Post-mortem interaction between methidathion and human serum albumin in blood

Abstract

Methidathion [3-(dimethoxyphosphinothioylsulfanylmethyl)-5-methoxy-1,3,4-thiadiazol-2-one; hereinafter DMTP], one of the most widely used organophosphorus pesticides, has been detected in some clinical cases of accidental exposure and suicide in Japan. It has been reported that DMTP concentration is decreased in blood. In this study, it is difficult to recover DMTP in the free form because DMTP is bound to human serum albumin (HSA). We detected DMTP adducts in HSA by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). The mass spectra showed that DMTP was preferably bound to the lysine (K), tyrosine (Y), and cysteinylproline (CP) residues of HSA. The concentrations of K-adduct, DMTP-Y-adduct and DMTP-CP-adduct were increased in vitro in a dose-dependent fashion when DMTP concentration was lower than the lethal dose. Furthermore, the DMTP-Y-adduct and DMTP-CP-adduct were also detected in post-mortem blood of an autopsied subject who died by intentional DMTP ingestion. The results suggested that the DMTP-Y-adduct and DMTP-CP-adduct could be used as a biomarker of DMTP poisoning, and the decrease concentration of DMTP in blood after death could be determined on the basis of the concentration of the DMTP-CP-adduct in blood.

INTRODUCTION

Methidathion [3-(dimethoxyphosphinothioylsulfanylmethyl)-5-methoxy-1,3,4-thiadiazol-2-one; hereinafter DMTP] is a broad-spectrum organophosphorus pesticide marketed around the world (Fig. 1a). DMTP consists of an O,O-dimethylthiophospho moiety and a thiadiazole moiety linked by a phosphorus-sulfur bond, and acts as an insecticide by inhibiting acetylcholine esterase (AChE). This structural feature is common to insecticides having the same toxicity (Fukuto, 1990). The inhibition of AChE by DMTP results in the accumulation of acetylcholine (ACh) in the synaptic cleft, and this mechanism is responsible for the high acute toxicity of DMTP in human and animals. DMTP ranks tenth among the compounds contributing to poisoning deaths due to accidents and suicide by pesticide ingestion in Japan (Kudo et al., 2010), and its lethal concentration in blood ranges from 1.8 to 66 μg/mL (Takayasu et al., 2012). In forensic toxicology, the accurate determination of drug concentration in post-mortem blood is imperative to assess addiction and death by poisoning. However, it has been reported that DMTP concentration in blood was decreased in vitro (Ageda et al., 2006). Thus, we assume that DMTP concentration in post-mortem blood cannot be accurately determined.

Fig. 1

Structures of methidathion (DMTP, a), malathion (b), and malathion adduct in HSA (K-adduct, c).

It is known that changes in blood drug concentration after death are attributed to the post-mortem redistribution of the drug to organs/tissues and the post-mortem decomposition by plasma and bacterial enzymes such as esterase (Peters and Steuer, 2018; Sastre et al., 2017). We have recently reported novel mechanisms for post-mortem changes caused by blood proteins (Yamagishi et al., 2021a, 2021b, 2021c). First, malathion, an organophosphorus pesticide, was specifically bound to human serum albumin (HSA), resulting in the decrease in blood concentration of its free form after death. Malathion (Fig. 1b) was preferably bound to the lysine (K) and cysteinylproline (CP) residues of HSA. The K-adduct and malathion-CP-adduct derived from malathion were increased in vitro in a dose-dependent fashion when malathion concentration was lower than the lethal dose. Furthermore, the K-adduct were also detected in post-mortem blood of an autopsied subject who died of intentional malathion ingestion. The determination of the K-adduct (Fig. 1c) would enable estimation of blood malathion concentration at the time of death. Second, the carbamate pesticide methomyl was specifically bound to hemoglobin (Hb), thereby resulting in the decrease in blood concentration of its free form after death. Furthermore, the anti-anxiety agent bromazepam was oxidatively degraded by Hb via the Fenton reaction. For the above mechanisms, one mechanism is speculated to explain changes in blood DMTP concentration after death. Organophosphorus compounds such as malathion, which have the same structural characteristics as DMTP, are bound to HSA and form amino acid adducts (Fu et al., 2020; John et al., 2018; Kranawetvogl et al., 2018; Yamagishi et al., 2021b). Thus, DMTP may also be bound to HSA. HSA is the most abundant protein in plasma and functions as a carrier protein for many endogenous and exogenous compounds in bloodstream (Er et al., 2013). Therefore, in this study, we intended to clarify the mechanisms underlying the interaction of DMTP with HSA.

We measured DMTP in HSA solution and evaluated DMTP-amino acid adducts in HSA by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). LC-Q/TOF-MS was used for the following reasons: (1) it met the analytical requirements for these evaluations, and (2) it was an effective technique for the evaluation of unidentified amino acid adducts because it provided the accurate molecular mass by TOF-MS and molecular information by tandem MS (Yamagishi et al., 2021a, 2021b).

Experimental

Ethics approval

This study was performed with approval from The Research Ethics Committee of the Graduate School of Pharmaceutical Sciences (Approval No. R001) and the School of Medicine (Approval No. 2819), Chiba University. Use of autopsy samples for analyses was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). We have included a disclaimer on our official website stating that we occasionally collect samples from autopsies for research use. Families who do not consent to it can freely opt out by informing us.

Chemicals

Acetonitrile (ACN, LC/MS grade) and distilled water (LC/MS grade) were purchased from Kanto Chemical (Tokyo, Japan). DMTP (TraceSure^®), formic acid (FA, 98–100%), 0.1 mol/L phosphate buffer (pH 7.4), 1 mol/L ammonium formate solution (HPLC grade) and trifluoroacetic acid (TFA, HPLC grade) were obtained from Fujifilm Wako Pure Chemical (Osaka, Japan). HSA, pronase, and Amicon ultra-0.5 centrifugal filter units with molecular weight cut-off of 10 kDa were purchased from Sigma-Aldrich (St. Louis, MO, USA). Diazepam-d5 was purchased from Hayashi Pure Chemical Ind., Ltd. (Osaka, Japan). Control human blood was purchased from BioIVT (London, UK).

Instrument

A liquid chromatograph hyphenated with a quadrupole/time-of-flight mass spectrometer was used. 5600+QTOF (AB Sciex, Foster City, CA, USA) was equipped with Nexera X2 (Shimadzu, Kyoto, Japan) as the liquid chromatograph. The electrospray ionization source parameters were as follows: ionspray voltage floating, 5500 V; temperature, 500ºC; ion source gas 1, 50 psi; ion source gas 2, 50 psi; curtain gas, 25 psi; declustering potential, 50 V; collision energy, 10 V; and m/z range, 100–1000. Chromatographic separation was accomplished with a CORTECS^® T3 C18 column (Waters, Milford, MA, USA, 100 × 2.1 mm I.D., 2.7 μm) maintained at 40°C. The mobile phase was composed of 0.1% formic acid and 10 mmol/L ammonium formate (A) and ACN (B), and was eluted in the following gradient: 0 to 12 min: 10–60% B; 12 to 18 min: 60–100% B; 18 to 23 min: 100% B; and 23 to 30 min: 10% B. The flow rate was 0.3 mL/min, and the injection volume was 2 or 20 μL.

Recovery of DMTP from reaction mixture with HSA

We used the reported concentrations of HSA (Yamagishi et al., 2021b). A 0.1 mL aliquot of a reaction mixture containing HSA (final concentration, 40 mg/mL) and DMTP (final concentration, 1 μg/mL) was incubated at 37ºC for 0 and 24 hr. After the incubation, diazepam-d5 (50 ng/mL) as the internal standard in 1.9 mL of ACN was added to the reaction mixture. The reaction mixture was vortexed, sonicated, and centrifuged at 10,000 x g for 10 min, and the supernatant was injected into the LC-Q/TOF-MS apparatus to determine DMTP concentration. The injection volume was 2 μL.

Detection, identification, and validation of DMTP adducts in HSA

A 0.1 mL aliquot of a reaction mixture containing 40 mg/mL HSA and 100 µg/mL DMTP in 0.1 mol/L phosphate buffer was incubated at 37°C for 24 hr. Enzyme digestion with pronase was performed according to previously reported methods (Yamagishi et al., 2021b). Briefly, 6 mg of pronase was added to the reaction mixture, and an overnight incubation was carried out at 37°C. To detect and identify DMTP adducts in HSA, 0.3 mL of 0.5% TFA was added to 0.1 mL of the reaction mixture after the second incubation. The reaction mixture was vortexed and centrifuged at 10,000 x g for 10 min, and the supernatant was injected into the LC-Q/TOF-MS apparatus. The injection volume was 2 μL.

For the validation of DMTP adducts in HSA, a 0.5 mL aliquot of a reaction mixture consisting 40 mg/mL HSA and 0.3, 1, 3, 10, 30, or 100 mg/mL DMTP in 0.1 mol/L phosphate buffer was incubated at 37°C for 24 hr. After the incubation, 0.5 mL of the reaction mixture was transferred into an ultrafiltration device (Amicon ultra-0.5 centrifugal filter unit) and centrifugation was performed at 10,000 x g for 10 min. The aliquot was washed three times with 0.3 mL of 0.1 mol/L phosphate buffer and centrifuged again at 10,000 x g for 10 min. Enzyme digestion with pronase was performed according to previously reported methods (Yamagishi et al., 2021b). Briefly, 30 mg of pronase was added to the reaction mixture, and an overnight incubation was carried out at 37°C. A 0.3 mL aliquot of 0.5% TFA was added to a 0.1 mL portion of the reaction mixture after the second incubation. The reaction mixture was vortexed and centrifuged at 10,000 x g for 3 min, and the supernatant was subjected to LC-Q/TOF-MS. The injection volume was 20 μL. Precursor ions of the lysine (K)-adduct, the DMTP-tyrosine (Y)-adduct, and the DMTP-cysteinylproline (CP)-adduct of the DMTP moiety were detected at m/z 271.1, m/z 306.1, and m/z 395.1, respectively. Product ions of the K-adduct, the DMTP-Y-adduct and the DMTP-CP-adduct were detected at m/z 271.0876, m/z 306.0560, and m/z 395.0512, respectively.

Detection of DMTP in post-mortem samples from DMTP poisoning subject and K-adduct, DMTP-Y-adduct and DMTP-CP-adducts in blood sample from DMTP poisoning subject

Femoral vein blood, heart blood, urine, and stomach content were collected from an 85-year-old man approximately 48 to 168 hr after death by intentional ingestion of DMTP-containing pesticide.

A 0.1 mL aliquot of the blood sample was transferred into an Amicon ultra-0.5 centrifugal filter unit. The aliquot was washed three times with 0.3 mL of 0.1 mol/L phosphate buffer and centrifuged at 10,000 x g for 10 min. 6 mg of pronase was added to the aliquot of the blood sample, and the blood sample was incubated at 37°C overnight. After 0.3 mL of 0.5% TFA was added to a 0.1 mL portion of the blood sample after the incubation, the aliquot was centrifuged again at 10,000 x g for 10 min. The reaction mixture was injected into the LC-Q/TOF-MS apparatus. The injection volume was 20 μL. Precursor ions of the K-adduct, the DMTP-Y-adduct, and the DMTP-CP-adduct were detected at m/z 271.1, m/z 306.1, and m/z 395.1, respectively. Product ions of the K-adduct, the DMTP-Y-adduct, and the DMTP-CP-adduct were detected at m/z 271.0876, m/z 306.0560, and m/z 395.0512, respectively.

Statistics

Data are expressed as means ± standard deviation. The Student’s t-test was performed for comparisons between two groups. Asterisks (* and **) denote significance at p < 0.05 and p < 0.01, respectively.

RESULTS

Stability of DMTP in HSA solution

DMTP concentration was decreased by incubation for 24 hr in 0.1 mol/L phosphate buffer, suggesting that a part of DMTP could be hydrolyzed in the buffer under the above-mentioned conditions. This experiment served as control (Fig. 2). DMTP concentration in 0.1 M phosphate buffer and HSA solution at 24 hr compared with that at 0 hr was 67.1% and 16.7%, respectively.

Fig. 2

Effect of HSA on DMTP concentration. One μg/mL DMTP was incubated in 0.1 mol/L phosphate buffer and HSA solution. The Student’s t-test was performed for comparisons between two groups. Double asterisks (**) indicate significant difference at p < 0.01.

Detection and identification of DMTP adducts in HSA

The difference analysis of the mass spectra of pronase-digested HSA amino acids and peptides with and without DMTP treatment was performed. Three specific ions detected in the DMTP-treated HSA were extracted at m/z 271.0865, 306.0562, and 395.0502. The first one at m/z 271.0865 was eluted at the retention time of 2.0 min and tentatively named DMTP-UK-1 (Fig. 3b). The second one at m/z 306.0562 was eluted at the retention time of 3.1 min and tentatively named DMTP-UK-2 (Fig. 3d). The third one at m/z 395.0502 was eluted at the retention time of 4.0 min and tentatively named DMTP-UK-3 (Fig. 3f). The peak appearing before 1 min seemed to be a non-specific signal and therefore, it was not taken into consideration.

Fig. 3

Elution profiles of ions extracted at m/z 271.0865 (a and b), 306.0562 (c and d), and 395.0502 (e and f) for pronase-digested HSA without DMTP treatment (a, c, and e) and with DMTP treatment (b, d, and f). Retention times of DMTP-UK-1, DMTP-UK-2, and DMTP-UK-3 were 2.0, 3.1, and 4.0, respectively.

The results of MS/MS analyses of DMTP-UK-1, DMTP-UK-2, and DMTP-UK-3 are shown in Figs. 4a, 4b, and 4c, respectively. In Fig. 4a, four ions were detected; the largest ion at m/z 271.0865 was the precursor ion (DMTP-UK-1), and this was renamed K1. Three fragment ions were detected at m/z 254.1014, 225.0808, and 208.0547, and these were named K2–K4 in decreasing order of fragment size. The assignments of these ions are summarized in Table 1. These assignments suggested that K-1 (DMTP-UK-1) was the K-adduct (Fig. 4a) in our previous study (Yamagishi et al., 2021b). In Fig. 4b, four ions were detected; the largest ion at m/z 306.0562 was the precursor ion (DMTP-UK-2), and this was renamed Y1. Three fragment ions were detected at m/z 289.0304, 260.0496, and 243.0230, and were these were named Y2–Y4 in decreasing order of fragment size. The assignments of these ions are sumarized in Table 2. The Δm/z values for Y1–Y4 were less than ± 3.7 ppm. These assignments unambiguously suggested that Y-1 (DMTP-UK-2) was 2-amino-3-(4-((dimethoxyphosphorothioyl)oxy)phenyl)propanoic acid, which was named the DMTP-Y-adduct (Fig. 4b). In Fig. 4c, one precursor and four fragment ions were detected. The ion detected at m/z 395.0502 was the precursor ion (DMTP-UK-3), and this was renamed CP1. Four fragment ions were detected at m/z 349.0428, 263.0502, 217.0618, and 145.0101, and these were named CP2–CP5 in decreasing order of fragment size. The assignments of these ions are sumarized in Table 3. These assignments suggested that CP-1 (DMTP-UK-3) was S-(((5-methoxy-2-oxo-1,3,4-thiadiazol-3(2H)-yl)methyl)thio)cysteinylproline, which was named the DMTP-CP-adduct (Fig. 4c).

Fig. 4

MS/MS spectra of unknown DMTP adducts detected in the positive ion mode. Unknown peaks DMTP-UK-1 (a), DMTP-UK-2 (b), and DMTP-UK-3 (c) detected in Fig. 3 were subjected to LC-Q/TOF-MS analysis and assigned.

Table 1. Assignment of precursor and product ions of K-adduct.

Peak label	Elemental composition	m/z theoretical	m/z measured	Δ m/z (ppm)
K1	C8H20N2O4PS	271.0876	271.0865	−4.1
K2	C8H17NO4PS	254.0610	254.1014	159.0
K3	C7H18N2O2PS	225.0821	225.0808	−5.8
K4	C7H15NO2PS	208.0556	208.0547	−4.3

Table 2. Assignment of precursor and product ions of DMTP-Y-adduct.

Peak label	Elemental composition	m/z theoretical	m/z measured	Δ m/z (ppm)
Y1	C12H19N4O5S3	306.0560	306.0562	0.7
Y2	C11H17N4O3S3	289.0294	289.0304	3.5
Y3	C9H15N2O3S2	260.0505	260.0496	−3.5
Y4	C4H5N2O2S	243.0239	243.0230	−3.7

Table 3. Assignment of precursor and product ions of DMTP-CP-adduct.

Peak label	Elemental composition	m/z theoretical	m/z measured	Δ m/z (ppm)
CP1	C12H19N4O5S3	395.0512	395.0502	−2.5
CP2	C11H17N4O3S3	349.0457	349.0428	−8.3
CP3	C9H15N2O3S2	263.0519	263.0502	−6.5
CP4	C8H13N2O3S	217.0641	217.0618	−10.6
CP5	C4H5N2O2S	145.0066	145.0101	24.1

Validation of LC-Q/TOF-MS and detection of DMTP in post-mortem samples from DMTP poisoning subject and K-adduct, DMTP-Y- and DMTP-CP-adduct in blood sample from DMTP poisoning subject

The K-adduct, DMTP-Y-adduct, and DMTP-CP-adducts were detectable at 3.0, 1.0, and 0.3 μg/mL or higher concentrations, respectively (Fig. 5). The relationship between DMTP concentration and the peak areas of the K-adduct, DMTP-Y-adduct and DMTP-CP-adducts showed good linearity, i.e., the correlation coefficients (r²) were 1.000, 0.994, and 0.998, respectively.

Fig. 5

Relationship between DMTP concentration and peak areas of K-adduct (a), DMTP-Y-adduct (b), and DMTP-CP-adduct (c).

DMTP concentrations in femoral vein blood, heart blood, and stomach content collected from an 85-year-old man who died of intentional DMTP ingestion were 3.6, 6.2 and 6,475 μg/mL, respectively. DMTP was not detected in urine (Table 4).

Table 4. DMTP concentrations in autopsied subject who died of intentional DMTP poisoning (μg/mL).

Femoral vein blood	Heart blood	Urine	Stomach content
3.6	6.2	N.D.	6,475

N.D.: not detected

Neither DMTP-Y- nor DMTP-CP-adduct was detected in control human blood (Figs. 6a and 6c). The DMTP-Y- and DMTP-CP-adducts were detected in blood collected from the autopsied subject mentioned above (Figs. 6b and 6d). On the other hand, the K-adduct was not detected in control human blood or blood collected from the autopsied subject mentioned above (data not shown). Decrease concentration of DMTP in this blood sample mentioned above after death was estimated to be 1.1 μg/mL on the basis of the amount of DMTP-CP-adduct in blood.

Fig. 6

Elution profiles of DMTP-Y-adduct and DMTP-CP-adduct in pronase-digested HSA from control human blood (a and c) and DMTP poisoning subject (b and d).

DISCUSSION

We speculated that other organophosphorus pesticides having structures in common with malathion would also produce similar adducts in HSA. Indeed, we found the mechanism by which HSA is implicated in the post-mortem metabolism of DMTP, namely, the direct interaction of DMTP with the amino acid residues of HSA. As the most abundant protein in blood plasma, HSA would probably play an important role in the disappearance of DMTP in post-mortem blood.

It has been reported that the phosphoryl moiety of DMTP is bound to the serine residue in AChE (Fukuto, 1990). The formation of HSA amino acid adducts with various substances such as 1,2-benzoquinone and ethylene oxide is also known (Rubino et al., 2009). Using LC-Q/TOF-MS, we found that DMTP formed adducts with the K, Y, and CP residues of HSA, namely, DMTP was divided into two moieties: the phosphorothionyl moiety, which was bound to the ε-amino group of the K and Y residues, and the alkylthiol moiety, which was bound to the thiol group of the CP residue. To our knowledge, this is the first observation that DMTP was bound to amino acids other than serine. These amino acid residues have also been reported to bind to other organophosphorus compounds (Fu et al., 2020; John et al., 2018; Kranawetvogl et al., 2018; Yamagishi et al., 2021b). Thus, these amino acid residues were considered to be of reactive with organophosphorus compounds and/or their metabolites. Because only one CP residue was found in the HSA sequence, we concluded that DMTP was specifically bound to ³⁴Cys in HSA. In contrast to the CP residue, many K and Y residues were found in the HSA sequence. A previous report has shown that organophosphorus compounds were preferably bound to several K and Y residues of HSA (Fu et al., 2020). We intend to clarify whether DMTP is bound to a specific K or Y residue in HSA in our future study.

DMTP concentration in post-mortem blood is important for determining death by DMTP poisoning. The K-adduct, DMTP-Y-adduct and DMTP-CP-adducts were specifically detected by LC-Q/TOF-MS, and their quantification was well validated. The lethal DMTP dose in blood was higher than 1.6 μg/mL (Takayasu et al., 2012), which meant that the detection of DMTP-Y-adduct and DMTP-CP-adduct instead of DMTP could be used to determine death by DMTP poisoning. In other words, both DMTP-Y-adduct and DMTP-CP-adduct could be used as a biomarker of DMTP poisoning, and the decrease of DMTP concentration after death could be calculated from the concentrations of the DMTP-Y-adduct and DMTP-CP-adduct. We recently found that the decrease of concentration of malathion, which is the same type of pesticide as DMTP, could be calculated from the concentration of a specific adduct in HSA (Yamagishi et al., 2021b).

Although measuring the inhibition of AChE activity in blood of an autopsied subject is effective to determine DMTP poisoning, a more precise method for the quantification of DMTP is desired to unambiguously determine the cause of death by DMTP poisoning. In this study, we found that blood DMTP concentration in DMTP poisoning subject was higher than the lethal DMTP dose (Table 4). On the other hand, the decrease concentration of DMTP (1.1 μg/mL) was calculated from the amount of the DMTP-CP-adduct. In the previous study, substantial amounts of DMTP was decreased in blood in vitro (Ageda et al., 2006). This proved that DMTP concentration in post-mortem blood could not be accurately determined when DMTP per se in post-mortem blood was measured, and the DMTP-CP-adduct could be used as a biomarker of DMTP poisoning in clinical practice.

In conclusion, DMTP concentration in post-mortem blood was decreased because DMTP formed adducts with the amino acid residues of HSA, such as lysine (K), tyrosine (Y), and cysteinylproline (CP). Because the DMTP-Y-adduct and DMTP-CP-adduct were detected in blood of autopsied subject who died of intentional DMTP ingestion, the DMTP-Y-adduct and DMTP-CP-adducts could be used as a biomarker of DMTP poisoning. In addition, we were able to calculate blood DMTP concentration on the basis of the amount of CP-adduct in blood. We expect that other organophosphorus pesticides having structures in common with DMTP and malathion would also produce similar adducts in HSA.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Shigeki Tsuneya for supplying samples of an autopsied subject who died of intentional malathion ingestion.

Conflict of interest

The authors declare that there is no conflict of interest.

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