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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.

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.

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., 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. Alterna- tive 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 concentra- tions in cardiac blood, brain tissue, and muscle tissue could be useful in the postmor- tem 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 con- centrations, indicating that cardiac blood can be useful in cases where femoral blood is not available. However, the measured concentrations should be interpreted with caution. KEYWORDS

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 (ADHD) contains 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 with our conventional multi-target ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method, determination of (R)- and (S)-amphetamine was performed by re-injecting the sample extract on a Phenomenex LUX® AMP chiral column using the same analytical instrument and mobile phases. The chiral separation was performed isocratic within a run time of 6 minutes. The analytical workflow was applied to blood samples from 5248 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 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 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.

Background and aim: We have received special request for nitrous oxide analysis on traffic case samples since the beginning of 2020. The gas can cause dizziness, disorientation and fainting spells that might affect the ability to drive, but it is not yet covered in the Danish traffic regulations regarding driving under the influence of drugs. A surge of national and international traffic incidents have heightened attention and the need for analysis of nitrous oxide in blood samples. We present a retrospective analysis of nitrous oxide analysis results to gain insight into the scope and characteristics of traffic cases involving living drivers suspected of driving under the influence of nitrous oxide. Methods: To accommodate police requests for testing, we have developed a semiquantitative headspace GC-MS analysis method for nitrous oxide in blood. Estimation of nitrous oxide concentrations is done by comparing sample results to freshly made calibration curves and additions of Xenon gas as internal standard. Positive samples have been defined as having estimated concentrations greater than 0.1 mL nitrous oxide per L blood. Nitrous oxide test results from traffic cases in Greater Copenhagen and Eastern Denmark have been assessed through a three-year (2020-2022) retrospective analysis. The prevalence of nitrous oxide has been compared to other commonly found drugs of abuse in traffic cases in Greater Copenhagen during the same period. Results: The case load for nitrous oxide analysis has been steadily increasing over the three-year period, with a total of 62 traffic case blood samples tested for the presence of nitrous oxide. In total, 52 of the 62 cases were positive and most offenders were young males. Nitrous oxide was the seventh most detected drug in blood samples from traffic cases in Greater Copenhagen in 2022. Conclusion: We present a simple and effective method for the semiquantitative forensic determination of driving under the infl

Aim Drug concentrations in peripheral blood are most often used in interpretation of toxicological results in postmortem cases. However, peripheral blood is not always available or suitable for analysis, especially in advanced stages of decomposition. Therefore, other matrices than peripheral blood need to be investigated in order to reveal the cause of death. However, there is no obvious second choice. In this study, we analyzed postmortem femoral blood, cardiac blood, brain tissue and muscle tissue in order to investigate if drug concentrations in these alternative matrices were comparable to femoral blood and thereby useful for toxicological interpretation. Method A total of 104 consecutive autopsies where toxicological investigation was included were performed in a four-month period. Femoral blood (n = 89), cardiac blood (n = 85), brain tissue (n = 91) and muscle tissue (n = 104) were analyzed in all cases when available. Quantification of 69 common drugs and metabolites of toxicological interest was performed with four different multi-analyte methods all employing automated sample preparation and UHPLC MS/MS in MRM mode. Results In 104 autopsy cases, we were able to detect a total of 53 drugs and metabolites. Alternative matrix to femoral blood concentration ratios were calculated for 47 analytes for cardiac blood, 49 analytes for brain tissue and 50 analytes for muscle tissue, respectively. However, about half of the analytes were only detected in < 5 cases. Median ratios of analytes detected in ≥ 5 cases were determined to 0.59–1.9 (median = 1.1) for cardiac blood to femoral blood, 0.57–2.5 (median = 1.5) for brain tissue to femoral blood and 0.67–7.0 (median = 1.4) for muscle tissue to femoral blood. The largest deviation in ratios was observed for THC in muscle tissue. Here muscle tissue to femoral blood ratios varied from 2.3 to 20 with a median of 7.0 (n = 10). It is, however, a well-known fact that quantitative analysis o

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.

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 exists a smaller number of drugs, typically acidic drugs, which require the use of negative electrospray ionization either via a separate injection 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 1717 whole-blood samples submitted between 2014 and 2019, 48 were positive for salicylic acid (1.1-1400 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+CH3CN+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.

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 foun

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.

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.

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.

Diabetes mellitus is a disease caused by a deficiency in (type 1) or inability to use insulin (type 2). Untreated it can lead to diabetic ketocidosis (DKA) – state with high levels of ketone bodies (acetone, acetoacetate, beta-hydroxybutyrate (BHB)). This state can be life threatening. Measurement of ketone bodies together with vitreous/urine glucose and glycosylated hemoglobin (HbA1C) are therefore essential to diagnose DKA-related deaths. All autopsy samples received at our department in the period 2016-2018 for toxicological investigations were analyzed for acetone, BHB, and vitreous glucose (N = 1394). In case of a high level of BHB, HbA1C and urine glucose were measured. Thirty two cases (2.3%) were concluded to be DKA-related deaths. Eleven (34%) of these had no known history of diabetes. BHB accounts for the major part of ketone bodies and is directly associated with the acidosis effect. Therefore, BHB is preferred to acetone when evaluating DKA and other ketoacidosis-related deaths. We compared acetone and BHB levels to evaluate if the easy acetone measurement could cover our needs for screening. We found that high BHB levels (>2000 μmol/L) were detected if the acetone cut off was set to 0.01 g/L. But, many samples would have low BHB < 3-500 μmol/L with this cut off, and many samples with raised BHB (500-1,200 μmol/L) would not be detected. We therefore recommend to screen all samples for BHB. In case of a high BHB (>1,000 μmol/L) vitreous/urine glucose and HbA1C must be measured to distinguish DKA from other types of ketoacidosis.

Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is an important analytical tool in the systematic toxicological analysis performed in forensic toxicology. However, some important compounds, such as the antiepileptic drug valproate (valproic acid; VPA), cannot be directly detected with positive electrospray ionization (ESI+ ) due to poor ionization. Here we demonstrate an omics-based retrospective analysis for the identification of indirect screening targets for VPA in whole blood with LC-ESI+ -HRMS. Analysis was performed utilizing data acquired across four years from LC-ESI+ -HRMS, with VPA results from a quantitative LC-MS/MS method. The combined data with VPA results were split into an exploration set (n = 68; 28% positive) and a test set (n = 37; 32% positive). Eight indirect targets for VPA were identified in the exploration set. The evaluation of these targets was confirmed with retrospective target analysis of the test set. Using a combination of two out of the eight indirect targets, we attained a sensitivity of 92% (n = 12; VPA concentration range: 4.4-29.7 mg/kg) and 100% specificity (n = 25) for VPA with LC-ESI+ -HRMS. VPA screening targets were identified with retrospective data analysis and could be appended to the existing screening procedure. A sensitive and specific screening with LC-ESI+ -HRMS was achieved with targets corresponding to the sodium adducts of C7 H14 O3 and C8 H14 O3 . Three chromatographic resolved isomer peaks were observed for the latter, and the consistently most intense peak was tentatively identified as 3-hydroxy-4-en-VPA.

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.

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 (C17H24N2O7). 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 s

Objectives: The objective of this study was to examine the frequency of psychoactive drugs and alcohol in drivers under suspicion of driving under the influence of drugs and alcohol in 2015 and 2016 in the eastern part of Denmark. The trends in the number of traffic cases sent for drug analysis since 2000 and alcohol analysis since 2011 are also discussed. Methods: Blood samples from drivers suspected of being under the influence of alcohol and/or medication and/or illicit drugs in 2015 and 2016 were investigated as requested by the police. The blood samples were screened for alcohol and/or tetrahydrocannabinol (THC) alone, for other drugs (covering all drugs, except THC, listed in the Danish list of narcotic drugs), or for THC and other drugs. Age and gender were also recorded. The number of drug traffic cases since 2000 and the number of alcohol cases since 2011 were extracted from our Laboratory Information Management System (LIMS). Results: In total, 11,493 traffic caseswere investigated. Alcohol and/or drugs exceeded the legal limit in 9,657 (84%) cases. Men constituted 95% of the drivers investigated for drugs and 88% of the alcohol cases. The drivers investigated for drugs consisted primarily of young men, whereas drivers investigated for alcohol were older. The frequency was higher for positive alcohol cases above the legal limit (87%) than for drug cases (76%) above the fixed concentration limit. THC (67–69%) was the most frequently detected drug above the legal limit, followed by cocaine (27–28.5%), amphetamine (17%), and clonazepam (6–7%) in both years.Morphine (5.4%), included among the 5 most frequent drugs in 2015, was replaced by methadone (4.6%) in 2016. Few new psychoactive drugs (NPS) were detected. The number of traffic cases sent for drug analysis has increased more than 30-fold since 2000–2006, and the number of traffic cases submitted in 2016 for drug analysis was h

There is limited knowledge on the global prescription and consumption patterns of therapeutic (TD) and illicit drugs (ID). Pooled urine analysis and wastewater-based epidemiology (WBE) has been used for local-based drug screening. It is, however, difficult to study the global epidemiology due to difficulties in obtaining samples. The aims of the study were to test the detectability of TD and ID in airplane wastewater samples categorized according to their geographical origin. Wastewater samples (n= 17) were collected from long-distance flights and prepared with enzymatic conjugate cleaving followed by either precipitation or solid phase extraction. Aliquots were analysed on various liquid chromatography – mass spectrometers. TDs were grouped according to their Anatomical Therapeutic Chemical (ATC) codes. Identification confidence was assigned to three levels based on variables including detection on multiple instruments and number of targets per compound. A total of 424 compounds were identified across all samples, distributed on 87 unique TD and 2 ID. Two principal components in a principal component analysis separated three clusters of wastewater samples corresponding to geographical origin of the airplanes with therapeutic subgroup ATC codes as variables. Airplane wastewater analysis is useful for identifying targets for WBE and toxicological analysis and explore drug use and abuse patterns.

There is limited knowledge on the global prescription and consumption patterns of therapeutic (TD) and illicit drugs (ID). Pooled urine analysis and wastewater-based epidemiology (WBE) has been used for local-based drug screening. It is, however, difficult to study the global epidemiology due to difficulties in obtaining samples. The aims of the study were to test the detectability of TD and ID in airplane wastewater samples categorized according to their geographical origin. Wastewater samples (n= 17) were collected from long-distance flights and prepared with enzymatic conjugate cleaving followed by either precipitation or solid phase extraction. Aliquots were analysed on various liquid chromatography – mass spectrometers. TDs were grouped according to their Anatomical Therapeutic Chemical (ATC) codes. Identification confidence was assigned to three levels based on variables including detection on multiple instruments and number of targets per compound. A total of 424 compounds were identified across all samples, distributed on 87 unique TD and 2 ID. Two principal components in a principal component analysis separated three clusters of wastewater samples corresponding to geographical origin of the airplanes with therapeutic subgroup ATC codes as variables. Airplane wastewater analysis is useful for identifying targets for WBE and toxicological analysis and explore drug use and abuse patterns.