefore, further experiments towards attaining enough sensitivity are required.
The proposed metabolic pathways of acetamiprid in rats areshown in Fig. 2.

The time-dependent changes in peak areas of acetamiprid
and its metabolites in rat plasma are shown in Fig. 3. Acet-

The LC-QTOF-MS proﬁles of acetamiprid and its four

15 min to 24 h after administration of acetamiprid (Fig. 3a).

metabolites are summarized in Table 1. Acetamiprid waseluted at a retention time of 3.19 min. The low-energy mass

M1, which was most strongly detected, increased overtime, reached a maximum at 4‒6 h, and was completely

spectrum showed an [M＋H] ion at m/z 223.0752. The
mass error between the measured and theoretical mass val-eliminated at 48 h (Fig. 3b). The levels of M2 increased
over time, reached a maximum at 12‒24 h, and were thenues was 0.90 ppm. The high-energy mass spectrum pro-completely eliminated at 72 h (Fig. 3c). M3 and M4 showed

vided a fragment ion at m/z 126.0109, which was formedby loss of C4H6N3. The mass error between measured andsimilar behaviors to M1 (Figs. 3d and 3e). Ford and Casidareported that there was an increase from 2.2 ppm acetami-

theoretical mass values was −1.59 ppm. These results

prid at 15 min to approximately 6 ppm at 60‒240 min when

revealed the high reliability of the identiﬁcation method.

acetamiprid was administered intraperitoneally at 10 mg/kg＋

19)

The metabolite M1, which was the most strongly

to mice . They also found that the levels of M2 peaked at

detected metabolite, was eluted at a retention time ofabout 30 min . The discrepancy in these results between

19)

37

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Medical Mass Spectrometry Vol. 2 No. 1

Fig. 2.

Proposed metabolic pathways for acetamiprid in rats.
19)

their study

and the present study may be due to different
22)

administration routes. Yeter and Aydin reported that the-carbamoyl-N-[(6-chloro-3-pyridyl)methyl]-Nmetabolite N′
methylacetamidine was not detected in decedents blood,consistent with our ﬁndings.

Conclusions

We investigated in vivo metabolic proﬁling for acetamiprid in rats using LC-QTOF-MS. LC-QTOF-MS enabled us
to monitor time-dependent changes in plasma levels of acetamiprid and its metabolites. The results from animal experiments showed that the four metabolites detected in the present study may be useful biomarkers for acetamiprid
exposure in humans.

Conﬂict of Interest
The authors declare that we have no conﬂict of interest.
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