Cocaine Metabolites Research Articles

Evaluation of a Point of Care Urine Drug Test System for Application in Outpatient Pain Management Clinics

Abstract Introduction/Objective Most states require testing for illicit and controlled substances in ambulatory, non-terminal pain management populations to ensure prescription compliance. Point of care (POC) urine drug testing (UDT) is a widely adopted testing method that assists with clinical decision making within minutes. However, these immunoassays may lack the sensitivity and specificity needed for potent medications such as fentanyl. This study compares the performance characteristics of a POC UDT to an in-house, high throughput, automated chemistry analyzer and the reference method, gas chromatography-mass spectrometry (GC-MS). Providers should be aware of the limitations of each methodology given the rise of synthetic opioid misuse, and the need to quantify multiple substances and metabolites within the same drug class. Methods/Case Report 100 residual deidentified patient urine samples from an ambulatory pain management population were analyzed using GC-MS, Profile®-V MEDTOX Scan® POCT device (Burlington, NC), and a Beckman Coulter AU480® Chemistry Analyzer using EMIT® II Plus reagents (Beckman Coulter; Brea, CA). Drug classes on the Profile®-V included: amphetamines, barbiturates, benzodiazepines, buprenorphine, cocaine metabolite, methamphetamine, methadone, opiates, oxycodone, cannabinoids and phencyclidine. Drug classes on the AU480® included the same except without a methamphetamine-specific immunoassay. Results (if a Case Study enter NA) The results from GC-MS showed that 99% (99/100) of the samples were positive for one or more of the drug classes. Overall, the AU480® was 90% sensitive and 98% specific while the MEDTOX Scan® was 87% sensitive and 98% specific. Both devices exhibited variable performance across drug classes. Neither the AU480® nor the MEDTOX Scan® detected fentanyl in five specimens. Conclusion POCT-UDS systems like the Profile®-V MEDTOX Scan® can provide good accuracy and may offer a time and cost benefit when compared to higher throughput analyzers like the AU-480®. Due to the low reference values of immunoassays, definitive testing such as GC-MS is recommended for use in the pain management population.

B-298 Quantitation and Abundance of Xylazine and Xylazine Metabolites in Human Urine by LC-MS/MS

Abstract Background The most common cause of drug overdose fatality in the US involves synthetic opioids such as fentanyl. Recently the Director of the White House Office of National Drug Control Policy designated fentanyl associated with xylazine as an emerging threat to the US, and a national response plan will include xylazine testing. Xylazine, a clonidine analog is not FDA-approved for use in humans but has been increasingly detected in unregulated illicit drugs supplies. Our understanding of xylazine detection, metabolism, and clinical impact on patients, however, is limited. The objectives of this study are to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to investigate the presence and abundance of xylazine metabolites in remnant urine samples submitted for clinical testing. Methods Remnant urine samples were identified when tested positive for xylazine during routine clinical LC-MS/MS testing. All experiments were performed using an Acquity HPLC coupled to a Waters TQS-micro triple quadrupole mass spectrometer. Urines were diluted with xylazine-D6 internal standard, hydrolyzed using β-glucuronidase and separated on a 100mm x 2.1mm, 2.6μm Kinetex C18 column (Phenomenex) with a 4 minute step gradient from 2% to 98% mobile phase B (0.1% formic acid and 2mM ammonium acetate in A: water; B: methanol) with a flow rate of 400 μl/min at 50°C. Xylazine and 4-OH-xylazine were detected in positive mode using multiple reaction monitoring, with transitions optimized from standards (Cayman Chemical and/or Sigma Aldrich), resulting in the following transitions: xylazine: 221>90, 221>164; 4-OH xylazine: 237>90, 237>137. Xylazine and 4-OH-xylazine were quantified using calibration curves prepared from 1 - 5,000 ng/mL in negative urine. Product ion scanning (PIS) was used to detect four additional xylazine metabolites, based on spectral matching to previously published full scan high resolution product ion spectra. The highest abundance fragment ions and identified retention times from PIS experiments were used to perform targeted detection for sulfone-xylazine (253>181, 253>147), oxo-xylazine (235>122, 235>114), OH-sulfone-xylazine (269>197, 269>163), and OH-oxo-xylazine (251>197, 251>148). Results Xylazine was detected at >=1 ng/mL in 38% (54/138) of fentanyl positive urines. A subset (n= 362) of urines that screened positive for opiates, amphetamines, or cocaine metabolite, were also tested but xylazine was detected in only 1 urine that did not contain fentanyl.Xylazine concentrations quantified in 61 remnant urines ranged from <1 to >5,000 ng/mL. 4-OH-xylazine concentrations ranged from <1 to 509 ng/mL. We observed five previously reported xylazine metabolites: 4-OH-xylazine, oxo-xylazine, sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine. The mean concentration ratio of 4-OH-xylazine-to-xylazine was 0.11. The mean peak area ratios (metabolite-to-xylazine) for sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine, and oxo-xylazine were 3.5, 3.8, 2.1, and <0.1, respectively. The proposed sulfone-xylazine and OH-oxo-xylazine metabolites were observed to have the most abundant signal of all detected metabolites, although there is currently no commercially available standard to facilitate quantification. Conclusions Xylazine and 4-OH-xylazine were quantitated in 61 patient urines. Four additional xylazine metabolites were observed and relative abundance to xylazine was estimated using peak areas. Future studies include quantitation of xylazine and xylazine metabolites in plasma and association of xylazine detection with clinical presentation and patient outcomes.

B-263 Analytical Performance Evaluation of Eleven Drug of Abuse Assays on the Atellica CI Analyzer

Abstract Background The Atellica® CI Analyzer is an automated, high-throughput integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM Assays. This study evaluated the analytical performance of the Atellica CH Amphetamines (Amp), Barbiturates (Brb), Benzodiazepine (Bnz), Cannabinoids THC (Thc), Cocaine Metabolite (Coc), Ecstasy (Xtc), Methadone (Mdn), Opiates (Op), Oxycodone (OXY), Phencyclidine (Pcp), and Propoxyphene (Ppx) Assays on the Atellica CI Analyzer. Methods Precision and method comparison (MC) studies were used as performance indicators. Precision studies were performed according to CLSI EP05-A3 using quality control (QC) samples consisting of contrived human urine samples. One aliquot of each QC was tested in duplicate in two runs per day ≥2 hours apart on each analyzer for ≥ 20 days. Precision for each assay was evaluated with one reagent lot on one system. Method comparison studies were performed with three reagent lots according to CLSI EP12-A2. Individual native and contrived human urine samples were analyzed using the Atellica CH Amp, Barb, Bnz, Thc, Coc, Xtc, Mdn, Op, OXY, Pcp, and Ppx Assays on both the Atellica CH and Atellica CI Analyzers. The results were assessed based on analyte values used for distinguishing positive (value ≥cutoff) and negative (value <cutoff) specimens. Results As shown in table below, repeatability and within-lab %CVs based on manufacturer arbitrary units (mAU) were <0.8% and <3.2%, respectively or qualitative interpretation for each replicate remained unchanged for all 80 precision testing measurements (OXY, PcP). Qualitative accuracy assessed by concordance analysis demonstrated ≥95% agreement between the Atellica CI Analyzer and Atellica CH Analyzer. Conclusions Evaluation of the Atellica CH Amp, Barb, Bnz, Thc, Coc, Xtc, Mdn, Op, OXY, Pcp, and Ppx Assays using the Atellica CI Analyzer demonstrated acceptable precision and equivalent performance compared to the same assays on the Atellica CH Analyzer.

B-291 Validation of a Simple Qualitative Immunoassay for 21 Drugs of Abuse in Meconium on a Single Biochip Array

Abstract Background Drugs use during pregnancy is a major social and medical issue, which may lead to significant pre- and post-natal complications, such as preterm birth and newborn mortality. Exposed newborns may have long-term behavioral, cognitive, and developmental problems. Accurate detection of in utero drug exposure is crucial for timely medical intervention for the newborn and the mother. Meconium is the ‘gold standard' matrix for detection of drugs in newborn which reflects drug exposure during pregnancy. This study aims to develop and optimize a clinical assay for simultaneous screening of 21 drugs in meconium using a custom biochip array. Methods Custom biochip array (Randox Laboratories Ltd.) included 21 discrete test regions (DTRs) for amphetamine, methamphetamine, opioids/opiates (targeting morphine, oxymorphone, oxycodone, hydrocodone, 6-monoacetyl morphine, tramadol, buprenorphine, fentanyl, and methadone), phencyclidine (PCP), cocaine (targeting benzoylecgonine), benzodiazepines (targeting oxazepam, lorazepam, and clonazepam), zolpidem, barbiturates (targeting phenobarbital), ethyl glucuronide, methylphenidate, and marijuana metabolite (targeting Delta-9-Carboxy-Tetrahydrocannabinol [THC-COOH]). Patient samples were obtained from the residual meconium samples that had been previously used for other tests. Blank meconium samples were spiked at in-house cutoff, negative and positive assay control levels. All samples were extracted in specimen diluent using Biotage® Lysera and centrifuged. The supernatant was analyzed on a fully automated Randox Evidence+® Analyzer. A robotic arm pipetted 60 µL sample on to a biochip and then the analyzer performed a competitive chemiluminescent immunoassay. The results were generated as relative light units (RLUs). Results The sample results were determined to be “positive” or “negative” based on average RLU of cutoff controls for each analyte on an assay. The intra-assay (CV ≤ 15%) and inter-assay (CV ≤ 18%) precision was 100% agreement for positive and negative controls and there was no observed carryover for any of the analytes. The sample recovery was excellent with minimal to no matrix suppression for all analytes. Accuracy was performed by comparing spiked analyte results from Randox Evidence+® Analyzer with the expected results and all the spiked samples matched (positive or negative). When authentic patient or spiked results were compared with external laboratory test results, some discordant results were obtained. However, the discrepancies could be explained based on the different cutoffs (e.g., the Randox assay had lower cutoffs) or the known assay cross reactivities within the analytes of same class. These deviations did not affect clinical utility of the assay. Conclusions We validated a novel and simple qualitative chemiluminescent immunoassay in meconium using a custom biochip array. Unlike other routine screening/ELISA methods, which require separate assays for detection of similar drug targets, the custom biochip array offers a convenient, rapid and efficient method that utilizes only a single sample preparation for the screening of all 21 drug targets. Therefore, this technology has potential to be utilized as high-throughput screening assay. Like all qualitative assays, the limitation of this test is that all positive results will need to be assessed quantitatively for a definitive interpretation.

B-274 Determining Novel Biomarker Candidates Associated With Heroin and Cocaine Exposure Using Urine Metabolomics

Abstract Background Substance abuse poses an escalating health risk in the United States. While acute drug exposure can be tracked using drugs and their metabolites as biomarkers in clinical settings, there is a notable absence of biomarkers for chronic drug exposure or addiction. The development and availability of such biomarkers could significantly enhance clinicians’ ability to identify patients at high risk of addiction, and more accurately predict disease progression and treatment responses. Methods Our study aimed to discover novel biomarkers associated with substance abuse by analyzing raw mass spectrometry (MS) data from urine comprehensive drug screening (UCDS) conducted at the University of Pittsburgh Medical Center (UPMC). The MS data, acquired through untargeted data collection using LC-qToF-MS at the UPMC Clinical Toxicology Laboratory, was initially preprocessed (including peak identification, alignment, and putative annotation) using MS-DIAL to generate a data matrix. This matrix, consisting of objects (cases) and features (m/z and retention time), was further normalized and analyzed using MetaboAnalyst. A combination of statistical methods, including point biserial correlation analysis, volcano plot, significance analysis of microarrays and metabolites (SAM), empirical Bayesian analysis of microarrays and metabolites (EBAM), partial least squares discriminant analysis (PLS-DA), and random forest (RF), were employed to select features significantly associated with the EIA-results. These features were subsequently evaluated for analyte identification using MS-FINDER. Results Our analysis identified nine significant features associated with 6MAM-EIA for heroin abuse, including 6-monoacetylmorphine itself and norfentanyl. For features associated with cocaine metabolite-EIA, 37 significant features were selected, including multiple cocaine metabolites, nicotine metabolites, and norfentanyl. Some of these selected features are likely products of in-source fragmentation or endogenous metabolites. Conclusions Our study has identified a set of potential biomarker candidates for illicit drug exposure. Notably, norfentanyl was found to be significantly associated with both 6MAM-EIA and cocaine metabolite-EIA, reflecting current trends in illicit drug use. Further chemical identification of these biomarkers is planned for future work.

Impact of reduced sampling frequency of illicit drug wastewater monitoring in the Netherlands

Drug consumption estimates are traditionally based on surveys or information from police seizures. Alternatively, residues of illicit drugs in untreated wastewater (influent) can be used to calculate mass loads and subsequently estimate drug consumption in the community throughout the week. For this purpose, wastewater is commonly sampled for seven consecutive days within the Sewage analysis CORe group Europe (SCORE), while other sampling schemes may be implemented in long-term studies outside this consortium. The current study demonstrates how sampling frequency of illicit drug residues in the influent of wastewater treatment plants (WWTPs) affects the derived weekly average. Thirty WWTPs were sampled over the course of 12 years and influents were analyzed for five drugs (metabolites): 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine, amphetamine, benzoylecgonine (a metabolite of cocaine), and 11-nor-9-Carboxy tetrahydrocannabinol (THC-COOH). Subsequently, small and large WWTPs were grouped with a threshold of 100,000 inhabitants. After data curation, standardized loads were calculated (mg/d per 1000 inhabitants). Weekly averages of loads of the drug residues were calculated based on six scenarios (sampling one to six weekdays) and compared to the weekly average in the control situation (sampling seven weekdays) in a Monte Carlo simulation. Results indicate that drug residues with more dynamic loads over a week require more frequent sampling. The analysis illustrates that a decreased sampling frequency (4 or 5 days per week) still leads to a representative weekly average for all drugs tested when a deviation up to a factor of 1.25 is deemed acceptable. However, knowledge on typical levels is necessary to define outliers. We therefore recommend to study dynamics in drug residue loads for WWTPs before reducing sampling frequency in long term monitoring programs.

Analysis of crude wastewater from two treatment plants in South Wales for 35 new psychoactive substances and cocaine, and cannabis

This study investigates the presence of new psychoactive substances (NPS) and their metabolites in two wastewater treatment plants (WWTPs) situated in South Wales, UK (WWTP-1 and WWTP-2). Analysis was conducted for 35 NPS and metabolites, along with the inclusion of benzoylecgonine (main cocaine metabolite) and cannabis, the most detected illicit substances. Benzoylecgonine was identified as the predominant substance in both WWTPs. Epidemiological calculations revealed the average population consumption of cocaine to be 3.88 mg/d/1000 inhabitants around WWTP-1 and 1.97 mg/d/1000 inhabitants for WWTP-2. The removal efficiency of benzoylecgonine across both WWTPs was observed at an average of 73%. Subsequent qualitative analyses on randomly selected wastewater samples detected medicinal compounds including buprenorphine, methadone, and codeine in both WWTPs. An additional experiment employing enzymatic hydrolysis revealed the presence of morphine, an increased presence of codeine, and 11-Nor-9-Carboxy-THC (THC-COOH) post-hydrolysis. These findings underscore the significant presence of illicit substances and medicinal compounds in wastewater systems with the absence of NPS within the South Wales area, highlighting the necessity for enhanced monitoring and treatment strategies to address public health and environmental concerns.

Comprehensive Evaluation of Cocaine and its Hydroxy Metabolites in Seized Cocaine and a Large Cohort of Hair Samples.

As cocaine is not only incorporated into hair via blood following ingestion but also by external contamination, hair samples are commonly tested for cocaine metabolites to prove ingestion. However, cocaine metabolites can also be present as degradation products in typical street cocaine samples. The present study investigates minor hydroxycocaine metabolites para- and meta-hydroxycocaine together with para- and meta-hydroxybenzoylecgonine in seized cocaine (n=200) and hair samples from routine casework (n=2,389). Analytical results of hair samples were interpreted using an established decision model for the differentiation between actual use and external contamination using metabolic ratios (metabolite to cocaine). They were further examined concerning background of request, hair color, body site of sample collection, sex, and metabolic ratios of the main metabolites (benzoylecgonine, norcocaine, and cocaethylene). All seized cocaine samples were positive for para- and meta-hydroxycocaine with a maximum percentage of 0.025 and 0.052 %, respectively; para- and meta-hydroxybenzoylecgonine were detected in 55 and 56 % of samples with a maximum percentage of 0.044 and 0.024 %, respectively. Analytical results of 424 hair samples (17.7 %) were interpreted as being predominantly from contamination; the majority of these samples were from traffic medicine cases (83.7 %). Metabolic ratios of minor hydroxycocaine metabolites were significantly higher in hair samples interpreted as originating from use than in samples interpreted as caused by contamination. Metabolic ratios for hydroxycocaines were significantly higher in forensic cases compared to abstinence controls and also in black hair compared to blond/gray hair. However, this was not the case for hydroxybenzoylecgonine metabolic ratios. No statistical difference was observed with regard to the donor's sex. Hydroxycocaine metabolic ratios increased significantly with increasing ratios of norcocaine and cocaethylene to cocaine, respectively. The study demonstrates that hydroxycocaine metabolites (including thresholds for their metabolic ratios) must be used for a reliable interpretation of positive cocaine results in hair samples.

Cocaine-derived hippuric acid activates mtDNA-STING signaling in alcoholic liver disease: Implications for alcohol and cocaine co-abuse

The simultaneous abuse of alcohol-cocaine is known to cause stronger and more unpredictable cellular damage in the liver, heart, and brain. However, the mechanistic crosstalk between cocaine and alcohol in liver injury remains unclear. The findings revealed cocaine-induced liver injury and inflammation in both marmosets and mice. Of note, co-administration of cocaine and ethanol in mice causes more severe liver damage than individual treatment. The metabolomic analysis confirmed that hippuric acid (HA) is the most abundant metabolite in marmoset serum after cocaine consumption and that is formed in primary marmoset hepatocytes. HA, a metabolite of cocaine, increases mitochondrial DNA leakage and subsequently increases the production of proinflammatory factors via STING signaling in Kupffer cells (KCs). In addition, conditioned media of cocaine-treated KC induced hepatocellular necrosis via alcohol-induced TNFR1. Finally, disruption of STING signaling in vivo ameliorated co-administration of alcohol- and cocaine-induced liver damage and inflammation. These findings postulate intervention of HA-STING-TNFR1 axis as a novel strategy for treatment of alcohol- and cocaine-induced excessive liver damage.Graphical

(Invited) Norfentanyl Detection with Carbon Nanotube-Based Field-Effect Transistors

The opioid crisis, propelled predominantly by synthetic opioids such as illicit fentanyl, constitutes a significant global public health threat. To meet the critical demand for a sensitive and portable analytical tool for detecting fentanyl exposure, we have developed two types of biosensors based on single-walled carbon nanotubes (SWCNTs).The first biosensor is a field-effect transistor (FET) device with semiconductor-enriched SWCNTs, functionalized with norfentanyl antibody.1 This antibody-SWCNT-FET biosensor exhibits ultrasensitive detection of norfentanyl, the primary inactive metabolite of fentanyl, in urine samples. Various sensor configurations were investigated to optimize sensing results. The use of a "reduced" antibody facilitated oriented immobilization, enhancing the proximity of the antigen-antibody interaction to the sensor surface and resulting in improved sensitivity. These norfentanyl biosensors demonstrate remarkable sensitivity, with a limit of detection in the fg/mL range in both calibration and synthetic urine samples, showcasing their potential as highly reliable detection tools.While capture probes like antibodies enhance chemical sensitivity and selectivity, the stability of nanotube-based biosensors is constrained by the activity of these capture probes. To address this limitation, we have explored an alternative approach involving sensor arrays consisting of different metal-organic frameworks (MOFs) on SWCNTs, which act as nonspecific receptors. With the assistance of machine learning-driven discrimination, such sensor arrays hold the potential to generate unique signals for different analytes.In pursuit of this objective, size-based norfentanyl detection was achieved using SWCNT@MOF composites in FET biosensors.2 Four SWCNT@MOF composites were synthesized, with SWCNT@UiO-67 demonstrating the highest sensing response to norfentanyl due to a size-matching effect within the MOF channel. Composites with smaller pore sizes such as SWCNT@UiO-66 and SWCNT@UiO-66-NH2 did not exhibit sensitivity improvement compared to bare SWCNT. We have also demonstrated selectivity of these biosensors for norfentanyl over norhydrocodone and benzoylecgonine, which are major metabolites of hydrocodone and cocaine, respectively.Moreover, we successfully applied our sensor fabrication with a flexible FET electrode and a portable sensing setup, showing significant potential for developing a portable device for on-site detection of fentanyl exposure with improved sensitivity. Therefore, we envision our norfentanyl biosensors can enhance the safety and security of both first responders and overdose victims and provide a platform technology that can contribute to the development of effective tools for addressing the opioid crisis.[1] Shao, W.; Zeng, Z.; Star A., An Ultrasensitive Norfentanyl Sensor Based on a Carbon Nanotube-Based Field-Effect Transistor for the Detection of Fentanyl Exposure. ACS Appl. Mater. Interfaces 2023, 15, 37784–37793.[2] Zeng, Z.; Islamov, M.; He, Y.; Day, B. A.; Rosi, N. L.; Wilmer, C. E.; Star, A., Size-Based Norfentanyl Detection with SWCNT @ UiO-MOF Composites. ACS Appl. Mater. Interfaces 2023, accepted.

Dilute-and-shoot approach for the high-throughput LC-MS/MS determination of illicit drugs in the field of wastewater-based epidemiology

The determination of illicit drugs in urban influent wastewater (IWW) enables the monitoring of spatial and temporal drug usage trends and assessment of community lifestyle habits. The increasing number of wastewater surveillance studies has emphasized the necessity for the development of rapid, high-throughput methods that maintain high quality data. This work evaluates the use of a dilute-and-shoot methodology, based on direct injection (DI) of centrifuged samples, as an alternative approach to the widely applied sample pre-treatment based on solid-phase extraction, for the liquid chromatography-tandem mass spectrometry determination of seven widely consumed illicit drugs and their metabolites in IWW (amphetamine; cocaine metabolite, benzoylecgonine; ketamine; 3,4-methylenedioxymethamphetamine (MDMA); methamphetamine; cannabis metabolite, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THCCOOH); heroin metabolite, 6-acetylmorphine (6-MAM)). Comparison of both approaches in terms of matrix effects, sensitivity and accuracy, demonstrates the DI method suitability to correctly quantify these analytes in IWW, with a limit of quantification lower than 30 ng L−1 for most compounds. After validation of the method and participation in an interlaboratory exercise, the DI method was applied to the analysis of 54 IWW samples collected from different Spanish wastewater treatment plants. Additionally, quality controls were incorporated in each analysis batch to support the DI method applicability and robustness. The use of a 10 μL-DI reduces time-consuming sample preparation, analysis time and measurement uncertainty. Moreover, it supports green chemistry by reducing the consumption of organic solvents and it facilitates logistics by collecting, transporting, and storing less sample volume. The methodology is therefore especially appropriate for monitoring illicit drugs in large wastewater-based epidemiology sampling campaigns or when fast near real-time results are needed.

Electrochemical detection of cocaine metabolites (benzoylecgonine and ecgonine) at the miniaturized electrified liquid-liquid interface

The main urinary cocaine metabolites are benzoylecgonine and ecgonine methyl ester. These molecules possess two chemical functional groups that can be charged within the conventional pH range, this is the tertiary amine group (both) and the carboxylic group (benzoylecgonine). We have found that in the pH range from 2 to 6 (for benzoylecgonine) and pH from 2 to 4 (for ecgonine methyl ester) both molecules can be detected at the electrified liquid-liquid interface (eLLI) by following the interfacial ion transfer reactions triggered in the presence of the concerned metabolites in the aqueous phase. The presence of the benzene ring in the benzoylecgonine structure increases its hydrophobicity (on the formal Galvani potential difference scale) by around 200 mV as compared with ecgonine methyl ester which transfer is overlaid with the inorganic cations crossing the LLI at the positive end of the potential window. Macroscopic eLLI was used during physicochemical ecgonine methyl ester and benzoylecgonine characterization as we have defined their diffusion coefficients, ion partition, and concentration fraction diagrams (only for the latter). The miniaturized platform was used during electroanalytical studies focused on benzoylecgonine giving a few µM limit of detection. For this purpose, we have used the fused silica capillary as the eLLI support. All our findings allowed us to use this system first to develop the electroanalytical protocol for benzoylecgonine quantification to finally detect it in the spiked urine sample with nearly 100 % recovery.

Simultaneous determination of ethyl glucuronide, cocaine, cocaethylene, and benzoylecgonine in hair by using LC–MS/MS

Alcohol and cocaine (COC) are commonly co-used drugs that cause addiction and have harmful effects. Their abuse may threaten the health of the abuser and public safety by causing serious accidents or crimes. The recidivism rate of drug-related crimes closely correlates with alcoholism. Several incidences of alcohol consumption in combination with drug abuse have been reported. Here, liquid chromatography tandem mass spectrometric method was developed to simultaneously analyze ethyl glucuronide (EtG), a metabolite of ethanol; COC; cocaethylene (CE), an alcohol-derived metabolite of COC; and benzoylecgonine (BZE), a major metabolite of COC, to determine the concurrent use of alcohol with COC. For pre-treatment, ultracentrifugation (5 min, 50,000 g) and mixed-mode anion exchange solid-phase extraction were used to increase the recovery of target compounds and minimize the matrix effect of hair. The lower limits of quantification were: 7 pg/mg (EtG), 2 pg/mg (COC), 10 pg/mg (CE), and 1 pg/mg (BZE). The correlation coefficient (r) of the calibration curve within the quantified range of target compounds was ≥ 0.9978. The intra- and inter-day accuracies were − 6.1–9.7% and − 9.3–8.3%, and intra- and inter-day precisions were 0.5–10.3% and 0.6–14.4%, respectively. The recovery, matrix effect, process efficiency, and autosampler stability were 89.2–104.8%, 81.6–105.4%, 81.5–107.1%, and 96.6–109.7%, respectively. The novel analytical method was validated with hair samples from individuals suspected of alcohol and COC use, and the method could distinguish between independent and concurrent use. Based on the findings, the analytical approach developed in this study is anticipated to be valuable in drug and alcohol dependence tests that require the simultaneous detection of alcohol and COC abuse.

Heat shock protein (HSP) and its correlation to cocaine-related death: a systematic review.

Investigating deaths related to chronic cocaine abuse can be a difficult task, particularly when they occur suddenly and without explanation. Cocaine abuse can trigger biological effects similar to physiological stressors, causing the body to produce heat-shock proteins (HSPs). However, there is still limited information on the specific levels of each HSP type. This systematic review aims to comprehensively collect and analyze all existing literature data regar-ding the relationship between HSPs and cocaine abuse to investigate whether HSPs can be utilized as forensic markers for accurately dia-gnosing cocaine-related deaths. The Authors conducted the literature search using PubMed and Scopus databases, searching for articles published between 1 January 1992 and 1 April 2024 using the text string: "heat shock protein" AND "cocaine". Twenty articles were collected, but only nine were included in the systematic review. The data gathered pertained to both human and murine species. The majority of the analyzed articles revealed an elevation in HSP25, HSP27, HSP60, HSP70, HSP72, and HSP73 levels in the brain, cerebellum, and liver, indicating cocaine-induced stress. The relationship between HSP and cocaine has been unclear over time. However, recent studies have shown that cocaine consumption leads to an increase in HSP levels, particularly in the central nervous system. This correlation can also be observed in certain types of liver cells that are capable of binding cocaine metabolites. In conclusion, HSP brain levels, along with other biomarkers, may be used to diagnose sudden, unexpected death related to cocaine abuse.

Comparing the acute toxicities of co-exposure to cocaine and ethanol versus cocaine alone.

Cocaine is commonly consumed with ethanol, which leads to the formation of cocaethylene through transesterification. Cocaethylene is an active metabolite of cocaine with a longer duration of action. Literature on the combined toxicity of cocaine, ethanol, and cocaethylene is conflicting. We aimed to compare the acute toxicities of co-exposure to cocaine and ethanol versus cocaine alone in Hong Kong. This was a retrospective study on acute cocaine toxicities reported to the Hong Kong Poison Control Center from 1 January 2010 to 22 January 2023. Cocaine exposure was confirmed by urine immunoassays/laboratory tests and ethanol co-ingestion was confirmed by blood ethanol concentrations. A serious outcome was defined as a National Poison Data System outcome moderate or above. Univariate analyses and multivariable logistic regression were performed to compare the associations of clinical outcomes with and without ethanol, followed by subgroup analyses of cases with complete data. We analyzed 109 patients (median age 29 years, 71% men, 68% Chinese), of whom 20 had confirmed ethanol co-ingestion (mean blood ethanol concentration 1350mg/L). Multivariable analysis showed that co-exposure to cocaine and ethanol was associated with a lower risk of serious outcomes (adjusted odds ratio 0.09, 95% confidence interval 0.01-0.77; p = 0.03) after adjusting for age, sex, ethnicity, route of cocaine administration, and physical health status. Subgroup analyses showed similar findings. In contrast to previous studies, we did not identify a higher risk of serious outcomes after co-exposure to cocaine and ethanol compared to cocaine alone in a predominantly Chinese cohort.

Infective Endocarditis in an Intravenous Drug User Leading to Myocardial Rupture and Hemopericardium.

Infective endocarditis (IE) is an infectious disorder of the innermost lining of the heart that can be fatal if left untreated. Infective endocarditis can spread beyond the endocardium into the myocardium and cause arrhythmias and myocardial wall rupture. Individuals with a history of intravenous drug use are at increased risk of developing IE and are at higher risk of dying, given their limited access to health care and adherence to treatment. A medicolegal autopsy was performed on a 30-year-old woman with a history of intravenous drug use and recent assault after a hospital admission during which she did not survive resuscitation. The cause of death was found to be myocardial rupture in the setting of transmural IE. Postmortem imaging showed hemopericardium which was identified grossly with valvular vegetations in the heart. A ventricular wall defect along with transmural abscess formation was identified. Perimortem toxicology was positive for fentanyl, methamphetamine, and benzoylecgonine, a metabolite of cocaine. Postmortem blood cultures were positive for coagulase-negative Staphylococci, Staphylococcus aureus, Candida tropicalis, and Viridians group Streptococci. Postmortem tissue cultures taken from the heart was positive for Candida glabrata and Streptococcus mitis. The decedent had significant risk factors for IE, such as intravenous drug use and a prior admission to hospital for IE. The organisms identified on culture are in-keeping with the gross findings of IE and the terminal event of myocardial rupture was likely the result of tissue damage resulting from IE.

Quantitative LC-MS/MS Analysis of Amphetamines and Cocaine in Human Urine.

We hereby present a fast and high-throughput LC-MS/MS assay for the simultaneous analysis of amphetamines and cocaine in human urine. The assay is used for confirmations following immunoassay urine drug screens as well as a quantitative assay to report actual urine concentrations in the range 30-10,000ng/mL for each of the seven analytes, namely, amphetamine; methamphetamine; phentermine; methylenedioxyamphetamine; 3,4-methylenedioxymethamphetamine; methylenedioxy-ethyl-amphetamine; and a cocaine metabolite, benzoylecgonine. The assay derives its efficacy from minimal sample preparation via dilute and shoot. The platform is based on reversed-phase liquid chromatography coupled to the TSQ Endura triple-quadrupole (QqQ) MS instrument for detection via electrospray ionization multiple-reaction monitoring MS. The quantitative analysis is based on the linear calibration whereby the instrument response for each analyte at a given concentration is normalized against stable isotope-labeled internal standard. In addition, the assay can be multiplexed across more than one LC channel to obtain high-sample throughput.

Wastewater analysis for new psychoactive substances and cocaine and cannabis in a Northern Ireland Prison

The global drug market has been significantly impacted by the emergence of new psychoactive substances, leading to challenges in creating effective legislative controls and their use within recreational drug consumption. This research explores the prevalence of new psychoactive substances and non-medicinal and medicinal compounds within a prison facility in Northern Ireland. Wastewater samples collected from seven different manholes within the prison were analysed for 37 target compounds including the two most found illicit substances: benzoylecgonine (primary metabolite of cocaine) and cannabis. Using solid phase extraction with Oasis HLB and liquid-chromatography-time-of-flight-mass spectrometry across a gradient of 9 min, our analysis revealed that benzoylecgonine was the sole compound consistently present in all collected samples. Following this finding, our target compound selection was broadened to encompass medicinal compounds and employing qualitative analysis we re-evaluated the samples and discovered the presence of buprenorphine, benzodiazepines, methadone, morphine, and codeine. Finally, the study explored the application of enzymatic beta-glucuronidase hydrolysis to the samples. This final phase yielded significant findings, indicating the presence of codeine and nordiazepam at higher peak intensities, thereby shedding light on the potential implications of this enzymatic process.

B-299 Liquid Chromatography-mass Spectrometry Measurement of Drugs of Abuse and Alcohol Biomarker Phosphatidylethanol (peth 16:0/18:1) Using Volumetric Dried Blood Spot Device

Abstract Background Reliable biomarkers have great significance in the investigation of alcohol consumption patterns and in the treatment of alcohol-induced diseases. Direct biomarkers to detect alcohol intake are non-oxidative products of ethanol metabolism (1, 2). Phosphatidylethanol (PEth), blood-based direct alcohol biomarker, represents a group of unnatural phospholipids formed on leukocytes and predominantly erythrocyte membranes. The presence of PEth in blood samples is an indisputable indicator of ethanol consumption (3, 4). Moreover, detecting drugs of abuse (DOA) including opiates, amphetamines, cocaine metabolites, benzodiazepines, THC-metabolites, MDMA, methadone, fentanyl etc. is important to establish patterns of illicit drug usage. In recent years, DBS has been gaining interest in plenty of research areas owing to ease of specimen collection and storage (5, 6).The main objective of this study was to determine the concentration of PEth-16:0 18:1 in DBS samples collected using a volumetric dried blood spot (DBS) device (HemaxisTM DB) and to identify/quantify drugs of abuse (including 119 parameters-Jasem Clinical Toxicology mixture) using another blood spot punch of the same DBS sample. Methods According to Jasem method, DBS samples were prepared by sampling from spiked 20 µL of blood drop located onto paraffin film. PEth-16:0 18:1 and DOA were extracted from entire blood spot of DBS specimens implementing two different extraction reagents at room temperature for 10 min then subjected to HPLC system equipped with Agilent 6470 TQ. The total run times from injection to injection for PEth and DOA were 4.0 min and 12.0 min respectively. Results The linearity and accuracy of the methods were evaluated using 5 DBS calibrators levels for PEth and 6 DBS calibrators levels for DOA. Linearity was confirmed in the range 10 to 1000 ng/mL (r² > 0.995) for PEth 16:0/18:1 and in the range 2 to 100 ng/mL for DOA (r² > 0.99). For two methods linearity, RSD% (inter-intraday) and accuracy were within the analytical acceptable ranges. Conclusion The measurement of blood PEth levels provides a precious tool to determine the chronological profile based on retrospective alcohol intake in a reliable way. Besides, simultaneous analysis of DOA containing 119 parameters ensures comprehensive analytical solution and clinically relevant information. Both of Jasem LC-MS/MS approaches are centred on simple sample preparation following non-invasive sample collection method. Prior to rapid extraction, applying volumetric DBS device assures to collect fixed volume of blood regardless of blood haematocrit level making the measurements more consistent.

A-377 The Selection and Implementation of POC Illicit Drug Toxicology Testing in Hamad Medical Corporation - Doha, Qatar

Abstract Background In 2013, Qatar launched the National Mental Health Strategy which is further supported by National Health Strategy 2018–2022 and Qatar National Vision 2030, where the mental health has been identified as a priority area of development. In doing so, the Ministry of Public Health worked closely with the Ministry of Interior in providing the appropriate treatment rehabilitation. With all considerable advantages of point-of-care testing (POCT) in rapid diagnosis, POC toxicological test has been implemented and used in screening of patients for drugs of abuse. In this study, we highlighted the challenges in selection and implementation process of POC toxicological test in Hamad Medical Corporation (HMC). Methods Due to unavailability of all required drug panels, it had to be customized and tested for the verification purpose before starting the evaluation process. Maintaining HMC procurements regulations and Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologist (CAP) standards for POC toxicology tests, two POC illicit drug toxicology screening tests has been evaluated, comparison study has been performed against the HMC central laboratory. POCT team conducted training session ensuring the appropriate practice in place. The Extendable Laboratory Information System (LIS) functionality has been utilized for seamless data management and reporting for the automated analyzer. Results QUIDEL triage meter pro is an automated drug testing analyzer which detects Amphetamines, Barbiturates, Benzodiazepines, Cannabinoids, Methadone Metabolite, Methamphetamine, Opiates, Tricyclic and Cocaine Metabolites, while the customized manual testing kit FASTEP detects Tramadol (TRA), Methylenedioxymeth-amphetamine (MDMA), Phencyclidine (PCP), Buprenorphine (BUP), Oxycodone (OXY), Fentanyl (FYL), Ethyl Glucuronide (EtG), Cotinine (COT), Pregabalin (PGB) and Gamma Hydroxybutyrate (GHB). Both analytical performances were verified. Rapid bedside testing performed for more than 100 patients in last 8 months during admission, observation, and pre- discharge process. There is An Obvious improvement observed in testing turnaround time compared to the laboratory, easy results interpretation and reporting as well as cost-effective. Conclusion The implementation of POC illicit drug toxicology testing in HMC aligned with core pillars of the Qatar vision 2030 to deliver the right treatment at the right time and place. Its enhancing patient outcomes and overall Satellite health care sector in Qatar.