Quantitative Estimation of 38 Illicit Psychostimulants in Blood by GC-APCI-QTOFMS with Nitrogen Chemiluminescence Detection Based on Three External Calibrators.

A method was developed for quantitative estimation of illicit psychostimulants in blood, with an emphasis on new psychoactive substances, based on gas chromatography nitrogen chemiluminescence detection coupled with atmospheric pressure chemical ionization quadrupole time-of-flight mass spectrometry (GC-NCD-APCI-QTOFMS). Quantitative estimation relied on the NCD's N-equimolar response to nitrogen, using amphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and methylenedioxypyrovalerone as external calibrators for prim-, sec- and tert- amines, respectively. After spiking with 38 stimulants at 3 concentration levels, the donor blood samples were submitted to liquid-liquid extraction at a basic pH followed by acylation with trifluoroacetic anhydride. All but 3 psychostimulants could be analyzed with a limit of quantification (LOQ) of 0.05 mg/L. At LOQ, the coefficient of variation (CV) values for between-day accuracy was 62.3-143.3% (mean, 93.5%; median, 88.5%) and precision 6.6-22.4% (mean, 15.8%; median, 16.1%). In addition, 11 post-mortem blood samples, containing 0.08-2.4 mg/L of amphetamine (n = 5), methamphetamine (n = 4) or MDMA (n = 4), were analyzed by the GC-NCD-APCI-QTOFMS method, and the results were compared with an established electron ionization GC-MS method with appropriate calibration. The agreement between the 2 methods was 62.5-117.3%. Regarding identification, the APCI source permitted detection of the intact precursor ion, or the respective acylation product, for all of the measured compounds. The GC-NCD-APCI-QTOFMS method developed here enables instant quantitative estimation of illicit psychostimulants in blood at reasonable accuracy, without the necessity of possessing the true reference standards for each analyte.

Toxic lifespan of the synthetic opioid U-47,700 in Finland verified by re-analysis of UPLC-TOF-MS data.

U-47,700 is a synthetic opioid that emerged on the novel psychoactive substance market a few years ago. After incorporating the substance into the urine UPLC-TOF-MS screening used in post-mortem toxicology, the drug was detected in 10 autopsy cases within routine case work. In all cases, the cause of death was accidental poisoning by U-47,700 alone or in combination with other psychoactive substances. The concentration of U-47,700 in the blood samples ranged between 0.15-2.0 mg/L with a median of 0.30 mg/L. In one of the cases with a U-47,700 concentration of 0.27 mg/L, no other psychoactive substances were detected. The stored TOF-MS analytical data from the year preceding the incorporation of U-47,700 into the screening was reprocessed in order to search for more positive cases. The data-independent acquisition of the original screening allowed for retrospective re-analysis of the full-scan data without additional experiments on the actual sample. The retrospective data-analysis revealed two additional cases positive for U-47,700. The first mention of U-47,700 on a Finnish internet discussion forum was in March 2015. After having been detected in several death cases, the drug was put under national control in November 2016 and the last fatality occurred in 2017. The toxic lifespan of U-47,700 thus lasted for approximately 2 years in Finland. Forensic and clinical laboratories need to rapidly adjust their screening procedures in order to adapt to the continuously expanding field of novel psychoactive substances. Retrospective data-analysis is a practical tool for monitoring the emergence of new substances onto the market.

Comparison of Post-targeted and Pre-targeted Urine Drug Screening by UHPLC-HR-QTOFMS.

The current illicit drug scene, with its unpredictable appearance of new psychoactive substances, challenges drug-testing laboratories. Ultra-high performance liquid chromatography-high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-HR-QTOFMS) provides an especially versatile analytical platform for responding to this continuous change. QTOFMS can be used to collect nonselective MS/MS by broadband data-independent acquisition (DIA), recording all product ions regardless of the precursor ion. Another approach is to collect selective MS/MS by data-dependent acquisition (DDA), using a narrow precursor mass window with preset criteria such as the presence of a particular ion among a precursor ion list. The present study compared methods based on these two modes of data acquisition on a single UHPLC-HR-QTOFMS instrument setup and using identical sample preparation. The DIA method relied on a post-targeted reverse database search and the DDA method on a spectrum library search, each comprising the same selection of 200 drugs of abuse. The performance between the methods was compared in terms of the limit of identification (LOI) and specificity. The median LOI of the DIA method (8 ng/mL) was lower than that of the DDA method (16 ng/mL). Among the 20 model compounds, a better LOI was obtained with DIA for 13 compounds. DIA was superior in resolving closely eluting and co-eluting isomeric and isobaric compounds. Comparison between the feasibility of DIA and DDA for casework was carried out by analyzing 50 authentic case urine samples. DIA produced 266 identifications involving 46 different substances, and DDA produced 225 identifications involving 42 substances. Moreover, substance identification by DIA was more straightforward and the method was easier to deploy in casework. Nonetheless, the DDA approach with substance-specific collision energies produced informative product ion spectra suitable for occasional confirmatory analyses.

Development of a GC-APCI-QTOFMS library for new psychoactive substances and comparison to a commercial ESI library.

Gas chromatography coupled to atmospheric pressure chemical ionization quadrupole time-of-flight mass spectrometry (GC-APCI-QTOFMS) was evaluated for the identification of new psychoactive substances (NPS). An in-house high mass resolution GC-APCI-QTOFMS test library was developed for 29 nitrogen-containing drugs belonging mostly to synthetic stimulants. The library was based on 12 intra-day measurements of each compound at three different collision energies, 10, 20 and 40 eV. The in-house library mass spectra were compared to mass spectra from a commercial library constructed by liquid chromatography-electrospray ionization (LC-ESI) QTOFMS. The reversed library search scores between the in-house GC-APCI library and the commercial LC-ESI library were compared once a week during a 5-week period by using data measured by GC-APCI-QTOFMS. The protonated molecule was found for all drugs in the full scan mode, and the drugs were successfully identified by both libraries in the targeted MS/MS mode. The GC-APCI library score averaged over all collision energies was as high as 94.4/100 with a high repeatability, while the LC-ESI library score was also high (89.7/100) with a repeatability only slightly worse. These results highlight the merits of GC-APCI-QTOFMS in the analysis of NPS even in situations where the reference standards are not immediately available, taking advantage of the accurate mass measurement of the protonated molecule and product ions, and comparison to existing soft-ionization mass spectral libraries. Graphical abstract Tandem mass spectra obtained from GC-APCI-QTOFMS are comparable to LC-ESI-QTOFMS library spectra.

Simultaneous identification and quantification of new psychoactive substances in blood by GC-APCI-QTOFMS coupled to nitrogen chemiluminescence detection without authentic reference standards.

A novel platform is introduced for simultaneous identification and quantification of new psychoactive substances (NPS) in blood matrix, without the necessity of using authentic reference standards. The instrumentation consisted of gas chromatography (GC) coupled to nitrogen chemiluminescence detection (NCD) and atmospheric pressure chemical ionization quadrupole time-of-flight mass spectrometry (APCI-QTOFMS). In this concept, the GC flow is divided in appropriate proportions between NCD for single-calibrant quantification, utilizing the detector's equimolar response to nitrogen, and QTOFMS for accurate mass-based identification. The principle was proven by analyzing five NPS, bupropion, desoxypipradrol (2-DPMP), mephedrone, methylone, and naphyrone, in sheep blood. The samples were spiked with the analytes post-extraction to avoid recovery considerations at this point. All the NPS studies produced a protonated molecule in APCI resulting in predictable fragmentation with high mass accuracy. The N-equimolarity of quantification by NCD was investigated by using external calibration with the secondary standard caffeine at five concentration levels between 0.17 and 1.7 mg/L in blood matrix as five replicates. The equimolarity was on average 98.7%, and the range of individual equimolarity determinations was 76.7-130.1%. The current analysis platform affords a promising approach to instant simultaneous qualitative and quantitative analysis of drugs in the absence of authentic reference standards, not only in forensic and clinical toxicology but also in other bioanalytical applications.

New psychoactive substances as part of polydrug abuse within opioid maintenance treatment revealed by comprehensive high-resolution mass spectrometric urine drug screening.

OBJECTIVE: At present, polydrug abuse comprises, besides traditional illicit drugs, new psychoactive substances (NPS) and non-prescribed psychotropic medicines (N-PPM). Polydrug abuse was comprehensively evaluated among opioid-dependent patients undergoing opioid maintenance treatment (OMT). METHODS: Two hundred consecutively collected urine samples from 82 OMT patients (52 male) treated with methadone or buprenorphine-naloxone medication were studied using a liquid chromatography/time-of-flight mass spectrometry screening method. The method enables simultaneous detection of hundreds of abused substances covering the traditional drugs of abuse and many NPS as well as N-PPM. RESULTS: Ninety-two (45.8%) samples were positive for the abused substances. Benzodiazepines (29.0%), amphetamines (19.5%), cannabinoids (17.0%), NPS (13.0%), N-PPM (9.0%), and opioids (9.0%) were detected in different combinations. The simultaneous occurrence of up to three groups of abused substances was common (40.0%), and in one sample, all six groups were found. The stimulant NPS alpha-pyrrolidinovalerophenone was found in 10.0% and the sedative N-PPM pregabalin in 4.0% of the samples. The patients were seldom aware of what particular NPS they had abused. CONCLUSIONS: A widespread occurrence of abused substances beyond the ordinary was revealed. Identifying these patients is essential as polydrug abuse is a safety risk to the patient and may cause attrition from OMT.

Patterns of drug abuse among drug users with regular and irregular attendance for treatment as detected by comprehensive UHPLC-HR-TOF-MS.

The most severe consequences of drug abuse include infectious diseases, overdoses, and drug-related deaths. As the range of toxicologically relevant compounds is continually changing due to the emergence of new psychoactive substances (NPS), laboratories are encountering analytical challenges. Current immunoassays are insufficient for determining the whole range of the drugs abused, and a broad-spectrum screening method is therefore needed. Here, the patterns of drug abuse in two groups of drug users were studied from urine samples using a comprehensive screening method based on high-resolution time-of-flight mass spectrometry. The two groups comprised drug abusers undergoing opioid maintenance treatment (OMT) or drug withdrawal therapy and routinely visiting a rehabilitation clinic, and drug abusers with irregular attendance at a harm reduction unit (HRU) and suspected of potential NPS abuse. Polydrug abuse was observed in both groups, but was more pronounced among the HRU subjects with a mean number of concurrent drugs per sample of 3.9, whereas among the regularly treated subjects the corresponding number was 2.1. NPS and pregabalin were more frequent among HRU subjects, and their abuse was always related to drug co-use. The most common drug combination for an HRU subject included amphetamine, cannabis, buprenorphine, benzodiazepine, and alpha-pyrrolidinovalerophenone. A typical set of drugs for treated subjects was buprenorphine, benzodiazepine, and occasionally amphetamine. Abuse of several concurrent drugs poses a higher risk of drug intoxication and a threat of premature termination of OMT. Since the subjects attending treatment used fewer concurrent drugs, this treatment could be valuable in reducing polydrug abuse.

Mitochondrial respiratory dysfunction due to the conversion of substituted cathinones to methylbenzamides in SH-SY5Y cells.

The increased use of cathinone-type designer drugs, known as legal highs, has led to concerns about their potential neurotoxicity due to their similarity to methamphetamine (METH). Therefore, closer investigations of their toxic effects are needed. We investigated the effects of the cathinones 4-methylmethcathinone (4-MMC) and 3,4-methylenedioxymethcathinone (MDMC) and the amphetamine METH on cytotoxicity and mitochondrial respiration in SH-SY5Y neuroblastoma cells. We also investigated the contribution of reactive species, dopamine, Bcl-2 and tumor necrosis factor α (TNFα) on toxicity. Finally, we investigated the effect of cathinone breakdown products using ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry and studied their involvement in toxicity. We observed dose-dependent increases in cytotoxicity and decreases in mitochondrial respiration following treatment with all cathinones and amphetamines. Glutathione depletion increases amphetamine, but not cathinone toxicity. Bcl-2 and TNFα pathways are involved in toxicity but dopamine levels are not. We also show that cathinones, but not amphetamines, spontaneously produce reactive species and cytotoxic methylbenzamide breakdown products when in aqueous solution. These results provide an important first insight into the mechanisms of cathinone cytotoxicity and pave the way for further studies on cathinone toxicity in vivo.

Fatal kavalactone intoxication by suicidal intravenous injection.

Kavalactones are a group of compounds found in kava, a beverage or extract prepared from the rhizome of the kava plant (Piper methysticum). Traditionally kava extracts have been used for their anxiolytic and sedative properties. Sales of kava extracts were severely restricted or prohibited in European countries in 2002 following several cases of serious hepatotoxicity. Here we report a case where high concentrations of kavalactones and ethanol were detected in post mortem femoral blood. An injection needle with a 10-mL syringe containing 7.5 mL of slightly yellowish liquid was found next to the victim, and there were numerous needle prints on both lower arms following the venous tracks. No evidence of other cause of death was found in the medico-legal investigation. The case was therefore classified as suicide using an injection of kavalactones intravenously together with alcohol poisoning.

Targeting misuse of 2-amino-N-ethyl-1-phenylbutane in urine samples: in vitro-in vivo correlation of metabolic profiles and development of LC-TOF-MS method.

A phenyethylamine derivative, 2-amino-N-ethyl-1-phenylbutane (2-AEPB), has recently been detected in doping control and drugs-of-abuse samples, and identified as a non-labelled ingredient in a dietary supplement. To facilitate efficient control of this substance we have studied the in vitro metabolic behaviour of 2-AEPB with human liver preparation, compared these results with in vivo pathways in human, and finally propose an analytical strategy to target the potential misuse of 2-AEPB for toxicological, forensic and doping control purposes. The major in vitro formed metabolites originated from desethylation (M1) and monohydroxylation (M2). A minor metabolite with hydroxylation/N-oxidation was also observed (M3). In vitro-in vivo correlation was studied in an excretion study with a single, oral dose of 2-AEPB-containing supplement. An unmodified substance was the most abundant target compound and detected until the last point of sample collection (72 h), and the detection of M1 (40 h) and M2 (27 h) demonstrated good correlation to in vitro results. In the study with authentic cases (n = 6), 2-AEPB and M1 were mainly found in free urinary fraction, whereas higher inter-individual variability was observed for M2. It was predominantly conjugated and already within this limited number of cases, the ratio between glucuronide- and sulpho-conjugated fractions varied significantly. As a conclusion, hydrolysis is not mandatory in the routine sample preparation, and as the separation can be based on either gas chromatography or liquid chromatography, this study verifies that routine mass spectrometric detection methods targeted to amphetamine derivatives can be easily extended to control the misuse of 2-AEPB.

Comparison between drug screening by immunoassay and ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry in post-mortem urine.

Immunoassay is currently the most common approach for urine drug screening. However, the continuous emergence of new psychoactive substances (NPS) and their low urinary concentrations have challenged the scope and sensitivity of immunoassays. Consequently, specialized toxicology laboratories rely more and more on mass spectrometry (MS) based techniques. Ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOF-MS) is an especially attractive technique for comprehensive drug screening. The objective was to compare the performances of immunoassay and UHPLC-HR-TOF-MS in terms of scope, flexibility, sensitivity, and reliability of substance identification. A total of 279 post-mortem urine samples were analyzed using a method representative of each technique. The immunoassay method was an Emit II Plus enzyme immunoassay for the following drug groups: amphetamines, benzodiazepines, buprenorphine, cannabis, and opiates. The UHPLC-HR-TOF-MS method was a recently published method covering hundreds of drugs: conventional drugs of abuse, abused prescription drugs, and NPS of various classes. UHPLC-HR-TOF-MS produced a lower number of false positive (FP) results for the drug groups covered by immunoassay. Many of the false negative (FN, n = 40) and FP (n = 22) immunoassay results were obviously due to the higher cut-off concentrations and interfering matrix, respectively. Moreover, the wider scope of UHPLC-HR-TOF-MS allowed detection of NPS and prescription drugs. UHPLC-HR-TOF-MS gave FP results related to a few particular substances. The future option of adjusting all compound-specific reporting parameters individually would allow the method's sensitivity and specificity to be fully exploited.

Keto amphetamine toxicity-focus on the redox reactivity of the cathinone designer drug mephedrone.

The ß-keto amphetamine (cathinone, ß-KA) designer drugs such as mephedrone (4-methylmethcathinone, 4-MMC) show a large degree of structural similarity to amphetamines like methamphetamine (METH). However, little is currently known about whether these substances also share the potential neurotoxic properties of their non-keto amphetamine counterparts, or what mechanisms could be involved. Here, we evaluate the cytotoxicity of ß-KAs in SH-SY5Y cells using lactate dehydrogenase (LDH) assays, assess the redox potential of a range of ß-KAs and non-keto amphetamines using the sensitive redox indicator 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1), and explore the effect of 4-MMC on the formation of protein adducts using ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) and on the mitochondrial respiratory chain using high-resolution respirometry. We show that treatment with ß-KAs increases LDH release. Further, we demonstrate that even under physiological pH, ß-KAs are effective and selective-as compared with their non-keto analogues-reductants in the presence of electron acceptors. Increased pH (range 7.6-8.0) greatly enhanced the reactivity up to sixfold. We found no evidence of protein adduct formation, suggesting the reactivity is due to direct electron transfer by the ß-KAs. Finally, we show that 4-MMC and METH produce dissimilar effects on the respiratory chain. Our results indicate that ß-KAs such as 4-MMC possess cytotoxic properties in vitro. Furthermore, in the presence of an electron-accepting redox partner, the ketone moiety of ß-KAs is vital for pH-dependent redox reactivity. Further work is needed to establish the importance of ß-KA redox properties and its potential toxicological importance in vivo.

A high-sensitivity ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method for screening synthetic cannabinoids and other drugs of abuse in urine.

The continuing emergence of designer drugs imposes high demands on the scope and sensitivity of toxicological drug screening procedures. An ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method was developed for screening and simultaneous confirmation of both designer drugs and other drugs of abuse in urine samples in a single run. The method covered selected synthetic cannabinoids and cathinones, amphetamines, natural cannabinoids, opioids, cocaine and other important drugs of abuse, together with their main urinary metabolites. The database consisted of 277 compounds with molecular formula and exact monoisotopic mass; retention time was included for 192 compounds, and primary and secondary qualifier ion exact mass for 191 and 95 compounds, respectively. Following a solid-phase extraction, separation was performed by UHPLC and mass analysis by HR-TOFMS. MS, and broad-band collision-induced dissociation data were acquired at m/z range 50-700. Compound identification was based on a reverse database search with acceptance criteria for retention time, precursor ion mass accuracy, isotopic pattern and abundance of qualifier ions. Mass resolving power in spiked urine samples was on average FWHM 23,500 and mass accuracy 0.3 mDa. The mean and median cut-off concentrations determined for 75 compounds were 4.2 and 1 ng/mL, respectively. The range of cut-off concentrations for synthetic cannabinoids was 0.2-60 ng/mL and for cathinones 0.7-15 ng/mL. The method proved to combine high sensitivity and a wide scope in a manner not previously reported in drugs of abuse screening. The method's feasibility was demonstrated with 50 authentic urine samples.

In silico and in vitro metabolism studies support identification of designer drugs in human urine by liquid chromatography/quadrupole-time-of-flight mass spectrometry.

Human phase I metabolism of four designer drugs, 2-desoxypipradrol (2-DPMP), 3,4-dimethylmethcathinone (3,4-DMMC), α-pyrrolidinovalerophenone (α-PVP), and methiopropamine (MPA), was studied using in silico and in vitro metabolite prediction. The metabolites were identified in drug abusers' urine samples using liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC/Q-TOF/MS). The aim of the study was to evaluate the ability of the in silico and in vitro methods to generate the main urinary metabolites found in vivo. Meteor 14.0.0 software (Lhasa Limited) was used for in silico metabolite prediction, and in vitro metabolites were produced in human liver microsomes (HLMs). 2-DPMP was metabolized by hydroxylation, dehydrogenation, and oxidation, resulting in six phase I metabolites. Six metabolites were identified for 3,4-DMMC formed via N-demethylation, reduction, hydroxylation, and oxidation reactions. α-PVP was found to undergo reduction, hydroxylation, dehydrogenation, and oxidation reactions, as well as degradation of the pyrrolidine ring, and seven phase I metabolites were identified. For MPA, the nor-MPA metabolite was detected. Meteor software predicted the main human urinary phase I metabolites of 3,4-DMMC, α-PVP, and MPA and two of the four main metabolites of 2-DPMP. It assisted in the identification of the previously unreported metabolic reactions for α-PVP. Eight of the 12 most abundant in vivo phase I metabolites were detected in the in vitro HLM experiments. In vitro tests serve as material for exploitation of in silico data when an authentic urine sample is not available. In silico and in vitro designer drug metabolism studies with LC/Q-TOF/MS produced sufficient metabolic information to support identification of the parent compound in vivo.

A rare case of serial killing by poisoning.

A case of serial killing by poisoning by a 59-year-old practical nurse is discussed. Following a report by an emergency-room doctor of an attempted murder, police performed an investigation into all deaths of patients in the nurse's care. Earlier, a medico-legal cause-of-death investigation had been performed on two of these cadavers, but in the other three cases the death certificate had been issued after a medical investigation only. In two of these latter cases, the body had been cremated, but fixed histological samples taken at medical autopsy were available, while in one case the person had died recently and the body was thereafter exhumed and autopsied. All of the suspected victims were older people who required nursing, and the nurse's course of action was consistent in all cases. In the absence of ordinary post-mortem toxicology samples in the medical cases, extraordinary evidence--paraffin-embedded liver tissue samples originally taken for histology at autopsy--was successfully recovered in two cases and analyzed for drugs. In all five cases, drugs not prescribed to the patient were detected, including digoxin, dixyrazine, citalopram, venlafaxine, and benzodiazepines (diazepam, chlordiazepoxide, temazepam, and oxazepam). The nurse was eventually found guilty of five murders by poisoning, five attempted murders, and three aggravated assaults. The nurse was sentenced to life imprisonment.

Combined drug screening and confirmation by liquid chromatography time-of-flight mass spectrometry with reverse database search.

A method based on in-source collision-induced dissociation (ISCID) liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) and reverse target database search was developed and evaluated for drug screening and confirmation in analytical toxicology context. An established LC-TOFMS screening method, in which identification relies solely on protonated molecule accurate mass measurement, isotopic pattern fit, and retention time (RT), was completed to include 1-3 qualifier ions for each analyte in the database. The qualifier ions for 431 compounds were selected from the experimental ISCID spectra, and their molecular formulae were assigned by applying SmartFormula3D and MSFragmenter software. Three qualifier ions were assigned for 64.5%, two or three for 81.4%, one for 14.8%, and none for 3.7% of the compounds studied. Comparison between ISCID LC-TOFMS and LC-TOFMS with 25 authentic autopsy urine samples showed an improved confidence level with the ISCID method, as isomeric interferences were excluded in most cases. However, some false negative (FN) results were obtained at low concentration levels close to the reporting criteria. The cut-off concentration of the ISCID method was 10-100 ng/mL with 80% of the 49 representative compounds tested, and the level was approximately two times higher than that obtained by LC ion trap MS. The presented method enables simultaneous screening and confirmation whenever at least one qualifier ion is available, as applying an accurate mass precursor ion and one product ion surpasses the standard of four identification points that is required by the current EU protocol.

Prediction of liquid chromatographic retention for differentiation of structural isomers.

A liquid chromatography (LC) retention time prediction software, ACD/ChromGenius, was employed to calculate retention times for structural isomers, which cannot be differentiated by accurate mass measurement techniques alone. For 486 drug compounds included in an in-house database for urine drug screening by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOFMS), a retention time knowledge base was created with the software. ACD/ChromGenius calculated retention times for compounds based on the drawn molecular structure and given chromatographic parameters. The ability of the software for compound identification was evaluated by calculating the retention order of the 118 isomers, in 50 isomer groups of 2-5 compounds each, included in the database. ACD/ChromGenius predicted the correct elution order for 68% (34) of isomer groups. Of the 16 groups for which the isomer elution order was incorrectly calculated, two were diastereomer pairs and thus difficult to distinguish using the software. Correlation between the calculated and experimental retention times in the knowledge base tested was moderate, r(2)=0.8533. The mean and median absolute errors were 1.12 min, and 0.84 min, respectively, and the standard deviation was 1.04 min. The information generated by ACD/ChromGenius, together with other in silico methods employing accurate mass data, makes the identification of substances more reliable. This study demonstrates an approach for tentatively identifying compounds in a large target database without a need for primary reference standards.

Current use of high-resolution mass spectrometry in drug screening relevant to clinical and forensic toxicology and doping control.

Clinical and forensic toxicology and doping control deal with hundreds or thousands of drugs that may cause poisoning or are abused, are illicit, or are prohibited in sports. Rapid and reliable screening for all these compounds of different chemical and pharmaceutical nature, preferably in a single analytical method, is a substantial effort for analytical toxicologists. Combined chromatography-mass spectrometry techniques with standardised reference libraries have been most commonly used for the purpose. In the last ten years, the focus has shifted from gas chromatography-mass spectrometry to liquid chromatography-mass spectrometry, because of progress in instrument technology and partly because of the polarity and low volatility of many new relevant substances. High-resolution mass spectrometry (HRMS), which enables accurate mass measurement at high resolving power, has recently evolved to the stage that is rapidly causing a shift from unit-resolution, quadrupole-dominated instrumentation. The main HRMS techniques today are time-of-flight mass spectrometry and Orbitrap Fourier-transform mass spectrometry. Both techniques enable a range of different drug-screening strategies that essentially rely on measuring a compound's or a fragment's mass with sufficiently high accuracy that its elemental composition can be determined directly. Accurate mass and isotopic pattern acts as a filter for confirming the identity of a compound or even identification of an unknown. High mass resolution is essential for improving confidence in accurate mass results in the analysis of complex biological samples. This review discusses recent applications of HRMS in analytical toxicology.

Evaluation of a high resolving power time-of-flight mass spectrometer for drug analysis in terms of resolving power and acquisition rate.

Liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) is applied increasingly to various fields of small molecule analysis. The moderate resolving power (RP) of standard TOFMS instruments poses a risk of false negative results when complex biological matrices are to be analyzed. In this study, the performance of a high resolving power TOFMS instrument (maXis by Bruker Daltonik, Bremen, Germany) was evaluated for drug analysis. By flow injection analysis of critical drug mixtures, including a total of 17 compounds with nominal masses of 212-415 Da and with mass differences of 8.8-23.5 mDa, RP varied from 34,400 to 51,900 (FWHM). The effect of acquisition rate on RP, mass accuracy, and isotopic pattern fit was studied by applying 1, 2, 5, 10, and 20 Hz acquisition rates in a 16 min gradient elution LC separation. All three variables were independent of the acquisition rate, with an average mass accuracy and isotopic pattern fit factor (mSigma) of 0.33 ppm and 5.9, respectively. The average relative standard deviation of RP was 1.8%, showing high repeatability. The performance was tested further with authentic urine extracts containing a co-eluting compound pair with a nominal mass of 296 Da and an 11.2 mDa mass difference. The authentic sample components were readily resolved and correctly identified by the automated data analysis. The average RP, mass accuracy, and isotopic pattern fit were 36,600, 0.9 ppm, and 7.3 mSigma, respectively.

Nicotine, alcohol, and drug findings in young adults in a population-based postmortem database.

INTRODUCTION: To obtain reliable information on nicotine and drug use through a population-based study, the prevalence of nicotine use in deceased young adults was studied in the Finnish postmortem toxicology database for a 3-year period. The nicotine user and non-nicotine user groups were compared by alcohol, drug, and drug-of-abuse findings and by the manner of death. METHODS: Nicotine users were identified based on detection of nicotine, cotinine, and/or trans-3'-hydroxycotinine in urine from a population-based sample of deceased young adults aged 15-34 years at the time of death (n = 1,623, ∼60% of all fatalities). Background information from case referrals was used to distinguish the abuse of medicines from their therapeutic use. The manner of death was taken from death certificates. RESULTS: Nicotine use was more common in young adults (75%) than among all cases in the database (55%). There were twice as many ethanol-positive cases in nicotine users (60%) than in non-nicotine users (30%). Nicotine use was common (70%-79%) among individuals on antipsychotics, antidepressants, anxiolytics, and/or hypnotics and sedatives. The proportion of nicotine users was also high among the drugs-of-abuse positive cases (85%). There were fewer deaths that were classified as natural in the nicotine users group. CONCLUSIONS: Among deceased young adults, nicotine use was two to three times as common as has been estimated for the corresponding living population (20%-30%). Nicotine use was also strongly associated with substance abuse and mental illnesses requiring pharmacotherapy. This group of young adults usually cannot be reached by traditional health surveys.