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.

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.

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.