Validation And Application Of A Liquid Chromatography Tandem Mass Spectrometry Method For Mitragynine In Postmortem Blood And Liver

Kratom is a tree originating in Southeast Asia, and has a history of usage among farmers. The leaves of the tree were chewed and used by farmers as a stimulant in order to work long days. Over the years, the popularity of the drug spread worldwide and has risen in recent years. In 2016, kratom was put on a Drugs of Concern list by the United States Drug Enforcement Administration (DEA). A lack of research including studies for mitragynine and 7-hydroxymitragynine has been observed, and there are even fewer postmortem case studies in the literature. The rise in kratom usage was noticed in the casework of the Office of the Chief Medical Examiner (OCME) Toxicology Laboratory in Oklahoma. The number of cases involving mitragynine, an alkaloid in kratom, has risen over recent years, but the OCME did not have a validated method for quantitation. The importance of this thesis project was to validate a method for the quantitation of mitragynine and qualitative identification of 7-hydroxymitragynine, assess the stability of the two alkaloids in three different storage conditions (ambient, refrigerated, and frozen), and examine real world cases coming through the OCME. The extraction utilized in this method was a phosphate buffer followed by filtering the samples using 3 mL columns containing diatomaceous earth. Extracts were then concentrated by drying them down, reconstituted, and washed with hexanes. The samples were then analyzed using liquid chromatography paired with tandem mass spectrometry (LC/MS-MS). The method for quantitation of mitragynine was validated with the criteria of interferences, bias, precision, ion suppression/enhancement, limit of detection, carryover, calibration model, and stability taken into consideration. The validation criteria for 7-hydroxymitragynine were interferences, ion suppression/enhancement, limit of detection, carryover, and stability. All criteria were met and deemed acceptable by the OCME lab’s Quality Assurance Program. The stability study demonstrated that mitragynine is more stable in refrigerator conditions as opposed to freezer, and that 7-hydroxymitragynine went through an approximate 20% decrease over 65 days in the refrigerator in blood. The case studies had ranges of 130-1,400 ng/mL for heart blood, 130-1,300 ng/mL for femoral blood, and 220-7,000 ng/mL for liver. Central/Peripheral (C/P) and Liver/Peripheral (L/P) ratios were calculated to identify the potential for mitragynine to undergo postmortem redistribution (PMR). The C/P and L/P ratios were an average of 1.0 and 2.7 respectively, suggesting that mitragynine does not experience significant PMR. This thesis project will be adding to the body of knowledge concerning mitragynine and 7-hydroxymitragynine which will help researchers and postmortem labs assess the storage conditions of their specimen and help with interpretation of toxicology results in death investigations.

Opioids such as morphine, codeine, and oxycodone are extracts or analogs isolated from a single source: the opium poppy. For a long time, it was believed to be nature's only source of opioids. But it now appears that biological diversity has evolved an alternative source of opioid compounds-those derived from the plant Mitragyna speciosa. This plan

Over the last decade, and particularly during the recent five years, a rapidly increasing number of novel psychoactive substance (NPSs), often marketed as “designer drugs”, “legal highs”, “herbal highs”, “research or intermediate chemicals” and “laboratory reagents”, has appeared on the drug market in an effort to bypass controlled substance legislation. NPSs encompass a wide range of different compounds and drug classes but had been dominated by synthetic cannabinomimetics and psychostimulatory synthetic cathinones, so-called β-keto amphetamines. Compounds from the later class were first detected in Europe in 2004, and since then 103 new cathinones have been identified and reported to the European Monitoring Centre for Drugs and Drug Addiction, with 57 during the last two years. Synthetic cathinones – novel addictive and stimulatory psychoactive substances is the first publication of this kind that provides readers with background on chemical structures, detection, prevalence and motivation of use of the very popular group of NPSs. This book also presents comprehensive overview of the mechanisms of action, pharmacological activity, and main metabolic pathways of synthetic cathinones, followed by a detailed discussion of the acute and chronic toxicity associated with the use of these substances. Written by international experts in the field, this multi-authored book is a valuable reference not only for scientists, clinicians and academics, but also for readers representing different professional background who are involved in educational-prophylactic activities directed to harm reduction of psychoactive compounds.

Analytical toxicologists are involved in the analysis of drugs and poisons in biological samples in different environments. Many scientists in the field of analytical toxicology have adopted LC-MS in their daily work, and this is illustrated by the increasing numbers of research papers published and presented at relevant conferences.

New Psychoactive Substances (NPS) is an umbrella term covering hundreds of substances across different drug groups. Many of these substances were originally developed for therapeutic use but have later appeared on the recreational drug market. The use of NPS has been associated with many outbreaks leading to hospitalizations and has been implicated in numerous fatalities worldwide. To be able to analytically detect drugs in a forensic setting is vital in the fight against the abuse of NPS. One of the most notable challenges in detection of NPS is the identification of major urinary metabolites for use as biomarkers. Furthermore, given the lack of reference standards in most metabolism studies, the major urinary metabolites can often only be tentatively determined. This thesis describes the synthesis and analysis of potential metabolites used to identify the exact structures of major metabolites of the synthetic cannabinoid AKB-48, fentanyl and five fentanyl analogues in authentic human urine samples and/or hepatocyte incubations. Synthetic targets were chosen based on previous metabolism studies by our research group. Subsequently, synthetic routes were developed to produce numerous potential metabolites across the studied NPS. The synthesized reference standards were analyzed by LC-QTOF-MS alongside hepatocyte drug incubations and authentic human urine samples. Comparison of the resulting analytical data was used to determine the exact structures of many metabolites. This includes urinary metabolites of AKB-48 with a single hydroxyl group situated on a secondary carbon of the adamantane moiety, or position 3 or 5 of the pentyl side chain. For the studied fentanyls, the ?-OH and the 4’-OH metabolites were abundant metabolites identified in hepatocyte incubations while the 4’-OH, 4’-OH-3’-OMe and 3’,4’-diOH were the favored metabolic motifs among the metabolites identified in urine. Additionally, a concise synthetic route to produce synthetic cannabinoid metabolites with the 4-OH-5F pentyl side chain motif was developed and demonstrated for four synthetic cannabinoids. These findings and the developed synthetic routes can be used to provide forensic toxicology laboratories with urinary biomarkers for drug detection. Moreover, the synthesized reference standards of major metabolites can be studied to better understand the toxicity of their parent drugs. Nya psykoaktiva substanser (NPS) är det officiella namnet för den grupp droger som tidigare har kallats för designerdroger, nätdroger eller internetdroger. NPS definieras som droger som utgör ett likvärdigt hot mot folkhälsan som droger som återfinns på Förenta Nationernas narkotikakonventioner men som själva inte återfinns under dessa konventioner. Det finns hundratals olika rapporterade NPS spridda över olika droggrupper, såsom syntetiska cannabinoider och syntetiska opioider. Vissa av dessa droger syntetiserades ursprungligen i forskningssyfte, men tog sig senare in på den illegala drogmarknaden. De mer nyligen framtagna NPS är ofta designade att efterlikna effekterna av etablerade droger, såsom morfin eller ?9-THC, vilket är den huvudsakliga psykoaktiva substansen i cannabis. Användandet av NPS har associerats till många kluster av intoxikationer som har lett till hospitaliseringar. Många har även dött till följd av användandet av NPS. Inte minst i USA där en grupp av NPS kallad för fentanylanaloger är högst delaktig i den pågående opioidkrisen. Den ständiga inströmningen av nya NPS leder till att detektion av och diskriminering mellan dem utgör en svår utmaning för forensiska toxikologilaboratorier. Tillgången av lämpliga referenssubstanser möter inte deras efterfrågan, delvis på grund av att vilka biomarkörer som är optimala för drogdetektion inte alltid är uppenbart. Till exempel så är metabolismen av syntetiska cannabinoider i regel både snabb och omfattande. Av den anledningen kan användandet av modersubstansen som biomarkör vid analys av urin leda till falskt negativa resultat. Urin som biologisk matris har många fördelar jämfört med blod. Till exempel så har urin ett längre detektionsfönster och högre drogkoncentrationer. För att kunna identifiera optimala biomarkörer för droganalys av urinprover så måste drogernas metabolism utredas. De flesta metabolismstudier använder sig av humana levermikrosomer eller hepatocyter som inkuberas tillsammans med droger för att generera metaboliter in vitro. Urinprover från individer i vilkas blod det har återfunnits droger används också men tillgången är tyvärr begränsad. Dessa metaboliter separeras sedan med hjälp av kromatografiska tekniker och deras kemiska strukturer bestäms med hjälp av masspektrometri. Dock så är utvärderingen av masspektrometridata komplicerad och det är heller inte möjligt att skilja på vissa positionsisomerer genom att enbart analysera masspektrometridata. För att kunna möjliggöra exakt strukturutredning så krävs referensstandarder. Därför var målet med denna avhandling att addera syntes och analys av referensstandarder till de etablerade tillvägagångssätten att studera metabolismen av NPS. Ett stort antal potentiella metaboliter av AKB-48 och andra syntetiska cannabinoider samt av fentanyl och fentanylanaloger syntetiserades. Genom att använda dessa referensstandarder i metabolismstudier så kunde de exakta kemiska strukturerna för många metaboliter bestämmas. Dessutom så identifierades mönster i de metaboliska profilerna bland fentanyl och fentanylanaloger. Dessa mönster kan användas för att förbättra predikteringen av metaboliter för andra nuvarande och kommande fentanylanaloger. Dessa resultat samt de utvecklade syntesvägarna kan nyttjas i framställningen av referenssubstanser i syfte att användas som biomarkörer för att i urin kunna detektera drogmissbruk. Referenssubstanserna kan även användas för att studera metaboliternas farmakologiska egenskaper vilket kan leda till en djupare förståelse kring toxiciteten hos modersubstanserna.

The ACMT National Case Conference (NCC) is a monthly discussion of novel or interesting cases in medical toxicology. Participation is through online webinar, and the conferences are recorded to allow for review at any time. The cases in this book are taken from recordings of NCC with edits and revisions by contributors and editors to demonstrate educational points. The majority of the case information is from the original recording and represents actual patient presentations. However, some of the details have been changed and fictional information added to enhance the educational value. This volume covers a broad range of toxicological topics, and specialty guidance is offered at the end of every case to aid non-toxicologists. The dilemmas are applicable to both academic and clinical medicine. A list of relevant questions is also provided for each case. Subjects include common toxicological problems, rare presentations of common problems, common problems with controversial treatments or difficult diagnoses, and rare problems. Case Studies in Medical Toxicology from the American College of Medical Toxicology is a detailed reference text on specific toxicological issues and also serves as a practical review for those taking board exams. As a result, this volume is an important and necessary resource for medical students, residents, and fellows, as well as primary-care physicians, intensivists, and toxicologistsCase Studies in Medical Toxicology from the American College of Medical Toxicology is a detailed reference text on specific toxicological issues and also serves as a practical review for those taking board exams. As a result, this volume is an important and necessary resource for medical students, residents, and fellows, as well as primary-care physicians, intensivists, and toxicologists. All proceeds from this book will be donated to the Medical Toxicology Foundation.

One of the major areas of emphasis in academia in recent years has been interdisciplinary research, a trend that promises new insights and innovations rooted in cross-disciplinary collaboration. The book titled "INTERDISCIPLINARY RESEARCH AND INNOVATION" is an edited book, this book is designed to help to understand the tools required for stepping beyond traditional disciplinary boundaries and applying knowledge and insights from multiple fields. Relentlessly focused on practical applications, the book will enable to plan and execute their own interdisciplinary research projects. This book will be ideal for students, academicians, professionals, researchers and Scientists. Furthermore it could be used as course material at universities covering the topics.