Kanju Saka

Determination of amobarbital and phenobarbital in serum by gas chromatography–mass spectrometry with addition of formic acid to the solvent

A rapid and accurate method for quantification of amobarbital and phenobarbital was developed using gas chromatography-mass spectrometry (GC-MS) without derivatization. Though the compounds measured without derivatization showed low sensitivity because of adsorption, addition of 3% formic acid to the solvent improved the sensitivity for the analytes. Taking account of matrix effect, solid-phase and liquid-liquid extraction from serum were examined. The correlation coefficients of the calibration curves were 0.9995 or better, and the accuracy and precision of intraday and interday assays were in line with Food and Drug Administration (FDA) criteria.

Autopsy report on pseudo-Bartter syndrome with renal calcification induced by diuretics and diet pills.

A woman in her mid-forties had repeated vomiting and diarrhoea accompanied by muscle weakness soon after she started taking seven different diet pills imported from Thailand. After she had taken the pills for 8 days, respiratory depression progressed rapidly to arrest. Blood tests at the Emergency Department showed severe hypokalaemia with metabolic alkalosis. We diagnosed that she had developed pseudo-Bartter syndrome from the findings based on ionic abnormalities and high renin and aldosterone levels, and hyperplasia of the juxtaglomerular apparatus. A postmortem blood analysis indicated subtherapeutic levels of furosemide. We concluded that the patient died from pseudo-Bartter syndrome, which was triggered by chronic self-administration of furosemide and aggravated by the diet pills. This is the first pseudo-Bartter syndrome autopsy report to show histological localisation of calcification in the kidneys.

Relationship between the matrix effect and the physicochemical properties of analytes in gas chromatography

The phenomenon “matrix-induced chromatographic response enhancement” (matrix effect) causes quantitative errors in gas chromatography (GC) analyses. This effect varies according to the analyte nature, matrix type and concentration, and GC-system parameters. By focusing on the physicochemical properties of analytes, a predictive model was developed for the matrix effect using quantitative structure–property relationships. Experimental values of the matrix effect were determined for 58 compounds in a serum extract obtained from solid-phase extraction as the matrix. Eight molecular descriptors were selected, and the matrix-effect model was developed by multiple linear regression. The developed model predicted values for the matrix effect without any further experimental measurements. It also indicated that the molecular polarity (particularly H-bond donors) and volume of the analyte increase the matrix effect, while hydrophobicity and increasing number of nonpolar carbon atoms in the analyte decrease the matrix effect. The model was applied to the analysis of barbiturates. The predicted values indicated that N-methylation decreases the matrix effect, and the relative predicted values were effective for the selection of an internal standard. The obtained insight into the matrix effect and the prediction data will be helpful for developing quantitative analysis strategies.

Identification of active ingredients in dietary supplements using non-destructive mass spectrometry and liquid chromatography–mass spectrometry

A mid-forties woman purchased seven different dietary supplements from Thailand on the internet and subsequently died after taking these supplements. Since there were no ingredient labels on the supplements, we identified the active ingredients using direct analysis in real time-mass spectrometry (DART-MS), direct exposure probe-MS (DEP-MS), and liquid chromatography-MS (LC-MS). DART-MS gives exact molecular weights and DEP-MS shows the fragmentation of a molecule by electron ionization. Analyses using these two instruments are rapid and do not require extraction of the sample. The compounds predicted by DART-MS and DEP-MS were confirmed by LC-MS and the active ingredients of the seven dietary supplements were identified.

Analysis of Tolfenpyrad and its Metabolites in Plasma in a Tolfenpyrad Poisoning Case

Tolfenpyrad (TFP) is a pesticide that was first approved in 2002 in Japan under the trade name of Hachi-hachi. Analyses of TFP and its major metabolite, 4-[4-[(4-chloro-3-ethyl-1-methylpyrazol-5-yl)carbonylaminomethyl]phenoxy]benzoic acid (PTCA), in plasma obtained from a cadaver suspected to have died of TFP poisoning, were conducted by liquid chromatography-mass spectrometry. The existence of TFP and PTCA was confirmed by scan mode and quantitative analysis was performed by selected ion monitoring mode. Calibration curves showed good linearity over the range of 0.1-4 and 0.25-4 µg/mL, and concentrations were estimated to be 1.97 ± 0.02 and 2.88 ± 0.04 µg/mL for TFP and PTCA, respectively. The plasma extract was further examined to find other metabolites using quadrupole time-of-flight MS, and the results revealed three more metabolites, which were suggested to be hydroxy-TFP, dehydro-TFP and hydroxy-PTCA. Plausible metabolic pathways of TFP in humans are: (i) oxidation of the methyl group on the benzene ring, and (ii) hydroxylation followed by dehydration at the ethyl group on the pyrazole ring.

Fatal Poisoning with Both Dichlorvos and Phenthoate

Organophosphates are widely used as pesticides. However, organophosphates are occasionally orally ingested to commit suicide. In this case, a man in his late 80s committed suicide by ingesting both dichlorvos and phenthoate. Autopsy findings revealed a characteristic volatile odor from his mouth, stomach, lungs, liver, and kidneys. The esophageal mucosa was denatured and had lost elasticity. Serum cholinesterase activity was 9 IU/L. Toxicological analyses performed using high‐performance liquid chromatography–tandem mass spectrometry revealed that dichlorvos concentrations in the left and right cardiac blood samples were 11.6 and 4.6 μg/mL, respectively. Phenthoate concentrations in the left and right cardiac blood samples were 5.8 and 0.51 μg/mL, respectively. The total amounts of dichlorvos and phenthoate in the stomach were 7.35 and 4.55 g, respectively. The case history, autopsy findings, and toxicological analyses indicated that the cause of death was acute fatal poisoning after oral ingestion of both dichlorvos and phenthoate.

High-throughput functional evaluation of variants of unknown significance in ERBB2

Purpose: The advent of next-generation sequencing technologies has enabled the identification of several activating mutations of Erb-B2 receptor tyrosine kinase 2 (ERBB2) among various cancers. However, the significance of infrequent mutations has not been fully investigated. Herein, we comprehensively assessed the functional significance of the ERBB2 mutations in a high-throughput manner. Experimental Design: We evaluated the transforming activities and drug sensitivities of 55 nonsynonymous ERBB2 mutations using the mixed-all-nominated-in-one (MANO) method. Results: G776V, G778_S779insG, and L841V were newly revealed to be activating mutations. Although afatinib, neratinib, and osimertinib were shown to be effective against most of the ERBB2 mutations, only osimertinib demonstrated good efficacy against L755P and L755S mutations, the most common mutations in breast cancer. In contrast, afatinib and neratinib were predicted to be more effective than other inhibitors for the A775_776insYVMA mutation, the most frequent ERBB2 mutation in lung cancer. We surveyed the prevalence of concurrent ERBB2 mutation with gene amplification and found that approximately 30% of ERBB2-amplified urothelial carcinomas simultaneously carried ERBB2 mutations, altering their sensitivity to trastuzumab, an mAb against ERBB2. Furthermore, the MANO method was applied to evaluate the functional significance of 17 compound mutations within ERBB2 reported in the COSMIC database, revealing that compound mutations involving L755S were sensitive to osimertinib but insensitive to afatinib and neratinib. Conclusions: Several ERBB2 mutations showed varying sensitivities to ERBB2-targeted inhibitors. Our comprehensive assessment of ERBB2 mutations offers a fundamental database to help customize therapy for ERBB2-driven cancers. We identified several ERBB2 mutations as activating mutations related to tumorigenesis. In addition, our comprehensive evaluation revealed that several ERBB2 mutations showed varying sensitivities to ERBB2-targeted inhibitors, and thus, the functional significance of each variant should be interpreted precisely to design the best treatment for each patient. Clin Cancer Res; 24(20); 5112–22. ©2018 AACR.