Kiyotaka Usui

Rapid drug extraction from human whole blood using a modified QuEChERS extraction method

A modified QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction method followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the simultaneous determination of forensically important drugs and poisons (more than 90 compounds) in human whole blood. Because the QuEChERS method is commonly used for the analysis of pesticide residues in foods, we customized the QuEChERS method for forensic use. This extraction method consists essentially of two steps: (1) extraction/partitioning and (2) dispersive-solid phase extraction. In step 1, three-fold diluted blood was mixed with an internal standard (D5-diazepam for basic drugs or D5-phenobarbital for acidic drugs) solution, a QuEChERS pre-packed extraction kit (containing magnesium sulfate and sodium acetate) and a stainless steel bead, then partitioned into three layers by centrifugation. In step 2, the top layer (acetonitrile) was transferred into a centrifuge tube containing a dispersive-solid phase sorbent (containing primary secondary amine, end-capped octadecylsilane, and magnesium sulfate) and mixed for purification. After the centrifugation, supernatant was injected into LC-MS/MS. The QuEChERS method was applied in an autopsy case and we confirmed that this method can easily extract various types of drugs and metabolites from human whole blood. The combination of the modified QuEChERS method and LC-MS/MS could enable technicians inexperienced in forensic toxicological analysis to acquire reliable data quickly and easily.

Rapid determination of disulfoton and its oxidative metabolites in human whole blood and urine using QuEChERS extraction and liquid chromatography–tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of disulfoton and five of its oxidative metabolites (disulfoton-sulfoxide, disulfoton-sulfone, demeton-S, demeton-S-sulfoxide and demeton-S-sulfone) in human whole blood and urine. Extraction was undertaken using a QuEChERS method, which is commonly used in food analysis. D10-Disulfoton was used as the internal standard. Separation was carried out using a CAPCELL-PAK MG II column (35×2.0 mm i.d., 5 μm, Shiseido) with a mobile phase of 10 m mol/L ammonium formate and methanol. This method was applied in an autopsy case, and disulfoton and its oxidative metabolites were successfully detected in both blood and urine. The concentrations of disulfoton in the blood and urine were 360 and 23.8 ng/mL, respectively. There was a relatively low concentration of demeton-S in both the blood (4.0 ng/mL) and urine (45.7 ng/mL). To date, there have been no reported cases of detection of demeton-S in human samples.

Simultaneous determination of 11 aconitum alkaloids in human serum and urine using liquid chromatography–tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of four aconitines (aconitine, mesaconitine, hypaconitine, jesaconitine) and seven of their hydrolysis products (benzoylmesaconine, benzoylhypaconine, 14-O-anisoylaconine, benzoylaconine, aconine, mesaconine, hypaconine) in human serum and urine samples. Extraction was undertaken using a mixed-mode cation-exchange cartridge (OASIS MCX), and D(5)-aconitine was used as an internal standard. Separation of aconitum alkaloids was carried out using an L-column ODS with the mobile phase consisting of 10mM ammonium formate and methanol. The intra- and inter-day precisions were 0.3% to 9.9% and 3.2% to 12.8%, respectively. Intra- and inter-day accuracies were -14.1% to 7.3%, and -10.6% to 8.3%, respectively. The limit of detection and limit of quantification of analytes were 0.04-0.38 ng/mL and 0.12-1.15 ng/mL respectively. This method was applied in an autopsy case and successfully detected aconitines and their metabolites as well as some anti-psychiatric drugs.

Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case

We report here the quantitative analysis of cathinone-type designer drug 3,4-dimethylmethcathinone (3,4-DMMC) in blood and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a fatal case. Abuse of 3,4-DMMC is widespread and a global issue. However, to date, there have been no reports of 3,4-DMMC-related deaths. We encountered a death in which 3,4-DMMC was thought to play a causative role, and successfully identified this designer drug from biological samples by using LC-MS/MS and QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction method. For standard samples, detection of 3,4-DMMC in human blood and urine samples in the calibration range (5-400 ng/mL) was successful with recoveries of 85.9-89.4% (blood) and 95.8-101% (urine), limits of detection of 1.03 (blood) and 1.37 ng/mL (urine) and limits of quantification of 5.00 (blood) and 5.38 ng/mL (urine). The concentrations of 3,4-DMMC in blood (external iliac vein) and urine in the case were 27 mg/L and 7.6 mg/L, respectively. Some metabolites, including 3,4-dimethylcathione (DMC) and β-ketone reduced metabolites (β-OH-DMMC and β-OH-DMC), were detected in both blood and urine.

Clinical and toxicological findings of acute intoxication with synthetic cannabinoids and cathinones

Reporting of the analytical and clinical findings of synthetic cannabinoids and cathinones is essential in carrying out a complete clinical assessment of new psychoactive substances.

Acute cholinergic syndrome in a patient with mild Alzheimer’s type dementia who had applied a large number of rivastigmine transdermal patches on her body

Abstract Case presentation: A 91-year-old woman was transferred to our Emergency Medical Center and Poison Center with somnolence, hypertension (186/61 mm Hg), and repeated vomiting. Three hours later, 10 transdermal patches, each containing 18 mg of rivastigmine (9.5 mg/24 h), were found on her lower back and both thighs, when miosis, facial and trunk sweating, enhanced bowel sound, hypertension, and sinus tachycardia were noted. She was diagnosed with acute cholinergic syndrome due to rivastigmine poisoning. Her hypertension and sinus tachycardia peaked 8 and 5 h after all the patches were removed, respectively. Her symptoms subsided spontaneously after 17 h. Discussion: In the present case, our patient was presented with acute cholinergic syndrome due to carbamate intoxication after massive transdermal exposure to rivastigmine. Toxicological analysis revealed a remarkably high estimated serum rivastigmine concentration (150.6 ng/ml) and notably low serum butyrylcholinesterase activity (35 IU/l) on admission, with a markedly prolonged calculated elimination half-life of 6.5 h. Conclusions: Emergency physicians should consider acetylcholinesterase inhibitor exposure (e.g., rivastigmine) when patients are present with acute cholinergic syndrome.

Unexpectedly high blood concentration of bisoprolol after an incorrect prescription: A case report

An elderly person died of uncontrolled bradycardia in a hospital. The doctor had prescribed 1.35 mg of bisoprolol fumarate orally, but a nurse mistakenly gave the patient 10 mg of the drug 9 hours prior to her death. Bisoprolol was detected in her blood by liquid chromatography-mass spectrometry at a concentration of 176 ng/mL. Even if the patient had chronic heart failure, this concentration is double the expected value. This patient was found to have a mutation within cytochrome P2D6, with thymidine substituted for cytosine at position 100 and cytosine for guanine at position 4180, causing proline to serine and threonine to serine amino acid substitutions. This mutation in the intermediate metabolizer allele reportedly reduces enzyme activity by half. However, in addition to the type of cytochrome P450 allelic variant, the amount of enzyme product influences metabolism of this drug. In this case, the high blood concentration of bisoprolol was only partly attributable to an error in prescription; its concentration was inexplicably high.

Toxicokinetics of the Synthetic Cathinone α-Pyrrolidinohexanophenone

Synthetic cathinones inhibit monoamine transporters, such as serotonin, norepinephrine, and dopamine transporters, and act on the central nervous system via increasing synaptic concentrations of monoamines. These compounds, which are highly addictive and potentially poisonous, are new psychoactive substances. In this study, we investigated the toxicokinetics of the synthetic cathinone, α-pyrrolidinohexanophenone (α-PHP), and assessed the relationship between the toxicokinetics and the long-term clinical symptoms induced by α-PHP in a male patient. The patient (39 years old) suddenly started uttering inarticulate words and demonstrating incomprehensible behavior in his house, and was brought to the emergency department of Iwate Medical University hospital. He presented with psychotic symptoms, such as hallucinations and delusion; however, his vital signs were normal. The hallucinations and delusion improved by the third day of hospitalization. Toxicological analysis was performed using liquid chromatography-tandem mass spectrometry with QuEChERS extraction. α-PHP was detected in his serum at a concentration of 175 ng/mL on his arrival at the hospital. His serum concentrations of α-PHP were serially determined and their natural logarithms were plotted against time after arrival. Although serum concentrations at early time points were lacking, the obtained curve was consistent with a two-compartment model and indicated a serum elimination half-life of 37 h. The long-lasting psychotic symptoms induced by synthetic cathinones appear to be correlated with their toxicokinetic characteristics, such as their long half-lives. Finally, interpreting the toxicokinetics of synthetic cathinones may provide useful information for the toxicological assessment of new psychoactive substances for forensic and clinical purposes.

A Retrospective Study on the Epidemiological and Clinical Features of Emergency Patients with Large or Massive Consumption of Caffeinated Supplements or Energy Drinks in Japan

Objective We conducted a retrospective study on the epidemiological and clinical features of patients with acute caffeine poisoning in Japan. Methods Letters requesting participation were sent to 264 emergency departments of hospitals, and questionnaires were mailed to those that agreed to participate. Patients Participants were patients transported to emergency departments of hospitals between April 2011 and March 2016 after consuming large or massive amounts of caffeinated supplements and/or energy drinks (caffeine dose ≥1.0 g). Results We surveyed 101 patients from 38 emergency departments. Since April 2013, the number of patients has markedly increased. Of these young patients (median age, 25 years), 53 were men, and 97 had consumed caffeine in tablet form. Estimated caffeine doses (n=93) ranged from 1.2 to 82.6 g (median, 7.2 g). Serum caffeine levels on admission (n=17) ranged from 2.0 to 530.0 μg/mL (median level, 106.0 μg/mL). Common abnormal vital signs and laboratory data on admission included tachypnea, tachycardia, depressed consciousness, hypercreatinekinasemia, hyperglycemia, hypokalemia, hypophosphatemia, and hyperlactatemia. Common signs and symptoms in the clinical course included nausea, vomiting, excitement/agitation, and sinus tachycardia. Seven patients (6.9%) who had consumed ≥6.0 g of caffeine, or whose serum caffeine levels on admission were ≥200 μg/mL, developed cardiac arrest. Ninety-seven patients (96.0%) recovered completely, but 3 patients (3.0%) died. Conclusion The present analysis of data from more than 100 emergency patients revealed clinical features of moderate to fatal caffeine poisoning. We recommend highlighting the toxicity risks associated with ingesting highly caffeinated tablets.

Postmortem Computed Tomographic Analysis of Death Caused by Oral Drug Intoxication

Traditional autopsy has changed little in the past century. In Japan, the rate of forensic autopsy in cases of unusual death is very low. Therefore, multi-slice computed tomography (CT) has been used to obtain imaging data instead of or in addition to autopsy in suspicious forensic cases. In our institute, postmortem multi-slice CT has been performed since 2009, and by 2014 there were over 1,000 cases. Our extensive experience with postmortem CT shows that in many cases of death by drug overdose, stomach contents exhibit high X-ray absorption. This article reviews the relationship between CT findings of stomach contents and toxicological analysis results in 23 cases of death by drug overdose. All cases (12 females and 11 males, aged 44 ± 11 years) known to have orally ingested drugs were included in this study. We assessed the slices of all stomach areas on consecutive axial CT images. Twenty cases (87%) showed high X-ray absorption in the stomach, while the other three did not demonstrate radio-dense stomach contents even though drug analysis detected lethal concentrations of drugs in the blood. In conclusion, drugs were frequently, but not always, visualized as contents with high X-ray absorption in the stomach. Postmortem gastric CT images can provide useful information in cases of oral drug intoxication if there are empty drug packages or a suicide note at the death scene. However, precise determination of the cause of death requires full autopsy in cases where there is no indication of suicide at the death scene.