Relationship between opiates and asthma in the determination of death.

Several studies have shown an association between asthma and opiate abuse. This retrospective study aims to analyse the demographic, toxicological, and seasonal differences in asthmatic and non-asthmatic subjects who died of opiates. In addition, the relationship between toxicological levels of opiates and histologic grade of lung inflammation is examined. Deaths from 2013 to 2018 involving opiates as the primary cause of death in Cook County, Illinois (USA) were reviewed. Twenty-six cases of opiate deaths of individuals with a history of asthma and lung histology slides available were identified. In comparison, 40 cases of deaths due to opiates only were analysed. A check-list system for the evaluation of the grade of microscopic inflammation in asthma was developed. We found statistically significant differences between the asthmatics and the non-asthmatics regarding demography (age and race) and toxicology (6-MAM presence). In particular, the "opiate and asthma group" was mainly composed of African-American subjects, in contrast with the "opiate group", consisting mostly of Caucasian. The mean age was significantly higher in the "opiate and asthma group" compared with the "opiate group". A greater presence of 6-MAM was detected in the "opiate group" compared with the "opiate and asthma group". While we expected to find that low opiate levels would lead to deaths in asthmatics and, in particular, that lower opiate concentrations would cause deaths in subjects with higher grades of histologic inflammation, our study suggests that the quantity of drug and the level of inflammation are not statistically significant in the determination of death. We, therefore, recommend histologic examination of the lungs to evaluate for asthma, particularly in suspected low-level opiate-related deaths, to help further clarify any relationship between asthma and opiate use.

Quantification of methadone, buprenorphine, naloxone, opioids, and their derivates in whole blood by liquid chromatography-high-resolution mass spectrometry: Analysis of their involvement in fatal forensic cases.

Opioids represent a broad family of compounds that can be used in several indications: analgesics, antitussives, opioid substitution therapy (e.g. methadone, buprenorphine…). When these products are misused, they are often addictive. Thus, we aimed to develop an analytical method able to rapidly quantify several opiates and opioids (6-monoacetylmorphine, buprenorphine, codeine, dihydrocodeine, 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine, ethylmorphine, heroin, methadone, morphine, nalbuphine, naloxone, norbuprenorphine, norcodeine, norpropoxyphene, oxycodone and propoxyphene) in whole blood by ultra-high performance liquid chromatography combined with high resolution mass spectrometry (UHPLC-HRMS). The validated assay requires only 100 µL of the blood sample. The sample is prepared by a rapid liquid-liquid extraction using 5% zinc sulfate (W/V), methanol and acetonitrile. Calibration curves range from 0.98 to 1000 µg/L, except for buprenorphine (0.39-100 µg/L) and norbuprenorphine (0.20-100 µg/L). Inter- and intra-analytical accuracy was less than 15%. Therefore, we describe the development and full validation of an accurate, sensitive and precise assay using UHPLC-HRMS for the analysis of opioids in whole blood. After validation, this new assay is successfully applied on a routine laboratory application basis.

Optimization of cloned enzyme donor immunoassay cut-offs for drugs of abuse in post-mortem whole blood.

INTRODUCTION: Immunoassay (IA) tests are not widely applied in post-mortem samples, since they are based on technologies requiring relatively non-viscous specimens, and compounds originating from the degradation of proteins and lipids during the post-mortem interval can alter the efficiency of the test. However, since the extraction techniques for IA tests are normally rapid and low-cost, IA could be used as near-body drug-screening for the classes of drugs most commonly found in Italy and Europe. In this study, semi-quantitative results on post-mortem whole blood samples obtained through CEDIA analysis (cannabinoids, cocaine, amphetamine compounds, opiates and methadone), were compared with results of confirmatory analysis obtained using GC-MS. Screening cut-offs for all drugs were retrospectively optimized. METHODS: Post-mortem whole blood samples from autopsy cases of suspected fatal intoxication were collected over 3 years. Samples were initially analyzed through CEDIA (CEDIA, ILab 650, Werfen). Confirmatory analyses were then performed by GC-MS (QP 2010 Plus, Shimadzu). Screening cut-offs were retrospectively optimized using Receiver Operating Characteristic (ROC) analysis. RESULTS: CEDIA results were available for 125 samples. Two-hundred-eighty-nine (289) positive screening results were found. Among these, 162 positive confirmation results were obtained. Optimized screening cut-offs were as follows: 6.5ng/ml for THC; 4.2ng/ml for THC-COOH; 12.0ng/ml for cocaine; 6.6ng/ml for benzoylecgonine; 6.4ng/ml for opiates; 2.0ng/ml for methadone. Analysis of ROC-curves showed a satisfying degree of separation in all tests except for amphetamine compounds, with areas under the curve (AUC) between 0.915 (THC) and 0.999 (for benzoylecgonine and methadone). DISCUSSION: The results of the study showed that CEDIA screening at the optimized cut-offs exhibits a very high sensitivity and good specificity and positive predictive value (PPV) for cannabinoids, cocaine and metabolites, opiates and methadone. A high number of false positives (n=19) for amphetamine compounds was observed at the optimized cut-off, resulting in a very low PPV, which is also influenced by the very low number of TP (n=4). CONCLUSION: The results of the study show that the CEDIA is a valuable screening test on post-mortem whole blood for cannabinoids, cocaine and metabolites, opiates and methadone, but it is not recommended for amphetamine compounds, due to the high number of false positives. The strengths of the study are the large sample size, the inclusion of post-mortem cases only and the high level of sensitivity and specificity obtained at the optimized cut-offs.

Determination of opiates in whole blood using microextraction by packed sorbent and gas chromatography-tandem mass spectrometry.

In Portugal, and worldwide, the abuse of psychoactive substances is increasing, with a significant incidence of deaths related to their consumption. Opiates are one of the most prevalent group of substances in that context, and they are responsible for a significant impact of the mentioned harms. Therefore, it becomes necessary to equip labs with faster and effective methods to identify and quantify these substances. This work describes the development and validation of a novel analytical method for the simultaneous determination of morphine, codeine and 6-monoacetylmorphine in blood samples by gas chromatography-tandem mass spectrometry (GC-MS/MS), using microextraction by packed sorbent (MEPS) for sample preparation. Before the MEPS procedure, a precipitation step with acetonitrile was performed. The MEPS parameters were optimized using the fractional factorial planning (2k-1) and surface response methodology. The final optimized conditions were: number of strokes (20), amount of formic acid in the washing solution (3.36%), number of washes of the sorbent (1), amount of ammonium hydroxide in the elution solution (2.36%) and number of elution cycles (11). After the extraction procedure, the analytes were derivatized with MSTFA with 5% TMS. Using a sample volume of 250 µL, the method was validated according to internationally accepted standards. The method proved to be linear in the range of 5-1000 ng/mL with coefficients of determination greater than 0.99 for all analytes. Intra-and inter-day precision and accuracy were in accordance with the above-mentioned criteria, presenting coefficients of variation ≤15% and relative errors within a range of ± 15% of the theoretical concentration. The absolute recoveries ranged from 6 to 23%. The validated method was applied to the analysis of real samples, being an advantageous tool for the detection of those substances in blood. This is the first time that GCMS/MS with MEPS was used for the determination of these compounds in biological fluids.

A Gas Chromatography-Triple Quadrupole Mass Spectrometry Assay for the Quantification of Opiates in Human Blood Samples.

A simplified protein precipitation method in combination with gas chromatography-triple quadrupole mass spectrometry (GC-MS-MS) analysis was developed and validated for the simultaneous determination of codeine, morphine, 6-acetylmorphine (6-MAM), hydrocodone and hydromorphone in human blood samples. A protein precipitation with 10% trichloroacetic acid followed by solid-phase extraction using a mixed-mode cartridge was used to separate the analyte from the blood samples. A BSTFA + 1% TMCS was used for derivatization of opiates prior to the analysis. Codeine-D3, morphine-D3, 6-acetylmorphine-D3 and hydrocodone-D6 were used as internal standards. The GC-MS-MS was operated under multiple-reaction monitoring mode using electron ionization technique. The transition ions used for quantitation were 371 → 234 for codeine, 429 → 146 for morphine, 399 → 287 for 6-MAM, 299 → 228 for hydrocodone and 357 → 314 for hydromorphone. The method was linear over the concentration range 2.5-1000 ng/mL for all analytes, except hydrocodone which was linear over 5-1000 ng/mL with a correlation coefficient (r2) = 0.99. The limit of detection was 1.0 ng/mL for all compounds except hydrocodone which was 2.5 ng/mL. The limit of quantitation was 2.5 ng/mL for all compounds except hydrocodone which was 5.0 ng/mL. The precision (% RSD) was within 1.26-14.81 and the accuracy (% Bias) was within -6.29-10.93% for all compounds. The method successfully analyzed morphine (305 ng/mL) and 6-acetylmorphine (6-MAM) (2.3 ng/mL) in a human blood sample received from an opiate user.

Determination of 12 commonly found compounds in DUID cases in whole blood using fully automated supported liquid extraction and UHPLC-MS/MS.

A high-throughput UHPLC-MS/MS method for the most frequently found compounds; tetrahydrocannabinol (THC), amphetamine, methamphetamine, MDMA, clonazepam, diazepam, nordiazepam, oxazepam, alprazolam, nitrazepam, morphine, and codeine, in driving under the influence of drugs (DUID) cases in whole blood, is presented. Automated sample preparation by 96-well supported liquid extraction (SLE) plates with ethyl acetate + heptane (80 + 20, v/v) as organic solvent was carried out on a Freedom Evo 200 platform from Tecan. An aliquot of 100 µL whole blood was used. Sample preparation time for 96 samples was 1.5 h. Compounds were separated with gradient elution on a C18 column (50 × 2.1 mm, 1.7 µm) with a mobile phase consisting of 5 mM pH 10.2 ammonium formate and methanol. The run time was 4.5 min and 1 µL was injected on an Acquity UPLC I-Class system with a Xevo TQS tandem-quadrupole mass spectrometer in multiple-reaction monitoring mode (MRM) from Waters. Isotope labelled, 13C, internal standards (ISs) were used for all compounds except for alprazolam and morphine, which had deuterated analogs. Quantification was carried out with calibrators without whole blood matrix. Full validation was carried out according to international guidelines, and a new approach for evaluation of process efficiency (PE) has been presented. Linear or quadratic weighted (1/x) calibration curves were used with R2 ≥ 0.999. The method showed satisfactory deviations ±16% when compared to the existing methods, and satisfactory agreement with proficiency testing control samples (z-score -1.6 to 1.8, n = 16 samples). The precision, estimated as the relative standard deviation (RSD) of the concentration difference between results from two independent analyses of authentic whole blood samples, was ≤7.2% in antemortem and ≤9.3% in postmortem samples. Recovery was ≥85% for all the compounds, except morphine ≥62% and THC ≥ 50%. PE was satisfactory for all the compounds with low variation in IS response, RSD ≤ 16% (THC 27%) in antemortem samples and ≤34% (THC 66%) in postmortem samples. To the best of our knowledge, this is the first automated 96-well SLE UHPLC-MS/MS method developed for the simultaneous determination of these 12 compounds in whole blood covering the concentration ranges found in forensic samples. The method has been used in routine work during the last ten months, analysing about 9900 antemortem and 1000 postmortem whole blood samples, and has proven to be robust and reliable.

Activity-Based Concept to Screen Biological Matrices for Opiates and (Synthetic) Opioids.

BACKGROUND: Detection of new highly potent synthetic opioids is challenging as new compounds enter the market. Here we present a novel screening method for the detection of opiates and (synthetic) opioids based on their activity. METHODS: A cell-based system was set up in which activation of the µ-opioid receptor (MOR) led to recruitment of ß-arrestin 2, resulting in functional complementation of a split NanoLuc luciferase and allowing readout via bioluminescence. Assay performance was evaluated on 107 postmortem blood samples. Blood (500 µL) was extracted via solid-phase extraction. Following evaporation and reconstitution in 100 µL of Opti-MEM® I, 20 µL was analyzed in the bioassay. RESULTS: In 8 samples containing synthetic opioids, in which no positive signal was obtained in the bioassay, quadrupole time-of-flight mass spectrometry revealed the MOR antagonist naloxone, which can prevent receptor activation. Hence, further evaluation did not include these samples. For U-47700 (74.5-547 ng/mL) and furanyl fentanyl (<1-38.8 ng/mL), detection was 100% (8/8) for U-47700 and 95% (21/22) for furanyl fentanyl. An analytical specificity of 93% (55/59) was obtained for the opioid negatives. From an additional 10 samples found to contain other opioids, 5 were correctly scored positive. Nondetection in 5 cases could be explained by very low concentrations (<1 ng/mL alfentanil/sufentanil) or presence of inactive enantiomers. CONCLUSIONS: The MOR reporter assay allows rapid identification of opioid activity in blood. Although the cooccurrence of opioid antagonists is currently a limitation, the bioassay's high detection capability, specificity, and untargeted nature may render it a useful first-line screening tool to investigate potential opioid intoxications.

Comparison of Biological Screening and Diagnostic Indicators to Detect In Utero Opiate and Cocaine Exposure Among Mother-Infant Dyads.

BACKGROUND: Opioid and cocaine antenatal substance use can result in significant obstetric and pediatric health implications. Accurate detection of in utero-exposed neonates can improve patient care and health outcomes. Therefore, the effectiveness of mother-infant biological and diagnostic indicators collected at labor and delivery to provide accurate detection of in utero opiate and cocaine exposure was assessed. METHODS: A retrospective medical chart review included 335 mother-infant dyads exposed to antenatal substances who were delivered between January 2009 and March 2014. Mother-infant dyads were a subset of a larger retrospective cohort of 560 substance-using mothers, who had a valid meconium drug screen (MDS) and anesthesia before delivery. Alternative biological and diagnostic indicators of maternal urine drug screens (UDS), maternal substance use International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM) codes, and neonatal exposure diagnostic ICD-9-CM codes were compared against MDS. Data were analyzed using classification accuracy measures. RESULTS: Compared with MDS, maternal UDS had the highest sensitivity [0.52, 95% confidence interval (CI), 0.39-0.65] and specificity (0.88, 95% CI, 0.79-0.97) to detect intrauterine opiate exposure. Maternal substance use diagnosis had the highest sensitivity (0.39, 95% CI, 0.16-0.61) and maternal UDS had the highest specificity (1.00, 95% CI, 0.99-1.00) to detect intrauterine cocaine exposure. Cocaine exposure had significantly higher accuracy scores across detection methods compared with opiate exposure. CONCLUSIONS: Alternative indicators collected at delivery were ineffective at identifying in utero substance exposure, especially neonatal-exposed ICD-9-CM codes. Low sensitivity scores indicate that many exposed neonates could be misdiagnosed or left untreated. Accurate antenatal exposure identification at delivery is an important form of tertiary assessment that warrants the development of improved screening methodology and standardization of hospital biological drug testing.

Presence of Alcohol, Cocaine, and Other Drugs in Suicide and Motor Vehicle Crash Decedents Ages 18 to 54.

BACKGROUND: Use of alcohol and select other drugs confer risk for injury deaths, yet how such use compares in different types of injury deaths including suicide and fatal motor vehicle collisions (MVCs) is unclear. METHODS: Individuals in New Mexico ages 18 to 54 that died in 2012 by suicide or MVC were analyzed. Toxicology results were used to code the presence of alcohol and the presence of 1 or more drugs including cocaine, opiate (oxycodone, heroin, etc.), or amphetamine or methamphetamine, yielding a 4-category variable: Alcohol + Drug, Alcohol (without drug), Drug (without alcohol), and Neither (ref). Suicides were compared to MVCs (ref) using unconditional logistic regression analyses adjusted for sex, age, and ethnicity. Poisoning suicides were removed prior to analyses to exclude cases where the drugs may have been used to hasten death. RESULTS: Analyses were based on 185 suicides and 161 MVCs. Alcohol + Drug was more likely in suicide decedents, AOR (95% CI) = 4.33 (1.70, 11.03). Alcohol (without drug) and Drug (without alcohol) did not differ between the groups. Uniquely, all suicides that were positive for cocaine were also positive for alcohol. As follow-up, similar results were obtained in a post hoc analysis that limited the drug exposure variable to cocaine: Alcohol + Cocaine, AOR (95% CI) = 4.69 (1.59, 13.88). CONCLUSIONS: The co-presence of alcohol and 1 or more drugs of abuse, particularly cocaine, may be more likely in suicide deaths compared to MVCs. Results may inform prevention efforts targeting specific substances and types of injury.

Rapid screening for drugs of abuse in biological fluids by ultra high performance liquid chromatography/Orbitrap mass spectrometry.

We present a UPLC(®)-High Resolution Mass Spectrometric method to simultaneously screen for nineteen benzodiazepines, twelve opiates, cocaine and three metabolites, and three "Z-drug" hypnotic sedatives in both blood and urine specimens. Sample processing consists of a high-speed, high-temperature enzymatic hydrolysis for urine samples followed by a rapid supported liquid extraction (SLE). The combination of ultra-high resolution chromatography with high resolution mass spectrometry allows all 38 analytes to be uniquely detected with a ten minute analytical run. Limits of detection for all target analytes are 3ng/mL or better, with only 0.3mL of specimen used for analysis. The combination of low sample volume with fast processing and analysis makes this method a suitable replacement for immunoassay screening of the targeted drug classes, while providing far superior specificity and better limits of detection than can routinely be obtained by immunoassay.

[Analytical validation of a chromatographic method dedicated to search and identify natural and semi-synthetic opiates]. / Validation analytique d'une méthode chromatographique destinée à rechercher et à identifier les opiacés naturels ou (semi) synthétiques.

The identification of a product absorbed by an opiate consumer is sometimes problematic since there is no specific biomarker for all molecules. We developed an ultra-high pressure liquid chromatography coupled to tandem mass spectrometry technique which allows the identification and the quantification of 25 opiates in plasma. The sample preparation consists in a solid-phase extraction on Oasis MCX cartridges (Waters). The method has been validated according to FDA criteria completely for 21 substances and with some reservations for the remaining 4 analytes. This method has been applied to 80 patients treated at the University Hospital of Liege for whom the screening of opiates was positive. The identification of the product consumed was effective in 86% of cases.

Dispersive liquid-liquid microextraction followed by high-performance liquid chromatography-ultraviolet detection to determination of opium alkaloids in human plasma.

A novel, simple, rapid and sensitive dispersive liquid-liquid microextraction method based on the solidification of floating organic drop (DLLME-SFO) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was used to determine opium alkaloids in human plasma. During the extraction procedure, plasma protein was precipitated by using a mixture of zinc sulfate solution and acetonitrile. Some effective parameters on extraction were studied and optimized. Under the optimum conditions (extraction solvent: 30.0 µl 1-undecanol; disperser solvent: 470 µl acetone; pH: 9; salt addition: 1%(w/v) NaCl and extraction time: 0.5 min), calibration curves are linear in the range of 1.5-1000 µgl(-1) and limit of detections (LODs) are in the range of 0.5-5 µgl(-1). The relative standard deviations (RSDs) for 100 µgl(-1) of morphine and codeine, 10.0 µgl(-1) of papaverine and 20.0 µgl(-1) of noscapine in diluted human plasma are in the range of 4.3-7.4% (n=5). Finally, the method was successfully applied in the determination of opium alkaloids in the actual human plasma samples. The relative recoveries of plasma samples spiked with alkaloids are 88-110.5%. The obtained results show that DLLME-SFO combined with HPLC-UV is a fast and simple method for the determination of opium alkaloids in human plasma.

[Occurrence of psychoactive substance in driver's blood samples in the materials of Poznan University of Medical Sciences, Department of Forensic Medicine]. / Obecnosc srodków psychoaktywnych we krwi uczestników ruchu drogowego na podstawie materialów katedry i Zakladu Medycyny Sadowej Uniwersytetu Medycznego w Poznaniu.

Recently it has been observed increasing number of drugged drivers in Wielkopolska region. In the period from 2006 to 2009 a total number of 2473 blood samples collected from drivers (2141 from alive suspects and 332 from deceased) and examined for psychoactive agents in the Department of Forensic Medicine Poznan University of Medical Sciences. Positive results were obtained in 68.8% and 22.3% of blood samples taken from living men and women respectively. In the group of deceased 7.3% (male) and 8.4% (female) were found positive. The most frequently detected substance were cannabinoids - 57.8%, then amphetamines and its analogues 18.8%, benzodiazepines 5.8%, opiates 3.4% and cocaine benzoiloecgonine 0.9%. In 13.0% concomitant use of amphetamine and cannabinoids were reported.

The detection of various opiates and benzodiazepines by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry.

A technique using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC x GC/TOFMS) is applied to qualitative and quantitative drug testing. Human serum was 'spiked' with known quantities of benzodiazepines and a 'street heroin' mixture including some of the major metabolites and impurities. The sample components were extracted from the matrix by solid-phase extraction (SPE). Constituents containing polar hydroxyl and/or secondary amine groups were derivatised with N-methyl-N-(tert-butyldimethyl)trifluoroacetamide (MTBSTFA) to improve the chromatographic performance. An orthogonal separation of the matrix constituents was achieved by coupling a DB-5ms (5% phenyl) to a BPX50 (50% phenyl) GC column. The eluant was focused onto the second column by a twin-stage cryo-modulator. Rapid 6 s modulation times were achieved by transfer from a 30 m x 0.25 mm (length x internal diameter) to a 2 m x 0.1 mm column. TOFMS with rapid spectral acquisition (< or =500 spectra/s) was employed in the mass range m/z 40-650. A clean mass spectrum was obtained for each analyte using mass spectral deconvolution software. The sensitivity and repeatability of the method were evaluated by the preparation of calibration standards for two benzodiazepines, flunitrazepam and its major metabolite 7-aminoflunitrazepam (7-amino-FN), in the concentration range 5-1000 ng/mL. The limits of detection (LODs) and limits of quantitation (LOQs), calculated by repeat injections (x10) of the lowest standard, were 1.6 and 5.4 ng/mL (flunitrazepam); 2.5 and 8.5 ng/mL (7-amino-FN), respectively. There is scope to extend this protocol to screen a large number of drugs and metabolites stored in a library database.

Method for quantification of opioids and their metabolites in autopsy blood by liquid chromatography-tandem mass spectrometry.

A method using liquid chromatography-electrospray ionization-tandem mass spectrometry was developed and validated for the determination of morphine, codeine, hydromorphone, dihydrocodeine, oxycodone, buprenorphine, and naloxone with their metabolites morphine-3-glucuronide, morphine-6-glucuronide, normorphine, 6-acetylmorphine, 6-acetylcodeine, codeine-6-glucuronide, norcodeine, hydromorphine-3-glucuronide, dihydrocodeine-6-glucuronide, dihydromorphine, dihydromorphine-3-glucuronide, dihydromorphine-6-glucuronide, oxymorphone, norbuprenorphine, buprenorphine-3-glucuronide, norbuprenorphine-3-glucuronide, and naloxone-3-glucuronide in human whole blood. Polar metabolites (glucuronides) and other analytes were extracted by SPE using Bond Elut C18. Chromatographic separation was performed on a Phenomenex Synergi reversed-phase column with gradient elution based on a mobile phase consisting of 10mM ammonium formate adjusted to pH 3 and acetonitrile. Intraday and interday precision for all analytes were between 0.6% and 13.8%, and recoveries were between 80.3% and 101.4%. Calibration curves were linear for all analytes over the concentration range 5-400 ng/mL, and correlation coefficients (R(2)) were better than 0.999. Limits of detection and quantitation were 0.16-1.2 ng/mL and 0.5-4.09 ng/mL, respectively. The method described consolidates previous work on opioids and their metabolites published in the literature and is the first to include the detection of naloxone-3-glucuronide. The method has been applied in routine postmortem cases after opiate overdose with the threefold purpose of providing interpretive information on the cause and type of death (rapid, sub-acute, or delayed death) and to distinguish heroin, morphine, and codeine users.