Metabolomics of methadone: clinical and forensic toxicological implications and variability of dose response

Methadone is a full μ-opioid receptor agonist used in the treatment of heroin addiction. It is commercialized as a racemic mixture with considerable variability in the pharmacokinetics and pharmacodynamics between individuals that can affect dose-response and toxicological profile. This review aims to discuss metabolomics of methadone, namely by presenting all major and minor metabolites and pharmacokinetic drug interactions. The main mechanism for methadone metabolism is hepatic through the cytochrome P450, specifically isoenzymes 2B6, 3A4 and 2D6. Firstly, methadone is N-demethylated and cyclize to form its major 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP) metabolites. Several alternate minor pathways have been described namely various methadol metabolites, which proved to be active.

It is expected that knowing the metabolomics of methadone may provide further insights, attempting a personalized therapy aiming to attain effective blood concentrations. The historical record is therefore especially important when investigating clinical and forensic cases related to methadone administration, since interindividual responses are known to vary considerably.     

Collection of biological samples in forensic toxicology

Forensic toxicology is the study and practice of the application of toxicology to the purposes of the law. The relevance of any finding is determined, in the first instance, by the nature and integrity of the specimen(s) submitted for analysis. This means that there are several specific challenges to select and collect specimens for ante-mortem and post-mortem toxicology investigation. Post-mortem specimens may be numerous and can endow some special difficulties compared to clinical specimens, namely those resulting from autolytic and putrefactive changes. Storage stability is also an important issue to be considered during the pre-analytic phase, since its consideration should facilitate the assessment of sample quality and the analytical result obtained from that sample. The knowledge on degradation mechanisms and methods to increase storage stability may enable the forensic toxicologist to circumvent possible difficulties. Therefore, advantages and limitations of specimen preservation procedures are thoroughfully discussed in this review. Presently, harmonized protocols for sampling in suspected intoxications would have obvious utility. In the present article an overview is given on sampling procedures for routinely collected specimens as well as on alternative specimens that may provide additional information on the route and timing of exposure to a specific xenobiotic. Last, but not least, a discussion on possible bias that can influence the interpretation of toxicological results is provided. This comprehensive review article is intented as a significant help for forensic toxicologists to accomplish their frequently overwhelming mission.     

UPLC-MS/MS法测定中兽药中非法添加物金刚烷胺

目的建立检测中兽药中金刚烷胺残留的UPLC-MS/MS方法。方法样品经提取稀释后,采用ACQUITY UPLCTMBEH C18色谱柱为分离柱,质谱正离子扫描测定。结果金刚烷胺在5～100 ng·mL^-1与峰面积线性关系良好（r^2=0.999 2）;样品检出限为1μg·mL^-1,定量限为2μg·mL^-1。在40、50、60 mg·mL^-1回收率为85%～115%,批内批间变异系数均〈10%。结论本方法快速、灵敏、重现性好,适用于中兽药中非法添加金刚烷胺的检测。     

Progress of liquid chromatography-mass spectrometry in clinical and forensic toxicology

The use of liquid chromatography-mass spectrometry (LC-MS) has recently exploded in various analytic fields, including toxicology and therapeutic drug monitoring (although still far behind pharmacokinetics). There is no doubt that LC-MS is currently competing with gas chromatography (GC)-MS for the status of the reference analytic technique in toxicology. This review presents, for the nonspecialist reader, the principles, advantages, and drawbacks of LC-MS systems using atmospheric pressure interfaces. It also gives an overview of the analytic methods for xenobiotics that could be set up with these instruments for clinical or forensic toxicology. In particular, as far as quantitative techniques are concerned, this review tries to underline the large number and variety of drugs or classes of drugs (drugs of abuse, therapeutic drugs) or toxic compounds (e.g., pesticides) that can be readily determined using such instruments, the respective merits of the different ionization sources, and the improvements brought about by tandem MS. It also discusses new applications of LC-MS in the field of toxicology, such as "general unknown" screening procedures and mass spectral libraries using LC-atmospheric pressure ionization (API)-MS or MS-MS, presenting the different solutions proposed to overcome the naturally low fragmentation power of API sources. Finally, the opportunities afforded by the most recent or proposed instrument designs are addressed.     

The index line as a new aid in clinical and forensic toxicology

On behalf of the German Federal Ministry for Research and Technology, we investigated the use of electronic data processing for clinical toxicological purposes. Initially, sufficient funds were available for a comprehensive approach to the problem and programs covering the following areas were established:

1. A casuistic data program;
2. an identification program for wild fruits;
3. a program for the identification of tablets by their shapes

Due to a subsequent lack of funds, it was necessary to develop a partial solution: this solution is what we call the Index Line. The Index Line—limited data on poisonings—should enable the user to receive information by telex from the German Institute for Medical Documentation and Information, i.e., information on “who has what, where”. As a first step the continuous Index Line registration of all cases of poisoning recorded at the poison control centers in Munich, Freiburg, Hamburg, and Nürnberg as well as at the State Institute for Food, Pharmaceutical and Forensic Chemistry in Berlin was founded in 1975. To participate in the program, complete instructions are necessary.     

Linking human and veterinary health: Trends, directions and initiatives

The objective of this brief article is to provide an overview of some of the important harmonization efforts that are currently under way within the animal health community. Topics include: scientific networks and interdisciplinary communication: organizations that address animal-related public health concerns; the role of the veterinary pharmaceutical scientist within human health-oriented professional organizations; recent publications pertaining to veterinary pharmacology, pharmaceutics and therapeutics; and the role of global networking in veterinary product research and development.     

Metabolism of psilocybin and psilocin: clinical and forensic toxicological relevance

Psilocybin and psilocin are controlled substances in many countries. These are the two main hallucinogenic compounds of the “magic mushrooms” and both act as agonists or partial agonists at 5-hydroxytryptamine (5-HT)_2A subtype receptors. During the last few years, psilocybin and psilocin have gained therapeutic relevance but considerable physiological variability between individuals that can influence dose-response and toxicological profile has been reported. This review aims to discuss metabolism of psilocybin and psilocin, by presenting all major and minor psychoactive metabolites. Psilocybin is primarily a pro-drug that is dephosphorylated by alkaline phosphatase to active metabolite psilocin. This last is then further metabolized, psilocin-O-glucuronide being the main urinary metabolite with clinical and forensic relevance in diagnosis.     

Toxicogenomics and clinical toxicology: An example of the connection between basic and applied sciences

The relationship between basic research and its potential clinical applications is often a difficult subject. Clinical toxicology has always been very dependent on experimental research whose usefulness has been impaired by the existence of huge differences in the toxicity expression of different substances, inter- and intra-species which make it difficult to predict clinical effects in humans. The new methods in molecular biology developed in the last decades are furnishing very useful tools to study some of the more relevant molecules implied in toxicokinetic and toxicodynamic processes. We aim to show some meaningful examples of how recent research developments with genes and proteins have clear applications to understand significant clinical matters, such as inter-individual variations in susceptibility to chemicals, and other phenomena related to the way some substances act to induce variations in the expression and functionality of these targets.     

Progress of capillary electrophoresis in therapeutic drug monitoring and clinical and forensic toxicology

During the past decade, capillary electrophoresis (CE) emerged as a promising, effective, and economical approach for the analysis of licit and illicit drugs and their metabolites in biologic samples. This review provides an overview of the principles of CE, the features of CE instrumentation, and the key aspects of CE-based drug assays that were developed for therapeutic drug monitoring (TDM), clinical and forensic toxicology, and assessment of drug metabolism and pharmacokinetics. CE performed in fused-silica capillaries has sufficiently matured and can thus be applied routinely, whereas chip-based instrumentation comprising fully integrated assays is still in development. Despite the attractive advantages of electrokinetic capillary technology, relatively few CE-based assays for TDM and for drug screening of clinical and forensic interest have been adopted in the routine arena. The lack of complete systems designed for unattended operation, the reluctance of bioanalysts to replace a satisfactory existing method, and tight budgets are believed to have hindered the widespread replacement of older (mainly chromatographic) technology. Another limitation of CE is that this technique is somewhat less sensitive than other analytic techniques used for drug analysis in biologic fluids. New instrumental developments featuring user-friendly software and the introduction of assay kits, however, should increase the number of validated CE drug tests becoming used on a routine basis.     

Drugs in nails: physiology, pharmacokinetics and forensic toxicology

In recent years, drug analysis in keratinised matrices, such as hair and nails, has received considerable attention because of several advantages over drug testing methodologies employing body fluids, such as urine or serum. For example, keratinic matrices, such as finger- and toenails, can accumulate drugs during long term exposure. Drugs are incorporated into nails by a double mechanism: (i) deposition into the root of the growing nail via the blood flow in the nail matrix; and (ii) incorporation via the nail bed during growth from the lunula to the beginning of the free margin. Together, these account for a wide retrospective window of drug detection. Nails can provide a good forensic matrix for the detection of drugs of abuse. Indeed, the international literature has reported the use of nail analysis in postmortem detection of drugs of abuse, drug testing in the workplace and drug screening to detect prenatal exposure, even though further studies are needed for correct interpretation of the data obtained. Another application of drug analysis in nails consists of the possibility of detecting the presence of an antimycotic at the site of action during antifungal therapy for patients with onychomycosis. When available, this evidence has permitted drug treatment of a shorter duration and reduced toxicity. However, so far the potential of drug monitoring in nails still lacks harmonisation and validation of analytical methodologies and a better comprehension of the possible correlation between drug concentrations in the matrix and period of exposure.     

Mass Spectrometry Strategies for Clinical Metabolomics and Lipidomics in Psychiatry,Neurology, and Neuro-Oncology

Metabolomics research has the potential to provide biomarkers for the detection of disease, for subtyping complex disease populations, for monitoring disease progression and therapy, and for defining new molecular targets for therapeutic intervention. These potentials are far from being realized because of a number of technical, conceptual, financial, and bioinformatics issues. Mass spectrometry provides analytical platforms that address the technical barriers to success in metabolomics research; however, the limited commercial availability of analytical and stable isotope standards has created a bottleneck for the absolute quantitation of a number of metabolites. Conceptual and financial factors contribute to the generation of statistically under-powered clinical studies, whereas bioinformatics issues result in the publication of a large number of unidentified metabolites. The path forward in this field involves targeted metabolomics analyses of large control and patient populations to define both the normal range of a defined metabolite and the potential heterogeneity (eg, bimodal) in complex patient populations. This approach requires that metabolomics research groups, in addition to developing a number of analytical platforms, build sufficient chemistry resources to supply the analytical standards required for absolute metabolite quantitation. Examples of metabolomics evaluations of sulfur amino-acid metabolism in psychiatry, neurology, and neuro-oncology and of lipidomics in neurology will be reviewed.     

坤泰胶囊改善卵巢早衰的毒理及临床研究进展

卵巢早衰是由多种致病因素引起的卵巢功能衰竭性疾病,发病率呈逐年上升趋势,严重影响了女性的正常生活。坤泰胶囊由《伤寒论》中的古方化裁而来,能够有效调节女性内分泌水平、改善卵巢功能、治疗卵巢功能衰退,可单用或与性激素联合用于卵巢早衰的治疗。大鼠毒理学研究证实坤泰胶囊无明显生殖毒性,大量临床研究也表明其安全性较好。对坤泰胶囊改善卵巢早衰的动物毒理和临床研究进展进行综述,为其在临床上推广应用提供依据。     

坤泰胶囊改善卵巢早衰的毒理及临床研究进展

卵巢早衰是由多种致病因素引起的卵巢功能衰竭性疾病,发病率呈逐年上升趋势,严重影响了女性的正常生活。坤泰胶囊由《伤寒论》中的古方化裁而来,能够有效调节女性内分泌水平、改善卵巢功能、治疗卵巢功能衰退,可单用或与性激素联合用于卵巢早衰的治疗。大鼠毒理学研究证实坤泰胶囊无明显生殖毒性,大量临床研究也表明其安全性较好。对坤泰胶囊改善卵巢早衰的动物毒理和临床研究进展进行综述,为其在临床上推广应用提供依据。     

Web based information on clinical toxicology for the United Kingdom: uptake and utilization of TOXBASE in 2000

AIMS: To examine the use and uptake of TOXBASE, an Internet database for point of care provision of poisons information in the United Kingdom during its first calendar year of web-based access. METHODS: Interrogation of the database software to examine: use by different types of user and geographical origin; profile of ingredient and product access; time of access to the system; profile of access to other parts of the database. RESULTS: Registered users of the system increased in the first full year of operation (1224 new users) and usage of the system increased to 111 410 sessions with 190 223 product monograph accesses in 2000. Major users were hospitals, in particular accident and emergency departments. NHS Direct, a public access information service staffed by nurses, also made increasing use of the system. Usage per head of population was highest in Northern Ireland and Scotland, and least in southern England. Ingredients accessed most frequently were similar in all four countries of the UK. Times of use of the system reflect clinical activity, with hospitals making many accesses during night-time hours. The most popular parts of the database other than poisons information were those dealing with childhood poisoning, information on decontamination procedures, teratology information and slang terms for drugs of abuse. CONCLUSIONS: This Internet system has been widely used in its first full year of operation. The provision of clinically relevant, up to date, information at the point of delivery of patient care is now possible using this approach. It has wide implications for the provision of other types of therapeutic information in clinical areas. Web-based technology represents an opportunity for clinical pharmacologists to provide therapeutic information for clinical colleagues at the bedside.     

Computational chemistry, systems biology and toxicology. Harnessing the chemistry of life: revolutionizing toxicology. a commentary

There is a continuing interest in, and increasing imperatives for, the development of alternative methods for toxicological evaluations that do not require the use of animals. Although a significant investment has resulted in some achievements, progress has been patchy and there remain many challenges. Among the most significant hurdles is developing non-animal methods that would permit assessment of the potential for a chemical or drug to cause adverse health effects following repeated systemic exposure. Developing approaches to address this challenge has been one of the objectives of the European Partnership for Alternative Approaches to Animal Testing (EPAA). The EPAA is a unique partnership between the European Commission and industry that has interests in all aspects of reducing, refining and replacing the use of animals (the '3Rs'). One possible strategy that emerged from a broad scientific debate sponsored by the EPAA was the opportunity for developing entirely new paradigms for toxicity testing based upon harnessing the increasing power of computational chemistry in combination with advanced systems biology. This brief commentary summarizes a workshop organized by the EPAA in 2010, that had the ambitious title of 'Harnessing the Chemistry of Life: Revolutionizing Toxicology'. At that workshop international experts in chemistry, systems biology and toxicology sought to map out how best developments in these sciences could be exploited to design new strategies for toxicity testing using adverse effects in the liver as an initial focus of attention. Here we describe the workshop design and outputs, the primary purpose being to stimulate debate about the need to align different areas of science with toxicology if new and truly innovative approaches to toxicity testing are to be developed.     

Ethylene glycol: Evidence of glucuronidation in vivo shown by analysis of clinical toxicology samples

In the search for improved laboratory methods for the diagnosis of ethylene glycol poisoning, the in vivo formation of a glucuronide metabolite of ethylene glycol was hypothesized. Chemically pure standards of the β‐O‐glucuronide of ethylene glycol (EG‐GLUC) and a deuterated analog (d₄‐EG‐GLUC) were synthesized. A high‐performance liquid chromatography and tandem mass spectrometry method for determination of EG‐GLUC in serum after ultrafiltration was validated. Inter‐assay precision (%RSD) was 3.9% to 15.1% and inter‐assay %bias was ?2.8% to 12.2%. The measuring range was 2–100 μmol/L (0.48–24 mg/L). Specificity testing showed no endogenous amounts in routine clinical samples (n = 40). The method was used to analyze authentic, clinical serum samples (n = 31) from patients intoxicated with ethylene glycol. EG‐GLUC was quantified in 15 of these samples, with a mean concentration of 6.5 μmol/L (1.6 mg/L), ranging from 2.3 to 15.6 μmol/L (0.55 to 3.7 mg/L). In five samples, EG‐GLUC was detected below the limit of quantification (2 μmol/L) and it was below the limit of detection in 11 samples (1 μmol/L). Compared to the millimolar concentrations of ethylene glycol present in blood after intoxications and potentially available for conjugation, the concentrations of EG‐GLUC found in clinical serum samples are very low, but comparable to concentrations of ethyl glucuronide after medium dose ethanol intake. In theory, EG‐GLUC has a potential value as a biomarker for ethylene glycol intake, but the pharmacokinetic properties, in vivo/vitro stability and the biosynthetic pathways of EG‐GLUC must be further studied in a larger number of patients and other biological matrices.