Development of two fully automated quick, easy, cheap, effective, rugged, and safe pretreatment methods for the extraction of psychotropic drugs from whole blood samples.

Quick, easy, cheap, effective, rugged, and safe extraction strategies are becoming increasingly adopted in various analytical fields to determine drugs in biological specimens. In the present study, we developed two fully automated quick, easy, cheap, effective, rugged, and safe extraction methods based on acetonitrile salting-out assisted liquid-liquid extraction (method 1) and acetonitrile salting-out assisted liquid-liquid extraction followed by dispersive solid-phase extraction (method 2) using a commercially available automated liquid-liquid extraction system. We applied these methods to the extraction of 14 psychotropic drugs (11 benzodiazepines and carbamazepine, quetiapine, and zolpidem) from whole blood samples. Both methods prior to liquid chromatography-tandem mass spectrometry analysis exhibited high linearity of calibration curves (correlation coefficients, > 0.9997), ppt level detection sensitivities, and satisfactory precisions (< 8.6% relative standard deviation), accuracies (within ± 16% relative error), and matrix effects (81-111%). Method 1 provided higher recovery rates (80-91%) than method 2 (72-86%), whereas method 2 provided higher detection sensitivities (limits of detection, 0.003-0.094 ng/mL) than method 1 (0.025-0.47 ng/mL) owing to the effectiveness of its dispersive solid-phase extraction cleanup step. These fully automated extraction methods realize reliable, labor-saving, user-friendly, and hygienic extraction of target analytes from whole blood samples.

Direct analysis of biodegradable chelating agents based on liquid chromatography/electrospray ionization mass spectrometry using a metal-free hydrophilic interaction liquid chromatographic column.

Recently, biodegradable aminopolycarboxylic acid chelating agents have attracted attention as an alternative to environmentally persistent chelating agents such as ethylenediamine-N,N,N',N'-tetraacetic acid. However, the detection of chelating agents requires complexation with metals or derivatization by esterification reagents, and their direct detection using the currently available analytical methods still represents a challenge. Herein, we describe a direct analytical method for the biodegradable chelating agents ethylenediamine-N,N'-disuccinic acid, 3-hydroxy-2,2'-iminodisuccinic acid, methylglycine-N,N'-diacetic acid, and N,N-bis(carboxymethyl)-L-glutamic acid, via ultra-performance liquid chromatography/electrospray ionization quadrupole/time-of-flight mass spectrometry. Satisfactory retention and separation with a good peak shape were successfully achieved using a metal-free hydrophilic interaction liquid chromatographic column. The calibration curves showed good linearity in the range of 1.0-50 µM with correlation coefficients greater than 0.9988. The detection limits ranged from 0.04 to 0.12 µM. Furthermore, the developed method could be applied to the quantitative analysis of the four chelating agents in biodegradation and photodegradation experiments at the laboratory level. The proposed method, which offers the advantages of quickness, sensitivity, and requiring no complicated pretreatment steps, is expected to contribute significantly to the practical analysis of chelating agents in environmental water samples.

Easily Operable Quantification Method of 21 Plant-Derived Alkaloids in Human Serum by Automatic Sample Preparation and Liquid Chromatography-Tandem Mass Spectrometry.

In this study, we developed an easily operable quantification method for 21 plant-derived alkaloids in human serum by automatic sample preparation and liquid chromatography-tandem mass spectrometry. We designed to perform parallel sample preparation by a developed apparatus, which increased sample throughput. We conducted an automatic sample preparation through de-proteinization with 0.1% formic acid in methanol and achieved recovery rates of 89-107% (2.0-14% RSD) for all targeted analytes, demonstrating its high repeatability. The method validation results were satisfactory as follows: the linearity (r 2) of each calibration curve ranged from 0.978 to 1.000; the inter- and intra-day accuracies were 89.0-125% and 82.1-110%, respectively; the inter- and intra-day precisions were below 13% and 10%, respectively. Additionally, the lower limits of detection and quantification were 0.0044-0.047 and 0.013-0.14 ng/mL, respectively. Finally, the developed method was applied to pseudo-protoveratrine A poisoning serum and pseudo-colchicine poisoning serum, which were prepared by diluting acute-poisoning mice serum with human serum. Our method successfully quantitated protoveratrine A (0.15-0.25 ng/mL) and colchicine (4.8-6.0 ng/mL). Thus, our method is essential for prompt clinical treatment and critical care on patient in acute intoxication cases caused by plant-derived alkaloids. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-022-04212-5.

Regioisomer Differentiation of Ring-Substituted Chloromethcathinones and Bromomethcathinones Using Gas Chromatography/Electron Ionization-Triple Quadrupole Energy-Resolved Mass Spectrometry.

Positional isomers o-, m-, and p-chloromethcathinones (CMCs) and m- and p-bromomethcathinones (BMCs) were effectively differentiated using gas chromatography (GC) and energy-resolved mass spectrometry (ERMS) analyses. GC demonstrated that the free bases of CMC and BMC isomers were simultaneously baseline-separated at a slow column heating rate (5 °C/min) using a conventional low-polar capillary column. ERMS showed that the trifluoroacetyl derivatives of the positional isomers differed in mass spectral abundances of both halophenyl and halobenzoyl cations. Moreover, the logarithmic plots of the abundance ratio of the two cations as a function of the collision energy (CE) exhibited marked differences among the isomers at each CE, following the order of ortho < para < meta for CMCs and para < meta for BMCs. The performed theoretical calculations of dissociation energy agreed well with the ERMS measurements. The GC and ERMS methodologies enabled unambiguous and reliable differentiation of CMC and BMC isomers. The developed approach is expected to significantly contribute to the accurate structural identification of new psychoactive substances in forensic, toxicological, and clinical fields.

Differentiation of o-, m-, and p-fluoro-α-pyrrolidinopropiophenones by Triton B-mediated one-pot reaction.

Positional isomer differentiation is crucial for the analysis of forensic drugs. Presently, it is difficult to distinguish among ortho, meta, and para positional isomers of ring-fluorinated synthetic cathinones, a major class of new psychoactive substances (NPSs), because they exhibit similar chromatographic properties and mass spectral patterns. We describe herein that the ring-fluorinated synthetic cathinone positional isomers, viz. o-, m-, and p-fluoro-α-pyrrolidinopropiophenones (o-, m-, and p-FPPPs), can be discriminated by their benzyltrimethylammonium hydroxide (Triton B)-mediated one-pot reaction with methanol at ambient temperature, followed by chromatographic and mass spectral analyses of the corresponding products. For p-FPPP, fluorine was nucleophilically substituted by the methoxy group to afford p-methoxy-α-pyrrolidinopropiophenone, while o- and m-FPPPs afforded the corresponding FPPP-enamine-pyrrolidine adducts, which allowed the above positional isomers to be unambiguously differentiated by comparing the reaction product chromatograms and mass spectra. The adopted approach, which does not require excess heating or use of metallic catalysts and features the advantages of simplicity and convenience, is expected to contribute toward practical NPS identification.

Energy-resolved mass spectrometry for differentiation of the fluorine substitution position on the phenyl ring of fluoromethcathinones.

A reliable method for structural analysis is crucial for the forensic investigation of new psychoactive substances (NPSs). Towards this end, mass spectrometry is one of the most efficient and facile methods for the identification of NPSs. However, the differentiation among 2-, 3-, and 4-fluoromethcathinones (o-, m-, and p-FMCs), which are ring-fluorinated positional isomers part of the major class of NPSs referred to as synthetic cathinones, remains a challenge. This is mostly due to their similar retention properties and nearly identical full scan mass spectra, which hinder their identification. In this study, we describe a novel and practical method for differentiating the fluorine substitution position on the phenyl ring of FMCs, based on energy-resolved mass spectrometry (ERMS) using an electron ionization-triple quadrupole mass spectrometer. ERMS measurements showed that the three FMC positional isomers exhibited differences in relative abundances of both the fluorophenyl cation (m/z 95) and the fluorobenzoyl cation (m/z 123). The logarithmic plots of the abundance ratio of these two cations (m/z 95 to m/z 123) as a function of the collision energy (CE) followed the order of o-FMC < p-FMC < m-FMC at each CE, which allowed the three isomers to be unambiguously and reliably differentiated. The theoretical dissociation energy calculations confirmed the relationship obtained by ERMS analyses, and additional ERMS measurements of methylmethcathinone positional isomers showed that the differences in abundance among the FMCs were attributed to the differences in their collision-induced dissociation reactivities arising from the halogen-induced resonance effects on the phenyl ring. Moreover, the method for differentiation described herein was successfully applied to the actual samples containing seized drugs. We expect that the described methodology will also contribute significantly to the reliable and accurate structural identification of NPSs in the fields of therapeutic, clinical, and forensic toxicology.

Molecularly imprinted polymer solid-phase extraction of synthetic cathinones from urine and whole blood samples.

In forensic drug analysis, extractive pretreatment is required prior to instrumental analysis to ensure successful detection of the target compounds. However, conventional extraction methods such as hydrophilic polymer-based solid-phase extraction and liquid-liquid extraction are unsuitable for an emerging class of new psychoactive substances, namely, synthetic cathinones, because they exhibit a lack of class selectivity and increased risk of target analyte decomposition during extraction. To address these issues, we describe a highly class-selective sample clean-up method for the extraction of synthetic cathinones from urine and whole blood samples, exploiting a molecularly imprinted polymer solid-phase extraction cartridge. In terms of the influence of the synthetic cathinone molecular structure on the extraction recovery, we showed that while longer alkyl side chains slightly reduced the extraction efficiency, substituent variation on the aromatic ring exerted no effect. Molecularly imprinted polymer solid-phase extraction of 11 synthetic cathinones from urine samples yielded higher recoveries than the two conventional extraction methods, and smaller matrix effect was observed than that with hydrophilic polymer-based solid-phase extraction. Molecularly imprinted polymer solid-phase extraction from whole blood samples gave recoveries comparable to those of urine samples. Therefore, the proposed method is applicable for the extraction and quantitative determination of synthetic cathinones in biological samples.

Differentiation of AB-FUBINACA and its five positional isomers using liquid chromatography-electrospray ionization-linear ion trap mass spectrometry and triple quadrupole mass spectrometry.

PURPOSE: Positional isomer differentiation is crucial for forensic analysis. The aim of this study was to differentiate AB-FUBINACA positional isomers using liquid chromatography (LC)-electrospray ionization (ESI)-linear ion trap mass spectrometry (LIT-MS) and LC-ESI-triple quadrupole mass spectrometry (QqQ-MS). METHODS: AB-FUBINACA, its two fluorine positional isomers on the phenyl ring, and three methyl positional isomers in the carboxamide side chain were analyzed by LC-ESI-LIT-MS and LC-ESI-QqQ-MS. RESULTS: Four of the positional isomers, excluding AB-FUBINACA and its 3-fluorobenzyl isomer, were chromatographically separated on an ODS column in isocratic mode. ESI-LIT-MS could discriminate only three isomers, i.e., the 2-fluorobenzyl isomer, the N-(1-amino-2-methyl-1-oxobutan-2-yl) isomer, and the N-(1-amino-1-oxobutan-2-yl)-N-methyl isomer, based on their characteristic product ions observed at the MS3 stage in negative mode. ESI-QqQ-MS differentiated all six isomers in terms of the relative abundances of the product ions that contained the isomeric moieties involved in collision-induced dissociation reactions. The six isomers were more clearly and significantly differentiated upon comparison of the logarithmic values of the product ion abundance ratios as a function of collision energy. CONCLUSIONS: The present LC-MS methodologies were useful for the differentiation of a series of AB-FUBINACA positional isomers.

Differentiation of AB-FUBINACA positional isomers by the abundance of product ions using electron ionization-triple quadrupole mass spectrometry.

Mass spectrometric differentiation of structural isomers is important for the analysis of forensic samples. Presently, there is no mass spectrometric method for differentiating halogen positional isomers of cannabimimetic compounds. We describe here a novel and practical method for differentiating one of these compounds, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA (para)), and its fluoro positional (ortho and meta) isomers in the phenyl ring by electron ionization-triple quadrupole mass spectrometry. It was found that the three isomers differed in the relative abundance of the ion at m/z 109 and 253 in the product ion spectra, while the detected product ions were identical. The logarithmic values of the abundance ratio of the ions at m/z 109 to 253 (ln(A109 /A253 )) were in the order meta < ortho < para and increased linearly with collision energy. The differences in abundances were attributed to differences in the dissociation reactivity between the indazole moiety and the fluorobenzyl group because of the halogen-positional effect on the phenyl ring. Our methodology, which is based on the abundance of the product ions in mass spectra, should be applicable to determination of the structures of other newly encountered designer drugs. Copyright © 2016 John Wiley & Sons, Ltd.

Highly Sensitive Detection of Organophosphorus Pesticides Using 5,10,15,20-Tetrakis(4-hydroxyphenyl)porphyrin.

We describe a unique UV-visible absorption spectral property of 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (THPP) in the presence of organophosphorus (OP) pesticides. Upon titrating each 16 among total 40 different OP pesticides, the Soret band was significantly red-shifted, and a very intense Q band appeared. They were attributed to the diprotonation of THPP. A suitable solvent for this reaction was determined to be methanol. THPP would become a potential sensor molecule used to detect OP pesticides with high sensitivity in the concentration range of 10(-6) - 10(-4) M.

Characterization of the chemical diversity of glycosylated mycosporine-like amino acids in the terrestrial cyanobacterium Nostoc commune.

Mycosporine-like amino acids (MAAs) are UV-absorbing pigments, and structurally unique glycosylated MAAs are found in the terrestrial cyanobacterium Nostoc commune. In this study, we examined two genotypes of N.commune colonies with different water extract UV-absorption spectra. We found structurally distinct MAAs in each genotype. The water extract from genotype A showed a UV-absorbing spectrum with an absorption maximum at 335nm. The extract contained the following compounds: 7-O-(ß-arabinopyranosyl)-porphyra-334 (478Da), pentose-bound shinorine (464Da), hexose-bound porphyra-334 (508Da) and porphyra-334 (346Da). The water extract from genotype B showed a characteristic UV-absorbing spectrum with double absorption maxima at 312 and 340nm. The extract contained hybrid MAAs (1050Da and 880Da) with two distinct chromophores of 3-aminocyclohexen-1-one and 1,3-diaminocyclohexen linked to 2-O-(ß-xylopyranosyl)-ß-galactopyranoside. A novel 273-Da MAA with an absorption maximum at 310nm was also identified in genotype B. The MAA consisted of a 3-aminocyclohexen-1-one linked to a γ-aminobutyric acid chain. These MAAs had potent radical scavenging activities in vitro and the results confirmed that the MAAs have multiple roles as a UV protectant and an antioxidant relevant to anhydrobiosis in N. commune. The two genotypes of N. commune exclusively produced their own characteristic glycosylated MAAs, which supports that MAA composition could be a chemotaxonomic marker for the classification of N. commune.

Fatal and non-fatal cases of lime sulfide exposure and pathogenetic mechanisms underlying pancreatic injury: Case reports with an animal experiment.

Lime sulfide poisoning by the oral route is rarely encountered in the practice of forensic science, whereas hydrogen sulfide poisoning is seen frequently. We report here two cases of fatal lime sulfide poisoning with several related cases and in addition induced histological damage with acute inflammation in animal models under at similar concentrations. We also evaluated sulfide and thiosulfate concentrations and speculated as to the cause of pancreatic damage in these cases.

Analysis of 11-nor-Δ9 -tetrahydrocannabinol-9-carboxylic acid and its glucuronide in urine by capillary electrophoresis/mass spectrometry.

Δ(9) -Tetrahydrocannabinol is the primary psychoactive component in cannabis, one of the most commonly used illicit drugs in the world. This paper describes a simple and rapid method for direct analysis of major metabolites of Δ(9) -tetrahydrocannabinol; 11-nor-Δ(9) -tetrahydrocannabinol-9-carboxylic acid and its glucuronide in urine by capillary electrophoresis/mass spectrometry. The only pretreatment needed for a urine sample was dilution with methanol containing an internal standard and centrifugation. Electrophoresis was carried out in an untreated fused-silica capillary (50 µm i.d. × 85 cm) filled with 40 m m ammonium formate (pH 6.4). An analysis could be completed within 10 min. For both compounds, the assay was linear over the range 0.1-10 µg/mL in urine with correlation coefficients (r(2) ) >0.99 and the limit of detection was 0.5 pg (10 nL injection). The detection yields and reproducibilities were determined at three different concentrations (0.1, 0.5 and 2 µg/mL in urine). The mean detection yields were 60-99%. The intra- and inter-day relative standard deviations of migration times were 0.063-0.19 and 0.18-0.36%, and those of peak areas were 4.2-18 and 5.9-25%, respectively. The proposed method successfully analyzed the urine samples of cannabis users.

Analysis of phosphorus-containing amino acid-type herbicides by sheathless capillary electrophoresis/electrospray ionization-mass spectrometry using a high sensitivity porous sprayer.

We describe a new practical capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) method for the forensic analysis of phosphorus-containing amino acid-type herbicides, glyphosate (GLYP), glufosinate (GLUF) and bialaphos (BIAL). A new sheathless interface, a high sensitivity porous sprayer (HSPS), was used in this study. The limits of detections of GLYP, GLUF and BIAL were 7.6, 0.61 and 0.57 pg, respectively. These values were 4-36 times lower than these obtained by conventional CE/ESI-MS using a sheath liquid. The developed method was successfully applied to the analysis of beverages spiked with the herbicides.

Simultaneous chiral analysis of methamphetamine and related compounds by capillary electrophoresis/mass spectrometry using anionic cyclodextrin.

A capillary electrophoresis/mass spectrometry method for the simultaneous chiral analysis of enantiomers of methamphetamine (MA), amphetamine (AP), dimethylamphetamine (DMA), ephedrine (EP), norephedrine (NE) and methylephedrine (ME) in urine has been developed. The background electrolyte was 1 M formic acid (pH 1.7). Using 0.85 mM heptakis(2,6-diacethyl-6-sulfato)-beta-cyclodextrin as the chiral selector, the 12 enantiomers were completely separated within 25 min. The detection limits were 0.01 microg mL(-1) for the enantiomers of MA, AP, DMA, EP and ME, and 0.02 microg mL(-1) for the enantiomers of NE using selected ion monitoring. The reproducibilities of within-run (n = 4) for the migration times and peak areas of the standard mixture were under 0.58% and 7.83%, respectively. The calibration curves of the peak areas of the 12 enantiomers were linear in the range of 0.05 - 10 microg mL(-1). This method was applicable to the analysis of urine samples.

Simultaneous chiral determination of methamphetamine and its metabolites in urine by capillary electrophoresis-mass spectrometry.

A capillary electrophoresis-mass spectrometry method for the simultaneous chiral determination of enantiomers of methamphetamine (MA), amphetamine (AP), dimethylamphetamine (DMA) and p-hydroxymethamphetamine (pOHMA), in urine has been developed. The internal standards used were 2-phenylethylamine and 1-amino4-phenylbutane. The electrolyte was 1 M formic acid (pH 2.2). The chiral selector, which was added to the electrolyte, was a mixture of 3 mM beta-cyclodextrin and 10 mM heptakis(2,6-di-O-methyl)-beta-cyclodextrin. The detection limits were 0.03 microg ml(-1) for the enantiomers of MA and AP and 0.05 microg ml(-1) for the enantiomers of pOHMA using selected ion monitoring. In the analysis of healthy adult urine samples spiked with MA, AP and pOHMA, the precision of within-run assays (n = 4) for the migration time after correction with two internal standards were under 0.04%, and the detection yields utilizing solid phase extraction were 95-105%. This method was applicable to the analysis of urine samples of MA addicts and DMA addicts.

Analysis of methamphetamine and its metabolites in hair.

Methamphetamine (MA) is a sympathomimetic amine whose abuse has become a serious problem in Japan, Korea, Taiwan and other Southeast Asian countries. The use of hair for the determination of MA use has become more commonplace. The maximum period in which MA and its main metabolites (amphetamine and p-hydroxymethamphetamine) can be detected in urine is about 10 days after its use. However, proof of MA use is possible in hair even several years after its use if the part of the hair that grew in the period of its use is available. In addition, segmental analysis of hair is capable of clarifying the history of MA abuse. This paper reviews the clean-up, extraction, analytical method and distribution of MA and its metabolites in hair from reports published in the last 20 years.

Analysis of reaction products of morphine and codeine with hydrogen peroxide by high-performance liquid chromatography/mass spectrometry.

Changes in the chemical structures of morphine and codeine in the presence of hydrogen peroxide, a major component of hair dye and decolorant treatments, were examined with high-performance liquid chromatography/mass spectrometry (LC/MS). A mixture of morphine and hydrogen peroxide solution, after incubation at 39 degrees C for 24 h, produced two reaction products (hydroxymorphines). When codeine was used in place of morphine, one reaction product (hydroxycodeine) was produced, in which the benzene ring was hydroxylated.

Analysis of reaction products of cocaine and hydrogen peroxide by high-performance liquid chromatography/mass spectrometry.

The change of chemical structure of cocaine in the presence of hydrogen peroxide, a main component of hair dye and decolorant treatments, was studied. High-performance liquid chromatography/mass spectrometry (LC/MS) was used for the separation and identification of cocaine derivatives. After a mixture of cocaine and hydrogen peroxide solutions was incubated at 39 degrees C (this temperature is commonly used when the hair is treated with hair dye or decolorant) for 24 h, six reaction products were detected by LC/MS. Two of them were ecgonine methyl ester and benzoylecgonine, which are metabolites of cocaine. The other reaction products were assumed to be ortho-, meta- and para-hydroxycocaines and dihydroxycocaine, in each of which the benzene ring was hydroxylated by the reaction. These five reaction products (except for dihydroxycocaine) were found immediately after mixing cocaine and hydrogen peroxide. Therefore, the above reaction products might be present in the hair of cocaine users that had treated their hair with hair dye or decolorant.