Comparison of Spectrophotometric Methods for the Determination of Carboxyhemoglobin in Postmortem Blood.

Carbon monoxide (CO) poisoning presents an interesting challenge for postmortem toxicology laboratories. The discontinuation of the CO-oximeter manufactured by Instrumentation Laboratories has left many forensic laboratories without this simple but reliable choice for the analysis of CO in blood. A comparable alternative that is quick and simple is analysis using a standard ultraviolet-visible spectrophotometer, which offers sufficient precision and accuracy for the measurement of percentage carboxyhemoglobin (%COHb) in postmortem blood. The hurdle for laboratories then becomes the selection of an appropriate spectrophotometric method since a variety of procedures have been published over the years. Four methods were evaluated based on literature findings and/or the appropriateness for postmortem testing. These methods are based on (i) relating %COHb to the ratio of COHb and Hb absorbance of a two-component system (two wavelengths), (ii) the multicomponent analysis of all hemoglobin species, (iii) the multicomponent analysis of a two-component system, and (iv) derivative spectroscopy. While all four methods performed similarly in terms of typical validation requirements, the ability to more effectively handle decomposed samples and the ease of sample preparation afforded (v) the multicomponent analysis of a two-component system-the most suitable for routine postmortem testing.

Method Validation of Seven Synthetic Cathinones by LC-MS/MS Analysis and the Prevalence of N-Ethylpentylone in Postmortem Casework.

N-ethylpentylone (NEP, ephylone, bk-EBDP) was the most prevalent synthetic cathinone detected by the Miami-Dade Medical Examiner Toxicology Laboratory from 2016-2018. There is limited information regarding the toxicity of NEP, however the few published reports suggest that NEP can cause serious toxic effects and sudden death. The purpose of this publication is to describe a validated LC-MS/MS method for seven synthetic cathinones (methylone, ethylone, butylone, dibutylone, α-PVP, pentylone, and NEP) and to present a detailed summary regarding the presence of NEP in postmortem casework at the Miami-Dade Medical Examiner Department. Post-mortem iliac blood, serum, liver, and brain specimens were prepared by solid-phase extraction with analysis by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). Analyte linearity was established from 0.01 to 0.5 mg/L on a six-point calibration curve. A total of 101 NEP quantitations were performed using this method. Concentrations in postmortem case samples ranged from 0.01 mg/L to 2.7 mg/L. Iliac blood concentrations averaged 0.312 mg/L with a median of 0.137 mg/L (n=72) across all causes and manners of death. Approximately half of the cases were homicides in which the decedent was the victim of gunshot wounds or stabbing. Two of the three highest concentration cases of NEP (2.7 mg/L and 1.7 mg/L) involved 38-year old white males who were tasered by police prior to death. The psychostimulant effect of NEP may result in an excited delirium and/or hallucinogenic state. The concentration of NEP detected in accidental intoxication and polydrug cases overlapped with those attributed to other causes, including homicides and police involved deaths.

Forty-Three Fatalities Involving the Synthetic Cannabinoid, 5-Fluoro-ADB: Forensic Pathology and Toxicology Implications.

Forty-three fatalities involving the potent synthetic cannabinoid, 5-Fluoro-ADB, are summarized. For each case, a description of the terminal event, autopsy findings, cause of death, qualitative identification of 5-Fluoro-ADB and its ester hydrolysis metabolite, 5-Fluoro-ADB metabolite 7, in urine, and the quantitative values obtained in the blood specimens are outlined. Central blood concentrations ranged from 0.010 to 2.2 ng/mL for 5-Fluoro-ADB and 2.0 to 166 ng/mL for 5-Fluoro-ADB metabolite 7. Peripheral blood concentrations ranged from 0.010 to 0.77 ng/mL and 2.0 to 110 ng/mL for 5-Fluoro-ADB and 5-Fluoro-ADB metabolite 7, respectively. The majority of cases resulted in central to peripheral blood concentration ratios greater than 1 for 5-Fluoro-ADB (58%) and 5-Fluoro-ADB metabolite 7 (71%) suggesting that postmortem redistribution occurs to some extent. Combining the increased cardiac weight and/or gastric volume and toxicology data identifying 5-Fluoro-ADB, it is hypothesized that abuse of this substance may precipitate a dysrhythmia and cause sudden death.

Identification and Quantification of 5-Fluoro ADB and the 5-Fluoro ADB Ester Hydrolysis Metabolite in Postmortem Blood Samples by LC-MS/MS.

5-Fluoro ADB, also known as 5-fluoro MDMB-PINACA, is a potent synthetic cannabinoid that is an agonist to the human cannabinoid CB1 and CB2 receptors. Adverse physiological and psychological effects that have resulted in hospitalization and/or death have been associated with 5-Fluoro ADB use. In addition, analytical confirmation of 5-Fluoro ADB use has been reported in both forensic human performance toxicology and postmortem cases. An analytical method for the identification and quantification of 5-fluoro ADB and the 5-fluoro ADB ester hydrolysis metabolite in human blood samples by liquid chromatography-tandem mass spectrometry was created and validated. The linear range of this assay was determined to be 0.01-10 ng/mL for 5-fluoro ADB and 10-500 ng/mL for the 5-fluoro ADB ester hydrolysis metabolite. The method met both precision and accuracy requirements. Endogenous and exogenous interferences were not observed. Ion suppression exceeding 25% was observed for 5-fluoro ADB. However, additional experiments were performed to ensure that the observed suppression did not affect other method validation parameters such as limit of detection and accuracy. Blood samples from 36 postmortem cases were analyzed utilizing this methodology. The average blood concentration of 5-fluoro ADB was 0.29 ng/mL in central blood specimens and 0.05 ng/mL in peripheral blood specimens. The average blood concentration of the 5-fluoro ADB ester hydrolysis metabolite was 49 ng/mL in central blood specimens and 21 ng/mL in peripheral blood specimens. A serum sample was also analyzed and had a serum concentration of 0.12 ng/mL for 5-fluoro ADB and 42 ng/mL for the 5-fluoro ADB ester hydrolysis metabolite. As the concentration of the 5-fluoro ADB ester hydrolysis metabolite was found at a greater concentration than that of 5-fluoro ADB, this metabolite may be a useful marker to monitor in an attempt to confirm 5-fluoro ADB use in toxicological investigations.

4-Fluoromethylphenidate: Fatal Intoxication Involving a Previously Unreported Novel Psychoactive Substance in the USA.

The (±)-threo-4-fluoromethylphenidate (4F-MPH) is a fluorinated analog of the prescription central nervous system stimulant medication, methylphenidate. This novel psychoactive substance was first detected in drug paraphernalia at the Miami-Dade County Medical Examiner Department Toxicology Laboratory in 2016 but was not detected in a biological specimen until 2018. Limited literature is available on 4F-MPH, with predominate literature being published out of Europe, and no known toxicities reported in the USA. Post-mortem specimens were screened using both gas chromatography mass spectrometry and liquid chromatography ion trap mass spectrometry (LC-Ion Trap-MSn). In addition, a validated method for the quantification of 4F-MPH was developed using liquid chromatography-tandem mass spectrometry (LC-MS-MS), with a linear range of 0.01-0.500 mg/L and acceptable validation criteria including precision, bias, carry-over, linearity and endogenous/exogenous interferences. In addition to the detection of 4F-MPH, 3-methoxy-PCP, amphetamine, methamphetamine, 6-monoacetylmorphine, morphine, codeine and tetrahydrocannabinol were also identified in the decedent. A single source of blood was collected (femoral vein) and quantified in all blood tubes used for collection, with concentrations varying from 0.012 to 0.05 mg/L. Additional specimens available for screening included gastric contents and urine. An additional peak having the same targeted ions and transitions as 4F-MPH was identified in both the LC-Ion Trap-MSn screening procedure and the LC-MS-MS quantitative procedure. This peak suggests the presence of a structural isomer, possibly (±)-erythro-4-fluoromethylphenidate, which cannot be confirmed due to there being no available certified reference material. This case report presents the first time that 4F-MPH was detected in a decedent, as well as the first time 4F-MPH has been listed in the official cause of death of a decedent in Florida.

To Test or Not To Test?: The Value of Toxicology in a Delayed Overdose Death.

A case demonstrating the necessity of thorough death investigation processes where toxicology plays an active role is presented. A 33-year-old white man presented to the emergency room in respiratory distress after an overdose episode where he was revived on the scene by fire rescue. His condition continued to deteriorate and he expired 6 days after the initial incident. No admission specimens were available for testing; however, there were specimens drawn 4 and 5 days after the incident. Drug paraphernalia from the scene was obtained by the laboratory through collaboration with local law enforcement. Drug paraphernalia was initially tested in the laboratory and after obtaining the results, the antemortem and postmortem specimens were tested identifying mitragynine and U-47700, among other drugs. These results indicate the value in obtaining and testing drug paraphernalia, and the value of testing antemortem specimens even in the event of a delay.

Quantitative Analysis of Fentanyl and Six Fentanyl Analogs in Postmortem Specimens by UHPLC-MS-MS.

Since 2013, opioid-related deaths have increased in Miami-Dade County, FL due to fentanyl and fentanyl analogs in the local heroin supply. From 2014 to 2015, a near 600% increase in fentanyl and fentanyl analog-related deaths was observed, followed by another 200% increase in 2016. In addition to fentanyl, six fentanyl analogs were identified in this time period: ß-hydroxythiofentanyl, acetyl fentanyl, furanyl fentanyl, carfentanil, butyryl fentanyl and para-fluoroisobutyryl fentanyl. As a result, an analytical method to quantify these compounds in postmortem biological fluids and tissues using solid-phase extraction with analysis by ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS) was developed and validated. All analytes except carfentanil were linear from 1 to 50 ng/mL with a limit of detection (LOD) of 0.5 ng/mL; carfentanil was linear from 0.2 to 10 ng/mL with a LOD of 0.1 ng/mL. For all analytes, bias was within ±14%, intra- and inter-day precision were ≤5% and extraction efficiency was >87%. Dilution integrity was assessed at 1:1, 1:4 and 1:9 (v/v) ratios; percent error from target was <12% for all analytes. No exogenous interferences were observed. Analytes were stable in preserved whole blood stored at 4°C for 9 months; extracted samples were stable in the refrigerated autosampler (15°C) for up to 72 h. The ability to distinguish the isomer pairs isobutyryl/butyryl fentanyl and p-fluoroisobutyryl/p-fluorobutyryl fentanyl is achieved by this method. The method was applied to 312 cases in which fentanyl and/or fentanyl analogs were previously identified. Postmortem concentrations in blood ranged from <0.2 to 0.73 ng/mL for carfentanil and from 30.6 to 91.7 ng/mL for p-fluoroisobutyryl fentanyl in cases in which these analytes were listed as the sole cause of death.

Qualitative Identification of Fentanyl Analogs and Other Opioids in Postmortem Cases by UHPLC-Ion Trap-MSn.

Since 2013, the Miami-Dade County Medical Examiner Department has experienced an increase in the number of opioid-related deaths. The majority of cases coincided with the introduction of fentanyl into the local heroin supply. From 2014 to 2015, Miami-Dade County experienced a near 600% increase in fentanyl-related deaths, followed by an additional 200% increase in 2016. In 2015, two novel fentanyl analogs were identified in medical examiner cases: beta-hydroxythiofentanyl and acetyl fentanyl. In 2016, four additional fentanyl analogs emerged: para-fluoroisobutyryl fentanyl, butyryl fentanyl, furanyl fentanyl and carfentanil, as well as the synthetic opioid U-47700. In order to address this epidemic, a method was developed and validated to identify 44 opioid-related and analgesic compounds in postmortem samples using ultra high performance liquid chromatography ion trap mass spectrometry with MSn capabilities. The limit of detection for all compounds ranged from 0.1 to 5 ng/mL, with a majority having MS3 spectral fragmentation. Blood, urine, liver or brain specimens from ~500 postmortem cases were submitted for analysis based on case history and/or initial screening results. Of those cases, 375 were positive for illicit fentanyl and/or one or more fentanyl analogs. Due to the potency of these compounds, they were almost always included in the cause of death. Worth emphasizing and extremely alarming is the detection of carfentanil in 134 cases, 104 of which were initially missed by gas chromatography mass spectrometry. By incorporating this sensitive, highly specific, and evolving screening procedure into the workflow, the toxicology laboratory continues to effectively assist the medical examiners in determining the cause and manner of death of decedents in Miami-Dade County.

Determination of 32 cathinone derivatives and other designer drugs in serum by comprehensive LC-QQQ-MS/MS analysis.

Recently, clandestine drug lab operators have attempted to bypass controlled substances laws and regulations with "designer" compounds chemically and pharmacologically similar to controlled substances. For example, "bath salts" have erupted onto the scene as "legal highs" containing cathinone analogs that have produced severe side effects in users worldwide. These products have sparked concern among law enforcement agencies, and emergency bans have been placed on the sale of such items. Despite the increasing number of designer drugs available, there are few comprehensive screening techniques for their detection and quantification in biological specimens. The liquid chromatography triple quadrupole tandem mass spectrometry (LC-QQQ-MS/MS) method presented here encompasses over thirty important compounds within the phenethylamine, tryptamine, and piperazine designer drug classes. Analytes were determined by LC-QQQ-MS/MS in the multiple-reaction monitoring mode after mixed-mode solid-phase extraction. The bioanalytical method was fully validated according to recommended international guidelines. The assay was selective for all analytes with acceptable accuracy and precision. Limits of quantification were in the range of 1-10 ng/mL for each compound with limits of detection near 10 pg/mL. In order to evaluate its applicability in a forensic toxicological setting, the validated method was used to analyze post-mortem specimens from two cases that were suspected of containing designer drugs. The method was able to identify and quantify seven of these compounds at concentrations as low as 11 ng/mL. The method should have wide applicability for rapid screening of important new drugs of abuse at high sensitivity in both post- and ante-mortem forensic analysis.

Fatality due to acute alpha-methyltryptamine intoxication.

In February 2003, the Miami-Dade County Medical Examiner Department reported the first known death in the country related to alpha-methyltryptamine (AMT). AMT is an indole analogue of amphetamine investigated in the 1960s as an antidepressant, stimulant, and monoamine oxidase inhibitor. Today, AMT is recognized as a powerful psychedelic drug among high school and college-aged men and women. Its popularity is partly due to the multitude of anecdotal websites discussing AMT as well as its legality and availability for purchase via the Internet prior to April 2003. Emergency designation of AMT as a Schedule 1 controlled substance by the Drug Enforcement Administration occurred shortly after the death in Miami-Dade County. The case in Miami involved a young college student who, prior to death, advised his roommate that he was "taking hallucinating drugs" and as a result had "discovered the secret of the universe". Approximately 12 h later, the roommate discovered the deceased lying in bed unresponsive. An empty 1-g vial of AMT was recovered from the scene and sent to the toxicology laboratory. Initial screening of urine by enzyme-multiplied immunoassay technique was positive for amphetamines, and the basic drug blood screen detected a small peak later identified by mass spectrometry as AMT. For quantitation, AMT was isolated using solid-phase extraction, derivatized with pentafluoropropionic anhydride, and analyzed using gas chromatography-mass spectrometry. Quantitative analysis was based upon m/z 276, 303, and 466 for AMT and m/z 306, 333, and 496 for the internal standard, 5-methoxy-alpha-methyltryptamine. A linear calibration curve from 50 to 500 ng/mL was used to calculate the concentration of AMT in the samples and controls. Blood, tissue, and gastric specimens were diluted to bring the observed concentration within the limits of the standard curve. Matrix matched controls were extracted and analyzed with each run. Postmortem iliac vein blood revealed 2.0 mg/L, gastric contents (48 g collected at autopsy) contained 9.6 mg total of AMT, liver contained 24.7 mg/kg, and the brain contained 7.8 mg/kg. An additional Medical Examiner case from another jurisdiction revealed 1.5 mg/L in antemortem serum.

Fatal cold medication intoxication in an infant.

The case history and toxicological findings of an infant fatality involving pseudoephedrine, brompheniramine, and dextromethorphan are presented. Concentrations of brompheniramine and dextromethorphan were measured in both postmortem blood and liver specimens using a gas chromatograph equipped with a nitrogen-phosphorus detector. Brompheniramine and dextromethorphan were 0.40 mg/L and 0.50 mg/L, respectively, in the blood sample and 0.16 mg/kg and 0.57 mg/kg in the liver sample. The concentration of pseudoephedrine in blood and liver specimens was measured using gas chromatography-mass spectrometry and was determined to be 14.4 mg/L in the blood and 16 mg/kg in the liver. Additionally, a baby bottle allegedly administered to the infant was collected as evidence and sent to the Medical Examiner's Office for evaluation. The amounts of total brompheniramine, dextromethorphan, and pseudoephedrine remaining in the baby bottle were 1.4 mg, 9.4 mg, and 40 mg, respectively.