Injection of oral medication into the skin confirmed by infrared spectroscopy.

"Skin popping" refers to the practice of injecting drugs, most commonly heroin, subcutaneously or into granulation tissue. Pharmaceutical tablets meant for oral consumption are modified into solutions for injection. Excipients-inactive substances that serve as vehicles for medication-are often not filtered out before injection and result in abscess formation, granulomatous inflammation, and scarring. Common excipients used in the production of pharmaceutical tablets include starch, microcrystalline cellulose, magnesium stearate, silica, and polyvinylpyrrolidone (PVP). Identification of these exogenous materials is valuable in confirming the diagnosis of skin popping, especially when patients may not be forthcoming about their drug use. We present a case of subcutaneous oral medication injection in which PVP and cellulose were identified by Fourier transform infrared spectroscopy. Considering the variable cutaneous manifestations of injection drug abuse, recognition of histopathologic and chemical characteristics of exogenous material from oral medications is helpful for diagnosis and intervention.

Detection of trifluridine in tumors of patients with metastatic colorectal cancer treated with trifluridine/tipiracil.

PURPOSE: Trifluridine (FTD) is the active component of the nucleoside chemotherapeutic drug trifluridine/tipiracil (FTD/TPI), which is approved worldwide for the treatment of patients with metastatic gastrointestinal cancer. FTD exerts cytotoxic effects via its incorporation into DNA, but FTD has not been detected in the tumor specimens of patients. The purpose of this study was to detect FTD in tumors resected from metastatic colorectal cancer (mCRC) patients who were administered FTD/TPI. Another purpose was to investigate the turnover rate of FTD in tumors and bone marrow in a mouse model. METHODS: Tumors and normal tissue specimens were obtained from mCRC patients who were administered FTD/TPI or placebo at Kyushu University Hospital. Tumors and bone marrow were resected from mice with peritoneal dissemination treated with FTD/TPI. To detect and quantitate FTD incorporated into DNA, immunohistochemical staining of paraffin-embedded specimens (IHC-p staining) and slot-blot analysis of DNA purified from these tissues were performed using an anti-BrdU antibody. IHC-p staining of proliferation and apoptosis markers was also performed. RESULTS: FTD was detected in metastatic tumors obtained from mCRC patients who were administered FTD/TPI, but who had discontinued the treatment several weeks before surgery. In a peritoneal dissemination mouse model, FTD was still detected in tumors 13 days after the cessation of FTD/TPI treatment, but had disappeared from bone marrow within 6 days. CONCLUSION: These results indicate that FTD persists longer in tumors than in bone marrow, which may cause a sustained antitumor effect with tolerable hematotoxicity.

Determination of New Psychoactive Substances in Whole Blood Using Microwave Fast Derivatization and Gas Chromatography/Mass Spectrometry.

The production and consumption of new psychoactive substances (NPSs) has been raising a major concern worldwide. Due to easy access and available information, many NPSs continue to be synthesized with an alarming increase of those available to purchase, despite all the control efforts created. A new analytical method was developed and validated to determine a group of phenethylamines and synthetic cathinones: cathinone, flephedrone, buphedrone, 4-MTA, α-PVP, methylone, 2C-P, ethylone, pentylone, MDPV and bromo-dragonFLY in whole blood. A mixed-mode solid phase extraction was applied to 250 µL of sample, and the extracts were derivatized with fast microwave technique before being analyzed by gas chromatography-mass spectrometry (GC-MS). The validation procedure followed the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines with parameters that included selectivity, linearity, limits of detection and quantification, intra- and inter-day precision and accuracy, recoveries and stability. The method presented linearity between 5 and 500 ng/mL for cathinone, buphedrone, 4-MTA, methylone, 2C-P and bromo-dragonFLY, 10-500 ng/mL for flephedrone, ethylone, pentylone and MDPV, and 40-500 ng/mL for α-PVP, with determination coefficients above 0.99 for all analytes. Recoveries ranged between 70.3% and 116.6%, and regarding intra- and inter-day precision, the relative mean errors were typically lower than 8.6%. The method was successfully applied to over 100 authentic samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal.

Determination of methadone and EDDP in oral fluid using the dried saliva spots sampling approach and gas chromatography-tandem mass spectrometry.

The present work describes the development and validation of a novel approach to determine methadone (MTD) and its main metabolite (EDDP) in oral fluid samples, using the dried saliva spots (DSS) sampling approach and gas chromatography-tandem mass spectrometry (GC-MS/MS). Oral fluid samples (50 µL) were applied into Whatman™ 903 protein saver filter paper cards and were allowed to dry overnight. The extraction was carried out by immersion of the spot in 1 mL of isopropyl alcohol with agitation for 1 min. Afterwards, the extract was centrifuged for 15 min at 3500 rpm and the supernatant evaporated to dryness and reconstituted with 50 µL of methanol. The procedure was considered linear in the range of 10 to 250 ng/mL for both compounds, with determination coefficients greater than 0.99. Intra- and inter-day precision and accuracy revealed coefficients of variation (CVs) lower than 15% at the studied concentrations, with mean relative errors within ± 15% of the nominal concentrations. Recoveries ranged from 45 to 74%. The limits of detection and quantification were 5 and 10 ng/mL respectively for both analytes. All studied parameters complied with the defined criteria and the method enabled the successful determination of MTD and EDDP in oral fluid samples from patients undergoing opiate substitution/maintenance therapy.

Discrimination between chewing of coca leaves or drinking of coca tea and smoking of "paco" (coca paste) by hair analysis. A preliminary study of possibilities and limitations.

BACKGROUND: Hair analysis is a suitable way to discriminate between coca chewers and consumers of manufactured cocaine using the coca alkaloids hygrine (HYG) and cuscohygrine (CUS) as markers. In the present preliminary study it was examined whether CUS and HYG can be detected in hair of occasional and moderate coca chewers or coca tea drinkers, whether CUS and HYG appear in hair of PACO consumers (smoking coca paste waste), and whether anhydroecgonine methyl ester (AEME) is a useful cocaine smoking marker in this context. METHOD: Three groups were included: 10 volunteers from Buenos Aires with occasional or moderate chewing of coca leaves or drinking coca tea, 20 Argentinean PACO smokers and 8 German cocaine users. The hair samples (1-4 segments) were analyzed by a validated LC-MS/MS method for cocaine (COC), norcocaine (NC), benzoylecgonine (BE), ecgonine methyl ester (EME), cocaethylene (CE), cinnamoylcocaine (CIN), tropacocaine (TRO), AEME, CUS and HYG. For comparison, eight samples of coca leaves or coca tea were analyzed. RESULTS: Only low concentrations of COC were found in hair of seven occasional users of coca leaves or coca tea (0.010-0.051 ng/mg). For three moderate chewers of coca leaves all compounds were detected including AEME but except TRO. The hair samples of PACO smokers contained much higher concentrations of COC (0.027-341 ng/mg, mean 37.4 ng/mg) and its metabolites. CUS was not found in these samples but traces of HYG were seen in 8 of 37 hair segments. AEME as a marker for coca smoking was detected in hair of 15 smokers. In comparison to COC, the concentrations of EME and CIN were higher for PACO smokers than for German cocaine consumers. AEME (56 ± 20 µg/g) was detected in all coca leave and coca tea samples which explains the detection of this substance in hair of coca chewers. Therefore, its use for differentiation between coca chewers and PACO smokers is limited. CONCLUSION: CUS remains to be the most suitable marker in hair for chewing coca leaves or drinking coca tea more frequently than two times per month since it does not appear in hair of Argentinean PACO smokers and German cocaine users. Contrary to a previous proposal, the ratios CIN/COC and EME/COC appeared not to be applicable as criteria for this purpose because of the higher concentration of these alkaloids in hair of PACO smokers. More research is needed to assess the value of AEME in hair of South American coca leave or cocaine users.

Human in vivo metabolism study of LGD-4033.

Selective androgen receptor modulators (SARMs) are an emerging class of therapeutics targeted to cachexia, sarcopenia, and hypogonadism treatment. LGD-4033 is a SARM which has been included on the Prohibited List annually released by the World Anti-Doping Agency (WADA). The aim of the present work was the investigation of the metabolism of LGD-4033 in a human excretion study after administration of an LGD-4033 supplement, the determination of the metabolites' excretion profiles with special interest in the determination of its long-term metabolites, and the comparison of the excretion time of the phase I and phase II metabolites. The results were also compared to those derived from previous LGD-4033 studies concerning both in vitro and in vivo experiments. Supplement containing LGD-4033 was administered to one human male volunteer and urine samples were collected up to almost 21 days. Analysis of the hydrolyzed (with ß-glucuronidase) as well as of the non-hydrolyzed samples was performed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) in negative ionization mode and revealed that, in both cases, the two isomers of the dihydroxylated metabolite (M5) were preferred target metabolites. The gluco-conjugated parent LGD-4033 and its gluco-conjugated metabolites M1 and M2 can be also considered as useful target analytes in non-hydrolyzed samples. The study also presents two trihydroxylated metabolites (M6) identified for the first time in human urine; one of them was recently reported in an LGD-4033 metabolism study in horse urine and plasma.

[The determination of pyrovaleron in the urine by gas chromatography in the combination with the method of extractive chilling-out and centrifugation]. / Gazokhromatograficheskoe opredelenie pirovalerona v moche metodom ékstraktsionnogo vymorazhivaniia v sochetanii s tsentrifugirovaniem.

The express-method for the determination of pyrovaleron in the urine based on the combination with the method of extractive freezing-out and centrifugation of the samples as the preliminary stage of the preparation of a biological object for the analysis. The identification and quantitative determination of the substance of interest were performed using gas chromatography with nitrogen-selective detection. The preparation of the samples was carried out as a single-step procedure no longer than 30 min in duration. The limit of alpha-pyrovaleron detection in the urine was estimated at 1 mcg/ml. Its concentration after extraction from the urine increased by a factor of more than nine.

Behaviour of hygrine and cuscohygrine in illicit cocaine production establishes their use as markers for chewing coca leaves in contrast with cocaine abuse.

Hygrine (HYG) and cuscohygrine (CUS) are natural alkaloids of coca leaves but are not found in illicit cocaine seizures. Therefore, they were proposed as markers for coca chewing in contrast to cocaine abuse in urine and hair testing. In order to examine at which step of the illegal cocaine production these compounds are lost, coca leaves were processed according to an authentic procedure by extraction with lime and kerosene, re-extraction with sulphuric acid, and precipitation of coca paste with ammonia. Non-extracted and extracted coca leaves, acidic extract and coca paste were analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for cocaine, ecgonine methyl ester (EME), cinnamoylcocaine (CIN), HYG, and CUS. It follows from the results that under these conditions, HYG and CUS are extracted only to a minor extent by kerosene and are not precipitated from the acidic re-extract in the coca paste. Due to this behaviour in illegal cocaine production, they fulfil the conditions as markers for coca chewing in an optimal way. However, for unambiguous discrimination between coca chewing and cocaine abuse in human samples, additional markers of manufactured cocaine are required. Copyright © 2016 John Wiley & Sons, Ltd.

Reference region modeling approaches for amphetamine challenge studies with [11C]FLB 457 and PET.

Detecting fluctuations in synaptic dopamine levels in extrastriatal brain regions with [(11)C]FLB 457 and positron emission tomography (PET) is a valuable tool for studying dopaminergic dysfunction in psychiatric disorders. The evaluation of reference region modeling approaches would eliminate the need to obtain arterial input function data. Our goal was to explore the use of reference region models to estimate amphetamine-induced changes in [(11)C]FLB 457 dopamine D2/D3 binding. Six healthy tobacco smokers were imaged with [(11)C]FLB 457 at baseline and at 3 hours after amphetamine (0.4 to 0.5 mg/kg, per os) administration. Simplified reference tissue models, SRTM and SRTM2, were evaluated against the 2-tissue compartmental model (2TC) to estimate [(11)C]FLB 457 binding in extrastriatal regions of interest (ROIs), using the cerebellum as a reference region. No changes in distribution volume were observed in the cerebellum between scan conditions. SRTM and SRTM2 underestimated binding, compared with 2TC, in ROIs by 26% and 9%, respectively, with consistent bias between the baseline and postamphetamine scans. Postamphetamine, [(11)C]FLB 457 binding significantly decreased across several brain regions as measured with SRTM and SRTM2; no significant change was detected with 2TC. These data support the sensitivity of [(11)C]FLB 457 for measuring amphetamine-induced dopamine release in extrastriatal regions with SRTM and SRTM2.

Intracerebral antioxidant ability of mature rats after neonatal hypoxic-ischemic brain injury estimated using the microdialysis-electron spin resonance method.

AIM: The intracerebral antioxidant ability of mature rats after neonatal hypoxic-ischemic (HI) brain injury was estimated using the microdialysis-electron spin resonance method. MATERIAL AND METHODS: Seven-day-old Wistar rats were subjected to a modified Levine's procedure for producing HI brain injury. After HI insult, pups were returned and reared with their dams. Seven weeks after HI insult, their intracerebral antioxidant abilities were measured using the microdialysis-electron spin resonance method after the intraperitoneal injection of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl. Ascorbic acid, L-cysteine, and glutathione (GSH) were also determined. The rats without HI insult were used as a control. RESULTS: The decay rate of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl in the non-ligated side of the cerebral hemisphere of the HI group was significantly larger than that of the control group. The amounts of ascorbic acid in the perfusate from the non-ligated side of the HI group were about four times larger than those of the control group. The amounts of L-cysteine and GSH of the HI group were about 10 times larger than those of the control group. CONCLUSIONS: The antioxidant ability in the non-ligated sides of the cerebral hemispheres of the mature rats 7 weeks after neonatal HI insult was higher than that of the control group. Higher amounts of ascorbic acid and GSH supported the higher antioxidant ability. The increase of the intracerebral antioxidant ability of the non-ligated side indicates the compensation of motor function for the lost side. The present results should offer important insights into the prognosis for hypoxic-ischemic encephalopathy.

Possibilities for discrimination between chewing of coca leaves and abuse of cocaine by hair analysis including hygrine, cuscohygrine, cinnamoylcocaine and cocaine metabolite/cocaine ratios.

Contrary to the illegal use of any form of manufactured cocaine, chewing of coca leaves and drinking of coca tea are allowed and are very common and socially integrated in several South American countries. Because of this different legal state, an analytical method for discrimination between use of coca leaves and abuse of processed cocaine preparations is required. In this study, the applicability of hair analysis for this purpose was examined. Hair samples from 26 Argentinean coca chewers and 22 German cocaine users were analysed for cocaine (COC), norcocaine (NC), benzoylecgonine (BE), ecgonine methyl ester (EME), cocaethylene (CE), cinnamoylcocaine (CIN), tropacocaine (TRO), cuscohygrine (CUS) and hygrine (HYG) by hydrophilic interaction liquid chromatography (HILIC) in combination with triplequad mass spectrometry (MS/MS) and hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS). The following concentrations (range, median, ng/mg) were determined in hair of the coca chewers: COC 0.085-75.5, 17.0; NC 0.03-1.15, 0.12; BE 0.046-35.5, 6.1; EME 0.014-6.0, 0.66; CE 0.00-13.8, 0.38; CIN 0.005-16.8, 0.79; TRO 0.02-0.16, 0.023; CUS 0.026-26.7, 0.31. In lack of a reference substance, only qualitative data were obtained for HYG, and two metabolites of CUS were detected which were not found in hair of the cocaine users. For interpretation, the concentrations of the metabolites and of the coca alkaloids in relation to cocaine were statistically compared between coca chewers and cocaine users. By analysis of variance (ANOVA) significant differences were found for all analytes (α = 0.000 to 0.030) with the exception of TRO (α = 0.218). The ratios CUS/COC, CIN/COC and EME/COC appeared to be the most suitable criteria for discrimination between both groups with the means and medians 5-fold to 10-fold higher for coca chewers and a low overlap of the ranges between both groups. The same was qualitatively found for HYG. However, these criteria cannot exclude cocaine use in addition to coca chewing. In this regard screening for typical cutting agents can be helpful and led to the detection of levamisole (21×), lidocaine (6×) and paracetamol (3×) in the 22 samples from German cocaine users, whereas no levamisole, lidocaine (3×) and paracetamol (1×) were found in hair from the Argentinean coca chewers. These criteria have to be confirmed for South American cocaine consumers including smokers of coca paste and may be different because of different composition of the drug and other use habits.

A study on photodegradation of methadone, EDDP, and other drugs of abuse in hair exposed to controlled UVB radiation.

The drug content of hair may be affected by washing, chemical or thermal treatments, the use of cosmetics, or exposure to the environment. Knowledge concerning the effect of natural or artificial light on drug content in hair can be helpful to the forensic toxicologist, in particular when investigating drug concentrations above or below pre-determined cut-offs. The photodegradation of methadone and its metabolite, 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was studied in authentic positive hair samples by comparing drug concentrations determined by liquid chromatrography-high resolution mass spectrometry before and after exposure to UVB light (in vivo study). The same approach was applied in order to investigate the light sensitivity of opiates (6-monoacetylmorphine and morphine) and cocainics (cocaine and benzoylecgonine) in true positive hair. The yields of photodegradation were calculated for each drug class in eight different positive hair samples irradiated by UVB at 300 J/cm(2) obtaining averages, ranges and standard deviations. In parallel, the photostability of all the compounds as 10(-5) -10(-4) M standard solutions in methanol were examined by means of UVB light irradiation in the range 0-100 J/cm(2) followed by UV/Vis spectroscopic analysis and direct infusion electrospray ionization-high resolution mass spectrometry (in vitro study). In hair, methadone was shown to be significantly affected by light (photodegradation of 55% on average), while its metabolite EDDP proved to be more photostable (17%). 6-monoacetylmorphine, morphine, benzoylecgonine, and cocaine were more photostable than methadone in vivo (on average, 21%, 17%, 20%, and 11% of degradation, respectively). When irradiated in standard solutions, the target molecules exhibited a larger photodegradation than in vivo with the exception of cocaine (photodegradation for methadone up to 70%, 6-monoacetylmorphine and morphine up to 90%, benzoylecgonine up to 67%, cocaine up to 15%). Some factors possibly affecting the yields of photodegradation in hair and partially explaining the differences observed between the in vivo and the in vitro studies were also investigated, such as the colour of hair (the role of melanin) and the integrity of the keratin matrix.

Sensomics analysis of key hazelnut odorants (Corylus avellana L. 'Tonda Gentile') using comprehensive two-dimensional gas chromatography in combination with time-of-flight mass spectrometry (GC×GC-TOF-MS).

Comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) has been used a few times to identify and quantitate single aroma-active compounds, but the capability of this technique to monitor a complete set of key odorants evoking the aroma of a given food in one run has not been exploited so far. A fast, multiodorant analysis using GC×GC-TOF-MS in combination with stable isotope dilution assays (SIDA) was developed to quantitate the entire set of aroma compounds, the sensometabolome, of raw and roasted hazelnuts ( Corylus avellana L. 'Tonda Gentile') previously established by GC-olfactometry. The capability of the method to evaluate the aroma contribution of each sensometabolite was evaluated by introducing a new term, the limit of odor activity value (LOAV), indicating whether a given aroma compound can be determined down to an odor activity value (OAV) of 1 (odor activity value = ratio of concentration to odor threshold). The advantage of the new method was proven by comparing the performance parameters with a traditional one-dimensional approach using GC-ion trap mass-spectrometry (GC-IT-MS). The results showed that the detector linearity and sensitivity of GC×GC-TOF-MS was on average higher by a factor of 10 compared to GC-IT-MS, thus enabling the quantitation of the aroma relevant amounts of 22 key odorants of hazelnuts in one run of the 30 aroma-active compounds. Seven novel isotopically labeled internal standards were synthesized to meet the analytical requirements defined by electron impact ionization in TOF-MS, that is, to keep the label. On the basis of the quantitative results obtained, it was possible to closely mimic the aroma of raw and roasted 'Tonda Gentile' hazelnuts by preparing an aroma recombinate containing the key odorants at their natural concentrations occurring in the nuts.

Hygrine and cuscohygrine as possible markers to distinguish coca chewing from cocaine abuse in workplace drug testing.

Cocaine abuse is widespread all over the world, and is performed generally by sniffing, injecting or smoking cocaine or crack. The distinction between the recreational use of cocaine from the practice of the so called "coqueo" is still an issue in those countries where this habit is diffused and where it is not considered an addiction, by this reason is necessary to develop a method for to distinguish the coca chewers and cocaine abusers. The use of an unique marker to distinguish between cocaine abuse and chewing of coca leaves is of fundamental importance in those countries where this habit is diffused. Certain alkaloids of the leaves of Erythroxylum coca are lost during the process of extraction/purification of cocaine and it is not possible to find them neither in seizures of chlorhidrate of cocaine nor urine samples of cocaine abusers. These markers are the hygrine and cuscohygrine that are present in the leaves of E. coca. A fast GC/MS method involving a liquid:liquid extraction procedure with tertbutylmethylether (TBME) is proposed for the determination of some alkaloids in cocaine leaves, cocaine seizures and biological samples. All specimens were alkalinized to pH 9 with a carbonate/bicarbonate buffer and then extracted with TBME. The analysis was carry out by GC/MS with electron impact at 70 eV and in full scan mode. The results demonstrate that hygrine and cuscohygrine are not found neither in the urine of cocaine abusers nor in cocaine seizures. For this reason this compounds could be considered as markers of coca chewing. This developed method permits to distinguish coca chewing from cocaine abuse in workplace drug testing through the analysis of urine samples.

Methadone concentrations in blood, plasma, and oral fluid determined by isotope-dilution gas chromatography-mass spectrometry.

Methadone (MTD) is widely used for detoxification of heroin addicts and also in pain management programs. Information about the distribution of methadone between blood, plasma, and alternative specimens, such as oral fluid (OF), is needed in clinical, forensic, and traffic medicine when analytical results are interpreted. We determined MTD and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in blood, plasma, blood cells, and OF by gas chromatography-mass spectrometry (GC-MS) after adding deuterium-labeled internal standards. The analytical limits of quantitation for MTD and EDDP by this method were 20 and 3 ng/mL, respectively. The amounts of MTD and EDDP were higher in plasma (80.4 % and 76.5 %) compared with blood cells (19.6 % and 23.5 %) and we found that repeated washing of blood cells with phosphate-buffered saline increased the amounts in plasma (93.6 % and 88.6 %). Mean plasma/blood concentration ratios of MTD and EDDP in spiked samples (N = 5) were 1.27 and 1.21, respectively. In clinical samples from patients (N = 46), the concentrations of MTD in plasma and whole blood were highly correlated (r = 0.92, p < 0.001) and mean (median) plasma/blood distribution ratios were 1.43 (1.41). The correlations between MTD in OF and plasma (r = 0.46) and OF and blood (r = 0.52) were also statistically significant (p < 0.001) and the mean OF/plasma and OF/blood distribution ratios were 0.55 and 0.77, respectively. The MTD concentration in OF decreased as salivary pH increased (more basic). These results will prove useful in clinical and forensic medicine when MTD concentrations in alternative specimens are compared and contrasted.

Evaluation of the presence of drugs of abuse in tap waters.

A total of seventy samples of drinking water were tested for non-controlled and illicit drugs. Of these, 43 were from Spanish cities, 15 from seven other European countries, three from Japan and nine from seven different Latin American countries. The most frequently detected compounds were caffeine, nicotine, cotinine, cocaine and its metabolite benzoylecgonine, methadone and its metabolite EDDP. The mean concentrations of non-controlled drugs were: for caffeine 50 and 19 ng L(-1), in Spanish and worldwide drinking water respectively and for nicotine 13 and 19 ng L(-1). Illicit drugs were sparsely present and usually at ultratrace level (<1 ng L(-1)). For example, cocaine has mean values of 0.4 (Spain) and 0.3 ng L(-1) (worldwide), whereas for benzoylecgonine, these mean values were 0.4 and 1.8 ng L(-1), respectively. Higher concentrations of benzoylecgonine were found in Latin American samples (up to 15 ng L(-1)). No opiates were identified in any sample but the presence of methadone and EDDP was frequently detected. Total mean values for EDDP were 0.4 ng L(-1) (Spain) and 0.3 ng L(-1) (worldwide). Very few samples tested positive for amphetamines, in line with the reactivity of chlorine with these compounds. No cannabinoids, LSD, ketamine, fentanyl and PCP were detected.

The metabolism of [14C]-zibotentan (ZD4054) in rat, dog and human, the loss of the radiolabel and the identification of an anomalous peak, derived from the animal feed.

This paper presents an overview of a cross-species investigation of the metabolic fate of [(14)C]-zibotentan (ZD4054), with particular focus on the main analytical challenges encountered during the study. A combination of detection methods were used including HPLC coupled to UV, RAD and/or MS(MS), and (1)H NMR spectroscopy. The objective was to characterise and identify the major metabolites found in the circulation and excreta of rat and dog for comparison with those produced in human. Initial investigations in rat, using [(14)C]-labelled zibotentan positioned on the oxadiazole ring and HPLC-UV-RAD analysis, revealed seven labelled resolved metabolite peaks. Parallel analysis by HPLC-UV-MS (with in-source fragmentation) uncovered two additional metabolites, indicating loss of the radiolabel during biotransformation. Hence, in subsequent studies in rat, dog and human, dual-radiolabelled zibotentan was employed with the (14)C-label positioned on the pyridine ring, which was shown to be less prone to metabolism. A total of 12 metabolites were found in the excreta and plasma in all species. One of these metabolites was found in the circulation in humans, which warranted further investigations. Characterisation of the isolated human circulating metabolite by (1)H NMR was complicated by the co-extraction of a matrix component with a similar UV-chromophore to zibotentan, which was identified as daidzein, an isoflavone derived from the animal feed.

Comparative EPR study of different macrophage types stimulated for superoxide and nitric oxide production.

Despite the major impact of ROS on human health, their quantification remains difficult and requires an analytical approach, such as the EPR spin trap technique. In this study, a comparative EPR analysis of different macrophage types stimulated for superoxide and nitric oxide production was performed. U937 monocytes, J774A.1, RAW 264.7 and primary mouse (PMM) macrophages were included. In contrast to the U937 cells, all macrophages produced significant EPR signals after stimulation. The use of PMA as stimulator and CM-H as spin probe led to the highest response in EPR signals for detection of O(2)(.-) as nitroxide radical. A combination of LPS and IFN-gamma and the spin trap [Fe(DETC)(2)] turned out to be the best combination for the production and detection of intracellular NO spin adducts. In conclusion, this study established practical experimental conditions for the EPR analysis of O(2)(.-) and NO produced by different types of activated macrophages.

Exposure-response of 1,2:3,4-diepoxybutane-specific N-terminal valine adducts in mice and rats after inhalation exposure to 1,3-butadiene.

1,3-Butadiene (BD) is a known rodent and human carcinogen that is metabolized mainly by P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol. The individual epoxides vary up to 200-fold in their mutagenic potency, with DEB being the most mutagenic metabolite. It is important to understand the internal formation of the individual epoxides to assign the relative risk for each metabolite and to understand the molecular mechanisms responsible for extensive species differences in carcinogenicity. This study presents a comprehensive exposure-response for the formation of the DEB-specific N,N-(2,3-dihydroxy-1,4-butadiyl)valine (pyr-Val) in mice and rats. Using nano-ultra high pressure liquid chromatography-tandem-mass spectrometry allowed analysis of pyr-Val in mice and rats exposed to BD as low as 0.1 and 0.5 ppm BD, respectively, and demonstrated significant differences in the amounts and exposure-response of pyr-Val formation. Mice formed 10- to 60-fold more pyr-Val compared to rats at similar exposures. The formation of pyr-Val increased with exposures, and the formation was most efficient with regard to formation per parts per million BD at low exposures. While formation at higher exposures appeared linear in mice, in rats formation saturated at exposures > or = 200 ppm for 10 days. In rats, amounts of pyr-Val were lower after 20 days than after 10 days of exposure, suggesting that the lifespan of rat erythrocytes may be shortened following exposure to BD. This research supports the hypothesis that the lower susceptibility of rats to BD-induced carcinogenesis results from greatly reduced formation of DEB following exposure to BD.

Phylogenetic and chemical diversity of three chemotypes of bloom-forming lyngbya species (Cyanobacteria: Oscillatoriales) from reefs of southeastern Florida.

The cyanobacterial genus Lyngbya includes free-living, benthic, filamentous cyanobacteria that form periodic nuisance blooms in lagoons, reefs, and estuaries. Lyngbya spp. are prolific producers of biologically active compounds that deter grazers and help blooms persist in the marine environment. Here, our investigations reveal the presence of three distinct Lyngbya species on nearshore reefs in Broward County, FL, sampled in 2006 and 2007. With a combination of morphological measurements, molecular biology techniques, and natural products chemistry, we associated these three Lyngbya species with three distinct Lyngbya chemotypes. One species, identified as Lyngbya cf. confervoides via morphological measurements and 16S rRNA gene sequencing, produces a diverse array of bioactive peptides and depsipeptides. Our results indicate that the other two Lyngbya species produce either microcolins A and B or curacin D and dragonamides C and D. Results from screening for the biosynthetic capacity for curacin production among the three Lyngbya chemotypes in this study correlated that capacity with the presence of curacin D. Our work on these bloom-forming Lyngbya species emphasizes the significant phylogenetic and chemical diversity of the marine cyanobacteria on southern Florida reefs and identifies some of the genetic components of those differences.