Determination of the exogenous character of testosterone in bovine urine by gas chromatography-combustion-isotope ratio mass spectrometry.

A new approach was developed in order to control testosterone abuse in animal production. A gas chromatographic-combustion-isotope ratio mass spectrometric (GC-C-IRMS) method was used to distinguish the exogenous character from the endogenous character of the main metabolites of testosterone (epitestosterone and etiocholanolone) in cattle urine. This method is based on a comparison between the carbon isotope ratio (13C/12C) of testosterone metabolites and those of testosterone endogenous precursors. After urinary steroid purification, extracts were acetylated with acetic anhydride and injected into the GC-C-IRMS system. In order to validate the method, testosterone enanthate was administered to a 4 year old cow. The 13C/12C isotope ratios of testosterone exogenous metabolites appeared to be significantly different to the 13C/12C precursor ratios and were detected until 3 weeks after the anabolic administration. These preliminary results appear to be promising for the difficult control of natural hormones in livestock.     

Targeted phase II metabolites profiling as new screening strategy to investigate natural steroid abuse in animal breeding

The use of steroid hormones as growth promoters in cattle is banned within the European Union since 1988 but can still be fraudulently employed in animal breeding farms for anabolic purposes. While efficient targeted confirmatory methods have been implemented in control laboratories for many years, fast and reliable screening methods are still required, especially in the case of natural hormones abuse, but more globally for new "fishing" strategies allowing to reveal the use of even unknown anabolic agents. The development of focused profiling or untargeted metabolomic approaches is thus emerging in this context. The present study was a focused profiling study using steroids phase II metabolites, with the aim to get a better understanding of the steroid metabolism disruptions after exogenous administration of androstenedione and finally reveal potential biomarkers signing its administration. A sample preparation procedure was first developed, based on a separation of 31 glucuronide and sulphate conjugate compounds using an anion exchange SPE system. Each fraction was then analysed by UPLC-MS/MS in MRM mode showing a rapid (between 4h and 4 days after treatment) and huge excretion of several direct metabolites of androstenedione such as etiocholanolone-glucuronide or epiandrosterone-sulphate.     

Use of isotope ratio mass spectrometry to differentiate between endogenous steroids and synthetic homologues in cattle: A review

Although substantial technical advances have been achieved during the past decades to extend and facilitate the analysis of growth promoters in cattle, the detection of abuse of synthetic analogs of naturally occurring hormones has remained a challenging issue. When it became clear that the exogenous origin of steroid hormones could be traced based on the ¹³C/¹²C isotope ratio of the substances, GC/C/IRMS has been successfully implemented to this aim since the end of the past century. However, due to the costly character of the instrumental setup, the susceptibility of the equipment to errors and the complex and time consuming sample preparation, this method is up until now only applied by a limited number of laboratories. In this review, the general principles as well as the practical application of GC/C/IRMS to differentiate between endogenous steroids and exogenously synthesized homologous compounds in cattle will be discussed in detail, and will be placed next to other existing and to be developed methods based on isotope ratio mass spectrometry. Finally, the link will be made with the field of sports doping, where GC/C/IRMS has been established within the World Anti-Doping Agency (WADA) approved methods as the official technique to differentiate between exogenous and endogenous steroids over the past few years.     

Study of traditional Chinese animal drugs using FT-IR and 2D-IR correlation spectroscopy

Using FT-IR and 2D-IR correlation spectroscopy, the intensity changes and their sequence of amide I and II bands of four traditional Chinese animal drugs (Cornu Cervi Pantotrichum, Cornu Saigae Tataricae, Scorpio and Hirud) under thermal perturbation are studied, and component of Ca(3)(PO(4))(2) in the drug of Cornu Cervi Pantotrichum and sulfates components in the drug of scorpion are identified. The drug of Cornu Cervi Pantotrichum contains inorganic salt Ca(3)(PO(4))(2) and the drug of Scorpio contains sulfates. It is assigned that the bands of 604 and 561 cm(-1) belong to the component of Ca(3)(PO(4))(2), and the bands of 637 and 615 cm(-1) belong to sulfates. Organic components of these drugs respond to the thermal perturbation far stronger than that of the inorganic components. The intensities of amide I and II bands in the drugs, except for amide II band in drug Scorpio, change strongly. For the drugs of Cornu Cervi Pantotrichum, Cornu Saigae Tataricae and Hirudo, the intensity changes of amide II band occurs prior to that of amide I band. The C-N bond in the product operator(3)(4) conjugative system converts from double bond to single bond, but the C=O bond remains double bond during the heating process. On the other hand, amide II vibrations, which may involve much more of the hydrogen bonded local structures of amide groups in the system compared to the C=O dominated amide I vibration, may undergo thermally induced changes at a temperature much lower than the other mode. The traditional Chinese animal drugs can be identified rapidly and non-separately by using FT-IR and 2D-IR correlation spectroscopy.     

Hair Analysis as Method for Determination of Level of Drugs and Pharmaceutical in Human Body: Review of Chromatographic Procedures

Abstract

Interest in hair analysis as an alternativ or complementary approach to urinalysis for drug abuse detection has grown in recent years. Hair analysis can be particularly advantageous for drugs and their enantiomers.

More than hundred pharmaceuticals, drugs of abuse agents are reported to be detectable in human and animal hair. This article reviews the aalysis of drugs and drug metabolites by chromatographic procedures, incuding the pretreatment steps, and the xtraction methods. Tihe eneral tendency in the last years, to highly sophisticated techiques gas chromatography–mass spectrometry (GC–MS–NCI), high pressure liquid chromatography–mass spectrometry (HPLC–MS), gas chromatography–mass spectrometry–mass spectrometry (GC–MS–MS) well illustrates this constant fight for sensitivity.     

Effect of preparation and preservation procedures on carbon and nitrogen stable isotope determinations from zooplankton

A literature survey of zooplankton stable isotope studies revealed inconsistencies between authors concerning (a) fixation and (b) allowance for gut clearance of zooplankton prior to delta13C and delta15N determinations. To address whether commonly used preservation techniques induce changes in stable isotope values, fresh lake zooplankton (control) were compared with preserved (ethanol, methanol, formaldehyde, gluteraldehyde, frozen and shock frozen) material. Differences of up to 1.1 per thousand for carbon and 1.5 per thousand for nitrogen isotopic signatures were found. Even freezing, the most frequently used method identified from the literature, caused significant changes compared with the control. We advocate the use of fresh material prepared immediately whenever possible, or complementary testing of the preservative method to be used. Larger organisms are routinely eviscerated, or specific tissues are dissected, and analysed for stable isotopes to reduce errors introduced via the gut contents. Yet zooplankton gut clearance is rarely performed: the gut content assumed to be negligible relative to organism mass. Experimental determinations of relative gut mass, from both original and compiled data, range from 1-26% for different zooplankton species. Using reported isotopic values of basal resources from natural systems, we calculated that, when analysing bulk zooplankton, inclusion of the gut mass may introduce substantial errors of >3 per thousand. Thus it appears prudent to perform the simple procedure of gut clearance, especially for copepod species.     

Stability of analytes in biosamples—an important issue in clinical and forensic toxicology?

Knowledge of the stability of drugs in biological samples is important for the interpretation of toxicological findings. This paper reviews data on the stability of drugs in blood, plasma, or serum. Since such data have already been reviewed for classic drugs of abuse, the focus here is on newer drugs of abuse and on therapeutic drugs. Key information about the conditions of the stability experiments will be provided and the following drugs or drug classes are covered: amphetamines, amphetamine-derived, piperazine-derived, and phenethylamine-derived designer drugs, antidepressants, neuroleptics, anti-HIV drugs, antiepileptics, cardiovascular drugs, and others. In addition, aspects of stability experiments and their evaluations are discussed. The data presented show that the majority of drugs are stable in blood, plasma, or serum samples under the conditions usually encountered in a clinical or forensic toxicology laboratory. Instability usually only occurs for drugs carrying ester moieties, sulfur atoms, or other easily oxidized or reduced structures. Nevertheless, clinical or forensic specimens should always be stored at least in the refrigerator and preferably at -20 degrees C or lower to avoid any degradation. Finally, results obtained from biosamples that have been stored at room temperature for a longer time should be interpreted with great care and partial degradation should always be considered.     

Development and application of stable carbon isotope analysis to the detection of cortisol administration in cattle

The use of anabolic agents in food-producing animals has been prohibited within the EU since 1988. The control of the illegal use of natural steroid hormones in cattle is still an exciting analytical challenge as no definitive method and nonambiguous analytical criteria are available. We have used gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to demonstrate the administration of cortisol to cattle. The method consisted of an efficient combination between OASIS HLB solid-phase extraction (SPE), oxidation, SiOH SPE and semi-preparative high-performance liquid chromatography (HPLC) for glucocorticoid purification. By comparison of the (13)C/(12)C isotopic ratio of the oxidised product of cortisol, i.e. 5 beta-androstane-3,11,17-trione (5 beta AAT), with an endogenous reference compound (ERC), dehydroepiandrosterone (DHEA), the differentiation of cortisol metabolite origin, either endogenous or exogenous, has been achieved. After treatment of an animal, the delta(13)C(VPDB) values of 5 beta AAT reached -30 to -32 per thousand, whereas the delta(13)C(VPDB) values of DHEA remained at -25 per thousand. A significant difference in the delta(13)C(VPDB) values between DHEA and 5 beta AAT was measurable over a period of 3 days after a single administration of cortisol to the animal.     

基于核苷类化合物HILIC-ESI-TOF/MS分析的海洋动物药指纹图谱方法

探讨了核苷类化合物指纹图谱用于不同海洋动物药真伪鉴别的可行性, 为贵重动物药的鉴别提供了一种新方法. 采用亲水色谱-电喷雾飞行时间质谱(HILIC-ESI-TOF/MS)对不同海洋动物药中的16种核苷类化合物进行分析, 构建了基于16种核苷类化合物的特征指纹图谱, 结合相似度分析和聚类分析, 用于不同海洋动物药的鉴别. 结果表明, 基于核苷类化合物HILIC-ESI-TOF/MS分析的指纹图谱能反映不同海洋动物药各自的固有特征, 结合相似度分析和聚类分析可实现对不同海洋动物药的正确区分. 说明核苷类化合物指纹图谱有望成为动物药鉴别的新方法.     

Investigation of the feeding effect on the 13C/12C isotope ratio of the hormones in bovine urine using gas chromatography/combustion isotope ratio mass spectrometry

The effect of the feeding on the 13C/12C isotope ratio of four endogenous steroid hormones testosterone (T), epi-testosterone (epi-T), dehydroepiandrosterone (DHEA) and etiocholanolone (ETIO) in bovine urine was investigated. An analytical method to determine the accurate isotope ratio was developed including an extensive clean up followed by enrichment of the analytes in two steps of HPLC fractionation. Feeding experiments with four young animals were performed using C3 and C4 plants (grass, maize silage, hay, etc.) over a time period of about 280 days. One cattle was used as a control animal with no change of its diet over the full period. The detection of the 13C/12C isotope ratio of the acetylated extracts was performed by gas chromatography/combustion isotope ratio mass spectrometry. After the first change of the feeding from C4 to C3 plants significant changes of the delta 13C % values were observed from the -19 to -23% level to the -24 to -32% level for etiocholanolone and epi-testosterone in urine of three animals, whereas the DHEA values remained under the level of the two metabolites. Testosterone could not be detected with GC-C-IRMS due to its low concentration in young animals. After the second change of the diet from C3 to C4 plants (after 222 days), the measured delta 13C % values have been stabilised at the original level. The results show that in case of the feeding with only C3 plants the endogenous delta values of -32% can be reached. In this case the contribution of exogenous material with a delta value of -32% could not be detected independently of the concentration. If the diet contains C4 plants the difference or the ratio of the delta 13C % values becomes the determinant in the discriminatory power. For validation of the method a human and a cattle were treated with testosterone and the delta 13C % values were measured in incurred human and cattle urine.     

A high-sensitivity ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method for screening synthetic cannabinoids and other drugs of abuse in urine

The continuing emergence of designer drugs imposes high demands on the scope and sensitivity of toxicological drug screening procedures. An ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method was developed for screening and simultaneous confirmation of both designer drugs and other drugs of abuse in urine samples in a single run. The method covered selected synthetic cannabinoids and cathinones, amphetamines, natural cannabinoids, opioids, cocaine and other important drugs of abuse, together with their main urinary metabolites. The database consisted of 277 compounds with molecular formula and exact monoisotopic mass; retention time was included for 192 compounds, and primary and secondary qualifier ion exact mass for 191 and 95 compounds, respectively. Following a solid-phase extraction, separation was performed by UHPLC and mass analysis by HR-TOFMS. MS, and broad-band collision-induced dissociation data were acquired at m/z range 50–700. Compound identification was based on a reverse database search with acceptance criteria for retention time, precursor ion mass accuracy, isotopic pattern and abundance of qualifier ions. Mass resolving power in spiked urine samples was on average FWHM 23,500 and mass accuracy 0.3 mDa. The mean and median cut-off concentrations determined for 75 compounds were 4.2 and 1 ng/mL, respectively. The range of cut-off concentrations for synthetic cannabinoids was 0.2–60 ng/mL and for cathinones 0.7–15 ng/mL. The method proved to combine high sensitivity and a wide scope in a manner not previously reported in drugs of abuse screening. The method’s feasibility was demonstrated with 50 authentic urine samples.

Hydropyrolysis of steroids: a preparative step for compound-specific carbon isotope ratio analysis

Compound-specific stable carbon isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry is an important method for the detection of steroid abuse in athletes. However, steroids in their natural form exhibit poor chromatographic resolution, while derivatization adds carbon thereby corrupting the starting stable isotopic composition. Hydropyrolysis is a new approach, which defunctionalizes steroids but leaves their carbon skeleton intact. The process improves chromatography, allowing the faithful measurement of carbon isotope ratios and enabling a more effective apportionment for the source of steroids and their metabolites.     

Rapid gas chromatographic analysis of drugs of forensic interest.

High-speed gas chromatographic (GC) screening for drugs of forensic relevance is performed using a commercial Flash GC instrument in which the chromatographic column is resistively heated at rates of up to 30 degrees C/s. Temperature programming conditions are varied in an experiment designed to evaluate trade-offs between resolution and analysis time for a mixture of 19 drugs of abuse. All 19 components can be separated with excellent resolution in 90 s. Specific analytes can be analyzed even faster; for example, amphetamine analysis is completed in less than 20 s. Case studies of confiscated street drugs containing amphetamine, cocaine, and heroin are analyzed to evaluate the retention time repeatability. Ten replicate injections over a 2-day period for 3 different drug samples achieved retention time relative standard deviations in the range of 0.48 to 0.81%.     

Ecopharmacology: Deliberated or casual dispersion of pharmaceutical principles, phytosanitary, personal health care and veterinary products in environment needs a multivariate analysis or expert systems for the control, the measure and the remediation

The increasing human and animal use and abuse of drugs as well as of personal health care and gross domestic products, involve disposal and waste problems and, as a consequence, affect the environmental condition. Actually most of the active principles are complex synthesised organic molecules that react, inside human or animal body, by specific biochemical reactions that in no case can reach a 100% yield and produce residues that could be more noxious of the starting compounds. Just reading the indication sheet accompanying any drugs, it is easy to state that no drug can be considered healthy, so, their use constitutes a serious pollution source. The full awareness of this relatively new environmental problem let many researchers to face it from different point of view. Current studies are considering the sources of these substances in the environment, the effects on human health as well as on the flora and fauna species, the recalcitrance and possible degradation methods, analytical techniques able to determine them and their metabolites even at low concentrations and in complex matrices.Literature on the subject continuously increases and a comparison of all data became more and more difficult both for a single drug and for different ones based on the same or different active principles. This is a typical case in which chemometrics can extract a full information in the easier way, so the design of a European database coupled to suitable expert system software should be strongly suggested.     

Metabonomic study of biochemical changes in urinary of type 2 diabetes mellitus patients after the treatment of sulfonylurea antidiabetic drugs based on ultra‐performance liquid chromatography/mass spectrometry

A metabonomic study on biochemical changes in the urine of type 2 diabetes mellitus (T2DM) patients after the treatment of sulfonylurea (SU) antidiabetic drugs was performed. An ultra‐performance liquid chromatography/mass spectrometry (UPLC/MS) method was used to generate metabolic fingerprints for the metabonomic analysis of urinary samples obtained from 20 T2DM patients without any drug treatment and 20 T2DM patients treated with SU antidiabetic drugs and 20 normal glucose tolerance subjects. The resulting data were subjected to chemometric analysis (principal component analysis and partial least squares discriminant analysis) to investigate the effect of SU antidiabetic drugs on urinary metabolite profiles of T2DM patients. Biomarkers such as xanthine, phenylalanine, tryptophan, hippurate, phenylacetylglutamine, carnitine C8:1, carnitine C10:3, uric acid and citrate were found to be responsible for the separation of T2DM and SU‐treated groups, which indicates a potential effect of SU on energy metabolism, Tricarboxylic acid (TCA) cycle, gut microflora metabolism and oxidative stress. The study may be helpful to the understanding of the action of mechanism of SU antidiabetic drugs. Copyright © 2014 John Wiley & Sons, Ltd.     

Application of stable carbon isotope analysis to the detection of 17beta-estradiol administration to cattle

The use of anabolic agents in food producing animals is prohibited within the EU since 1988 (96/22/EC directive). The control of the illegal use of natural steroid hormones in cattle is still an exciting analytical challenge as far as no definitive method and non-ambiguous analytical criteria are available. The ability of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to demonstrate the administration of 17beta-estradiol to bovine has been investigated in this paper. By comparison of 13C/12C isotopic ratio of main urinary estradiol metabolite, i.e. 17alpha-estradiol, with two endogenous reference compounds (ERCs), i.e. dehydroepiandrosterone (DHEA) and 5-androstene-3beta,17alpha-diol, the differentiation of estradiol metabolite origin, either endogenous or exogenous, has been proved to be achievable. After treatment, the delta(13)C(VPDB)-values of 17alpha-estradiol reached -27 per thousand to -29 per thousand, whereas delta13CVPDB-values of DHEA remained between -13 per thousand and -20 per thousand depending on the diet, maize and grass, respectively. A significant difference of delta13CVPDB between ERCs and 17alpha-estradiol was measurable over a period of 2 weeks after estradiol ester administration to the animal.     

Development of a dual luciferase reporter screening assay for the detection of synthetic glucocorticoids in animal tissues

Synthetic glucocorticoids belong to the most frequently administered drugs in livestock production. These synthetic hormones are employed for therapeutic purposes against inflammatory reactions, disorders of the musculoskeletal system, bovine ketosis and many other diseases of farm animals. A widespread illegal use of synthetic glucocorticoids to improve feed intake and weight gain has also been observed. To enforce the residue limits imposed on glucocorticoid drugs and preclude their illicit administration as growth promoters, it is necessary to establish high throughput analytical methods that can be applied to the screening of animal tissues. Here, we developed a dual luciferase reporter assay that detects residues or contaminants with glucocorticoid activity. This screening assay is performed by transfection of human cell lines with two reporter constructs followed by the measurement of two distinct luminescence signals, one of which serves as internal control to correct for assay variabilities and unspecific matrix effects. The limit of detection (1.25 microg for dexamethasone in liver) depends on the biological potency of each synthetic glucocorticoid but, with all drugs tested, the maximal response reaches a 20 to 30 fold induction of luciferase activity. In combination with an appropriate sample clean-up method (recovery of 82%), this luciferase assay has been applied to the analysis of liver samples from calves treated with a single therapeutic injection of either dexamethasone or flumethasone. Thus, the dual luciferase reporter assay provides a new screening tool to detect unwanted glucocorticoid activities in animal tissues or other crude biological samples without knowledge of the precise chemical entity of the parent compounds or their metabolites.     

Natural Products Inhibitors of Monoamine Oxidases—Potential New Drug Leads for Neuroprotection,Neurological Disorders,and Neuroblastoma

Monoamine oxidase inhibitors (MAOIs) are an important class of drugs prescribed for treatment of depression and other neurological disorders. Evidence has suggested that patients with atypical depression preferentially respond to natural product MAOIs. This review presents a comprehensive survey of the natural products, predominantly from plant sources, as potential new MAOI drug leads. The psychoactive properties of several traditionally used plants and herbal formulations were attributed to their MAOI constituents. MAO inhibitory constituents may also be responsible for neuroprotective effects of natural products. Different classes of MAOIs were identified from the natural product sources with non-selective as well as selective inhibition of MAO-A and -B. Selective reversible natural product MAOIs may be safer alternatives to the conventional MAOI drugs. Characterization of MAO inhibitory constituents of natural products traditionally used as psychoactive preparations or for treatment of neurological disorders may help in understanding the mechanism of action, optimization of these preparations for desired bioactive properties, and improvement of the therapeutic potential. Potential therapeutic application of natural product MAOIs for treatment of neuroblastoma is also discussed.     

Fishing potential antitumor agents from natural plant extracts pool by dialysis and G-quadruplex recognition

Screening G-quadruplex ligands from natural plants is important because the ligands may be potential antitumor drugs. A new screening strategy is proposed based on the combination of dialysis and G-quadruplex recognition technique which could separates G-quadruplex ligand from natural extracts and elucidate the structure of this ligand. This result offers a novel approach to obtain active antitumor compounds.     

The Current Directions of Searching for Antiparasitic Drugs

Parasitic diseases are still a huge problem for mankind. They are becoming the main cause of chronic diseases in the world. Migration of the population, pollution of the natural environment, and climate changes cause the rapid spread of diseases. Additionally, a growing resistance of parasites to drugs is observed. Many research groups are looking for effective antiparasitic drugs with low side effects. In this work, we present the current trends in the search for antiparasitic drugs. We report known drugs used in other disease entities with proven antiparasitic activity and research on new chemical structures that may be potential drugs in parasitic diseases. The described investigations of antiparasitic compounds can be helpful for further drug development.