Evaluation and applicability of Alere iCup DX 14 for rapid postmortem urine drug screening at autopsy

Performing point‐of‐care urine drug screen testing at autopsy by a forensic pathologist may provide an early indication of the presence of analytes of interest during autopsy. An evaluation for the screening of 14 classes of common drugs of abuse in postmortem urine by the point‐of‐care screening device, Alere iCup DX 14, is presented. One hundred ninety postmortem urine samples were screened with the iCup occurring at autopsy by the forensic pathologist. Positive and negative results obtained from the screening kit were evaluated against confirmatory test results obtained using routine forensic toxicology analyses that employed LC‐MS/MS and GC‐MS to detect a combination of over 85 common drugs of abuse and medications. Sensitivity for each respective iCup drug class ranged from 66% (buprenorphine) to 100% (methadone, tricyclic antidepressants). Specificity for each respective iCup drug class ranged from 89% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, 3,4‐methylenedioxymethamphetamine, methadone). Positive predictive values ranged from 44% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, methylenedioxymethamphetamine, methadone), while negative predictive values ranged from 96% (methamphetamine) to 100% (barbiturates, methadone, tricyclic antidepressants). A high false‐positive rate was yielded by the benzodiazepine class. The lack of fentanyl screening in the point‐of‐care device is a significant limitation considering its prolific prevalence in forensic casework. The results obtained in the study should be acknowledged when considering the use of the Alere iCup DX 14 in the context of postmortem casework to help indicate potential drug use contemporaneously with autopsy and when requiring such preliminary results prior to the release of a final forensic toxicology report.     

Evaluation of the Abbott TDx-radiative energy attenuation (REA) ethanol assay in a study of 1105 forensic whole blood specimens

In a preliminary study to determine the applicability of the Abbott radiative energy attenuation (REA) method for the quantification of ethanol in whole blood specimens it was concluded that a larger number of samples was required to evaluate the method, particularly for use in forensic toxicology applications. In this study, 573 blood specimens from suspected driving while intoxicated individuals (DWI blood) and 532 postmortem blood specimens (PM blood) were analyzed by the REA method and a headspace gas chromatographic method (GC) currently used in this laboratory. "Negative" specimens (less than 10 mg/dL by GC) and "positive" specimens (greater than or equal to 10 mg/dL by GC) in each category were analyzed. Linear regression analysis comparing the REA values with the GC values was performed for each type of blood specimen. The equation obtained for DWI blood specimens was REA = 0.943 GC + 1.54; the equation for PM blood specimens was REA = 0.980 GC + 2.76. The correlation coefficient for each group was greater than 0.99. The data suggested that a limit of detection of 10 mg/dL could be applied for DWI blood specimens, while 20 mg/dL would be recommended as the limit of detection for PM blood specimens.     

Review of analytical methods for screening and quantification of fentanyl analogs and novel synthetic opioids in biological specimens

Fentanyl, fentanyl analogs, and other novel synthetic opioids (NSO), including nitazene analogs, prevail in forensic toxicology casework. Analytical methods for identifying these drugs in biological specimens need to be robust, sensitive, and specific. Isomers, new analogs, and slight differences in structural modifications necessitate the use of high-resolution mass spectrometry (HRMS), especially as a non-targeted screening method designed to detect newly emerging drugs. Traditional forensic toxicology workflows, such as immunoassay and gas chromatography mass spectrometry (GC–MS), are generally not sensitive enough for detection of NSOs due to observed low (sub-μg/L) concentrations. For this review, the authors tabulated, reviewed, and summarized analytical methods from 2010–2022 for screening and quantification of fentanyl analogs and other NSOs in biological specimens using a variety of different instruments and sample preparation approaches. Limits of detection or quantification for 105 methods were included and compared to published standards and guidelines for suggested scope and sensitivity in forensic toxicology casework. Methods were summarized by instrument for screening and quantitative methods for fentanyl analogs and for nitazenes and other NSO. Toxicological testing for fentanyl analogs and NSOs is increasingly and most commonly being conducted using a variety of liquid chromatography mass spectrometry (LC–MS)-based techniques. Most of the recent analytical methods reviewed exhibited limits of detection well below 1 μg/L to detect low concentrations of increasingly potent drugs. In addition, it was observed that most newly developed methods are now using smaller sample volumes which is achievable due to the sensitivity increase gained by new technology and new instrumentation.     

Automatic screening in postmortem toxicology

The systematic analysis of postmortem samples is one of the most challenging tasks in forensic toxicology. For determining cause of death, analysis of different tissues can be indispensable. Automation of these analyses would increase reproducibility and therefore lead to more reliable and comparable results. Recent developments in analytical toxicology and the availability of automation devices for various analytical stages, such as sampling, preliminary testing, sample extraction, chromatographic separation, identification, and data processing are examined and discussed. At present only parts of the analytical procedure have been automated-however, the goal should be the integration of these parts into a single, continuous process. Currently, only one "fully-automated" procedure for the comprehensive screening of blood and urine (excluding sample pretreatment, which remains separate) has been published. But it can be expected that automation of analytical procedures in forensic toxicology will indeed progress, even with regard to the very complex screening of postmortem samples.     

Screening for cocaine metabolite fails to detect an intoxication

Testing for the presence of cocaine (COC) is common in postmortem and clinical laboratories. COC use may be detected by screening urine specimens for COC metabolite. In the forensic arena, screening positive results are confirmed by a more specific and sensitive technique, such as gas chromatography-mass spectrometry. This article reports the case of an individual who died of COC intoxication but whose immunoassay screen (EMIT) for COC metabolite was negative. Gas chromatography-mass spectrometry analysis of the urine detected benzoylecgonine (BE) at a concentration of 75 ng/mL and COC at 55 ng/mL. These concentrations explain the negative screening result since the cutoff concentration of the assay was 300 ng/mL for BE. The reported cross reactivity with COC was 25,000 ng/mL. However, heart blood concentrations of COC and BE were 18,330 and 8640 ng/mL, respectively. The results from this case provide evidence that an EMIT test alone may fail to detect COC use. Individuals utilizing results of drug screening by immunoassay must be aware of the limitations of this testing methodology.     

Performance of immunoassays in screening for opiates,cannabinoids and amphetamines in post-mortem blood

Several immunoassay methods for screening of abused drugs in whole blood were evaluated in post-mortem forensic toxicology. Blood samples known to be positive or negative for opiates, cannabinoids or amphetamines by gas chromatography-mass spectrometry (GC-MS) were analysed by EMIT II Plus and EMIT d.a.u., Syva RapidTest and Triage 8 after acetone precipitation. In these experiments, the EMIT immunoassay method was modified by using the Dade Behring VIVA analyser to detect substances more sensitively. Low concentrations of abused drugs were detected in blood samples. The sensitivities of the modified EMIT method for opiates, cannabinoids and amphetamines were 100, 86 and 98%, respectively, whereas the values were below 86% with the other methods. The specificities of all immunoassay methods for opiates and cannabinoids were 83% or above but 51-85% for amphetamines. Sample rejection occurred in a few cases with the EMIT amphetamine assays. The modified EMIT immunoassay system presented here seems to be useful for screening of drugs of abuse in post-mortem blood samples, especially when urine is not available.     

Elevated morphine concentrations determined during infant death investigations: artifacts of withdrawal of care

Abstract:  Three cases are reported of elevated postmortem blood morphine concentrations (189–3036 ng/mL) that were observed during the course of death investigations involving three children ranging in age from 1 week to 2 years, all of whom underwent withdrawal of life support. In all three cases, the presence of opiates in postmortem blood was indicated by immunoassay (ELISA) and quantitative confirmatory analysis of free morphine concentrations in postmortem blood was performed by solid-phase extraction followed by gas chromatography/mass spectrometry (GC/MS) in the selected ion monitoring mode. While the practice of withdrawing life support from terminally ill patients, with the accompanying administration of narcotics/analgesics has been reported in the medical literature, it has not been adequately described in the forensic literature. The implications of this practice on the forensic toxicological interpretation of morphine findings are discussed. To our knowledge, this is the first report of postmortem morphine concentrations arising directly from administration in conjunction with withdrawal of care in pediatric patients.     

Determination of a newly encountered designer drug "p-methoxyethylamphetamine" and its metabolites in human urine and blood

A newly synthesized designer drug, para-methoxyethylamphetamine (PMEA) was unexpectedly detected in the postmortem specimens of fatality involving drug intoxication in 2005, Japan. For unequivocal identification, the isomeric discrimination of PMEA and its positional-isomers was performed by GC/MS with the trifluoroacetylation. In order to prove the intake of PMEA, the characteristic metabolites of PMEA were also identified by GC/MS analysis of the urine specimen with trifluoroacetylation. As a result, para-methoxyamphetamine, para-hydroxyethylamphetamine (POHEA) and para-hydroxyamphetamine were identified as the major metabolites of PMEA. For the quantitative analyses of PMEA and its three metabolites in body fluids, an automated column-switching LC/MS procedure was developed, and applied to the postmortem blood and urine specimens. In this fatal case, blood concentration of PMEA was estimated to be 12.2 microg/mL and this level seemed extremely high in comparison with lethal blood-levels of its analogues, representing acute-intoxication of the victim. Based on the quantitative results, PMEA was found to be extensively metabolized to POHEA via O-demethylation, partly followed by its conjugation.     

Liquid/solid extraction on diatomaceous earth for drug analysis in postmortem blood

Absorption extraction on diatomaceous earth was examined and found to be compatible with typical postmortem blood specimens encountered in forensic toxicology. The effects of solvent, solvent volume, eluate flow rate, and pH on drug recovery and extract quality were investigated. It was concluded that the best method was not necessarily that with the highest recoveries, but that considerations of extract quality were also required. The optimized method was compared with a single-step liquid/liquid extraction method and found to be superior in terms of ease of operation, extract quality, and absolute recovery. The results indicated also that although useful for screening, quantitative methods using liquid/solid extraction may be prone to error.     

Validity testing of commercial urine cocaine metabolite assays: IV. Evaluation of the EMIT d.a.u. cocaine metabolite assay in a quantitative mode for detection of cocaine metabolite

The EMIT d.a.u. cocaine metabolite assay (EMIT dau) was evaluated in a quantitative mode for analysis of clinical specimens obtained after controlled cocaine administration to human subjects. The quantitative results showed high concordance with those of gas chromatography/mass spectrometry (GC/MS) assays of the same specimens for benzoylecgonine, and no false positive or false negative results were obtained. The evaluation also included analysis of standardized solutions containing benzoylecgonine, cocaine, and other cocaine metabolites and isomers. The EMIT dau antibody demonstrated high selectivity for benzoylecgonine. The precision was somewhat less than that reported earlier for other commercial cocaine metabolite immunoassays. Quantitation of initial screening results from EMIT dau testing can serve as a useful guide for confirmation by GC/MS in forensic science urine testing.     

Screening postmortem whole blood for oxycodone by ELISA response ratios

The objective of this study was to investigate the accuracy of screening postmortem whole blood for oxycodone using the ratio of the oxycodone immunoassay response to the response for the specimen obtained with a general opiate-class immunoassay. Fifty eight specimens which were negative for opiates and 158 postmortem whole blood specimens positive for opiates including 66 specimens known to contain oxycodone were assayed. Specimens were diluted 1:5 with assay buffer and analyzed by both the Neogen Oxymorphone/Oxycodone ELISA and the Neogen Opiate Group ELISA (Neogen Corporation, Lexington KY). The oxycodone equivalents in ng/mL from the Oxymorphone/Oxycodone ELISA were divided by the morphine equivalents in ng/mL from the Opiates ELISA to obtain an Oxycodone/Opiates Response Ratio. This ratio was compared with the GC/MS data for all specimens and for opiate positive specimens. Receiver Operating Characteristic (ROC) analysis suggested that optimum relative response ratio was 2.0. The sensitivity of the ELISA response ratio for the presence of oxycodone at a response ratio cutoff of 2.0 was 89.4% +/- 3.8% and the specificity was 88.1% +/- 3.2%. Specimens with a ratio of 2.0 or higher had a greater than 50% probability (positive predictive value) of containing oxycodone in a population with a greater than 15% prevalence of oxycodone.     

Automated headspace solid-phase microextraction and capillary gas chromatography analysis of ethanol in postmortem specimens

Solid-phase microextraction (SPME) is a relatively new solventless sample preparation technique that allows simultaneous sampling, extraction, pre-concentration, and introduction of analytes from a sample matrix in a single procedure. This methodology has been used for the analysis of several drugs of forensic toxicology interest including volatile compounds. This paper describes a methodology for analysis of ethanol and other volatile compounds using automatic headspace solid-phase microextraction (HS-SPME) and capillary gas chromatography in postmortem specimens. The methodology was initially developed using standard solutions of acetaldehyde, acetone, methanol, and ethanol. Isobutanol was used as internal standard. Postmortem samples of blood, urine, and vitreous humor were obtained during medico-legal autopsies. To date, there are no published paper regarding alcohol analysis in vitreous humor specimens using HS-SPME and limited literature analyzing blood and urine samples. HS-SPME analysis showed that, under optimized conditions, ethanol and isobutanol (internal standard) were well-separated from other volatile compounds such as acetaldehyde, acetone, and methanol considered to be potential interferents in ethanol analysis. The calibration curves for each volatile compound demonstrated good linearity throughout the concentration range from 0.001 to 1.0 g/dl and the detection limit of ethanol in the studied specimens was approximately 0.0001 g/dl.     

Correlation of the incidence of cocaine and cocaethylene in hair and postmortem biologic samples

Hair samples are useful as a matrix for drug testing because drugs can be detected in hair for longer periods than in blood or urine. The authors report a prospective comparison of the detection of cocaine and cocaethylene in routine postmortem biologic specimens to the detection of cocaine and cocaethylene in hair. The authors collected hair samples from various areas of the head in 53 autopsy cases, prepared them, and analyzed them by gas chromatography/mass spectrometry (GC/MS) for cocaine and cocaethylene. The authors compared the results of hair analysis with the results of toxicologic analysis performed on routine postmortem samples by enzyme multiplied immunoassay technique and GC/MS. Cocaine was found in either biologic fluids or in hair in 16 of 53 samples tested. Nine samples were positive for cocaine in both biologic fluids and hair. Five samples contained cocaine only in biologic fluids, and two contained cocaine only in hair. Cocaethylene was present in two cases. Drug screening of hair provides additional information in some autopsy cases, but the authors have not made hair analysis a routine practice. It may prove useful to save hair samples in all cases for later analysis if warranted by additional history or autopsy findings.     

The FAA's postmortem forensic toxicology self-evaluated proficiency test program: the first seven years

Existing proficiency-testing (PT) programs do not address the complexity of postmortem forensic toxicology. These programs do not include decomposed samples and solid tissues. Therefore, the Federal Aviation Administration in July 1991 started such a needed PT program. This program is used to: (i) professionally develop and maintain technical currency on a voluntary, interlaboratory, and self-evaluation basis, and (ii) quantifiably assess methods in the absence and presence of interfering substances. There are currently about 30 laboratories in the program. Functioning under various governmental/non-governmental agencies and academic institutions, these laboratories represent a broad cross-section of the country. PT samples are distributed quarterly, and result summaries are sent to the participants, while maintaining their anonymity. Since the inception of the program, 28 PT samples encompassing whole blood, plasma, urine, kidney, or liver, with (or without) drugs, metabolites, and common chemicals (nicotine, caffeine, beta-phenylethylamine, etc.) have been analyzed by the participants. Analytical findings were generally consistent with the anticipated values, but they were dependent on the nature and conditions of the specimens and types of the added analytes. Some incidences of false positives of concern were noted, as well. This PT program is one of the few programs recommended by the American Board of Forensic Toxicology in which laboratories may participate for their accreditation by the Board. It is anticipated that this PT program will continue to play a critical part in supporting the quality assurance/quality control (QA/QC) component of forensic toxicology, thereby enhancing operational performance.     

An unusual fatality in a child due to oxycodone

An unusual fatality secondary to oxycodone in a child is reported. A 2-year-old female child was conveyed to a local hospital after exhibiting signs of rubbing of the mouth and staggering. A hospital toxicological immunoassay screen for drugs of abuse and tricyclic antidepressants was performed on a urine sample and reported as negative. She was discharged and found unresponsive the next morning. She was conveyed to a second hospital in full cardiopulmonary arrest and despite resuscitative efforts, was pronounced dead upon arrival. An autopsy was performed and postmortem specimens were submitted and screened for drugs using mainly chromatographic techniques. Quantitation was achieved by gas chromatography with nitrogen phosphorus detection. Confirmation was performed by gas chromatography/mass spectrometry. Oxycodone was the only drug detected in the following concentrations: heart blood, 1.36 mg/L; gastric contents, 7.33 mg in 33 mL (222.34 mg/L); liver, 0.2 mg/kg; and urine, 47.23 mg/L (47,230 ng/mL). In addition, immunoassay testing of the urine was positive for the opiate class of drugs. This case report demonstrates an unusual cause of death in a young child with emphasis on potential limitation in hospital urine screening tests and the importance of complete forensic toxicological testing in all child deaths.     

Acidic and neutral drugs screen in blood with quantitation using microbore high-performance liquid chromatography–diode array detection and capillary gas chromatography–flame ionization detection

Blood previously acidified with aqueous saturated ammonium chloride solution was extracted with ethyl acetate. The dried extract was subjected to acetonitrile–hexane partition. The acetonitrile portion was analysed for the presence of acidic and neutral drugs by HPLC–DAD (200 mm×2.1 mm I.D. microbore ODS-Hypersil column) and GC–FID (25 m narrow-bore×0.25 mm I.D. HP-5 column with 0.33 μm film thickness). The protocol was found to be suitable for both clinical toxicology (including emergency toxicology) and postmortem toxicology. At least 66 drugs of interest were unequivocally identified by RRTs (HPLC) and UV spectra (DAD) match while another 12 were unequivocally identified by double RRTs match (HPLC and GC). Quantitation was facilitated by incorporating calibration blood standards in each assay batch. The five drugs most commonly encountered in clinical blood specimens (1150 cases) were: paracetamol (47.4% of the cases); chlormezanone (6.6%), theophylline (1.74%), naproxen (1.65%) and mefenamic acid (1.56%). The following drugs were detected in toxicologically significant quantities in postmortem blood specimens (245 cases): phenobarbitone (1.22% of the cases), naproxen (0.82%), chlormezanone (0.82%), theophylline (0.82%), carbamazepine (0.41%) and paracetamol (0.41%).     

The determination of drugs of abuse in whole blood by means of FPIA and EMIT-dau immunoassays--a comparative study

Six groups of common drugs of abuse (cannabinoids, benzoylecgonine, opiates, barbiturates, benzodiazepines and amphetamines) were determined in whole blood after acetone precipitation, using enzyme multiplied immunoassay (EMIT dau) and fluorescence polarisation immunoassay (FPIA--Abbott TDx and ADx) methods. Both methods, designed primarily for urine, allowed the determination of all above mentioned class of drugs but amphetamine. Only 1 ml of a pre- or postmortem blood sample was needed. The sensitivity of cannabinoids determination was higher by FPIA. The FPIA method gave more precise results, particularly in the case of autopsy blood. The method was applied for drug screening in autopsy and police blood samples. The results (both positive and negative) were in agreement with those obtained with chromatographic methods.     

Comparison of results for quantitative determination of morphine by radioimmunoassay, enzyme immunoassay, and spectrofluorometry.

The quantitative results (accuracy and precision) for determination of opiates by radioimmunoassay (RIA), enzyme immunoassay (EMIT), and spectrofluorometry on split samples are compared. A variety of physiological samples were studied, including random urine from a methadone maintenance clinic and postmortem urine, blood, bile, brain, and lung tissue from heroin-induced or heroin-related deaths. The opiate concentrations detected by the two immunoassay methods were in good agreement with each other in the absence of interfering substances which are believed to react with the antimorphine antibodies. The immunoassay results were in agreement within the relative standard deviation with the fluorometry results in 55% of the urine samples and 80% of the blood samples. The immunological methods are superior to fluorometry for quantitation of morphine in urine samples due to quenching interferences in fluorometry from urine. They were comparable to fluorometry for quantitation of morphine in blood samples.     

Morbus Fahr--considerations on a case of sudden death

This paper presents 21 cases related to cyanide intoxication by oral ingestion. Cyanide concentrations in biological specimens are especially different from the type of postmortem specimens, and very important in interpreting the cause of death in postmortem forensic toxicology. Besides the detection of cyanide in autopsy specimens, the autopsy findings were unremarkable. Biological samples (0.2mL or equal to less than 10μg of cyanide) were analyzed colorimetrically for cyanide. In a series of 21 cyanide fatalities, the concentration ranges (mean±SD) of cyanide in heart blood, peripheral blood and gastric contents were 0.1-248.6mg/L (38.1±56.6mg/L), 0.3-212.4mg/L (17.1±45.1mg/L) and 2.0-6398.0mg/kg (859.0±1486.2mg/kg), respectively. The ranges of the heart/peripheral blood concentration ratio and gastric contents/peripheral blood concentration ratio were 0.3-10.6 (mean 3.4) and 3.4-402.4 (mean 86.0), respectively. From the difference of cyanide concentration and the concentration ratio of cyanide in different types of postmortem specimens, the possibility of the postmortem redistribution of cyanide and death by oral ingestion of cyanide could be confirmed. We reported cyanide fatal cases along with a review of literature.