Elisabeth Leere Øiestad

The aim of this study was to assess and benchmark plastic consumption in sample preparation for forensic analysis, alongside the development of an LC–MS method for ketamine analogs in whole blood, with various sustainability-related scores and parameters examined throughout. Ketamine analogs are emerging psychoactive substances associated with intoxication and fatalities globally. An analytical method was developed for determining ketamine and eight of its analogs (DCK, DMXE, 2-FDCK, 3-MeO-PCE, MXE, MXiPr, MXPr, O-PCE) in human whole blood. Focus was placed on traditional analytical parameters, but also the consumption of plastics and reagents, an overlooked aspect of clinical and forensic chemistry. We evaluated three sample preparation techniques: protein precipitation (PPT), liquid-liquid extraction (LLE), and electromembrane extraction (EME). While PPT and LLE are well-established techniques used in clinical settings, EME (i.e. electrophoresis across an oil membrane) is a less established but highly promising approach. All three sample preparation approaches demonstrated similar performance, with recoveries above 85% and matrix effects averaging approximately 100%. The EME approach was subsequently refined using a Box-Behnken-based design of experiment, and the method was validated according to the American Academy of Forensic Sciences guidelines. All validated parameters were within the limits, suggesting that the tunable EME approach is a potentially valuable tool in clinical chemistry. The amount of plastic consumables per 100 samples was calculated as being 511 g for PPT, 864 g for LLE, and 303 g for EME. Correspondingly, the amount of organic solvent consumption per sample was 470 µL, 1570 µL, and 210 µL. The AGREEprep scores (ranges from 0 to 1, 1 is best) were 0.48 ± 0.04, 0.36 ± 0.04, and 0.55 ± 0.05. EME fared best in greenness. However, the investigated approaches have a significant use of single-use consumables, inviting for sample preparation approaches that can reduce the plastic footprint toward more sustainable clinical chemistry. The rounded value of PPT (arguably the most used approach for related samples) is 0.5 kg per 100 samples, and we propose this number be used as a benchmark for plastic consumption in today´s sample preparation. This may serve as a practical reference point when developing and evaluating future sample preparation strategies.

On-site drug screening of oral fluid samples has gained attention because of its convenience and rapid results. The aim of this investigation was to compare the results of preliminary screening for drugs in oral fluid samples collected from suspected drug-impaired drivers using DrugWipe 6S and WipeAlyser reader with the results obtained from blood samples. Additionally, we compared the DrugWipe test results with findings of drug traces detected within the used DrugWipe devices. Police officers selected a sample of 355 suspected drug-impaired drivers in 2023. They used DrugWipe 6S for preliminary drug screening of drivers. After the field drug testing of oral fluid, the apprehended drivers were brought to a physician for the collection of blood samples. The collected samples (DrugWipe devices and blood samples) were submitted to the Norwegian National Forensic Toxicology Laboratory for analysis. The proportion of positive DrugWipe results that were unconfirmed when analysing blood samples was 82% for opiates, 75% for cocaine, and ∼19%–20% for amphetamines, cannabis, and benzodiazepines. The proportion of negative DrugWipe results that were found positive in blood samples was for cannabis and benzodiazepines ∼13%–14%, and for other drugs <3%. Detected drug traces in the used DrugWipe devices corresponded well with DrugWipe readouts for cannabis, amphetamines, and cocaine. The lack of correspondence between DrugWipe test results for cocaine and findings in blood may be due to the fact that the concentration of cocaine in saliva is often much higher than in blood, and the DrugWipe test is very sensitive. In addition, degradation and elimination of cocaine before the blood sample is taken may contribute to cocaine concentrations below the cut-off concentration in blood. For opiates and benzodiazepines, traces of drugs were found in relatively few DrugWipe devices. Many unconfirmed positives for opiates were most likely due to cross-reaction with substances in ‘snus’ (snuff tobacco).

Objective: People regularly contact emergency medicine services concerned that they have been exposed to drink spiking, i.e., exposure to drugs without their knowledge or permission. We identified drugs in blood and urine samples from patients suspecting exposure to drink spiking, with special consideration for drugs not reported taken by the patient (unreported drugs). Methods: From September 2018 to May 2019, we collected blood and urine samples from patients 16 years or older presenting at an emergency clinic in Oslo, Norway, within 48 hours of suspected exposure to drink spiking. We also collected information on ethanol ingestion and drugs taken. Blood samples were analyzed for 20 classical recreational drugs using ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) and an automated enzymatic method for ethanol. Urine samples were analyzed using immunoassay methods and a specific gas chromatography mass spectrometry (GCMS) method for gammahydroxybutyrate (GHB). Results: From 100 included patients (median age 24 years, 62 females), we collected 100 blood samples and 72 urine samples. Median time since exposure was 5 hours. Unreported drugs were found in 15 patients. Unreported drugs in the blood samples were clonazepam in 3, methylenedioxymethamphetamine (MDMA) in 3, amphetamine in 2, tetrahydrocannabinol (THC) in 2, tramadol in 1, cocaine in 1, and methamphetamine in 1. Unreported drugs in the urine samples were cocaine in 5, amphetamine in 4, ecstasy in 3, and cannabis in 2. Ethanol was found in 69 patients, all reporting ethanol ingestion. Median blood ethanol concentration was higher in patients with no unreported drugs detected, 1.00‰ (interquartile range (IQR) 0–1.52) vs. 0‰ (IQR 0–0.46) (p<0.001). GHB was not detected. Conclusion: Unreported drugs, possibly used for drink spiking, were found in 15% of patients. Blood ethanol concentration was higher when no unreported drugs were found. GHB was not detected in any patient.

Hair analysis can provide chronological insights into past drug use for months to years after drug administration. In comparison to analyses from other biological matrices, such as blood and urine, sample pretreatment is often tedious and not environmental friendly. In this study, we present a more environmental friendly approach to hair analysis using micropulverized hair and electromembrane extraction for the efficient extraction of 15 drugs of abuse, prescription drugs, and metabolites from hair. The optimized extraction method, involving micropulverization, demonstrated comparable yields to the standard approach of cutting and overnight incubation. A 15-min extraction method using a commercial electromembrane extraction prototype was developed and validated according to forensic guidelines, using only 10 µL of organic solvent per sample. The final method, employing HPLC–MS–MS with a biphenyl column, exhibited good linearity, precision, and sensitivity. An AgreePrep assessment comparing the environmental impact of our method with the standard routine method, involving overnight incubation and conventional liquid–liquid extraction, was conducted. This is the first time micropulverized hair has been subjected to electromembrane extraction.

Illegal amphetamine is usually composed of a racemic mixture of the two enantiomers (S)- and (R)-amphetamine. When amphetamine is used in medical treatment, however, the more potent (S)-amphetamine enantiomer is used. Enantiomer specific analysis of (S)- and (R)-amphetamine is therefore used to separate legal medical use from illegal recreational use. The aim of the present study was to describe our experience with enantiomer specific analysis of amphetamine in urine and oral fluid, as well as blood, and examine whether the distribution of the two enantiomers seems to be the same in the different matrices. We investigated 1722 urine samples and 1977 oral fluid samples from prison inmates, and 652 blood samples from suspected drugged drivers, where prescription of amphetamine was reported. Analyses were performed using UHPLC–MS-MS. The enantiomer separation was achieved by using a chiral column and results from the method validation are reported. Samples containing less than 60% (S)-amphetamine were interpreted as representing illegal use of amphetamine. The distribution of the two enantiomers was compared between the different matrices. In urine and oral fluid, the mean amount of (S)-amphetamine was 45.2% and 43.7%, respectively, while in blood, the mean amount of (S)-amphetamine was 45.8%. There was no statistically significant difference in the amount of (S)-amphetamine between urine and oral fluid samples and between urine and blood samples, but the difference was significant in blood compared to oral fluid samples (p < 0.001). Comparison of urine and oral fluid between similar populations indicated that enantiomers of amphetamine can be interpreted in the same way, although marginally higher amounts of (R)-amphetamine may occur in oral fluid. Oral fluid, having several advantages, especially during collection, could be a preferred matrix in testing for illegal amphetamine intake in users of medical amphetamine.

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.

The use of oral fluid as sample matrix has gained significance in the analysis of drugs of abuse due to its non-invasive nature. In this study, the 13 opioids morphine, oxycodone, codeine, O-desmethyl tramadol, ethylmorphine, tramadol, pethidine, ketobemidone, buprenorphine, fentanyl, cyclopropylfentanyl, etonitazepyne, and methadone were extracted from oral fluid using electromembrane extraction based on conductive vials prior to analysis with ultra-high performance liquid chromatography–tandem mass spectrometry. Oral fluid was collected using Quantisal collection kits. By applying voltage, target analytes were extracted from oral fluid samples diluted with 0.1% formic acid, across a liquid membrane and into a 300 μL 0.1% (v/v) formic acid solution. The liquid membrane comprised 8 μL membrane solvent immobilized in the pores of a flat porous polypropylene membrane. The membrane solvent was a mixture of 6-methylcoumarin, thymol, and 2-nitrophenyloctyl ether. The composition of the membrane solvent was found to be the most important parameter to achieve simultaneous extraction of all target opioids, which had predicted log P values in the range from 0.7 to 5.0. The method was validated in accordance to the guidelines by the European Medical Agency with satisfactory results. Intra- and inter-day precision and bias were within guideline limits of ± 15% for 12 of 13 compounds. Extraction recoveries ranged from 39 to 104% (CV ≤ 23%). Internal standard normalized matrix effects were in the range from 88 to 103% (CV ≤ 5%). Quantitative results of authentic oral fluid samples were in accordance with a routine screening method, and external quality control samples for both hydrophilic and lipophilic compounds were within acceptable limits.

Separation and quantification of amphetamine enantiomers are commonly used to distinguish between consumption of prescription amphetamine (mostly S‐amphetamine) and illicit forms of the drug (racemate). In this study, electromembrane extraction with prototype conductive vials was combined with ultra‐high performance supercritical fluid chromatography (UHPSFC‐MS/MS) to quantify R‐ and S‐amphetamine in urine. Amphetamine was extracted from 100 μL urine, diluted with 25 μL internal standard solution and 175 μL 130 mM formic acid, across a supported liquid membrane (SLM) consisting of 9 μL of a 1:1(w/w) mixture of 2‐nitrophenyloctyl ether (NPOE) and bis(2‐ethylhexyl)phosphite (DEHPi) into an acceptor phase containing 300 μL 130 mM formic acid. The extraction was facilitated by the application of 30 V for 15 min. Enantiomeric separation was achieved using UHPSFC‐MS/MS with a chiral stationary phase. The calibration range was 50–10,000 ng/mL for each enantiomer. The between‐assay CV was ≤5%, within‐assay CV ≤ 1.5%, and bias within ±2%. Recoveries were 83%–90% (CV ≤ 6%), and internal standard corrected matrix effects were 99–105 (CV ≤ 2%). The matrix effects ranged from 96% to 98% (CV ≤ 8%) when not corrected by the internal standard. The EME method was compared with a chiral routine method that employed liquid–liquid extraction (LLE) for sample preparation. Assay results were in agreement with the routine method, and the mean deviation between methods was 3%, ranging from −21% to 31%. Finally, sample preparation greenness was assessed using the AGREEprep tool, which resulted in a greenness score of 0.54 for conductive vial EME, opposed to 0.47 for semi‐automated 96‐well LLE.