Development of a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry for human urine (RaDPi-U).

Drug screening tests are mandatory in the search for drugs in forensic biological samples, and immunological methods and mass spectrometry (e.g., gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry) are commonly used for that purpose. However, these methods have some drawbacks, and developing new screening methods is required. In this study, we develop a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry (PESI-MS/MS), which is an ambient ionization mass spectrometry method, for human urine, named RaDPi-U. RaDPi-U is carried out in three steps: (1) mixing urine with internal standard (IS) solution and ethanol, followed by vortexing; (2) pipetting the mixture onto a sample plate for PESI; and (3) rapid-fire analysis by PESI-MS/MS. RaDPi-U targets 40 forensically important drugs, which include illegal drugs, hypnotics, and psychoactive substances. The analytical results were obtained within 3 min because of the above-mentioned simple workflow of RaDPi-U. The calibration curves of each analyte were constructed using the IS method, and they were quantitatively valid, resulting in good linearity (0.972-0.999) with a satisfactory lower limit of detection and lower limit of quantitation (0.01-7.1 ng/mL and 0.02-21 ng/mL, respectively). Further, both trueness and precisions were 28% or less, demonstrating the high reliability and repeatability of the method. Finally, we applied RaDPi-U to three postmortem urine specimens and successfully detected different drugs in each urine sample. The practicality of the method is proven, and RaDPi-U will be a strong tool as a rapid-fire drug screening method not only in forensic toxicology but also in clinical toxicology.

Metabolomics and Data-Driven Bioinformatics Revealed Key Maternal Metabolites Related to Fetal Lethality via Di(2-ethylhexyl)phthalate Exposure in Pregnant Mice.

We performed serum metabolome analysis of di(2-ethylhexyl)phthalate (DEHP)-exposed and control pregnant mice. Pregnant mice (n = 5) were fed a DEHP-containing diet (0.1% or 0.2% DEHP) or a normal diet (control) from gestational days 0-18. After maternal exposure to 0.2% DEHP there were no surviving fetuses, indicating its strong fetal lethality. There were no significant differences in the numbers of fetuses and placentas between the 0.1% DEHP and control groups, although fetal viability differed significantly between them, suggesting that maternal exposure to 0.1% DEHP could inhibit fetal growth. Metabolomics successfully detected 169 metabolites in serum. Principal component analysis (PCA) demonstrated that the three groups were clearly separated on PCA score plots. The biological interpretation of PC1 was fetal lethality, whereas PC2 meant metabolic alteration of pregnant mice via DEHP exposure without fetal lethality. In particular, the first component was significantly correlated with fetal viability, demonstrating that maternal metabolome changes via DEHP exposure were strongly related to fetal lethality. Levels of some amino acids were significantly increased in the DEHP-exposed groups, whereas those of some fatty acids, nicotinic acid, and 1,5-anhydroglucitol were significantly decreased in the DEHP groups. DEHP-induced increases in glycine levels could cause fetal neurological disorders, and decreases in nicotinic acid could inhibit fetal growth. In addition, a machine-learning Random forest could determine 16 potential biomarkers of DEHP exposure, and data-driven network analysis revealed that nicotinic acid was the most influential hub metabolite in the metabolic network. These findings will be useful for understanding the effects of DEHP on the maternal metabolome in pregnancy and their relationship to fetal lethality.

RECiQ: A Rapid and Easy Method for Determining Cyanide Intoxication by Cyanide and 2-Aminothiazoline-4-carboxylic Acid Quantification in the Human Blood Using Probe Electrospray Ionization Tandem Mass Spectrometry.

In this study, we developed a rapid and easy method to determine cyanide (CN) intoxication by quantification of CN and 2-aminothiazoline-4-carboxylic acid (ATCA), which is a new and reliable indicator of CN exposure, in the human blood using probe electrospray ionization tandem mass spectrometry (PESI/MS/MS) named RECiQ. For CN, we applied the previously reported one-pot derivatization method using 2,3-naphthalenedialdehyde and taurine, which can directly derivatize CN in the blood. The analytical conditions of the CN derivatization were optimized as a 10 min reaction time at room temperature. In contrast, ATCA could be directly detected in the blood by PESI/MS/MS. We developed quantitative methods for the derivatized CN and ATCA using an internal standard method and validated them using quality control samples, demonstrating that the linearities of each calibration curve were greater than 0.995, and intra- and interday precisions and accuracies were 5.1-15 and 1.1-14%, respectively. Moreover, the lower limit of detections for CN and ATCA were 42 and 43 ng/mL, respectively. Finally, we applied RECiQ to three postmortem blood specimens obtained from victims of fire incidents, which resulted in the successful quantification of CN and ATCA in all samples. As PESI/MS/MS can be completed within 0.5 min, and the sample volume requirement of RECiQ is only 2 µL of blood, these methods are useful not only for the rapid determination of CN exposure but also for the estimation of the CN intoxication levels during an autopsy.

Fatal intoxication by 5F-ADB and diphenidine: Detection, quantification, and investigation of their main metabolic pathways in humans by LC/MS/MS and LC/Q-TOFMS.

Despite the implementation of a new blanket scheduling system in 2013, new psychoactive substance (NPS) abuse remains a serious social concern in Japan. We present a fatal intoxication case involving 5F-ADB (methyl 2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamido]-3,3-dimethylbutanoate) and diphenidine. Postmortem blood screening by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOFMS) in the information-dependent acquisition mode only detected diphenidine. Further urinary screening using an in-house database containing NPS and metabolites detected not only diphenidine but also possible 5F-ADB metabolites; subsequent targeted screening by LC/tandem mass spectrometry (LC/MS/MS) allowed for the detection of a very low level of unchanged 5F-ADB in postmortem heart blood. Quantification by standard addition resulted in the postmortem blood concentrations being 0.19 ± 0.04 ng/mL for 5F-ADB and 12 ± 2.6 ng/mL for diphenidine. Investigation of the urinary metabolites revealed pathways involving ester hydrolysis (M1) and oxidative defluorination (M2), and further oxidation to the carboxylic acid (M3) for 5F-ADB. Mono- and di-hydroxylated diphenidine metabolites were also found. The present case demonstrates the importance of urinary metabolite screening for drugs with low blood concentration. Synthetic cannabinoids (SCs) fluorinated at the terminal N-alkyl position are known to show higher cannabinoid receptor affinity relative to their non-fluorinated analogues; 5F-ADB is no exception with high CB1 receptor activity and much greater potency than Δ9 -THC and other earlier SCs, thus we suspect its acute toxicity to be high compared to other structurally related SC analogues. The low blood concentration of 5F-ADB may be attributed to enzymatic and/or non-enzymatic degradation, and further investigation into these possibilities is underway.

Development of a mass spectrometric hydroxyl-position determination method for the hydroxyindole metabolites of JWH-018 by GC-MS/MS.

One of the many issues of designer drugs of abuse like synthetic cannabinoids (SCs) such as JWH-018 is that details on their metabolism has yet to be fully elucidated and that multiple metabolites exist. The presence of isomeric compounds poses further challenges in their identification. Our group has previously shown the effectiveness of gas chromatography-electron ionization-tandem mass spectrometry (GC-EI-MS/MS) in the mass spectrometric differentiation of the positional isomers of the naphthoylindole-type SC JWH-081, and speculated that the same approach could be used for the metabolite isomers. Using JWH-018 as a model SC, the aim of this study was to differentiate the positional isomers of its hydroxyindole metabolites by GC-MS/MS. Standard compounds of JWH-018 and its hydroxyindole metabolite positional isomers were first analyzed by GC-EI-MS in full scan mode, which was only able to differentiate the 4-hydroxyindole isomer. Further GC-MS/MS analysis was performed by selecting m/z 302 as the precursor ion. All four isomers produced characteristic product ions that enabled the differentiation between them. Using these ions, MRM analysis was performed on the urine of JWH-018 administered mice and determined the hydroxyl positions to be at the 6-position on the indole ring. GC-EI-MS/MS allowed for the regioisomeric differentiation of the hydroxyindole metabolite isomers of JWH-018. Furthermore, analysis of the fragmentation patterns suggests that the present method has high potential to be extended to hydroxyindole metabolites of other naphthoylindole type SCs in identifying the position of the hydroxyl group on the indole ring. Copyright © 2016 John Wiley & Sons, Ltd.

High-resolution mass spectrometric determination of the synthetic cannabinoids MAM-2201, AM-2201, AM-2232, and their metabolites in postmortem plasma and urine by LC/Q-TOFMS.

High-resolution mass spectrometry and accurate mass measurement by liquid chromatography/quadrupole-time of flight mass spectrometry (LC/Q-TOFMS) was applied to postmortem plasma and urine specimens from an autopsy of a fatal case involving synthetic cannabinoid use, resulting in the detection of three synthetic cannabinoids: MAM-2201, AM-1220, and AM-2232. We searched for their metabolites existing in postmortem plasma or urine by LC/Q-TOFMS and were able to detect N-dealkylated metabolites, defluorinated and further oxidized metabolites of MAM-2201, and some hydroxylated metabolites. Postmortem plasma concentrations of the parent drugs, N-dealkylated metabolites, and fluorinated and further oxidized metabolites of MAM-2201 were measured, and quantitation results revealed site differences between heart and femoral postmortem plasma concentrations of parent drugs and some metabolites, suggesting postmortem redistribution of the synthetic cannabinoids and their metabolites. Quantitation results suggest that defluorination is a major metabolic pathway for MAM-2201, and N-dealkylation is a common but minor pathway for the naphthoylindole-type synthetic cannabinoids in human.

Positional isomer differentiation of synthetic cannabinoid JWH-081 by GC-MS/MS.

Like many new designer drugs of abuse, synthetic cannabinoids (SC) have structural or positional isomers which may or may not all be regulated under law. Differences in acute toxicity may exist between isomers which impose further burden in the fields of forensic toxicology, medicine and legislation. Isomer differentiation therefore becomes crucial from these standpoints as new designer drugs continuously emerge with just minor positional modifications to their preexisting analogs. The aim of this study was to differentiate the positional isomers of JWH-081. Purchased standard compounds of JWH-081 and its positional isomers were analyzed by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) first in scan mode to investigate those isomers who could be differentiated by EI scan spectra. Isomers with identical or near-identical EI spectra were further subjected to GC-tandem mass spectrometry (MS/MS) analysis with appropriate precursor ions. EI scan was able to distinguish 3 of the 7 isomers: 2-methoxy, 7-methoxy and 8-methoxy. The remaining isomers exhibited near-identical spectra; hence, MS/MS was performed by selecting m/z 185 and 157 as precursor ions. 3-Methoxy and 5-methoxy isomers produced characteristic product ions that enabled the differentiation between them. Product ion spectrum of 6-methoxy isomer resembled that of JWH-081; however, the relative ion intensities were clearly different from one another. The combination of EI scan and MS/MS allowed for the regioisomeric differentiation of the targeted compounds in this study.