Toxicological profile using mass spectrometry in sudden cardiac arrest survivors admitted to a tertiary centre.

BACKGROUND: There has been no previous thorough toxicological examination of a cohort of patients with resuscitated sudden cardiac arrest. We aimed to determine the qualitative and quantitative drug composition in a resuscitated sudden cardiac arrest population, using forensic toxicology, with focus on prescribed, non-prescribed, and commonly abused drugs. METHODS: Individuals aged 18-90 years with resuscitated sudden cardiac arrest of presumed cardiac causes were prospectively included from a single tertiary center. Data from the sudden cardiac arrest hospitalization was collected from medical reports. Drugs used during resuscitation or before the blood sampling were identified and excluded in each patient. Mass spectrometry-based toxicology was performed to determine the absence or presence of most drugs and to quantify the findings. RESULTS: Among 186 consecutively enrolled resuscitated sudden cardiac arrest patients (median age 62 years, 83% male), 90% had a shockable rhythm, and were primarily caused by ischemic heart disease (66%). In total, 90 different drugs (excluding metabolites) were identified, and 82% of patients had at least one drug detected (median of 2 detected drugs (IQR:1-4)) (polypharmacy). Commonly abused drugs were present in 16%, and QT-prolonging drugs were present in 12%. Polypharmacy (≥5drugs) were found in 19% of patients. Importantly, none had potentially lethal concentrations of any drugs. CONCLUSION: In resuscitated sudden cardiac arrest patients with cardiac arrest of presumed cardiac cause, routine toxicological screening provides limited extra information. However, the role of polypharmacy in sudden cardiac arrest requires further investigation. No occult overdose-related cardiac arrests were identified.

Driving under the influence of nitrous oxide - A retrospective study of HS-GC-MS analysis in whole blood.

Since 2020, our lab has received blood samples from traffic cases involving suspicion of driving under the influence of nitrous oxide (N2O). While N2O analysis by gas chromatography (GC) has been around for decades, quantitative results in blood from drivers have been only scarcely reported. We present a three-year (2020-2022) retrospective study of N2O from traffic cases in Eastern Denmark with suspected involvement of N2O intake. Whole blood samples from traffic cases were analysed for N2O using headspace-GC-MS. Freshly made calibration curves and additions of xenon gas as an internal standard were used for calculation of N2O concentrations. Positive samples have been defined as having concentrations greater than 0.1 mL N2O/L blood. Over a three-year period, we have tested 62 traffic case blood samples for the presence of N2O. Despite the technical challenges associated with the analysis of N2O, we have found N2O in 52 of the samples. Calculated concentrations were in the range 0.1-48 mL N2O/L blood, which are similar to the few cases previously found in the literature.

Evidence of 11-Hydroxy-hexahydrocannabinol and 11-Nor-9-carboxy-hexahydrocannabinol as Novel Human Metabolites of Δ9-Tetrahydrocannabinol.

(-)-trans-Δ9-tetrahydrocannabinol (Δ9-THC) is the primary psychoactive compound in the Cannabis sativa plant. Δ9-THC undergoes extensive metabolism, with the main human phase I metabolites being 11-hydroxy-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH). Early animal studies have indicated that the 9-10 double bond may be reduced in vivo to yield 11-hydroxy-hexahydrocannabinol (11-OH-HHC) and 11-nor-9-carboxy-hexahydrocannabinol (HHC-COOH). These metabolites have not been confirmed in humans. In this study, we aimed to investigate whether this metabolic transformation occurs in humans. A range of cannabinoids and metabolites, including 11-OH-HHC and HHC-COOH, were measured in whole blood from 308 authentic forensic traffic cases, of which 222 were positive for Δ9-THC. HHC-COOH and 11-OH-HHC were detected in 84% and 15% of the Δ9-THC positive cases, respectively, and the estimated median concentration of HHC-COOH was 7%, relative to that of THC-COOH. To corroborate the in vivo findings, Δ9-THC and its metabolites 11-OH-THC and THC-COOH were incubated with pooled human liver microsomes. HHC-COOH was detected in both the Δ9-THC and 11-OH-THC incubations, while 11-OH-HHC was only detectable in the 11-OH-THC incubation. Hexahydrocannabinol was not detected in any of the incubations, indicating that it is 11-OH-THC or the corresponding aldehyde that undergoes double bond reduction with subsequent oxidation of the aliphatic alcohol to HHC-COOH. In summary, the presented data provide the first evidence of HHC-COOH and 11-OH-HHC being human phase I metabolites of Δ9-THC. These findings have implications for interpretation of analytical results from subjects exposed to Δ9-THC or HHC.

The peripheral endocannabinoid system and its association with biomarkers of inflammation in untreated patients with multiple sclerosis.

BACKGROUND AND PURPOSE: The endocannabinoid system (ECS) has been found altered in patients with multiple sclerosis (MS). However, whether the ECS alteration is present in the early stage of MS remains unknown. First, we aimed to compare the ECS profile between newly diagnosed MS patients and healthy controls (HCs). Next, we explored the association of the ECS, biomarkers of inflammation, and clinical parameters in newly diagnosed MS patients. METHODS: Whole blood gene expression of ECS components and levels of endocannabinoids in plasma were measured by real-time quantitative polymerase chain reaction and ultra-high-pressure liquid chromatography-mass spectrometry, respectively, in 66 untreated MS patients and 46 HCs. RESULTS: No differences were found in the gene expression or plasma levels of the selected ECS components between newly diagnosed MS patients and HCs. Interferon-γ, encoded by the gene IFNG, correlated positively (ρ = 0.60) with the expression of G protein-coupled receptor 55 (GPR55), and interleukin1ß (IL1B) correlated negatively (ρ = -0.50) with cannabinoid receptor 2 (CNR2) in HCs. CONCLUSIONS: We found no alteration in the peripheral ECS between untreated patients with MS and HC. Furthermore, our results indicate that the ECS has a minor overall involvement in the early stage of MS on inflammatory markers and clinical parameters when compared with HCs.

Scalable Analysis of Untargeted LC-HRMS Data by Means of SQL Database Archiving.

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is widely used to detect chemicals with a broad range of physiochemical properties in complex biological samples. However, the current data analysis strategies are not sufficiently scalable because of data complexity and amplitude. In this article, we report a novel data analysis strategy for HRMS data founded on structured query language database archiving. A database called ScreenDB was populated with parsed untargeted LC-HRMS data after peak deconvolution from forensic drug screening data. The data were acquired using the same analytical method over 8 years. ScreenDB currently holds data from around 40,000 data files, including forensic cases and quality control samples that can be readily sliced and diced across data layers. Long-term monitoring of system performance, retrospective data analysis for new targets, and identification of alternative analytical targets for poorly ionized analytes are examples of ScreenDB applications. These examples demonstrate that ScreenDB makes a significant improvement to forensic services and that the concept has potential for broad applications for all large-scale biomonitoring projects that rely on untargeted LC-HRMS data.

Suitability of cardiac blood, brain tissue, and muscle tissue as alternative matrices for toxicological evaluation in postmortem cases.

Drug concentrations in peripheral blood are often used to evaluate whether death was caused by drug intoxication. In some cases, peripheral blood is not available, and analytical results of alternative matrices should instead be used in the toxicological evaluation. However, reference concentrations of alternative matrices are few, which makes interpretation of results a challenge. In this study, concentrations of selected benzodiazepines, opioids, illicit drugs, and other commonly used drugs in postmortem femoral blood, cardiac blood, brain tissue, and muscle tissue are presented. Alternative matrix-to-femoral blood drug concentration ratios and correlations of blood and alternative matrix drug concentrations were calculated to examine which of the investigated alternative matrices were most suited to use for toxicological evaluation in cases where peripheral blood is not available. The results showed that concentrations in cardiac blood, brain tissue, and muscle tissue could be useful in the postmortem evaluation of most of the 19 selected analytes. In most cases, analytes were detected in all the alternative matrices. The median concentration ratios for the selected analytes in brain tissue, cardiac blood, and muscle tissue relative to femoral blood ranged from 0.57 to 3.42, 0.59 to 1.87, and 0.67 to 7.04, respectively. Overall, cardiac blood provided the concentrations most comparable with femoral blood concentrations, indicating that cardiac blood can be useful in cases where femoral blood is not available. However, the measured concentrations should be interpreted with caution.

Comparison of Comprehensive Screening Results in Postmortem Blood and Brain Tissue by UHPLC-QTOF-MS.

Alternative specimens collected during autopsies can be valuable in postmortem toxicology in cases where peripheral blood is not available. The applicability of brain tissue as an alternative matrix for drug screening by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry was investigated in this study. Results of the 50 most frequently detected drugs and metabolites of toxicological interest in blood and brain tissue samples from 1,719 autopsy cases were compared. Examination of the results in paired blood and brain tissue samples revealed that the two matrices were in general comparable, as the majority of the 50 analytes were observed in a high number of the examined cases in both blood and brain tissue. This demonstrates the potential of brain tissue as an alternative matrix for drug screening in postmortem toxicology or as a secondary matrix for confirmation.

Driving under the Influence of Amphetamine: Analytical Evaluation of Illegal or Prescription Drug Intake Using Chiral UHPLC-MS-MS.

Differentiation between consumption of illegal and prescription drugs remains an important aspect in forensic toxicology. While illicit amphetamine is most often racemic, the medicinal drugs marketed in Denmark for the treatment of attention-deficit hyperactivity disorder contain the pure (S)-enantiomer or a prodrug thereof. In this study, we present a simple and efficient analytical workflow to provide information about the origin of amphetamine consumed in forensic cases concerning driving under the influence of drugs (DUID). Following quantification of amphetamine and methamphetamine using our conventional multi-target ultra-high performance liquid chromatography-tandem mass spectrometry method, determination of (R)- and (S)-amphetamine was performed by reinjecting the sample extract on a Phenomenex LUX® AMP chiral column using the same analytical instrument and mobile phases. Chiral separation was performed isocratic within a run time of 6 min. The analytical workflow was applied to blood samples from 5,248 suspected DUID cases within a 2-year period. Amphetamine was detected in 18.7% of the samples, of which both enantiomers were detected in 89.5% of the cases, indicating the consumption of illegal racemic amphetamine. In 6.1% of the positive cases, both amphetamine and methamphetamine were detected, indicating either co-consumption of both amphetamines or consumption of methamphetamine. In the remaining 4.4%, only (S)-amphetamine was detected indicating the consumption of prescription drugs containing (S)-amphetamine or a prodrug thereof. Implementation of a simple and rapid chiral method in the conventional analytical workflow for routine forensic casework proved to be an efficient way to elucidate whether a positive amphetamine result originates from illegal or prescription drug consumption, without increasing turnaround time nor costs to any significant extent, as no additional sample preparation was required.

Overview of systematic toxicological analysis strategies and their coverage of substances in forensic toxicology.

Systematic toxicological analysis (STA) is the process of using an adequate analytical methodology to detect and identify as many potentially toxicologically relevant compounds as possible in biological samples. STA is an important part of everyday routine work within forensic toxicology, and several methods for STA have frequently been published and reviewed independently. However, the many drugs and other substances involved, as well as the constant emergence of new ones, may pose a major challenge in STA, which often demands a strategy involving multiple analytical methods in parallel. Such strategies have been published and evaluated less frequently despite their relevance in forensic toxicology. This mini-review briefly summarizes commonly applied methods for STA in forensic toxicology, including gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-MS (LC-MS) methods, and highlights some of their potential pitfalls. Second, it provides an overview of previously reported strategies to conduct STA, including a presentation of the STA strategy applied in the authors' laboratory. This involves broad drug screening by LC-high-resolution MS, supported by targeted screening and quantification using LC-tandem MS, headspace (HS)-GC-MS, HS-GC-flame ionization detector and other complementary methods. The STA strategy aims to cover as many potentially relevant drugs as possible and seeks to reduce potential pitfalls arising in forensic casework. The review underlines that not every substance can be identified in all circumstances even with a comprehensive STA strategy.

Exploring Enzymatic Hydrolysis of Urine Samples for Investigation of Drugs Associated with Drug-Facilitated Sexual Assault.

Analyzing urine is common in drug-facilitated sexual assault cases if the analysis of blood is not optimal. The efficient enzymatic pretreatment of urine is important for cleaving glucuronides and improving the detection of the parent drug. The aim was to investigate the efficiency of three ß-glucuronidases on eleven glucuronides relevant to DFSA at different incubation periods and temperatures. Human drug-free urine was fortified with 11 glucuronides, hydrolyzed with either ß-glucuronidase/arylsulfatase (Helix Pomatia), recombinant ß-glucuronidase B-One™ or recombinant ß-glucuronidase BGTurbo™ and incubated for 5, 10, 60 min, 18 h and 24 h at 20 °C/40 °C/55 °C before UHPLC-MS/MS analysis. The stability of 141 drugs and metabolites relevant to DFSA was investigated by incubating fortified urine under the same hydrolysis conditions. B-One™ showed efficient hydrolysis (>90%) of most glucuronides in 5 min at all temperatures, while BGTurbo™ showed a similar efficiency (>90%), but the optimal temperature (20-55 °C) and incubation time (5-60 min) varied among analytes. The ß-glucuronidase/arylsulfatase had the lowest efficiency and required the longest incubation (24 h) at 40-55 °C. The stability of 99% of 141 drugs and metabolites was not affected by incubation at 20-55 °C for 24 h. Recombinant enzymes show promising results for the simple and efficient hydrolysis of a broad panel of glucuronides relevant for DFSA.

Analytical reproducibility evaluated from duplicate measurements of authentic ante- and postmortem blood samples by LC-MS/MS compared with long-term imprecision estimates from quality control samples.

Precision (or imprecision) is one of the central performance parameters of all analytical methods. It is often evaluated during method validation in spiked samples, with a relatively low number of measurements typically during a short period. Validation results are well documented in the literature; however, evaluation of imprecision in authentic cases compared with long-term imprecision from quality control samples has not often been reported on. The aim of this study was to investigate imprecision estimated from duplicate measurements of ante- and postmortem blood samples and long-term imprecision estimates from quality control samples and to compare variations between them. Data were analyzed by using robust statistics, where results for the 29 analytes most frequently quantified by liquid chromatography-tandem mass spectrometry in ante- and postmortem blood samples were included. A total of 41,460 positive findings in authentic whole blood and 51,522 measurements from quality controls were investigated. Analysis was performed in duplicate on independent batches on two separate days. Overall, the imprecision estimated in quality control and the authentic samples were akin for most analytes. Ante- and postmortem blood samples showed similar imprecision for the majority of the analytes and were approximately the same level as long-term imprecision estimated from the quality control samples at low level, whereas relative imprecision of the quality control samples at high level were slightly lower than ante- and postmortem blood samples. The methods we evaluated showed satisfactory reproducibility and robustness for the investigated analytes.

A New Strategy for Efficient Retrospective Data Analyses for Designer Benzodiazepines in Large LC-HRMS Datasets.

The expanding and dynamic market of new psychoactive substances (NPSs) poses challenges for laboratories worldwide. The retrospective data analysis (RDA) of previously analyzed samples for new targets can be used to investigate analytes missed in the first data analysis. However, RDA has historically been unsuitable for routine evaluation because reprocessing and reevaluating large numbers of forensic samples are highly work- and time-consuming. In this project, we developed an efficient and scalable retrospective data analysis workflow that can easily be tailored and optimized for groups of NPSs. The objectives of the study were to establish a retrospective data analysis workflow for benzodiazepines in whole blood samples and apply it on previously analyzed driving-under-the-influence-of-drugs (DUID) cases. The RDA workflow was based on a training set of hits in ultrahigh-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (UHPLC-QTOF-MS) data files, corresponding to common benzodiazepines that also had been analyzed with a complementary UHPLC-tandem mass spectrometry (MS/MS) method. Quantitative results in the training set were used as the true condition to evaluate whether a hit in the UHPLC-QTOF-MS data file was true or false positive. The training set was used to evaluate and set filters. The RDA was used to extract information from 47 DBZDs in 13,514 UHPLC-QTOF-MS data files from DUID cases analyzed from 2014 to 2020, with filters on the retention time window, count level, and mass error. Sixteen designer and uncommon benzodiazepines (DBZDs) were detected, where 47 identifications had been confirmed by using complementary methods when the case was open (confirmed positive finding), and 43 targets were not reported when the case was open (tentative positive finding). The most common tentative and confirmed findings were etizolam (n = 26), phenazepam (n = 13), lorazepam (n = 9), and flualprazolam (n = 8). This method efficiently found DBZDs in previously acquired UHPLC-QTOF-MS data files, with only nine false-positive hits. When the standard of an emerging DBZD becomes available, all previously acquired DUID data files can be screened in less than 1 min. Being able to perform a fast and accurate retrospective data analysis across previously acquired data files is a major technological advancement in monitoring NPS abuse.

Metabolomics-driven determination of targets for salicylic acid and ibuprofen in positive electrospray ionization using LC-HRMS.

Due to the large number of basic therapeutic and illicit drugs, systematic toxicological analysis has widely been performed with liquid chromatography coupled to mass spectrometry using positive electrospray ionization. However, there exist a smaller number of drugs, typically acidic drugs, which require the use of negative electrospray ionization either via a separate analysis or polarity switching. Here, targets relating to salicylic acid and ibuprofen in positive electrospray ionization were determined through a metabolomics-driven retrospective investigation of forensic casework. Samples were previously screened using liquid chromatography coupled with high-resolution mass spectrometry with quantification of target analytes performed using liquid chromatography with tandem mass spectrometry. Of the 1,717 whole-blood samples submitted between 2014 and 2019, 48 were positive for salicylic acid (1.1-1,400 mg/kg) and 78 for ibuprofen (1-46 mg/kg). Based on the retrospective analysis, 19 and 90 targets were identified for salicylic acid and ibuprofen, respectively. For targets of salicylic acid, the protonated adduct of salicyluric acid ([M + H]+ , m/z 196.0605) was present in 89.6% (n = 32) of the salicylic acid positive cases, while the [M + HCOOH + CH3 CN + Ca - H]+ adduct (m/z 264.0179) of salicylic acid was present in all positive samples with concentrations above 66 mg/kg salicylic acid. Similarly, the [M + 2Na - H]+ adduct (m/z 251.1018) of ibuprofen was present in 98.7% (n = 77) of positive cases and was present in all samples with concentrations above 3 mg/kg ibuprofen.

Cocaine profiling method retrospectively developed with nontargeted discovery of markers using liquid chromatography with time-of-flight mass spectrometry data.

Illicit drug profiling performed by forensic laboratories assists law enforcement agencies through providing information about chemical and/or physical characteristics of seized specimens. In this article, a model was developed for the comparison of seized cocaine based on retrospective analysis of data generated from ultrahigh performance liquid chromatography with time-of-flight mass spectrometry (UHPLC-TOF-MS) comprehensive drug screening. A nontargeted approach to discover target compounds was employed, which generated 53 potential markers using data from cocaine positive samples. Twelve marker compounds were selected for the development of the final profiling model. The selection included a mixture of commonly used cocaine profiling targets and other cocaine-related compounds. Combinations of pretreatments and comparison metrics were assessed using receiver operating characteristic curves to determine the combination with the best discrimination between linked and unlinked populations. Using data from 382 linked and 34,519 unlinked distances, a classification model was developed using a combination of the standardization and normalization transformations with Canberra distance, resulting in a linked cut-off with a 0.5% false positive rate. The present study demonstrates the applicability of retrospectively developing a cocaine profiling model using data generated from UHPLC-TOF-MS nontargeted drug screening without pre-existing information about cocaine impurities. The developed workflow was not specific to cocaine and thus could potentially be applied to any seized drug in which there are both sufficient data and impurities present.

Development of a single retention time prediction model integrating multiple liquid chromatography systems: Application to new psychoactive substances.

Database-driven suspect screening has proven to be a useful tool to detect new psychoactive substances (NPS) outside the scope of targeted screening; however, the lack of retention times specific to a liquid chromatography (LC) system can result in a large number of false positives. A singular stream-lined, quantitative structure-retention relationship (QSRR)-based retention time prediction model integrating multiple LC systems with different elution conditions is presented using retention time data (n = 1281) from the online crowd-sourced database, HighResNPS. Modelling was performed using an artificial neural network (ANN), specifically a multi-layer perceptron (MLP), using four molecular descriptors and one-hot encoding of categorical labels. Evaluation of test set predictions (n = 193) yielded coefficient of determination (R2) and mean absolute error (MAE) values of 0.942 and 0.583 min, respectively. The model successfully differentiated between LC systems, predicting 54%, 81% and 97% of the test set within ±0.5, ±1 and ±2 min, respectively. Additionally, retention times for an analyte not previously observed by the model were predicted within ±1 min for each LC system. The developed model can be used to predict retention times for all analytes on HighResNPS for each participating laboratory's LC system to further support suspect screening.

Simple implementation of muscle tissue into routine workflow of blood analysis in forensic cases - A validated method for quantification of 29 drugs in postmortem blood and muscle samples by UHPLC-MS/MS.

Whole blood is most often the matrix of choice for postmortem analysis but it is not always available. In these cases, muscle tissue can be used as an alternative matrix. Therefore, an ultra-high-performance liquid chromatography-tandem mass spectrometry method for the quantification of 29 drugs and metabolites of toxicological interest in postmortem muscle tissue was developed and validated. Additionally, a validation of whole blood was carried out to compare the results from the two matrices. Solid-phase extraction was performed by an automated robotic system to minimize manual labour and risk of human errors, and increase robustness, sample throughput and sample traceability. The method was validated in terms of selectivity, matrix effect, extraction recovery, process efficiency, measuring range, lower limit of quantification, carry-over, stability, precision and accuracy. To correct for any inter-individual variability in matrix effects on analyte accuracy and precision, deuterated analogues of each analyte were used as internal standards. The lower limit of quantification in both blood and muscle homogenate ranged between 0.002 and 0.005 mg/kg, while the upper limit of quantification spanned from 0.20 to 1.0 mg/kg. Corrected with the 4-fold dilution factor, the corresponding concentrations in muscle tissue were 0.008-0.02 mg/kg at the lower limit of quantification and 0.80-4.0 mg/kg at the upper limit of quantification. The method showed acceptable precision and accuracy, with precision below 12% and accuracies ranging from 87% to 115% at up to 6 levels for all analytes in both matrices. In addition, comparison between calibration standards in spiked muscle homogenate and spiked blood showed that analyte concentrations in muscle samples could be quantified by using spiked blood samples as calibration standards with acceptable precision and accuracy when using deuterated analogues as internal standards. The investigation of matrix effects showed no great difference between blood and homogenates of non-decomposed and decomposed muscle tissue for most analytes. In the samples where high ion suppression or enhancement was observed, the results were corrected by the internal standards. Statistical comparison of quality control samples in blood and muscle tissue showed no obvious differences, and therefore muscle tissue was included in the routine method for analysis of blood samples and used in autopsy cases where no blood was available. By adding a semi-automated homogenization step before the remaining automated sample preparation, muscle tissue samples were easily incorporated into the workflow of the existing routine method. The present method has been successfully implemented in routine analysis of blood and muscle tissue since 2019.

Analysis of seized peptide and protein-based doping agents using four complimentary methods: Liquid chromatography coupled with time of flight mass spectrometry, liquid chromatography-ultraviolet, Bradford, and immunoassays.

Analysis and identification of seized doping-related products are important tasks for customs or forensic laboratories in order to prevent potentially dangerous and illegal compounds to go into circulation. At the Section of Forensic Chemistry in Copenhagen, we have a workflow consisting of four complimentary validated methods to identify common doping-related substances: liquid chromatography-ultraviolet (LC-UV), LC coupled with time of flight mass spectrometry (LC-TOF-MS), the colorimetric Bradford assay, and an immunoassay. The Bradford assay screens for peptide or proteins in the sample, and the immunoassay confirmed human chorionic gonadotropin (hCG). LC-UV was carried out with a C4 protein column for identification of peptides and proteins from a standard reference library, based on retention times and ratios between peak areas at 220, 254, and 280 nm. LC-TOF-MS was performed using a C18 column, and identification was based on comparison of the retention time and the accurate mass with those of reference standards. In 2019, we received 36 samples for peptide/protein analysis, all of which were tested using the LC-UV, LC-TOF-MS, and colorimetric method, and samples suspected of containing hCG were confirmed with an immunoassay. We found a total of 15 samples containing an illegal doping substance, 12 samples containing substances not prohibited by the Danish Doping List, and nine samples containing no peptides or proteins. In conclusion, the four complimentary methods constitute a suitable approach for identifying common peptide/protein doping substances in the day-to-day routine of a forensic laboratory, with limited sample preparation and interpretation of data.

How Should Organizations Respond to Racism Against Health Care Workers?

This case and commentary considers how organizations should respond to overt racism expressed by patients. The article considers the nature and scope of organizations' responsibilities to train both professional and nonprofessional staff and to enact zero-tolerance policies to address expressions of discrimination.

Identification of phenobarbital and other barbiturates in forensic drug screening using positive electrospray ionization liquid chromatography-high resolution mass spectrometry.

Comprehensive drug-screening performed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) enables identification of hundreds to thousands of drug compounds in a single analysis. Forensic drug screening is generally performed with positive electrospray ionization (ESI+ ), targeting basic drugs; however, a few toxicologically important drugs such as barbiturates, may require analysis by negative ESI. In this work, screening targets for barbiturates were determined using our LC-HRMS screening with ESI+ . For several years, our forensic whole blood samples have been analyzed using the LC-HRMS-ESI+ screening in parallel with a multi-target LC-MS/MS-ESI- method. From 2014 to 2018, 23 samples were positive for phenobarbital (0.5-81 mg/kg). Retrospective data analysis of 4816 blood samples (15 positive) revealed several potential screening targets for phenobarbital. The targets were tentatively identified by exact mass and isotopic pattern as uncommon adducts of phenobarbital and as a decomposition product of phenobarbital N-glucoside (C17 H24 N2 O7 ). Analysis of a test set containing eight positive (0.5-65 mg/kg phenobarbital) and 31 negative samples supported the use of the observed target m/z 323.0614 at 5.14 minutes, corresponding to the [M + HCOONa+Na]+ adduct of phenobarbital. The [M + HCOONa+Na]+ adduct was confirmed as a screening target for common barbiturates, by analysis of barbiturate reference standards in ESI+ /ESI- . The [M + HCOONa+Na]+ adduct allowed retrospective analysis with 91% sensitivity (n = 23) and 100% specificity (n = 4855) for phenobarbital in our existing LC-HRMS-ESI+ screening. The two negative results were the two whole-blood samples with the lowest phenobarbital concentration (<1.8 mg/kg). Thus, a specialized screening is not necessary and use of this adduct likely enables screening for other barbiturates.

Glycine-modified growth hormone secretagogues identified in seized doping material.

A number of unknown pharmaceutical preparations seized by Danish customs authorities were submitted for liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis. Comparison with reference standards unequivocally identified the content of the powders as analogs of the growth hormone secretagogues GHRP-2 (Pralmorelin), GHRP-6, Ipamorelin, and modified growth hormone releasing factor (modified GRF 1-29), which can be used as performance-enhancing substances in sports. In all cases, the detected modification involved the addition of an extra glycine amino acid at the N-terminus, and analytical methods targeting growth hormone secretagogues should hence be updated accordingly.