Accepted Manuscript

Case report

Detection of imidacloprid in biological fluids in a case of fatal insecticide in-toxication

Chiaki Fuke, Takumi Nagai, Kenji Ninomiya, Maki Fukasawa, Yoko Ihama,Tetsuji Miyazaki

PII: S1344-6223(13)00123-5DOI: http://dx.doi.org/10.1016/j.legalmed.2013.10.007Reference: LEGMED 1073

To appear in: Legal Medicine

Received Date: 17 December 2012Revised Date: 24 October 2013Accepted Date: 24 October 2013

Please cite this article as: Fuke, C., Nagai, T., Ninomiya, K., Fukasawa, M., Ihama, Y., Miyazaki, T., Detection ofimidacloprid in biological fluids in a case of fatal insecticide intoxication, Legal Medicine (2013), doi: http://dx.doi.org/10.1016/j.legalmed.2013.10.007

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Detection of imidacloprid in biological fluids in a case of fatal insecticide intoxication

Chiaki Fuke *, Takumi Nagai, Kenji Ninomiya, Maki Fukasawa, Yoko Ihama, Tetsuji

Miyazaki

Department of Forensic Medical Science, Graduate School of Medicine,

University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan

Abstract

Here, we describe a high-performance liquid chromatography/photodiode array

detector method for the detection of imidacloprid in biological fluids in a case of suicide

by ingestion of liquor mixed with Admire® Flowable insecticide (containing 20%

imidacloprid). A plastic bottle containing a cloudy liquid (concentration of ethanol in

the liquid was 150 mg/ml and that of imidacloprid was 50 mg/ml) was found near the

decedent. The biological fluids collected at autopsy were prepared by deproteinization

with acetonitrile. Zolpidem was used as an internal standard. The concentrations of

imidacloprid in femoral blood and cerebrospinal fluid were 105 µg/ml and 58.5 µg/ml,

respectively. Ethanol was also detected in the samples, with concentrations of 1.0 mg/ml

in femoral blood and 1.4 mg/ml in cerebrospinal fluid.

Key words: Imidacloprid, GC-MS, HPLC, Fatal intoxication, Poisoning

1. Introduction

Imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] is

a neonicotinoid insecticide in the chloronicotinyl nitroguanidine chemical family (Fig.

1). It was introduced in the Japanese market in 1992. Trade names for Bayer

CropScience’s imidacloprid products include Admire® and Hachikusan®.

Imidacloprid acts on several types of postsynaptic nicotinic acetylcholine receptors

(nAChRs) in the nervous system. In insects, these receptors are located exclusively

within the central nervous system. Following the binding of imidacloprid to the

nicotinic receptor, nerve impulses are spontaneously discharged at first; however,

subsequently, the neurons fail to propagate any signal [1].

There are various subtypes of mammalian nAChRs, all of which have a lower

binding affinity for imidacloprid than that observed in insects [1, 2]. Therefore, the

acute oral toxicity of imidacloprid in mammals is considered moderate [1]. For example,

the oral lethal dosage (LD50) in male rats is 420 mg/kg [3]. In mammals, imidacloprid is

mainly metabolized to 6-chloronicotinic acid and its glycine conjugate, and

5-hydroxyimidacloprid [1, 3].

Insecticide products containing imidacloprid are used worldwide; therefore,

occasionally, accidental intoxication or intentional self-intoxication with imidacloprid

occurs throughout the world [4-15]. Some fatal intoxication cases have been reported,

despite the relatively low toxicity of imidacloprid in mammals [6, 7, 9, 13, 14]. In this

report, we describe a high-performance liquid chromatography/photodiode array

detector (HPLC/PDA) detection method for the identification of imidacloprid in

biological fluids in a fatal intoxication case.

2. Case report

A man in his 70s, who was missing after a quarrel with his family, was found dead

near his ancestor’s grave. A plastic bottle containing a white liquid with an alcoholic

odor was found near the decedent. A medico-legal autopsy was performed 2 days after

the death. The man’s height was 154 cm and he weighed 56 kg. His face showed

congestion, and there were some viscous fluid in his mouth. There were no signs of

serious injury on the body. The heart, which weighed 430 g, showed left ventricular

hypertrophy without myocardial ischemic change or coronary artery stenosis. The left

and right lungs showed congestion and weighed 420 g and 610 g, respectively. Gastric

contents were a white viscous fluid (70 ml) with an unpleasant odor. Substantial

petechial bleeding was observed in the mucosa of the stomach and duodenum. The

abdomen had a surgical scar, and there were indications that the man had undergone

right hemicolectomy with an ileotransverse anastomosis in the abdominal cavity. There

was no evidence of a lethal disease.

3. Materials and methods

3. 1. Chemicals

Imidacloprid and 6-chloronicotinic acid were purchased from Wako (Osaka, Japan).

Zolpidem, used as an internal standard (IS), was purchased from Sigma-Aldrich, Japan

(Tokyo, Japan). Other chemicals used were HPLC grade or analytical grade. Water was

purified using a Milli-Q A10 system (Millipore, MA, USA).

3. 2. Biological samples

Samples (whole blood from the heart and femoral vein, cerebrospinal fluid, vitreous

humor, and urine) for analysis were collected at the time of autopsy. Frozen human

whole blood was purchased from KAC Co. LTD. (Kyoto, Japan). All samples were

stored at -30 °C prior to analysis.

3. 3. Stock solutions

Stock solutions of imidacloprid and 6-chloronicotinic acid were prepared in

methanol at 1.0 mg/ml. The IS working solution (1.0 mg/ml zolpidem) was also

prepared in methanol.

3. 4. Sample preparation

Ten microliters of the IS working solution were placed into a microtube (1.5 ml)

and evaporated to dryness under a stream of nitrogen gas. An aliquot (100 µl) of the

liquid specimen was added to the tube and vortex-mixed. Two hundred microliters of

acetonitrile were added to the tube while vortex-mixing. The mixture was centrifuged at

12000 g for 5 min. The supernatant was transferred to another microtube and evaporated

to dryness under a stream of nitrogen gas at 40 °C. The residue was dissolved in 100 µl

of 15% acetonitrile-water (v/v) and centrifuged at 12000 g for 5 min. The supernatant

was then transferred to the appropriate glass autosampler vial insert and injected into the

HPLC system.

3.5. HPLC conditions for the analysis of imidacloprid

The HPLC system consisted of an Alliance 2695 separation module and 2996

photodiode array detector (Waters, MA, USA). The analytical column used was an

XTerra○R MS C18 2.1 × 150 mm, 3.5 µm (Waters, MA, USA). Column temperature was

set at 40 °C. Chromatograms were monitored at 270 nm, and injection volume was 5 µl.

The mobile phase consisted of acetonitrile-20 mM potassium dihydrogen phosphate

buffer (pH 3.0) (15:85, v/v), and flow rate was maintained at 0.2 ml/min.

3. 6. Calibration curve and recovery

A calibration curve was obtained by injection of a mixture of the imidacloprid stock

solution and the IS working solution diluted with 15% acetonitrile-water (v/v) at the

concentration of imidacloprid 1.0, 5.0, 10.0, 50.0, 100.0 and 200.0 μg/ml, respectively,

and 100 μg/ml of IS. The recovery rates were calculated according to the following

formula; recovery rate (%) = (peak area of imidacloprid of added blood or specimen –

average peak area of not added blood or specimen) / peak area of imidacloprid of the

standard mixture x 100.

4. Results and discussion

The contents of the plastic bottle, heart blood, urine, and the gastric were initially

subjected to qualitative analysis. Screening was performed using gas

chromatography/mass spectrometry (GC/MS) and HPLC/PDA. Imidacloprid was

identified in contents of the plastic bottle, gastric content and heart blood by GC/MS

(Fig. 2). Due to the imidacloprid peak appeared as tailing peak on total ion

chromatogram of GC/MS analysis using DB-5 MS capillary column, GC/MS was not

suitable for the quantitative analysis of imidacloprid. Imidacloprid was identified in all

specimens by HPLC/PDA (Fig. 3). Other basic, acidic, or neutral drugs, or agricultural

chemicals were not detected in any specimen.

Under the HPLC conditions for imidacloprid detection, the peaks of

6-chloronicotinic acid, imidacloprid, and zolpidem (IS) appeared at 5.3, 9.4, and 22.7

min, respectively, on the chromatogram monitored at 270 nm (Fig. 3-A). A peak for

heart blood was observed at 9.4 min (Fig. 3-B), and the UV spectrum of this peak (Fig.

3-C) matched that of imidacloprid. 6-Chloronicotinic acid was not identified in any

specimen. 6-Chloronicotinic acid spiked to the heart blood at a concentration of 0.1

µg/ml was detectable. Absence of peaks interfering with the IS peak was also confirmed

(Fig. 3-B).

A linear calibration curve for imidacloprid was obtained in the concentration range

of 1.0 to 200.0 µg/ml (r2 = 0.999). The detection limit of imidacloprid in blood was 0.02

µg/ml (S/N = 3).

The method was validated by performing replicate analysis (n = 3) on the drug-free

blank blood spiked with imidacloprid (1.0, 10.0, and 100.0 µg/ml). Table 1 shows the

validation data and recovery yield for the imidacloprid analysis. The recovery yield of

the IS was 94.4 ± 3.2%. The accuracy of the estimated concentrations of each specimen

was validated by analysis of the specimen spiked with imidacloprid (50.0 µg/ml). The

detected concentrations of samples spiked with 50.0 µg/ml imidacloprid, compared to

the untreated samples, increased from 50.1 µg/ml to 52.3 µg/ml. These values displayed

acceptable error ranges (less than 10 %).

Table 2 summarizes the concentrations of imidacloprid in each specimen. The

values were corrected so that the differences between the values obtained by subtracting

the values of the non-spiked sample from the values of the imidacloprid-spiked sample

were equal to 50.0 µg/ml.

The imidacloprid concentration in the femoral blood of our subject (105 µg/ml) was

the highest among its concentrations in plasma or blood in all the cases of imidacloprid

intoxication reported thus far (Table 3). The imidacloprid insecticide product, Admire○R

Flowable (containing 20% imidacloprid), was found in the subject’s garden shed. About

half of the contents (100 ml) of the bottle were used. A plastic bottle containing a small

volume of a cloudy liquid (with an ethanol concentration of 150 mg/ml and an

imidacloprid concentration of 50 mg/ml) was found near the decedent. We suspected

that the relatively high concentration in this case (105 µg/ml) was because of very high

ingestion of imidacloprid and ingestion of a mixture of an imidacloprid-based

insecticide with an alcoholic beverage, leading to enhanced imidacloprid absorption.

Imidacloprid penetration through the blood–brain barrier is poor [1, 2]. In our case,

imidacloprid was detected in the cerebrospinal fluid, and its concentration (58.5 μg/ml)

was approximately half of that measured in the femoral blood (105 μg/ml). This finding

is in agreement with the results of a study conducted in mice by Ford and Casida [16].

Because the binding affinity of imidacloprid to vertebrate nAChRs has been shown to

be weaker than that in the case of insects, the toxicity due to imidacloprid is low in

mammals, while imidacloprid is highly selective against insects [1, 3]. However, we

considered the high imidacloprid concentrations in the body to be lethal.

A high concentration of imidacloprid was detected in the gastric contents. The

analysis performed for common toxic substances showed negative results, and no injury

or disease that could be considered a cause of death was found at autopsy. Due to the

absence of evidence for an alternative cause of death (macroscopic or histological) and

the results of the toxicological tests, we concluded that the cause of death in this case

was imidacloprid intoxication.

References

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Figure legends

Fig. 1. Structures of imidacloprid, 6-chloronicotinic acid, and zolpidem (internal

standard).

Fig. 2. Total ion chromatograms of GC/MS analysis of 100.0 μg/ml imidacloprid (A)

and ethylacetate extract of the heart blood (B), and the mass spectra of peak 1 (C) and

peak 2 (D).

Fig. 3. Representative chromatograms of (A) 10.0 μg/ml 6-chloronicotinic acid (1),

imidacloprid (2), and zolpidem (3, IS) containing standard solution, (B) a heart blood

sample, and (C) the UV spectrum of the peak observed at 9.4 min on the chromatogram

of the heart blood sample.

Table 1. Imidacloprid assay validation in various samples and recoveries.

Sample n Added Detected* CV Recovery*

(µg /ml) (µg /ml) (%) (%)

Blank blood 3 1.0 1.2±0.0 0.7 85.9±2.3

3 10.0 9.7±0.0 0.2 86.7±0.7

3 100.0 98.1±0.3 0.3 87.5±1.3

Heart blood 2 0.0 98.9±0.1 0.1

2 50.0 149.5±2.1 1.4 93.4±5.9

Femoral blood 2 0.0 100.9±4.3 4.3

2 50.0 148.7±0.4 0.3 94.7±4.3

Cerebrospinal fluid 2 0.0 59.7±0.3 0.5

2 50.0 110.7±3.6 3.2 101.8±6.4

Vitreous humor 2 0.0 51.3±0.2 0.4

2 50.0 102.9±1.1 1.1 103.6±8.3

Urine 2 0.0 28.4±0.1 0.4

2 50.0 80.7±0.8 1.0 105.2±1.7

* The value is average±standard deviation.

21

Table 2. Imidacloprid and ethanol concentrations in various specimens. Sample Imidacloprid Ethanol (µg/ml) (mg/ml) Heart blood 97.5* 0.8 Femoral blood 105* 1.0 Cerebrospinal fluid 58.5* 1.4 Vitreous humor 49.6* 1.3 Urine 27.0* 1.4 Gastric content 1000 1.4

* The value is the average of duplicate analysis.

22

Table 3. Imidacloprid concentrations in plasma or blood reported in previous literature

on human cases of imidacloprid insecticide intoxication.

Reference No. Age Sex Outcome Concentration Sampling time Origin of

after ingestion the sample

(µg/ml) (h)

5 95 M Survival 3 24 Serum

5 50 M Survival 15 3.5 Serum

5 89 F Survival 23 0.5 Serum

5 62 M Survival 30.7 - Serum

5 78 M Survival 84.9 6 Serum

6 33 M Death 12.5 PM Blood

6 66 M Death 2.05 PM Blood

10 35 F Survival 44.6 5 Plasma

10 in 33 cases Survival 0.02–51.25 Plasma

-: not written, PM: postmortem