Analysis of 62 synthetic cannabinoids by gas chromatography–mass spectrometry with photoionization

Gas chromatography–mass spectrometry (GC–MS) in electron ionization (EI) mode is one of the most commonly used techniques for analysis of synthetic cannabinoids, because the GC–EI-MS spectra contain characteristic fragment ions for identification of a compound; however, the information on its molecular ions is frequently lacking. To obtain such molecular ion information, GC–MS in chemical ionization (CI) mode is frequently used. However, GC–CI-MS requires a relatively tedious process using reagent gas such as methane or isobutane. In this study, we show that GC–MS in photoionization (PI) mode provided molecular ions in all spectra of 62 synthetic cannabinoids, and 35 of the 62 compounds showed only the molecular radical cations. Except for the 35 compounds, the PI spectra showed very simple patterns with the molecular peak plus only a few fragment peak(s). An advantage is that the ion source for GC–PI-MS can easily be used for GC–EI-MS as well. Therefore, GC–EI/PI-MS will be a useful tool for the identification of synthetic cannabinoids contained in a dubious product. To the best of our knowledge, this is the first report to use GC–PI-MS for analysis of synthetic cannabinoids.     

Analysis of cannabinoids in oral fluid by liquid chromatography–tandem mass spectrometry

A sensitive method was developed for quantifying a wide range of cannabinoids in oral fluid (OF) by liquid chromatography–tandem mass spectrometry (LC–MS/MS). These cannabinoids include ?⁹-tetrahydrocannabinol (THC), 11-hydroxy-?⁹-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-?⁹-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), ?⁹-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-?⁹-tetrahydrocannabinol glucuronide (THCCOOH-gluc), and ?⁹-tetrahydrocannabinol glucuronide (THC-gluc). Samples were collected using a Quantisal? device. The advantages of performing a liquid–liquid extraction (LLE) of KCl-saturated OF using heptane/ethyl acetate versus a solid-phase extraction (SPE) using HLB copolymer columns were determined. Chromatographic separation was achieved in 11.5 min on a Kinetex? column packed with 2.6-μm core–shell particles. Both positive (THC, 11-OH-THC, CBN, and CBD) and negative (THCCOOH, THC-gluc, THCCOOH-gluc, and THC-A) electrospray ionization modes were employed with multiple reaction monitoring using a high-end AB Sciex API 5000? triple quadrupole LC–MS/MS system. Unlike SPE, LLE failed to extract THC-gluc and THCCOOH-gluc. However, the LLE method was more sensitive for the detection of THCCOOH than the SPE method, wherein the limit of detection (LOD) and limit of quantification (LOQ) decreased from 100 to 50 pg/ml and from 500 to 80 pg/ml, respectively. The two extraction methods were successfully applied to OF samples collected from volunteers before and after they smoked a homemade cannabis joint. High levels of THC were measured soon after smoking, in addition to significant amounts of THC-A. Other cannabinoids were found in low concentrations. Glucuronide conjugate levels were lower than the method’s LOD for most samples. Incubation studies suggest that glucuronides could be enzymatically degraded by glucuronidase prior to OF collection.     

Simultaneous determination of new-generation antidepressants in plasma by gas chromatography–mass spectrometry

We have developed a gas chromatography–mass spectrometry (GC–MS) method for plasma for the determination of new-generation antidepressants, including olanzapine (antipsychotic used in bipolar disorder), and antidepressant selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine and its metabolite norfluoxetine, paroxetine, sertraline, venlafaxine, and mirtazapine. Sample preparation was performed by liquid–liquid extraction with tert-butyl methyl ether. Fluoxetine, norfluoxetine, sertraline, and paroxetine required subsequent derivatization with 1-(heptafluorobutyryl) imidazole (HFBI). The GC separation lasts a total of 23.76 min. Qualitative and quantitative analysis were performed using an electron-impact ionization gas chromatograph interfaced to a mass-selective detector in selected-ion monitoring mode to increase the sensitivity of the method. Method validation was performed taking into account linearity, sensitivity, selectivity, accuracy, precision, and recovery, achieving good results for all the parameters studied. Calibration curves were prepared in the range of 0.005–2 μg/ml (according to the therapeutic and toxic concentrations of each individual compound), with all correlation coefficients R ² > 0.99. The limit of quantification was between 0.005 and 0.1 μg/ml, depending on the compound, whereas the limit of detection ranged from 0.0025 to 0.05 μg/ml. The method is fast and simple, allowing the identification and quantification of some of the most widely used antidepressants at therapeutic or toxic concentrations, and may be useful in routine clinical and forensic toxicology analysis.     

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.

     

Quantitation of sibutramine in human hair using gas chromatography–isotope dilution tandem mass spectrometry

Forensic Toxicology - An analytical method for quantitation of sibutramine in human hair using gas chromatography (GC)–isotope dilution tandem mass spectrometry (MS/MS) was newly established....     

Regioisomeric separation of ring-substituted cathinones by liquid chromatography–mass spectrometry with a naphthylethyl column

Recently, various new psychoactive substances (NPSs) have been detected as components of “legal high” products. Synthetic cathinones are one of the most popular classes of NPSs, and are structurally related to the stimulants amphetamine and methamphetamine. In this study, we investigated the differentiation of 2-, 3-, and 4-ring-substituted regioisomers for ethylmethcathinones, fluoromethcathinones, ethylethcathinones and fluoroethcathinones by liquid chromatography–mass spectrometry (/mass spectrometry). At the first step, it was difficult to differentiate the regioisomers by product ion spectra for each cathinone group. Therefore, we had to differentiate them by retention times. Under all tested conditions, the 3- and 4-ring-substituted cathinones were not separated by a conventional octadecylsilyl bonded silica gel column, but complete chromatographic separation of all regioisomers was achieved under optimized conditions using a column with a naphthylethyl-bonded stationary phase. Using these conditions, an authentic “legal high” product containing a fluoromethcathinone was analyzed, and the active ingredient was 4-fluoromethcathinone clearly differentiated from 2- and 3-fluoromethcathinone. The proposed method enables the separation of regioisomeric ring-substituted cathinones without the need for pretreatment steps, such as derivatization or purification. To our knowledge, this is the first trial to use the naphthylethyl-bonded column to separate ring-substituted cathinone regioisomers completely. This column seems to have potential to be successfully used for separation of other types of compounds with ring-substituted regioisomer structures.     

Liquid chromatography–electrospray ionization mass spectrometry for simultaneous detection of mtDNA length and nucleotide polymorphisms

We demonstrate the applicability of ion-pair reversed-phase high-performance liquid chromatography—electrospray ionization time-of-flight mass spectrometry (ICEMS) for the simultaneous characterization of length and nucleotide polymorphisms. Two sections within the first (HVS-I) and second (HVS-II) hypervariable segments of the mitochondrial (mt)DNA control region were selected as targets, both containing poly-cytosine (C) tracts, which display length heteroplasmy at a substantial frequency in the population. The two mtDNA sections were simultaneously amplified and analyzed by ICEMS in 90 maternally unrelated mother–offspring pairs from Austria. The findings were confirmed by direct sequencing of the polymerase chain reaction products. For the detailed characterization of present-length heteroplasmic variants, the results retrieved through ICEMS were more informative compared with those derived from direct sequencing. Hence, ICEMS represents an interesting option for successful application in forensic science. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Chiral analysis of dextromethorphan and levomethorphan in human hair by liquid chromatography–tandem mass spectrometry

Forensic Toxicology - Methorphan exists in two enantiomeric forms including dextromethorphan and levomethorphan. Dextromethorphan is an over-the-counter antitussive drug, whereas levomethorphan is...     

Determination of antidepressants in whole blood using hollow-fiber liquid-phase microextraction and gas chromatography–mass spectrometry

A hollow-fiber liquid-phase microextraction (HF-LPME), used in three-phase mode, and combined with gas chromatography–mass spectrometry (GC–MS), was developed to quantify antidepressants and their major metabolites (amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, desmethylclomipramine, fluoxetine, and norfluoxetine) in whole blood samples, using their deuterated analogs as internal standards. The HF-LPME system comprised a disposable 8-cm polypropylene porous hollow fiber, 4.0 ml of sample solution (0.5 ml of blood added to 3.5 ml of 0.1 M NaOH: donor phase), dodecane (organic phase), and 0.1 M formic acid (acceptor phase) for extraction. After stirring the system, the acceptor phase was evaporated under a nitrogen stream and resuspended in 30 μl of methanol. Derivatization was not required. A 2.0-μl aliquot of this solution was injected into a GC–MS system. The method was validated after the optimization of several parameters that may influence the extraction efficiency. The limits of quantification for all antidepressants were below the therapeutic levels (20.0 ng/ml). The average intraday and interday precisions were within 9.7 and 9.8 %, respectively, for all analytes. The calibration curves were linear in the concentration range of 20–1,200 ng/ml. The developed method was applied to seven actual postmortem samples. Tricyclic antidepressants were detected in all of the analyzed cases. To our knowledge, this is the first demonstration of usefulness of HF-LPME for analysis of antidepressants in postmortem forensic cases.     

High-throughput determination of barbiturates in human plasma using on-line column-switching ultra-fast liquid chromatography–tandem mass spectrometry

A high-throughput method was developed for determinations of eight barbiturates (barbital, allobarbital, phenobarbital, cyclobarbital, amobarbital, secobarbital, thiopental, and thiamylal) in human plasma by on-line column-switching ultra-fast liquid chromatography–tandem mass spectrometry (MS–MS). Plasma samples (100 μl) spiked with the eight barbiturates and 5-(4-methylphenyl)-5-phenylhydantoin (internal standard) were diluted with 300 μl of 13.3 mM ammonium acetate/acetonitrile (33:67, v/v). After centrifugation and filtration, the clear supernatant was injected directly onto the extraction column (Oasis HLB cartridge column). The following procedure was fully automated. The analytes retained on the extraction column were eluted by backflushing of the extraction column and introduced onto the analytical column (Phenomenex Onyx monolithic C₁₈ column, 100 mm × 4.6 mm i.d.) by column switching. Quantification was performed by multiple reaction monitoring with negative-ion atmospheric pressure chemical ionization. Good peak separation and peak shapes of the eight drugs were achieved within an analysis time of 3 min, including the extraction time. All drugs spiked into plasma showed recoveries of 80–93 %. The regression equations for the eight drugs showed excellent linearities in the range of 10–5000 ng/ml of plasma, and the limits of detection ranged from 1.0 to 10 ng/ml. The lower and upper limits of quantitation were 10–50 ng/ml and 5000 ng/ml, respectively. Intraday and interday coefficients of variation for all the drugs were not >9.1 %. The accuracies of quantitation were 92.0–108 %. The method was successfully applied to determination of the level of amobarbital in human plasma after its oral administration to a volunteer.     

Simultaneous chiral impurity analysis of methamphetamine and its precursors by supercritical fluid chromatography–tandem mass spectrometry

Purpose

Impurity profiling of seized illicit methamphetamine (MA) provides information on MA manufacturing methods in clandestine laboratories, and this drug intelligence supports formulation of strategies to control MA abuse. In the present study, we developed a simultaneous chiral analysis method for MA and its precursors using supercritical fluid chromatography–tandem mass spectrometry equipped with an enantioselective stationary phase.

Methods

Chromatographic conditions were optimized by systematic investigation of the flow rate, temperature, back pressure, co-solvent, additive, and mobile phase composition. The ability of the developed method was evaluated using standard and authentic illicit MA.

Results

The use of a chiral selector in the stationary phase allowed for simultaneous chiral differentiation of MA and its precursors including ephedrine, norephedrine, chloropseudoephedrine, methylephedrine, dimethylamphetamine, and amphetamine. Sufficient limit of detection, repeatability of retention time, and linearity were achieved. A switching valve interfacing a chromatograph and a mass spectrometer enabled analyzing large amounts of MA directly. The application to the authentic illicit MA samples was achieved and revealed the existence of impurities, which was not detected by conventional gas chromatography–mass spectrometry.

Conclusions

The developed supercritical fluid chromatography–tandem mass spectrometry method could be a powerful analytical tool for MA impurity profiling.

     

Quantification of MDMB-4en-PINACA and ADB-BUTINACA in human hair by gas chromatography–tandem mass spectrometry

Forensic Toxicology - To test synthetic cannabinoid (SCs) in parent forms from living human, the hairs seems to be one of the best samples, because of the non-invasiveness upon their collection....     

Simple screening procedure for 72 synthetic cannabinoids in whole blood by liquid chromatography–tandem mass spectrometry

Purpose

In recent years, many synthetic cannabinoids (SCs) have appeared on the drug market. Despite the increasing number of SCs, there are few comprehensive screening methods for their detection in biological specimens. In this context, the purpose of this study was to develop a fast and simple liquid chromatography–tandem mass spectrometry screening procedure for detection and identification of SCs in whole blood.

Methods

The elaborated qualitative screening method allows the simultaneous detection and identification of 72 compounds from different chemical groups: naphthoylindoles, naphthoylindazoles, benzoylindoles, phenylacetylindoles, tetramethylcyclopropylindoles, indole-3-carboxylic acid esters, indole-3-carboxylic acid amides, indazole-3-carboxylic acid amides, and others. Whole-blood samples (0.2 mL) were precipitated with acetonitrile (0.6 mL). The separation was achieved with the gradient of the mobile phase composition (0.1% formic acid in acetonitrile and 0.1% formic acid in water) and the gradient of the flow rate (0.5–0.8 mL/min) in 16 min. Detection of all compounds was based on dynamic multiple reaction monitoring.

Results

Mass spectrometer parameters for all compounds were presented. All of the compounds were well-separated by their retention times and/or transitions. The limits of detection (LODs) for 50 compounds were in the range 0.01–0.48 ng/mL.

Conclusions

Estimated LODs make this assay suitable for the analysis of biological material. The procedure can be easily expanded for more substances, which is an indispensable advantage in the dynamically developing drug market. It can have wide application in various analytical forensic and clinical laboratories.

     

Analysis of ibotenic acid and muscimol in Amanita mushrooms by hydrophilic interaction liquid chromatography–tandem mass spectrometry

We have established the most modern method for analysis of ibotenic acid and muscimol in toxic Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Acivicin, an ibotenic acid analog and antitumor agent, was used as internal standard (IS). A target mushroom sample was homogenized in water/methanol (1:1) and mixed with a fixed concentration of IS. The target compounds and IS were purified with an Oasis MAX 3cc (60 mg) extraction cartridge. The eluate was subjected to hydrophilic interaction (TSK-GEL Amide-80 3 μm, 150 × 2.0 mm i.d. column) LC–MS–MS. The quantitation was made by multiple reaction monitoring (MRM). The ion transitions were: m/z 179→133.1 for IS, m/z 159→113.1 for ibotenic acid, and m/z 115→98.1 for muscimol. The elution was made in the gradient mode with 0.5 % formic acid aqueous solution (A) and 0.5 % formic acid in acetonitrile (B) from 90 % B to 80 % B in 1.85 min, and then in the isocratic elution mode with 20 % A/80 % B up to 10 min. The MRM chromatograms gave clear and symmetrical peaks for ibotenic acid, muscimol, and IS. Their recovery rates were 84.6–107 %. There was good linearity from 10 to 500 μg/g for both ibotenic acid and muscimol with correlation coefficients not <0.99. Intraday and interday accuracy and precision were also generally satisfactory. Using the above new method, the concentrations of ibotenic acid and muscimol were actually measured for a mushroom (most probably Amanita ibotengutake) obtained from a poisoning case; they were 210 and 107 μg/g, respectively. The novel points of our method are no requirement for derivatization before LC–MS–MS analysis, the use of anion exchange solid-phase extraction, the use of hydrophilic interaction column for LC separation, and the use of acivicin as IS.     

A gas chromatography–mass spectrometry based study on serum metabolomics in rats chronically poisoned with hydrogen sulfide

Hydrogen sulfide poisoning is a common occupational hazard, whose mortality and incidence rates are first and second, respectively, among occupational poisoning incidents in China. The main target organs of its toxicity are in the central nervous system and respiratory system. However, there are currently no specific direct tests that can be used to diagnose poisoned patients. In this study, we developed a serum metabonomic method using orthogonal partial least squares-discriminate analysis (OPLS-DA), based on gas chromatography–mass spectrometry (GC/MS) to evaluate the effect of chronic poisoning by hydrogen sulfide in rats. The OPLS-DA data demonstrated that the model group (n = 60) differed significantly from the control group (n = 30), suggesting that the metabolic profiles of the two groups are markedly different. Alterations in the levels of some metabolites such as citrate, galactose, lactate, mannose, inositol, urea, phosphate, alanine and valine were detected by OPLS-DA analysis. We observed changes in metabolic pathways including lipid metabolism, energy metabolism and amino metabolism in the model group. Our results indicate that GC/MS-based metabonomic methods may provide novel detection means for chronic hydrogen sulfide poisoning.     

Simultaneous and sensitive analysis of fourth-generation antidepressants in human plasma by ultra-performance liquid chromatography–tandem mass spectrometry

A sensitive method for simultaneous analysis of nine fourth-generation antidepressants in human plasma by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS–MS) is presented. The method was used with a Waters Acquity UPLC LC system, an Acquity TQD MS–MS system, and a Poroshell 120 EC-C18 separation column. For extraction of the target compounds, solid-phase extraction with Oasis HLB cartridges was used. All compounds were detected with retention times lower than 3 min. The calibration curves for the antidepressants spiked into human plasma showed similar good linearities in the range of subnanogram to nanogram per milliliter. The detection limits (signal-to-noise ratio = 3) were in the range of 0.2–100 pg/ml. The method gave satisfactory recovery rates, accuracy, and precision for quality control samples spiked with these drugs. To further validate the present method, 25 mg of milnacipran was orally administered to a healthy male volunteer, and the drug concentrations in plasma were measured in samples collected 0.5, 1, 2, 4, and 8 h after dosing. The concentrations were in the range of 31.2–56.8 ng/ml. To our knowledge, this is the most sensitive quantitative method for fourth-generation antidepressants so far reported, and should prove very useful in forensic and clinical toxicology. In addition, the quantitative analysis of tianeptine by LC–MS–MS is first described in this study.     

Determination of antidepressants in human postmortem blood,brain tissue,and hair using gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric (GC–MS) method in positive ion chemical ionization mode in combination with a solid phase extraction was optimized for new-generation antidepressants and their metabolites in postmortem blood, brain tissue, and hair. Twelve antidepressants and their active metabolites (i.e., mirtazapine, viloxazine, venlafaxine, citalopram, mianserin, reboxetine, fluoxetine, fluvoxamine, sertraline, maprotiline, melitracen, paroxetine, desmethylfluoxetine, desmethylmianserin, desmethylmirtazapine, desmethylsertraline, desmethylmaprotiline, desmethylcitalopram, and didesmethylcitalopram) could be quantified. In this article, in addition to the validation of the GC–MS method, four postmortem cases are discussed to demonstrate the usefulness of the described method in forensic toxicology. In these cases, sertraline, fluoxetine, citalopram, and trazodone in combination with their active metabolites were quantified. Blood concentrations ranged from subtherapeutic to toxic concentrations, while brain to plasma ratios ranged from 0.8 to 17. Hair concentrations ranged from 0.4 to 2.5 ng/mg depending on the compound and hair segment.     

Validated liquid chromatography–mass spectrometry method for the quantitation of N-substituted derivatives of 3,4-methylenedioxyamphetamine in rat urine

3,4-Methylenedioxyamphetamines (MDAs) are central nervous system stimulants that are widely diffusing into the illegal market. Their identification is often difficult because of the high structural variety of new compounds. We describe a method for identifying and quantitating four MDAs in rat urine; three of them are new designer drugs: 3,4-methylenedioxy-N-isopropylamphetamine (MDIP), 3,4-methylenedioxy-N-cyclopropylmethylamphetamine (MDCPM), and 3,4-methylenedioxy-N-benzylamphetamine (MDBZ). The well-known and well-studied 3,4-methylenedioxy-N-ethylamphetamine (MDEA) was included as a reference. The quantitative analysis was performed by liquid chromatography–mass spectrometry (LC–MS) using 2,3-dimethoxyphenethylamine-d ₃ (2,3-DMPEA-d ₃) as internal standard. Samples were extracted by solid-phase extraction before injection. Chromatographic separation was achieved using a C₁₈ column (150 × 2.1 mm i.d., particle size 3 μm) by gradient elution with a mixture of 0.1 % formic acid in water and acetonitrile. The step gradient elution required a total run time of about 25 min. Regression equations were linear over the tested concentration range (10–500 ng/ml). The limits of detection and quantitation were in the ranges of 4.20–10.5 ng/ml and 12.4–23.4 ng/ml, respectively. The intraday and interday precision showed relative standard deviation values of 5.20–14.1 %. This LC–MS method proved to be robust and reliable, and was successfully applied to the quantitation of each drug in rat urine after oral administration of each drug (1.0 mg/kg). To our knowledge, this is the first report to identify and quantitate MDIP, MDCPM, and MDBZ in biological samples by MS.