Distinguishing Between Cyclopropylfentanyl and Crotonylfentanyl by Methods Commonly Available in the Forensic Laboratory.

BACKGROUND: The opioid analgesic fentanyl and its analogues pose a major health concern due to its high potency and the increasing number of overdose deaths worldwide. The analogues of fentanyl may differ in potency, toxicity, and legal status, and it is therefore important to develop analytical methods for their correct identification. This can be challenging since many fentanyl analogues are structural isomers. Two fentanyl isomers that have been in the spotlight lately due to difficulties regarding separation and identification are cyclopropylfentanyl and crotonylfentanyl, which have been reported to display nearly identical fragmentation patterns and chromatographic behavior. METHODS: Chromatographic separation of cyclopropylfentanyl and crotonylfentanyl by ultra-high-performance liquid chromatography was investigated using 3 different stationary phases (high strength silica T3, ethylsiloxane/silica hybrid C18, and Kinetex biphenyl) using gradient elution with a mobile phase consisting of 10 mM ammonium formate pH 3.1 and MeOH. Detection was performed by tandem mass spectrometry. In addition, the major metabolites of the 2 compounds formed on incubation with human liver microsomes were identified by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis. RESULTS: Baseline separation of cyclopropylfentanyl and crotonylfentanyl was achieved on the ethylsiloxane/silica hybrid C18 column with retention times of 6.79 and 7.35 minutes, respectively. The major metabolites of the 2 analogues formed by human liver microsomes differed, with the main biotransformation being N-dealkylation and carboxylation for cyclopropylfentanyl and crotonylfentanyl, respectively. We demonstrated the usefulness of the 2 approaches by unambiguously identifying cyclopropylfentanyl, as well as its metabolites, in 2 authentic postmortem blood samples. CONCLUSIONS: In this study, we successfully demonstrated that cyclopropylfentanyl and crotonylfentanyl can be distinguished by methods commonly available in forensic laboratories.

Addressing the Fentanyl Analogue Epidemic by Multiplex UHPLC-MS/MS Analysis of Whole Blood.

BACKGROUND: Fentanyl and fentanyl analogues (fentanyls) are very potent opioids posing a serious threat to the public health. Thousands of overdose deaths across the world are caused by fentanyls, and the numbers are increasing. Rapid mapping of current trends in opioid abuse is necessary to accelerate preventive measures. To ensure this, there is a need for sensitive targeted multiplex MS/MS methods to pinpoint drugs of abuse. We present a fully validated UHPLC-MS/MS method for the determination of 26 fentanyls, including several structural isomers, and the opioid antagonist naloxone in human whole blood. METHODS: Blood samples were prepared by liquid-liquid extraction with ethyl acetate and heptane. The fentanyls were separated with UHPLC, using a Kinetex biphenyl column (2.1 × 100 mm, 1.7 µm; Phenomenex, Verløse, Denmark) with an acidic mobile phase. Quantification was performed by MS/MS. The method was validated according to SWGTOX guidelines. RESULTS: The developed method could successfully separate all 27 analytes, including 7 isomers, and was validated according to SWGTOX guidelines with very low limits of quantification (4-20 pg/mL). The applicability of the method was demonstrated by determination of fentanyls in postmortem blood samples from 2 cases. CONCLUSIONS: A selective, highly sensitive, and robust method for determination of a large panel of fentanyls and naloxone in blood was developed and validated. Naloxone was included to monitor use and efficacy of the opioid antidote in cases of fentanyl overdoses. The method demonstrated good ability to separate structural isomers, which is important to differentiate between the numerous available fentanyls with variable potency, toxicity, and legal status. The developed method can be used to identify fentanyls on the drug market to help combat the fentanyl crisis.

Rapid determination of designer benzodiazepines, benzodiazepines, and Z-hypnotics in whole blood using parallel artificial liquid membrane extraction and UHPLC-MS/MS.

Benzodiazepines (BZD) and Z-hypnotics are frequently analyzed in forensic laboratories, and in 2012, the designer benzodiazepines (DBZD) emerged on the illegal drug scene. DBZD represent a particular challenge demanding new analytical methods. In this work, parallel artificial liquid membrane extraction (PALME) is used for sample preparation of DBZD, BZD, and Z-hypnotics in whole blood prior to UHPLC-MS/MS analysis. PALME of BZD, DBZD, and Z-hypnotics was performed from whole blood samples, and the analytes were extracted across a supported liquid membrane (SLM) and into an acceptor solution of dimethyl sulfoxide and 200 mM formic acid (75:25, v/v). The method was validated according to EMA guidelines. The method was linear throughout the calibration range (R2 > 0.99). Intra- and inter-day accuracy and precision, as well as matrix effects, were within the guideline limit of ± 15%. LOD and LLOQ ranged from 0.10 to 5.0 ng mL-1 and 3.2 to 160 ng mL-1, respectively. Extraction recoveries were reproducible and above 52%. The method was specific, and the analytes were stable in the PALME extracts for 4 and 10 days at 10 and - 20 °C. No carry-over was observed within the calibration range. PALME and UHPLC-MS/MS for the determination of DBZD, BZD, and Z-hypnotics in whole blood are a green and low-cost alternative that provides high sample throughput (96-well format), extensive sample clean-up, good sensitivity, and high reproducibility. The presented method is also the first method incorporating analysis of DBZD, BZD, and Z-hypnotics in whole blood in one efficient analysis. Graphical abstract.

Driving under the influence of cocaine and MDMA: Relationship between blood concentrations and results from clinical test of impairment.

The general use of cocaine is increasing in recent years, while the trend for 3,4-methylenedioxymethamphetamine (MDMA) is less clear. The relationship between blood concentrations and impairment is poorly understood, which complicates interpretation. The aims of this study were to report prevalence and blood concentrations of cocaine and MDMA in drugged drivers, and to investigate the relationship between blood concentrations and impairment. Samples of whole blood were collected from apprehended drivers in the period 2000-2022, and a clinical test of impairment (CTI) was simultaneously performed. The samples were initially analyzed for cocaine and MDMA using gas chromatography-mass spectrometry (until 2009 and 2012, respectively), and later using ultra-high-performance liquid chromatography-tandem mass spectrometry. Overall, cocaine was detected in 2,331 cases and MDMA in 2,569 cases. There were 377 and 85 mono cases of cocaine and MDMA, respectively. In the mono cases, the median cocaine concentration was 0.09 mg/L (range: 0.02-1.15 mg/L), and 54% of the drivers were clinically impaired. The median MDMA concentration was 0.19 mg/L (range: 0.04-1.36 mg/L), and 38% were clinically impaired. There was a statistically significant difference in the median cocaine concentration between drivers assessed as not impaired (0.07 mg/L) and drivers assessed as impaired (0.10 mg/L) (P = 0.009). There was also a significant effect of the blood concentration of cocaine (adjusted odds ratio [aOR] = 6.42, 95% confidence interval [CI] = 1.13-36.53, P = 0.036) and driving during the evening/night-time (aOR = 2.17, 95% CI = 1.34-3.51, P = 0.002) on the probability of being assessed as impaired on the CTI. No significant differences were found for MDMA. Many drivers are not assessed as impaired on a CTI following cocaine or especially MDMA use. For cocaine, a relationship between blood concentrations and impairment was demonstrated, but this could not be shown for MDMA.

Metabolite markers for three synthetic tryptamines N-ethyl-N-propyltryptamine, 4-hydroxy-N-ethyl-N-propyltryptamine, and 5-methoxy-N-ethyl-N-propyltryptamine.

N-Ethyl-N-propyltryptamine (EPT), 4-hydroxy-N-ethyl-N-propyltryptamine (4-OH-EPT), and 5-methoxy-N-ethyl-N-propyltryptamine (5-MeO-EPT) are new psychoactive substances classified as tryptamines, sold online. Many tryptamines metabolize rapidly, and identifying the appropriate metabolites to reveal intake is essential. While the metabolism of 4-OH-EPT and 5-MeO-EPT are not previously described, EPT is known to form metabolites by indole ring hydroxylation among others. Based on general knowledge of metabolic patterns, 5-MeO-EPT is also expected to form ring hydroxylated EPT (5-OH-EPT). In the present study, the aim was to characterize the major metabolites of EPT, 4-OH-EPT, and 5-MeO-EPT, to provide markers for substance identification in forensic casework. The tryptamines were incubated with pooled human liver microsomes at 37°C for up to 4 h. The generated metabolites were separated and detected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis. The major in vitro EPT metabolites were formed by hydroxylation, N-dealkylation, and carbonylation. In comparison, 4-OH-EPT metabolism was dominated by double bond formation, N-dealkylation, hydroxylation, and carbonylation in vitro and hydroxylation or carbonylation combined with double bond loss, carbonylation, N-dealkylation, and hydroxylation in vivo. 5-MeO-EPT was metabolized by O-demethylation, hydroxylation, and N-dealkylation in vitro. The usefulness of the characterized metabolites in forensic casework was demonstrated by identification of unique metabolites for 4-OH-EPT in a human postmortem blood sample with suspected EPT or 4-OH-EPT intoxication.

High-throughput quantification of emerging "nitazene" benzimidazole opioid analogs by microextraction and UHPLC-MS-MS.

Benzimidazole opioids, often referred to as nitazenes, represent a subgroup of new psychoactive substances with a recent increase in fatal overdoses in the USA and Europe. With a variety of analogs emerging on the illicit drug market, forensic laboratories are challenged to identify these potent drugs. We here present a simple quantitative approach for the determination of nine nitazene analogs, namely, clonitazene, etodesnitazene, etonitazene, etonitazepyne, flunitazene, isotonitazene, metodesnitazene, metonitazene and protonitazene in whole blood using liquid-phase microextraction and electromembrane extraction in a 96-well format and liquid chromatography-tandem mass spectrometry. Green and efficient sample preparation was accomplished by liquid-phase microextraction in a 96-well format and resulted in high extraction yields for all analytes (>81%). Here, blood diluted with buffer (1:1, %v) was extracted from a donor compartment across a thin organic liquid membrane and into an aqueous acceptor solution. The acceptor solution was collected and directly injected into the analysis platform. Chromatographic separation was accomplished with a biphenyl column, allowing for a baseline separation of the structural isomers isotonitazene and protonitazene before detection by multiple reaction monitoring. Validation was performed according to Scientific Working Group of Forensic Toxicology guidelines. The calibration range was from 0.5 to 50 nM (except for protonitazene and clonitazene from 0.1 nM) with good linearity and limits of detection down to 0.01 nM. An AGREEprep assessment was performed to evaluate sample preparation greenness, with a final score of 0.71. Nitazenes represent a current threat to public health, and analytical methods that cover a wide range of these analogs are limited. Here, the described method may assist in the detection of nitazenes in whole blood and prevent these substances from being missed in postmortem investigations.

Comparative Study of Postmortem Concentrations of Benzodiazepines and Z-Hypnotics in Several Different Matrices.

Benzodiazepines and z-hypnotics are detected in the majority of fatal overdose cases in Norway, often in combination with other drugs of abuse, and their concentrations in peripheral blood (PB) might be important to elucidate the cause of death. In some forensic autopsies, PB is however not available. The aim of the present study was to compare concentrations of benzodiazepines and z-hypnotics in five alternative matrices to assess whether these concentrations are comparable to concentrations in PB. A total of 109 forensic autopsy cases were included. PB, cardiac blood (CB), pericardial fluid (PF), psoas muscle (PM), lateral vastus muscle (LVM) and vitreous humor (VH) from each case were analyzed using ultra high performance liquid chromatography--tandem mass spectrometry. We were able to detect clonazepam, 7-aminoclonazepam, flunitrazepam, 7-aminoflunitrazepam, nitrazepam, 7-aminonitrazepam, diazepam, nordiazepam, oxazepam, alprazolam, midazolam, zopiclone and zolpidem in all the analyzed matrices. Concentrations measured in VH were generally much lower than those of PB for all compounds except zopiclone. 7-Amino metabolite concentrations were high compared to the parent compounds, although less so for the muscle samples. Concentrations of the parent nitrobenzodiazepines in muscles were higher than those in PB, but for the other compounds, concentrations in muscle showed good correspondence with PB. Both CB and PF were viable alternative matrices for PB, although a larger variation and a tendency for higher concentrations in PF were observed. This study shows that CB, PM, LVM and PF can give comparable concentrations to PB for benzodiazepines and z-hypnotics, while VH was less suitable. The concentrations in alternative matrices must, however, be interpreted carefully.

Methadone, Buprenorphine, Oxycodone, Fentanyl and Tramadol in Multiple Postmortem Matrices.

Peripheral blood (PB) concentrations are generally preferred for postmortem toxicological interpretation, but some autopsy cases may lack blood for sampling due to decomposition or large traumas, etc. In such cases, other tissues or bodily fluids must be sampled; however, limited information exists on postmortem concentrations in matrices other than blood. Pericardial fluid (PF), muscle and vitreous humor (VH) have been suggested as alternatives to blood, but only a few studies have investigated the detection of opioids in these matrices. In this study, we aimed to investigate the detection of methadone, buprenorphine, oxycodone, fentanyl and tramadol in postmortem samples of PF, skeletal muscle and VH, in addition to PB and cardiac blood and if drug concentrations in these alternative matrices were comparable to those in PB and thereby useful for interpretation. In most of the 54 included cases, only one opioid was detected. Methadone, oxycodone, fentanyl and tramadol were detected in all of the alternative matrices in almost all cases, while buprenorphine was detected less often. For methadone, the concentrations in the alternative matrices, except in VH, were relatively similar to those in PB. Larger variations in concentrations were found for buprenorphine, oxycodone and tramadol. Quantitative analyses appeared useful for fentanyl, in all of the alternative matrices, but only four cases were included. Toxicological analyses of opioids in these alternative postmortem matrices can be useful for detection, but quantitative results must be interpreted with caution.

Selectivity and sensitivity of urine fentanyl test strips to detect fentanyl analogues in illicit drugs.

BACKGROUND: Urine fentanyl test strips have been employed to check street drugs for fentanyl and fentanyl analogue contamination, but there is limited evidence for the applicability of fentanyl strips for this purpose. We examined the ability of four commercially-available fentanyl test strips to detect fentanyl and a range of fentanyl analogues currently on the recreational drug market. METHODS: Four brands of fentanyl test strips (Rapid Response, One Step, Nal van Minden, and Rapid Self Test) were examined using single-component drug solutions containing fentanyl, 28 fentanyl analogues, four non-fentanyl synthetic opioids, or eight traditional drugs of abuse. The effect of co-presence of heroin or ascorbic acid on test results was also examined. RESULTS: All test strips detected fentanyl as well as 21-24 of the 28 fentanyl analogues tested. One of the test strip brands gave false positive results in the presence of ascorbic acid. CONCLUSIONS: Fentanyl test strips successfully detected the majority of fentanyl analogues tested. Drug solutions for testing should not be overly dilute, since the test results are highly concentration dependent. Fentanyl test strips have utility as a harm reduction tool, but they are no panacea for overdose since certain fentanyl analogues are not detected.

Distribution of tetrahydrocannabinol and cannabidiol in several different postmortem matrices.

Cannabis is the most widely used illicit substance worldwide. A limited number of studies have investigated whether tetrahydrocannabinol (THC) and cannabidiol (CBD) can be detected in other postmortem matrices than blood and urine. The aim of this study was to investigate the distribution of THC and CBD in several different postmortem matrices. Concentrations in peripheral blood were compared to those in cardiac blood, pericardial fluid, psoas muscle, vastus lateralis muscle, and vitreous humor. A total of 39 postmortem forensic autopsy cases were included. THC and CBD were analyzed using gas chromatography-mass spectrometry. We were able to detect both THC and CBD in most of the analyzed matrices. For vitreous humor, however, only approximately 50% of the cases were available for analysis, and only two were found to be positive. Median concentrations in peripheral blood were 0.0040 (0.00042-0.056) mg/L for THC and 0.0013 (0-0.023) mg/L for CBD. The concentration ratios between pericardial fluid and cardiac blood compared to peripheral blood were< 1 for both THC and CBD for the majority of the cases. For THC, a median ratio of 0.60 (0.063-7.2) and 0.65 (0.068-4.8) were found for pericardial fluid and cardiac blood, respectively, compared to peripheral blood, whereas for CBD the corresponding median ratios were 0.40 (0.010-1.9) and 0.80 (0.017-2.4). The THC concentrations in psoas muscle and vastus lateralis muscle were high compared to those in peripheral blood in several cases, and large variations in the muscles to peripheral blood concentration ratios were seen. This was also the case for CBD. Our study shows that THC and CBD can be detected in postmortem matrices other than peripheral blood, and results from other matrices might provide important information in forensic cases where peripheral blood is not available. However, vitreous humor was not suitable for detecting neither THC nor CBD.

Blood Concentrations of Designer Benzodiazepines: Relation to Impairment and Findings in Forensic Cases.

The use of designer benzodiazepines appears to be increasing in many countries, but data concerning blood concentrations are scarce, making interpretation of concentrations difficult. The aim of this study was to report blood concentrations of clonazolam, diclazepam, etizolam, flualprazolam, flubromazepam, flubromazolam and phenazepam and to investigate the relationship between blood concentrations and impairment. The concentration data are from blood samples collected from living cases (apprehended drivers and other drug offences) and medico-legal autopsies. The blood samples were analysed for the seven designer benzodiazepines mentioned above by ultra high performance liquid chromatography-tandem mass spectrometry. Positive cases from between 1 June 2016 and 30 September 2019 were included. Blood concentrations and the conclusion from a clinical test of impairment (when available) are reported. The presented seven benzodiazepines were detected in a total of 575 cases, where 554 of these cases concerned apprehended drivers or other criminal offenders. The number of findings and the median (range) concentrations were as follows: clonazolam, n = 22, 0.0041 mg/L (0.0017-0.053 mg/L); diclazepam, n = 334, 0.0096 mg/L (0.0016-0.25 mg/L); etizolam, n = 40, 0.054 mg/L (0.015-0.30 mg/L); flualprazolam, n = 10, 0.0080 mg/L (0.0033-0.056 mg/L); flubromazepam, n = 5, 0.037 mg/L (0.0070-0.70 mg/L); flubromazolam, n = 20, 0.0056 mg/L (0.0004-0.036 mg/L); and phenazepam, n = 138, 0.022 mg/L (0.0018-0.85 mg/L). A designer benzodiazepine was the only drug detected with relevance for impairment in 25 of the 554 living cases. The physician concluded with impairment in 19 of the 25 cases. Most of the concentrations in these cases were relatively similar to or higher than the median reported concentrations. The most frequent other drugs detected were amphetamine, tetrahydrocannabinol, clonazepam and methamphetamine. The presented blood concentrations can be helpful with the interpretation of cases involving one or more of these seven benzodiazepines. The results indicate that concentrations commonly observed in forensic cases are associated with impairment.

Determination of adrenaline, noradrenaline and corticosterone in rodent blood by ion pair reversed phase UHPLC-MS/MS.

A novel ion pair reversed phase ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of the stress hormones adrenaline, noradrenaline and corticosterone in rodent blood was developed and fully validated. Separations were performed on an Acquity HSS T3 column (2.1mm i.d.×100mm, 1.8µm) with gradient elution and a runtime of 5.5min. The retention of adrenaline and noradrenaline was substantially increased by employing the ion pair reagent heptafluorobutyric acid (HFBA). Ion pair reagents are usually added to the mobile phase only, but we demonstrate for the first time that including HFBA to the sample reconstitution solvent as well, has a major impact on the chromatography of these compounds. The stability of adrenaline and corticosterone in rodent blood was investigated using the surrogate analytes adrenaline-d3 and corticosterone-d8. The applicability of the described method was demonstrated by measuring the concentration of stress hormones in rodent blood samples.

Is Hair Analysis Useful in Postmortem Cases?

In postmortem cases, detection of drugs in blood is most relevant with regard to determining cause of death. However, it is sometimes also of interest to gain as much information as possible regarding the deceased's use of drugs in the period before death. The aim of this study was to compare results from analyses of a repertoire of psychoactive medicinal drugs in blood and hair samples from a larger material of postmortem cases. Hair samples in addition to blood were collected from 55 forensic autopsies and analyzed for a repertoire of 39 medicinal drugs (benzodiazepines, antidepressants and antipsychotics) using av fully validated liquid chromatography-tandem mass spectrometry method. In total, hair analyses gave information of the use of drugs not detected in blood in 47 of the 55 cases (85%). The most frequent single drugs detected in hair, but absent in blood, were benzodiazepines (64%), followed by antidepressants (35%). In each case, 1-10 (median two) single drugs were detected in hair, but absent in blood. In only two cases (4%), benzodiazepines were detected in blood and no benzodiazepines were detected in hair. In conclusion, hair analyses in addition to blood frequently indicate prior use of drugs that could yield important information about for instance unknown psychiatric diagnoses. In only a small number of cases lack of detections from the same drug class in hair might indicate reduced tolerance to drug effects.

Can measurements of heroin metabolites in post-mortem matrices other than peripheral blood indicate if death was rapid or delayed?

BACKGROUND: In heroin-related deaths, it is often of interest to determine the approximate time span between intake of heroin and death, and to decide whether heroin or other opioids have been administered. In some autopsy cases, peripheral blood cannot be sampled due to decomposition, injuries or burns. The aim of the present study was to investigate whether measurements of heroin metabolites in matrices other than peripheral blood can be used to differentiate between rapid and delayed heroin deaths, and if morphine/codeine ratios measured in other matrices can separate heroin from codeine intakes. METHODS: In this study, we included 51 forensic autopsy cases where morphine was detected in peripheral blood. Samples were collected from peripheral and cardiac blood, pericardial fluid, psoas and lateral vastus muscles, vitreous humor and urine. The opioid analysis included 6-acetylmorphine (6-AM), morphine, morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and codeine. Urine was only used for qualitative detection of 6-AM. 45 heroin-intake cases were divided into rapid deaths (n=24), based on the detection of 6-AM in blood, or delayed deaths (n=21), where 6-AM was detected in at least one other matrix but not in blood. An additional 6 cases were classified as codeine-intake cases, based on a morphine/codeine ratio below unity (<1) in peripheral blood, without detecting 6-AM in any matrix. RESULTS: The median morphine concentrations were significantly higher in the rapid compared with the delayed heroin deaths in all matrices (p=0.004 for vitreous humor and p<0.001 for the other matrices). In the rapid heroin deaths, the M3G/morphine concentration ratios were significantly lower than in the delayed deaths both in peripheral and cardiac blood (p<0.001), as well as in pericardial fluid (p<0.001) and vitreous humor (p=0.006), but not in muscle. The morphine/codeine ratios measured in cardiac blood, pericardial fluid and the two muscle samples resembled the ratios in peripheral blood, although codeine was less often detected in other matrices than peripheral blood. CONCLUSIONS: Measurements of heroin-metabolites in cardiac blood, pericardial fluid and vitreous humor provide information comparable to that of peripheral blood regarding rapid and delayed heroin deaths, e.g. M3G/morphine ratios <2 indicate a rapid death while ratios >3 indicate a delayed death. However, considerable overlap in results from rapid and delayed deaths was observed, and measurements in muscle appeared less useful. Furthermore, matrices other than peripheral blood can be used to investigate morphine/codeine ratios, but vitreous humor seems less suited.

Comparative Study of Postmortem Concentrations of Antidepressants in Several Different Matrices.

Peripheral blood (PB) is considered to be the golden standard for measuring postmortem drug concentrations. In several cases, PB is however not available, but information regarding drug findings might still be crucial in order to determine the cause of death. Antidepressants are frequently detected in postmortem samples from forensic toxicology cases, but the literature investigating concentrations in other matrices than peripheral and heart blood is limited.We here describe a study for comparison of concentrations for a large number of different drugs in six different matrices. A total of 173 postmortem cases were included in the study material. The results from 44 cases with findings of antidepressants (amitriptyline/nortriptyline, citalopram, mianserin, mirtazapine, paroxetine, sertraline, trimipramine and venlafaxine) are presented in this article. Concentrations in peripheral and cardiac blood (CB), pericardial fluid (PF), two muscle samples and vitreous humour (VH) are compared. Ratios between concentrations in different matrices have also been compiled from available literature.All the investigated antidepressants were detected in all different matrices, and comparable concentration levels were found in the different matrices with a few exceptions. Concentrations in VH were generally lower than in the other matrices, and in a few cases with low concentrations in blood the antidepressants were not detected in VH. For most of the cases, ratios of 0.5-2 were found between concentration in PB and that in the other matrices. Some deviant concentrations where however found.This study shows that CB, PF, muscle and VH can provide important indications of the corresponding concentrations in PB when PB is not available.

Metabolites of Heroin in Several Different Post-mortem Matrices.

In some forensic autopsies blood is not available, and other matrices are sampled for toxicological analysis. The aims of the present study were to examine whether heroin metabolites can be detected in different post-mortem matrices, and investigate whether analyses in other matrices can give useful information about concentrations in peripheral blood. Effects of ethanol on the metabolism and distribution of heroin metabolites were also investigated. We included 45 forensic autopsies where morphine was detected in peripheral blood, concomitantly with 6-acetylmorphine (6-AM) detected in any matrix. Samples were collected from peripheral blood, cardiac blood, pericardial fluid, psoas muscle, lateral vastus muscle, vitreous humor and urine. Opioid analysis included 6-AM, morphine, codeine, and morphine glucuronides. The 6-AM was most often detected in urine (n = 39) and vitreous humor (n = 38). The median morphine concentration ratio relative to peripheral blood was 1.3 (range 0-3.6) for cardiac blood, 1.4 (range 0.07-5.3) for pericardial fluid, 1.2 (range 0-19.2) for psoas muscle, 1.1 (range 0-1.7) for lateral vastus muscle and 0.4 (range 0.2-3.2) for vitreous humor. The number of 6-AM positive cases was significantly higher (P = 0.03) in the ethanol positive group (n = 6; 86%) compared to the ethanol negative group (n = 14; 37%) in peripheral blood. The distribution of heroin metabolites to the different matrices was not significantly different between the ethanol positive and the ethanol negative group. This study shows that toxicological analyses of several matrices could be useful in heroin-related deaths. Urine and vitreous humor are superior for detection of 6-AM, while concentrations of morphine could be assessed from peripheral or cardiac blood, pericardial fluid, psoas muscle and lateral vastus muscle.

Zopiclone concentrations in oral fluid and blood after, administration of therapeutic doses of zopiclone.

PURPOSE: Little is known about the relationship between concentrations in oral fluid (OF) and blood for the widely prescribed hypnotic drug zopiclone. The purpose of this study was to investigate the usefulness of OF zopiclone concentrations to predict blood zopiclone concentrations in order to introduce OF testing as an alternative to more cumbersome blood testing. METHODS: 16 healthy young male volunteers received capsules of either 5 or 10mg zopiclone on two different study days separated by at least one week. Blood and OF were collected simultaneously at baseline and 9 times after intake of zopiclone on each study day. In addition an OF sample was collected 24-81h after intake. Lunch was served between samples taken 2.5 and 3.5h after intake. All samples were analysed for zopiclone, and the cut-off was 10ng/ml in blood and 0.2ng/ml in OF-buffer mixture. RESULTS: Zopiclone was detected in all OF samples during the study day. After 24-81h, all subjects were also positive for zopiclone in OF, except from three subjects ingesting the 5mg dose. In a single case zopiclone was detected in a baseline OF sample 14days after intake on an earlier study day. Zopiclone was detected in both OF and blood in 231 OF/blood pairs, and a significant but weak correlation between OF and blood concentration was seen (R2 of 0.30). The median (range) zopiclone OF/blood concentration ratio (ZOBCR) for all samples were 3.3 (0.8-18). The ZOBCR decreased when the OF volume increased. After 30 of 31 given doses of zopiclone, the ZOBCR was higher in samples collected before lunch than samples collected after lunch. DISCUSSION: Vast intra- and interindividual differences in ZOBCR were found, and the correlation between OF and blood concentration was less pronounced than reported in former studies. In accordance with earlier studies we found a negative correlation between ZOBCR and OF volume. The ZOBCR decreases in relation to recent intake of a meal, probably because stimulated saliva production causes "dilution" of saliva. OF zopiclone concentration appeared unsuitable for estimation of blood zopiclone concentration. Due to long detection time, analysis of zopiclone in OF might be useful to detect non-recent, previous intake.

Oral fluid drug analysis in the age of new psychoactive substances.

Oral fluid has become an important matrix for drugs of abuse analysis. These days the applicability is challenged by the fact that an increasing number of new psychoactive drugs are coming on the market. Synthetic cannabinoids and synthetic cathinones have been the main drug classes, but the diversity is increasing and other drugs like piperazines, phenethylamines, tryptamines, designer opioids and designer benzodiazepines are becoming more prevalent. Many of the substances are very potent, and low doses ingested will lead to low concentrations in biological media, including oral fluid. This review will highlight the phenomenon of new psychoactive substances and review methods for oral fluid drug testing analysis using on-site tests, immunoassays and chromatographic methods.

Validated methods for determination of neurotransmitters and metabolites in rodent brain tissue and extracellular fluid by reversed phase UHPLC-MS/MS.

Fast and sensitive methods for simultaneous determination of dopamine (DA), the two DA-metabolites homovanillic acid (HVA) and 3-methoxytyramine (3-MT), serotonin (5-HT) and the 5-HT-metabolite 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE), acetylcholine (ACh), glutamic acid (Glu) and γ-aminobutyric acid (GABA) in rodent brain tissue (1.0-4000nM) and extracellular fluid (ECF) (0.5-2000nM) based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) have been developed. Of the three different sample preparation methods for brain tissue samples tested, a simple and rapid protein precipitation procedure with formic acid was found to give the best results. The neurotransmitters (NTs) and NT metabolites were separated using UHPLC with an Acquity UPLC HSS T3 C18 column (2.1×100mm, 1.8µm particle size) with acidic mobile phase. Gradient elution with methanol was used and quantification was performed using multiple reaction monitoring (MRM). The total run time was 5.2min including equilibration time. The methods were validated by determining calibration model, intra- and inter-day precision and accuracy, limit of detection (LOD), lower limit of quantification (LLOQ), matrix effects (ME), carry-over and stability. Surrogate analytes were used to enable determination of the recovery and ME of the endogenous analytes in brain tissue. The methods were applied for determination of NTs at basal levels in rodent brain ECF and brain tissue homogenate. The developed methods are valuable tools in the studies of mechanisms of drugs of abuse, and neurologic and psychiatric disease.