Hydrogen production using cocaine metabolite in microbial electrolysis cells.

In this study, the effects of cocaine metabolite, benzoylecgonine, commonly found in wastewater on hydrogen production were investigated using microbial electrolysis cells. Benzoylecgonine dissolved in synthetic urine and human urine containing benzoylecgonine were inoculated to evaluate hydrogen production performance in microbial electrolysis cells. Microbial electrolysis cells were inoculated with synthetic urine and human urine containing the cocaine metabolite benzoylecgonine for hydrogen gas production performance. Gas production was observed and measured daily by gas chromatography. GC-MS was used to analyze the compounds found in human urine before and after operation in microbial electrolysis cells. The metabolite's pH values and optical density in microbial electrolysis cells were analyzed spectrophotometrically. Hydrogen gas was successfully produced in microbial electrolysis cells (~ 5.5 mL) at the end of the 24th day in the presence of benzoylecgonine in synthetic urine. Human urine containing benzoylecgonine also generated hydrogen in microbial electrolysis cells. In conclusion, microbial electrolysis cells can be used to remove cocaine metabolites from contaminated wastewater generating hydrogen gas. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03805-7.

Toxicological Advancements in Cocaine Detection: A Review.

Cocaine, also known as methyl benzoylecgonine, is one of the most used drugs of abuse and one of the oldest; however, there has been a recent increase in the consumption of this substance. This trend has once again caught the attention of the scientific community. We discuss the current knowledge about this drug, focusing our attention on the forensic approach. Despite the fact that the cut-off of positivity to cocaine in drug tests is quite high, most current tests are able to detect much lower concentrations and could improve forensic sciences in both post-mortem investigations and in people screening. Immunological assays possessing substantial cross-reactivity to cocaine are particularly useful for screening oral fluid, hair, and post-mortem blood, where significant concentrations of the drug can be found. Liquid chromatography has now supplanted the previous techniques because it is very sensitive and specific and allows samples to be analyzed in a shorter time with only minimal sample preparation. Recent studies have focused on increased sensitivity, reduced processing times, and cheaper analysis.

Illicit drug consumption estimated using wastewater analysis and compared by settlement size in New Zealand.

Estimation of consumption of illicit drugs by wastewater-based epidemiology provides estimates of community drug-use patterns. This study describes monitoring data of three illicit drugs in New Zealand using wastewater-based epidemiology. Wastewater samples were collected at monthly intervals for larger (population ~ 50,000+) cities or in smaller towns where more data was required by authorities. In other smaller towns, samples were collected every 2 months. Samples were extracted and analysed for parent compounds and metabolites of methamphetamine, MDMA, cocaine, heroin and fentanyl consumption using solid-phase extraction followed by liquid chromatography coupled with tandem-mass spectrometry (LC-MS/MS) detection. Back calculations were performed to estimate the consumption of each drug in each catchment area. Methamphetamine was the drug measured with the highest estimated mean consumption rates (724 mg/1000 people per day) in New Zealand. North Island small urban settlements had the highest estimated mean methamphetamine consumption rates (1259 mg/1000 people/day). Cocaine had the lowest estimated consumption rates (9.4 mg/1000 people/day). The highest estimated mean cocaine consumption rate was in North Island major urban settlements (24.4 mg/1000 people/day). Major urban settlements had the highest estimated mean MDMA (420 mg/1000 people/day) and cocaine consumption rates (18.8 mg/1000 people/day). South Island medium urban settlements had unexpectedly high estimated mean consumption rates of MDMA (533 mg/1000 people/day) and cocaine (17.0 mg/1000 people/day). The higher-than-expected estimated cocaine consumption was from one medium urban settlement that is also a popular tourist destination in the South Island. Heroin biomarkers were not detected at any locations, and fentanyl was detected around or below the limit of reporting. This research provides information for appropriate responses for improved social and health investment to support social services associated with illicit drug consumption.

Tyrosine nitration of a humanized anti-cocaine mAb differentially affects ligand binding of cocaine and its metabolites.

Tetranitromethane was used to selectively modify tyrosine residues of a humanized anti-cocaine mAb (h2E2), under development for the treatment of cocaine use disorders. The effect of mild tyrosine nitration on the affinity of cocaine and two high affinity cocaine metabolites, cocaethylene and benzoylecgonine, was assessed using differential scanning fluorimetry to measure ligand affinities via ligand-induced thermal stabilization of the mAb antigen binding region. Nitrated tyrosine residues were identified by mass spectral analysis of thermolysin peptides. One objective was to understand the binding affinity differences observed for these three ligands, which are not explained by the published crystal structure of the h2E2 mAb Fab fragment co-crystalized with benzoylecgonine, since the carboxylic acid of benzoylecgonine that is esterified to form cocaine and cocaethylene is not in contact with the mAb. Importantly, the binding affinity of the cocaine metabolite benzoylecgonine was not decreased by mild nitration, whereas the binding affinities of cocaine and cocaethylene were decreased about two-fold. These ligands differ only in the substituent attached to the carboxylate moiety of the compound, with benzoylecgonine having an unesterified carboxylate, and cocaine and cocaethylene having methyl and ethyl esters, respectively, at this position. The results are consistent with nitration of light chain tyrosine residue 34, resulting in a less favorable interaction with cocaine and cocaethylene carboxylate esters, while not affecting binding of benzoylecgonine. Thus, light chain Tyr34 residue may have molecular interactions with cocaine and cocaethylene not present for benzoylecgonine, leading to the observed affinity differences for these three ligands.

The use of fly artifacts in a crime scene: Is there any application for forensic toxicology?

Fly artifacts (FA) are bloodstains resulting from insect activity at a crime scene, usually by feeding on human blood. Whether these artifactual stains might be useful for forensic toxicological investigations in cases of absence of conventional and unconventional matrices, for example, in cases concealment of the body or of extensive putrefaction, has not yet been investigated. The purpose of this study is to understand if FA trace evidence permits toxicological analysis when traditional matrices are not available. To this aim, FA experimentally produced by Calliphora vomitoria feeding on human blood of a cocaine and heroin user were collected from absorptive and non-absorptive material. FA material was analyzed by a new simple and fast LC-MS/MS method. Results were evaluated in terms of presence of the drug and relative amount of the detected molecules. From a qualitative point of view, the analysis of FA revealed all the substances originally detected in post-mortem blood in both cases. The ratios of cocaine/benzoylecgonine, codeine/morphine, and 6-monoacetylmorphine/morphine recovered in FA from cotton-textile materials and from non-absorptive surfaces were consistent with data resulted from original post-mortem blood. The preliminary study herein reported demonstrated that FA are extremely informative in case of cocaine and heroin users and merit further research in order to be applied in real caseworks.

Computational Design and Crystal Structure of a Highly Efficient Benzoylecgonine Hydrolase.

Benzoylecgonine (BZE) is the major toxic metabolite of cocaine and is responsible for the long-term cocaine-induced toxicity owing to its long residence time in humans. BZE is also the main contaminant following cocaine consumption. Here, we identified the bacterial cocaine esterase (CocE) as a BZE-metabolizing enzyme (BZEase), which can degrade BZE into biological inactive metabolites (ecgonine and benzoic acid). CocE was redesigned by a reactant-state-based enzyme design theory. An encouraging mutant denoted as BZEase2, presented a >400-fold improved catalytic efficiency against BZE compared with wild-type (WT) CocE. In vivo, a single dose of BZEase2 (1 mg kg-1 , IV) could eliminate nearly all BZE within only two minutes, suggesting the enzyme has the potential for cocaine overdose treatment and BZE elimination in the environment by accelerating BZE clearance. The crystal structure of a designed BZEase was also determined.

Simplified miniaturized analytical set-up based on molecularly imprinted polymer directly coupled to UV detection for the determination of benzoylecgonine in urine.

A simple, selective, and sensitive method involving a miniaturized solid phase extraction step based on a monolithic molecularly imprinted polymer (MIP) directly coupled on-line to UV detection was developed for the determination of benzoylecgonine (BZE) in complex biological samples. Monolithic MIPs were prepared into 100 µm internal diameter fused-silica capillaries either by thermal or photopolymerization. While leading to similar selectivities with respect to BZE, photopolymerization has made it possible to produce monoliths of different lengths that can be adapted to the targeted miniaturized application. The homogeneous morphology of these monolithic MIPs was evaluated by scanning electron microscopy prior to measuring their permeability. Their selectivity was evaluated leading to imprinting factors of 2.7 ± 0.1 for BZE and 4.0 ± 0.6 for cocaine (selected as template for the MIP synthesis) with polymers resulting from three independent syntheses, showing both the high selectivity of the MIPs and the reproducibility of their synthesis. After selecting the appropriate capillary length and the set-up configuration and optimizing the extraction protocol to promote selectivity, the extraction of BZE present in human urine samples spiked at 150, 250, and 500 ng mL-1 was successfully carried out on the monolithic MIP and coupled directly on-line with UV detection. The very clean-baseline of the resulting chromatograms revealing only the peak of interest for BZE illustrated the high selectivity brought by the monolithic MIP. Limits of detection and quantification of 56.4 ng mL-1 and 188.0 ng mL-1 were achieved in urine samples, respectively. It is therefore possible to achieve analytical threshold in accordance with the legislation on BZE detection in urine without the need for an additional chromatographic separation.

Sub-lethal toxicity and elimination of the cocaine metabolite, benzoylecgonine: a narrative review.

Cocaine abuse is a serious global public health and social problem, and cocaine detoxification remains a challenge. Benzoylecgonine (BE) is the main toxic metabolite after cocaine consumption, with a longer retention time in the body and environment than cocaine itself. According to many studies, the toxicity of BE to humans is as significant as cocaine itself. Moreover, BE is recognized as an addictive drug contaminant in the environment, especially the freshwater system, leading to worries of its ecotoxicity. Extensive studies on the adverse effects of BE on both humans and ecology have been conducted, showing a marked sub-lethal toxicity of BE to diverse organisms. To eliminate BE in vivo and in vitro, various elimination methods have been developed and their BE removal capacity were evaluated. In this review, we aimed to summarize information in the literature to understand better BE toxicity and elimination that may facilitate the clinical treatment of cocaine abuse. By studying the critical role of BE in cocaine abuse, we propose that the ideal treatment for cocaine abuse should not only detoxify cocaine itself but also remove or degrade BE. Emphasizing the necessity of developing effective BE elimination methods is significant for the development of potential clinical treatments and environmental protections.

Pharmacokinetic and pharmacodynamic analyses of cocaine and its metabolites in behaviorally divergent inbred mouse strains.

Cocaine (COC) is a psychostimulant with a high potential for abuse and addiction. Risk for COC use disorder is driven, in part, by genetic factors. Animal models of addiction-relevant behaviors have proven useful for studying both genetic and nongenetic contributions to drug response. In a previous study, we examined initial locomotor sensitivity to COC in genetically diverse inbred mouse strains. That work highlighted the relevance of pharmacokinetics (PK) in initial locomotor response to COC but was limited by a single dose and two sampling points. The objective of the present study was to characterize the PK and pharmacodynamics of COC and its metabolites (norcocaine and benzoylecgonine) in six inbred mouse strains (I/LnJ, C57BL/6J, FVB/NJ, BTBR T+ tf/J, LG/J and LP/J) that exhibit extreme locomotor responses to cocaine. Mice were administered COC at one of four doses and concentrations of cocaine, norcocaine and benzoylecgonine were analyzed in both plasma and brain tissue at 5 different time points. Initial locomotor sensitivity to COC was used as a pharmacodynamic endpoint. We developed an empirical population PK model that simultaneously characterizes cocaine, norcocaine and benzoylecgonine in plasma and brain tissues. We observed interstrain variability occurring in the brain compartment that may contribute to pharmacodynamic differences among select strains. Our current work paves the way for future studies to explore strain-specific pharmacokinetic differences and identify factors other than PK that are responsible for the diverse behavioral response to COC across these inbred mouse strains.

Effects of single and combined exposure to cocaine and benzoylecgonine on the oxidative status of Mytilus galloprovincialis.

The information concerning the effects of single and combined exposure to cocaine (COC) and its main metabolite, the benzoylecgonine (BE), towards marine organisms is still scant. Thus, the aim of this work was to compare the effects induced by 96 -hs exposure to a concentration of COC (500 ng/L) or BE (20 ng/L) and their mixture (500 ng/L of COC and 20 ng/L of BE) on Mytilus galloprovincialis. Oxidative stress biomarkers were applied on mussel gills and digestive gland, investigating changes in the amount of reactive oxygen species, activity of antioxidant (SOD, CAT and GPx) and detoxifying (GST) enzymes and lipid peroxidation. Independent exposure to COC and BE slightly altered mussel oxidative status in both the organs, while the mixture induced more marked responses compared to single molecules. Our results suggest the necessity to explore the toxicity of illicit drug mixtures to shed light on the risk of these molecules to marine organisms.

Comparison of Oral Fluid and Urine for Detection of Cocaine Abuse Using Liquid Chromatography with Tandem Mass Spectrometry.

BACKGROUND: Requests for urine (UR) and oral fluid (OF) drug testing at our institutions are increasing. However, few studies have assessed the accuracy of each matrix using paired specimens and LC-MS/MS. We compared OF and UR for detection of cocaine (COC) abuse in addiction medicine-psychiatry (AMP) clinics. METHODS: We measured COC and benzoylecgonine (BZE) in OF (limit of detection (LOD) 2.0 µg/L) and BZE in UR (LOD 5 µg/L) by LC-MS/MS in 258 paired samples, and compared the two matrices when higher UR cutoffs of 25, 50, and 150 µg/L were employed. RESULTS: UR detected more COC abuse than OF at the LOD (5 µg/L). BZE was detected in 63 UR specimens and COC and/or BZE in 40 OF specimens (29 OF+UR+, 11 OF+UR-, 34 OF-UR+). UR creatinine was lower in OF+UR- specimens. COC and BZE were detected in 88% (35/40) and 75% (30/40) of OF specimens, respectively. OF was equivalent to UR at detecting COC abuse using a 25 µg/L cutoff, and detected more COC abuse than UR using 50 and 150 µg/L cutoffs. The ratio of OF COC/BZE increased with decreasing UR BZE concentrations. CONCLUSIONS: We demonstrate that OF detects more COC abuse in an AMP setting when UR BZE cutoffs ≥ 50 µg/L are utilized, and that UR creatinine concentrations are significantly lower in specimens positive for COC and/or BZE in OF and negative for BZE in UR. The presence of only COC in OF and low concentrations of UR BZE likely indicates remote use of COC.

Blood and Plasma Volumetric Absorptive Microsampling (VAMS) Coupled to LC-MS/MS for the Forensic Assessment of Cocaine Consumption.

Reliable, feasible analytical methods are needed for forensic and anti-doping testing of cocaine and its most important metabolites, benzoylecgonine, ecgonine methyl ester, and cocaethylene (the active metabolite formed in the presence of ethanol). An innovative workflow is presented here, using minute amounts of dried blood or plasma obtained by volumetric absorptive microsampling (VAMS), followed by miniaturized pretreatment by dispersive pipette extraction (DPX) and LC-MS/MS analysis. After sampling 20 µL of blood or plasma with a VAMS device, the sample was dried, extracted, and loaded onto a DPX tip. The DPX pretreatment lasted less than one minute and after elution with methanol the sample was directly injected into the LC-MS/MS system. The chromatographic analysis was carried out on a C8 column, using a mobile phase containing aqueous formic acid and acetonitrile. Good extraction yield (> 85%), precision (relative standard deviation, RSD < 6.0%) and matrix effect (< 12%) values were obtained. Analyte stability was outstanding (recovery > 85% after 2 months at room temperature). The method was successfully applied to real blood and plasma VAMS, with results in very good agreement with those of fluid samples. The method seems suitable for the monitoring of concomitant cocaine and ethanol use by means of plasma or blood VAMS testing.

Structural analysis of free and liganded forms of the Fab fragment of a high-affinity anti-cocaine antibody, h2E2.

A high-affinity anti-cocaine monoclonal antibody, designated h2E2, is entering phase 1 clinical trials for cocaine abuse therapy. To gain insight into the molecular details of its structure that are important for binding cocaine and cocaine metabolites, the Fab fragment was generated and crystallized with and without ligand. Structures of the unliganded Fab and the Fab fragment bound to benzoylecgonine were determined, and were compared with each other and with other crystallized anti-cocaine antibodies. The affinity of the h2E2 antibody for cocaine is 4 nM, while that of the cocaine metabolite benzoylecgonine is 20 nM. Both are higher than the reported affinity for cocaine of the two previously crystallized anti-cocaine antibodies. Consistent with cocaine fluorescent quenching binding studies for the h2E2 mAb, four aromatic residues in the CDR regions of the Fab (TyrL32, TyrL96, TrpL91 and TrpH33) were found to be involved in ligand binding. The aromatic side chains surround and trap the tropane moiety of the ligand in the complex structure, forming significant van der Waals interactions which may account for the higher affinity observed for the h2E2 antibody. A water molecule mediates hydrogen bonding between the antibody and the carbonyl group of the benzoyl ester. The affinity of binding to h2E2 of benzoylecgonine differs only by a factor of five compared with that of cocaine; therefore, it is suggested that h2E2 would bind cocaine in the same way as observed in the Fab-benzoylecgonine complex, with minor rearrangements of some hypervariable segments of the antibody.

Seasonal monitoring of cocaine and benzoylecgonine in a subtropical coastal zone (Santos Bay, Brazil).

Illicit drugs and their metabolites represent a new class of emerging contaminants. These substances are continuously discharged into wastewater which have been detected in the aquatic environment in concentrations ranging from ng.L-1 to µg.L-1. Our study detected the occurrence of cocaine (COC) and benzoylecgonine (BE) in a subtropical coastal zone (Santos Bay, SP, Brazil) within one year. Water samples (surface and bottom) were collected from the Santos Submarine Sewage Outfall (SSOS) area. COC and BE were measured in the samples using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-ESI-MS/MS). Concentrations ranged from 12.18 to 203.6 ng.L-1 (COC) and 8.20 to 38.59 ng.L-1 (BE). Higher concentrations of COC were observed during the end of spring, following the population increase at summer season. COC and its metabolite occurrence in this coastal zone represent a threat to coastal organisms.

Disposable electrochemical immunosensor array for the multiplexed detection of the drug metabolites morphine, tetrahydrocannabinol and benzoylecgonine.

Heroin, marijuana and cocaine are widely abused drugs. Their use can be readily detected by analyzing urine for the metabolites morphine (MOR), tetrahydrocannabinol (THC) or benzoylecgonine (BZC). A multiplex immunosensor is described here for detection of MOR, THC and BZC using screen printed carbon array electrodes modified with gold nanoparticles. Antibodies against MOR, THC and BZC were immobilized on eight electrodes in a sensor array simultaneously, and a competitive assay was used for the detection. The free analytes in the sample compete with bovine serum albumin-conjugated analytes for the immobilized antibodies on the sensor surface. The array is capable of detecting the three drugs simultaneously within 20-40 min. The method has a high sensitivity, with detection limits as low as 1.2, 7.0, and 8.0 pg.mL-1 for MOR, THC and BZC, respectively. Cross reactivity testing was preformed to monitor any nonspecific binding. The results revealed good selectivity. Urine samples were spiked with the 3 drugs and tested with the multiplexed immunosensor. Recovery percentages ranged between 88 to 115%. Graphical abstract Schematic presentation of the multiplexed immunosensor for drugs of abuse,viz. tetrahydrocannabinol (THC), morphine (MOR), and benzoylecgonine (BZC)) by using an array of modified electrodes.

A laboratory-study on the analytical determination and removal processes of THC-COOH and bezoylecgonine in the activated sludge reactor.

The present study focused on 11-nor-9carboxy-Δ9-THC (THC-COOH) and Benzoylecgonine (BE), the most common metabolites of cannabis and cocaine, respectively, present in the domestic sewage entering the wastewater treatment plants. The aims of the study were: (1) to validate the analytical method of detection in wastewater and sludge; (2) to determine contribution of biodegradation and other processes to the removal in the biological reactor of the wastewater treatment plant (WWTP) and the response of biomass to different drug concentrations. The Ultra-Performance Liquid Chromatography coupled to tandem Mass Spectrometry method showed to be repeatable and reliable (recovery>75%; repeatability<10-15%; bias uncertainty<10) for measurements in wastewater; the ultrasound assisted extraction (USE) demonstrated to be reliable as pre-treatment of activated sludge solid phase. Both drugs were fully removed from the liquid phase in the lab-scale biological reactor within 24 h. Biodegradation was the main BE removal mechanism, and the first order kinetic model provided the best fitting of the experimental data. THC-COOH was mainly removed due to a combination of adsorption and biodegradation; adsorption was better described by the pseudo-second order kinetic model and the Freundlich isotherm. Both drugs at the higher concentrations caused inhibition of nitrogen oxidation and carbon removal.

An Unusually High Cocaine Blood Concentration in an Impaired Driving Investigation.

Cocaine is an illicit drug frequently encountered by forensic practitioners in driving under the influence of drugs (DUID) casework. Whole blood collected from a suspected drugged driver was found to contain 3.000 mg/L cocaine, 1.600 mg/L benzoylecgonine, and 0.260 mg/L methamphetamine. The high concentration of cocaine, while common in overdose death investigations, is unusual for an impaired driving case. Information from the officer revealed that the motorist swallowed cocaine during the traffic stop. Although a cocaine DUID charge could not be pursued, the blood methamphetamine concentration exceeded the State of Nevada "per se" limit for operating a motor vehicle. The motorist was successfully prosecuted for DUID based on his admission of using methamphetamine prior to driving and the blood methamphetamine result. This case highlights the importance of considering case history when interpreting laboratory results and the application of jurisdictional statutes as an approach to prosecuting suspected drug-impaired drivers.

Determination of Cocaine and Metabolites in Dried Blood Spots by LC-MS/MS.

Cocaine is one of the most common illegal drugs in Europe and other parts of the world. It is mainly metabolized to benzoylecgonine and ecgonine methyl ester but also to minor metabolites such as norcocaine and meta-hydroxy-benzoylecgonine. If ethanol is consumed simultaneously, cocaethylene is formed. Dried blood spots (DBS) are a minimally invasive sampling technique producing easy-to-ship specimens and have garnered increasing attention in forensic and clinical contexts in recent years. We hereby present a liquid chromatography/tandem mass spectrometry-based (LC-MS/MS) method for the quantification of cocaine, benzoylecgonine, ecgonine methyl ester, norcocaine, meta-hydroxy-benzoylecgonine, and cocaethylene in DBS.

Rapid Determination of Cocaine and Its Metabolite Benzoylecgonine in Hair by LC-MS/MS.

OBJECTIVES: To establish a rapid determination method with LC-MS/MS for cocaine and its metabolite benzoylecgonine in hair. METHODS: Deuterated internal standards （cocaine-D3 and benzoylecgonine-D8） were added to the decontaminated hair. After the extraction by ultrasonication with methanol, the compounds were separated by the Restek Allure PFP propyl column, and cocaine and benzoylecgonine were simultaneously analysed in multiple reaction monitoring mode. RESULTS: The cocaine and benzoylecgonine in hair showed a good linearity in the range of mass fraction between 0.02 and 10.00 ng/mg with the limits of detection of 0.01 ng/mg. CONCLUSIONS: The developed method is simple and rapid with a good selectivity, which is suitable for the determination of cocaine and its metabolite benzoylecgonine in hair.

A liquid chromatography-tandem mass spectrometry method for the determination of cocaine and metabolites in blood and in dried blood spots collected from postmortem samples and evaluation of the stability over a 3-month period.

The aims of this study were (1) to identify and quantify cocaine (COC), benzoylecgonine (BE), ecgonine methyl ester (EME), and cocaethylene (CE) in DBS; (2) to compare dried blood spots (DBSs) analytical results with the routine blood analyses; (3) to monitor analytes stability on DBS within a 3-month period. Eighty-five µL of blood from postmortem cases were put on a card for DBS analysis and kept in the dark, at room temperature. Samples were extracted through solid-phase extraction (SPE) cartridges and injected in the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The analytical procedure is simple, sensitive, and specific. Limits of detection (LODs) and quantification (LOQs) were calculated at 1.0 and 5.0 ng/mL(g) for COC and CE, and at 0.5 and 2 ng/mL for EME and BE, respectively. Validation parameters fulfilled all the acceptance criteria. Fifty-five postmortem cases were evaluated. Eighteen cases were positive for COC (44-2456 ng/mL) and BE (228-4700 ng/mL), 12 for EME (92-1500 ng/mL), and 11 cases for CE (11-273 ng/mL). Stability was evaluated on 8 cases collected in the period January 2017-January 2018. For each case, 5 DBSs were collected at T0. Four DBSs were analyzed within the 4 following weeks and 1 sample was analyzed after 3 months. The concentrations on DBSs, stored at room temperature, always matched the ones obtained on blood samples kept at -20°C (<20% variation, both at T0 and after 3 months). BE and COC concentrations remained stable after a 3-month storage, EME concentrations slightly increased after 3 weeks in the 2 analyzed samples, while CE provided a less homogeneous stability depending on the sample.