Simultaneous determination of water-soluble herbicides using hydrophilic interaction liquid chromatography-mass spectrometry.

PURPOSE: The analysis of water-soluble herbicides, including glyphosate (Glyp), glufosinate (Gluf), paraquat (PQ), and diquat (DQ), is time-consuming and expensive because they cannot be analyzed using general toxicological screening methods. Thus, this study aimed to develop a simple and rapid method to simultaneously analyze these compounds without any derivatization nor ion-pairing reagents. METHODS: The analytes were separated using hydrophilic interaction liquid chromatography and detected using tandem mass spectrometry. The developed method was applied to plant and biological samples assuming criminal damage and poisoning cases, respectively. RESULTS: All analytes were separated well and detected with good peak shapes. For plant samples, the herbicides were specifically detected from withered leaves using a simple extraction method. For biological samples, quantitative analysis was successfully validated, and the limit of quantification values of Glyp and Gluf were 0.2 µg/mL, and those of PQ and DQ were 1 ng/mL. CONCLUSION: The developed method had sufficient performance for practical forensic applications including poisoning cases and malicious uses to damage commercial crops.

No promising antidote 25 years after the Tokyo subway sarin attack: A review.

On the battlefields of Syria, many innocent civilians have been killed or injured by sarin poisoning. In Malaysia in February 2017, a North Korean man was assassinated with VX at Kuala Lumpur International Airport. In the face of such threats, a more effective antidote against organophosphonate acetylcholinesterase (AChE) inhibitors is needed, one that can freely penetrate into the central nervous system (CNS) through the blood-brain barrier (BBB). In the 1995 Tokyo subway sarin attack, which produced more than 6,000 victims, 2-pyridinealdoxime methiodide was the most commonly used antidote in hospitals, but it was unable to prevent CNS damage and no other oximes have been approved for use in Japan. Ultimately, 12 people died, and many victims had severe neurological injuries or sequelae. Although more than 25 years have passed since the incident, progress has been slow in the development of a new antidote that can penetrate the BBB, restore AChE activity in the CNS, and definitely prevent brain injury. From the perspectives of countering terrorism and protecting innocent people from nerve agent attacks, the search for nerve agent antidotes should be accelerated with the goals of improving both survival and quality of life. This review gives an overview of a series of our studies on the development of a new antidote since the Tokyo subway sarin attack and emphasizes that there is unfortunately still no promising antidote for saving the CNS in Japan.

Species identification of white false hellebore (Veratrum album subsp. oxysepalum) using real-time PCR.

Food poisoning is frequently caused by the accidental ingestion of toxic plants that possess strong morphological similarities to edible plants. False helleborine (Veratrum album) is one of the most common plants involved in such accidents. In cases of poisoning by toxic plants, rapid and accurate identification, usually based on the morphological or chemical analysis of plant parts, is required for appropriate medical treatment or forensic investigation. However, morphological examinations require experience in systematic botany because the samples are fragmentary, and chemical analysis of natural compounds can be difficult. In this study, we developed a TaqMan real-time PCR method using trnH-psbA and trnL-trnF that could be carried out in 30-60min. The lower detection limit was less than 10pg of DNA and the primer sets were specific to V. album and Veratrum stamineum. Mixed samples, cooked samples, and simulated gastric contents were successfully identified, and a multiplex assay of two regions was also possible. These results indicate that the TaqMan real-time PCR analysis is a very effective method to detect small samples of V. album and V. stamineum accurately and rapidly in poisoning cases.

The forgotten or underestimated relevance of biopharmaceutical-based assessments for the oral absorption studies of oxime reactivators.

INTRODUCTION: The absorption, distribution, metabolism, excretion and toxicity (ADME(T)) of oxime reactivators have been assessed with respect to their polarity, a fundamental requirement for their specific mechanism of action in the intoxication with organophosphorous compounds. The limitations of the therapeutic outcome have been associated not only with the severity of intoxication and to particularities of the toxicants, but also to the reduced lipophilicity and consequent restricted permeability across biological barriers. AREAS COVERED: This article inventories the plethora of mnemotic rules developed throughout the years for defining chemical spaces where drugs share one or more structural and ADME(T) characteristics. Their applicability to oxime is analyzed, especially in relation to intestinal absorption and brain distribution. Other aspects of oximes for antidotal outcome are also reviewed. EXPERT OPINION: The drugability rules are not applicable to oxime reactivators, because the increase in lipophicity and consequent improved permeability across biological barrier comes together with amplified (neuro)toxicity and reduced reactivating capacity. The available data suggest a high solubility and reduced metabolism, assigning the quaternary oximes to the fourth class of Biopharmaceutical Classification Systems. Reliance upon oral absorption data for designing safe centrally acting oximes can be of potential value, with adequate characterization of uptake-influx transporters interplay.

Genotyping of Toxic Pufferfish Based on Specific PCR-RFLP Products As Determined by Liquid Chromatography/Quadrupole-Orbitrap Hybrid Mass Spectrometry.

A method based on liquid chromatography-electrospray mass spectrometric analysis of the enzymatically digested amplicons derived from the mitochondrial 16S rRNA gene was established for the discrimination of toxic pufferfish. A MonoBis C18 narrow-bore silica monolith column (Kyoto Monotech) and a Q Exactive mass spectrometer (Thermo Fisher) were employed for separation and detection, respectively. Monoisotopic masses of the oligonucleotides were calculated using Protein Deconvolution 3.0 software (Thermo Fisher). Although a lock mass standard was not used, excellent accuracy (mass error, 0.83 ppm on average) and precision (relative standard deviation, 0.49 ppm on average) were achieved, and a mass accuracy of <2.8 ppm was maintained for at least 180 h without additional calibration. The present method was applied to 29 pufferfish samples, and results were consistent with Sanger sequencing.

Solid-phase extraction of phosphorous-containing amino acid herbicides from biological specimens with a zirconia-coated silica cartridge.

We report a rapid solid-phase extraction method for glyphosate (Glyp), glufosinate (Gluf), and bialaphos (Bial) using a zirconia-coated silica cartridge, which interacts specifically with phosphorous-containing amino acid herbicides (PAAHs). We extracted PAAHs from serum and urine samples. The PAAHs were derivatized with trimethyl orthoacetate-acetic acid and analyzed by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). The intra-day and inter-day accuracy was within ±13% RE, the intra-day and inter-day precision was less than 12% RSD, and the total recovery was more than 60% for Glyp and more than 80% for Gluf and Bial. The linearity ranges of the calibration curves of the serum samples were 0.2-10,000µg/mL for Glyp, 0.1-1000µg/L for Gluf, and 0.5-1000µg/L for Bial; and those of the urine samples were 0.4-20,000µg/L for Glyp, 0.2-2000µg/L for Gluf, and 0.1-2000µg/L for Bial. This range covers almost all the reported poisoning cases involving these compounds, from very mild to fatal cases. The present paper offers a universal cleanup method for PAAHs in serum and urine samples for clinical and forensic analysis.

Simultaneous determination of neonicotinoid insecticides in human serum and urine using diatomaceous earth-assisted extraction and liquid chromatography-tandem mass spectrometry.

A rapid and sensitive analytical method was developed for simultaneous determination of eight neonicotinoid insecticides (acetamiprid, clothianidin, dinotefuran, flonicamid, imidacloprid, nitenpyram, thiacloprid and thiamethoxam) and three specific metabolites of acetamiprid (N-desmethylacetamiprid, 5-(N-acetyl-N-methylaminomethyl)-2-chloropyridine and 5-(N-acetylaminomethyl)-2-chloropyridine) in human serum and urine. A diatomaceous earth-assisted extraction using Extrelut NT3 column with chloroform/2-propanol (3:1, v/v) as eluent was selected for the single step cleanup procedure for all the target compounds. Qualitative and quantitative analyses were conducted by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring mode. The limits of detection and the limits of quantification of eleven compounds were in the ranges of 0.1-0.2ng/mL and 0.5-10ng/mL for serum, 0.1-1ng/mL and 1-10ng/mL for urine, respectively. The extraction recoveries were between 80.9% and 101.8% for serum samples, 91.9% and 106% for urine samples. The intra-day RSDs and the inter-day RSDs were less than 11.5% and 13.2% for serum, less than 8.3% and 8.8% for urine. The proposed procedure will be suitable for forensic investigations of human poisoning cases with neonicotinoid insecticides. This is the first report of simultaneous determination of eight neonicotinoids in serum and urine samples.

Lipophilicity indices derived from the liquid chromatographic behavior observed under bimodal retention conditions (reversed phase/hydrophilic interaction): application to a representative set of pyridinium oximes.

The liquid chromatographic behavior observed under bimodal retention conditions (reversed phase and hydrophilic interaction) offers a new basis for the determination of some derived lipophilicity indices. The experiments were carried out on a representative group (30 compounds) of pyridinium oximes, therapeutically tested in acetylcholinesterase reactivation, covering a large range of lipophilic character. The chromatographic behavior was observed on a mixed mode acting stationary phase, resulting from covalent functionalization of high purity spherical silica with long chain alkyl groups terminated by a polar environment created through the vicinal diol substitution at the lasting carbon atoms (Acclaim Mixed Mode HILIC 1 column). Elution was achieved by combining different proportions of 5 mM ammonium formiate solutions in water and acetonitrile. The derived lipophilicity indices were compared with logP values resulting from different computational algorithms. The correlations between experimental and computed data sets are significant. To obtain a better insight on the transition from reversed phase to hydrophilic interaction retention mechanisms, the variation of the thermodynamic parameters determined through the van׳t Hoff approach was also discussed.

Liquid chromatography-tandem mass spectrometry method for determination of the pyridinium aldoxime 4-PAO in brain, liver, lung, and kidney.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated and applied to the in vitro determination of 4-[(hydroxyimino)methyl]-1-octylpyridinium cation (4-PAO), which can penetrate the blood-brain barrier and reactivate acetylcholinesterase (AChE) inhibited by alkylphosphonate in the brain, liver, lung, and kidney. The limit of detection (LOD) was 0.235 microg cation/g wet weight, and the quantification range and linearity of the calibration curve extended over a range of 0.470-941 microg cation/g wet weight. For the proof of applicability, when 4-PAO was administrated intravenously via the rat tail vein at 10% LD(50), we were able to quantify the 4-PAO concentration in the tissues: brain 7.60+/-1.32 microg cation/g wet weight (mean+/-SD, n=5), liver 26.8+/-2.82 microg cation/g, lung 76.4+/-24.9mugcation/g, and kidney 638+/-266 microg cation/g. In addition, the methods for 4-[(hydroxyimino)methyl]-1-decylpyridinium bromide (4-PAD) and 4-[(hydroxyimino)methyl]-1-(2-phenylethyl) pyridinium bromide (4-PAPE) were partly validated referring to the findings of the 4-PAO full validation. Thus, the LC-MS/MS method described in this study can be useful for quantification of pyridinium aldoxime methiodide (PAM)-type oximes in biological samples.

Effects of oximes on mitochondrial oxidase activity.

Oximes, including 2-pyridinealdoxime methiodide (2-PAM), are reactivators of acetylcholinesterase (AChE) inhibited by organophosphate poisoning. Unfortunately, their clinical use has been limited by their toxicity. To investigate the mechanism of this toxicity, the effects of oximes on the enzymes choline oxidase (ChOD) and cytochrome c oxidase (CyCOD) of the respiratory chain in mitochondria were examined. The oximes 2-PAM, obidoxime, and diacetylmonoxime significantly (P<0.01) inhibited ChOD activity, and the extent of inhibition correlated with the ability to reactivate inhibited AChE. When ChOD activity in mitochondrial extracts was tested, 2-PAM inhibited the activity by 75%, obidoxime and diacetylmonoxime did not significantly inhibit it, and 4-[(hydroxy-imino)methyl]-1-decylpyridinium bromide (4-PAD), which has greater toxicity, increased the amount of product generated in the assay to approximately 200% of normal levels. Similarly, 2-PAM inhibited the activity of CyCOD in mitochondrial extracts whereas obidoxime and diacetylmonoxime did not. One explanation for these findings is that, in addition to their inhibition of mitochondrial oxidases, the oximes may produce excessive reactive oxygen species such as H(2)O(2) in the mitochondrial fraction, which may account for some of their toxicity. This is a preliminary report related to the toxicities of oximes that may participate in the inactivation of mitochondrial oxidase enzymes. This hypothesis should be further investigated by in vivo study, including kinetic determination and free radical work.

Thin layer chromatography/fluorescence detection of 3,4-methylenedioxy-methamphetamine and related compounds.

A rapid and sensitive method for the detection of six methylenedioxylatedphenethylamines, 3,4-methylenedioxymethamphetamine (MDMA); 3,4-methylenedioxyamphetamine; 3,4-methylenedioxyethylamphetamine; N-methyl-1-(3,4-methylenedioxyphenyl)-2-butamine; N-methyl-1-(3,4-methylenedioxyphenyl)-3-butamine; and 3,4-methylenedioxydimethylamphetamine, by thin-layer chromatography with fluorescence detection is proposed. These compounds form fluorophores on the developing plate following spraying with a reagent consisting of sodium hypochlorite, potassium hexacyanoferrate (III), and sodium hydroxide, and heating for 3 min at 100 degrees C. Blue fluorescent spots were observed under ultraviolet light in a wavelength range of 250-400 nm. The detection limits for MDMA and the above related compounds were 50 ng. The proposed method was effectively applied to the detection of MDMA in urine samples.

Blood-brain barrier penetration of novel pyridinealdoxime methiodide (PAM)-type oximes examined by brain microdialysis with LC-MS/MS.

To develop a new reactivator of inhibited acetylcholinesterase (AChE) that can easily penetrate the blood-brain barrier (BBB), BBB penetration of 6 known and novel pyridinealdoxime methiodide (PAM)-type oximes (alkylPAMs) with relatively high reactivation activities was examined by in vivo rat brain microdialysis with liquid chromatography-mass spectrometry (LC-MS/MS). The 50% lethal dose (LD(50)) of alkylPAMs was intravenously determined for Wistar rats, then the limit of detection, quantification range and linearity of the calibration curve of the alkylPAMs in dialysate and blood were determined by LC-MS/MS. Following 10% LD(50) intravenous administration of the alkylPAMs, 4-[(hydroxyimino) methyl]-1-(2-phenylethyl) pyridinium bromide (4-PAPE) and 4-[(hydroxyimino) methyl]-1-octylpyridinium bromide (4-PAO) appeared in the dialysate. Striatal extracellular fluid/blood concentration ratios were 0.039+/-0.018 and 0.301+/-0.183 (mean+/-SEM), respectively, 1 h after treatment. This is the first report of BBB penetration of 4-PAPE, and the concentration ratio was smaller than that of 2-PAM. The mean BBB penetration of 4-PAO was approximately 30%, indicating that intravenous administration of 4-PAO may be effective for the reactivation of blocked cholinesterase in the brain. However, the toxicity of 4-PAO (LD(50); 8.89 mg/kg) was greater than that of 2-PAM. Further investigation is required to determine the effects of these alkylPAMs in organophosphate poisoning.

Rapid and sensitive determination of tryptophan, serotonin and psychoactive tryptamines by thin-layer chromatography/fluorescence detection.

A rapid, sensitive and selective method for the determination of tryptophan (Trp), serotonin (5-HT) and psychoactive tryptamines (PATs) by thin-layer chromatography (TLC) with fluorescence detection is proposed. These compounds form fluorophores on the developing plate by heating after spraying with sodium hypochlorite, hydrogen peroxide or potassium hexacyanoferrate(III)-sodium hydroxide reagent. Fluorescent spots (vivid blue) were observed by irradiation with ultraviolet light (365 nm). The detection limits of Trp, 5-HT and PATs were in the range from 0.01 microg to 0.06 microg. This method was effectively applied to the detection of confiscated PAT powder and PAT in abusers' urine samples.

New safe method for preparation of sarin-exposed human erythrocytes acetylcholinesterase using non-toxic and stable sarin analogue isopropyl p-nitrophenyl methylphosphonate and its application to evaluation of nerve agent antidotes.

INTRODUCTION: A non-toxic and stable sarin analogue, isopropyl p-nitrophenyl methylphosphonate (INMP), was synthesized for safe preparation of sarin-exposed acetylcholinesterase (AChE). RESULTS AND DISCUSSION: This agent was stable for years, able to be handled in an ordinary laboratory without special care, and its 50% inhibitory concentration (IC50) on 0.04 U/ml human erythrocytes AChE was 15 nM. This reagent was thought to be especially useful since it enables experiments that require sarin-inhibited AChE, such as the development of antidotes for sarin, in a usual laboratory. To demonstrate the usefulness of this method, 40 known and novel pyridinealdoxime methiodide (PAM)-type oxime antidotes were synthesized, and their reactivation activities to INMP-exposed AChE and structure-activities correlation were studied. CONCLUSION: Among the antidotes tested in this experiment except for 2-PAM, the compound found to have the highest reactivation activity, was the novel hydrophobic 2-PAM-type compound, 2-[(hydroxyimino)methyl]-1-[4-(tert-butyl)benzyl] pyridinium bromide.

Hydrolysis of an acetylthiocholine by pralidoxime iodide (2-PAM).

Pralidoxime iodide (2-PAM), an antidote approved for the reactivation of inhibited acetylcholinesterase (AChE) in organophosphate poisoning, dose-dependently hydrolyzed an acetylthiocholine iodide (ASCh). The AChE (0.3 U) activity inhibited by VX analog (ENMP, 0.1 microM) increased to approximately 200% of normal levels after a dosage of 5 mM 2-PAM (control 0.132+/-0.012 U/ml, 5 mM 0.253+/-0.026 U/ml). This result indicates that 2-PAM produced a thiocholine from the ASCh by hydrolysis. High-performance liquid chromatography (HPLC) analysis was then performed to further clarify the hydrolysis of ASCh with 2-PAM. It was clear that 2-PAM was converted to acetylated 2-PAM with acetic acid produced from ASCh by hydrolysis. Next, we tried to compare this esterase-like activity of 2-PAM with that of obidoxime, which is known as a strong reactivator of inhibited AChE, and with diacetylmonoxime, known as a weak reactivator. All of these oximes showed esterase-like activity, and their strengths were consistent with those of known reactivators of inhibited AChE. These results indicate that a great deal of the data obtained previously with ASCh relating to the effects of oximes must be rechecked. It is clear that oximes easily hydrolyze ASCh. We therefore strongly caution that the method of determining AChE activity with ASCh is not suitable for examining the effects of oximes.

Evaluation of a quantitative method for trace impurities in arsenous acids by synchrotron radiation X-ray fluorescence spectrometry and inductively coupled plasma-atomic emission spectrometry.

Thirteen arsenous acid samples of known origins and refining methods were collected. Each sample was subjected to quantitative analysis of any impurity elements present using synchrotron radiation X-ray fluorescence spectrometry (SR-XRF) and inductively coupled plasma-atomic emission spectrometry (ICP-AES). The trace elements selected were Sn, Sb and Bi for the reasons that they were considered not to be changed by their circumstances and that they showed high sensitivity to SR-XRF. These results obtained by both methods were compared and the correlation between these two methods was determined. The quantification of trace impurities obtained by SR-XRF using As as an internal standard showed good agreement with the results obtained by ICP-AES. The discrimination of refining method became possible by the comparison of these impurities' contents measured with non-destructive SR-XRF using several arsenous acid particles.

5-(4-Nitrophenyl)-2,4-pentadien-1-al (NPPD) used as a tracer by nonscientists at a simulated crime scene.

5-(4-Nitrophenyl)-2,4-pentadien-1-al (NPPD) was used as a tracer by nonscientists at a simulated crime scene. A card with both a plastic-coated smooth surface and a porous cellulose matrix paper surface was coated with a methanol solution containing 0.5mg/mL of NPPD. The card was touched with bare fingers and fingers covered by a cotton glove. A color-change protocol was then used to detect the presence of NPPD. The bare fingers or the fingers of gloves were swabbed with a cotton swab, or the parts of the glove that had touched the card were cut out. The swabs or the cloth pieces were dipped in methanol, a 0.1% methanol solution of naphthoresorcinol was added, and then concentrated hydrochloric acid was added. The observation of a red color at this point indicated a positive test. NPPD was easily observed in the experiments involving bare fingers, but no color change was observed from the swabbing of the cotton glove. However, when the cloth pieces cut from the fingers of the glove were subjected to the test, the red color was observed. In an attempt to enhance the sensitivity of the test, the volumes of the reagent solutions were reduced, but no improvement in sensitivity was obtained.

Discrimination of arsenous acids with comparison of trace elements contained by using synchrotron X-ray fluorescence spectrometry.

In the poisonous incident case occurred in Japan, clarification of the identity between seized poisons and retrieved from crime scene is strictly required from the court. In this case, arsenous acid was used as a poisonous material and, seized one from suspect's house was only twenty particles. The synchrotron X-ray fluorescence spectrometry by comparing the intensity ratios of L(alpha) line of four heavy metal, such as Bi, Sn, Sb, Se to K(beta) line of As was performed to overcome this problem. In this paper, the evaluation of this new method using 13 authentic arsenous acid samples, 4 of 13 were refined by Chinese method, 7 of 13 were refined by Japanese method (Sumitomo mining Co. Ltd. method), 2 of 13 were refined by German and Swiss method. As a result, by the comparison of the ratios of these four elements to As, these 13 samples were clearly classified to three products classes produced by different refining methods.

Evaluation of 5-(4-nitrophenyl)-2,4-pentadien-1-al (NPPD) as a tracer for shadowing pursuits.

The chemical compound 5-(4-nitrophenyl)-2,4-pentadien-1-al (NPPD), called "spy dust," was used in the Soviet Union as a shadowing pursuit, the act of following someone secretly, for investigating the activities of diplomatic personnel. It is also useful for counter-terrorism, and some criminal cases in the forensic science field. In this paper, it was synthesized and evaluated as a tracer for shadowing pursuits. The method for utilizing this reagent was very simple: it was dissolved in methanol (1 mg/mL) and sprayed on the restricted area. If the suspect was to enter this area or touched the sprayed material, NPPD was attached to the suspect's shoe surface or hands. The color examination was a two-steps process: first was the addition of 1 mL of a 0.1% naphthoresorcinol methanol solution to the methanol extracts of a methanol-contained cotton swab used to smear some surfaces of the suspect, and second, the addition of 1 mL of concentrated hydrochloric acid, which turned the solution dark red. The gamma max of the colored solution was 510 nm, measured by ultraviolet-vis spectroscopy. Detection limits for three methods were determined: a visual method (detection limit 100 ng/3 mL), an ultraviolet-visible spectrometric method (detection limit 10 ng/3 mL), and a selected-ion-monitoring gas chromatographic/mass spectrometric method (detection limit 300 pg/injection). The forensic utility of NPPD was demonstrated for two simulated cases: a theft case and a case where NPPD was used as a tracer to prove that an automobile had entered a restricted area. These examinations prove NPPD is a useful shadowing pursuit (spy dust) for the forensic science field.