Critical Design Strategies Supporting Optimized Drug Release from Polymer-Drug Conjugates.

The importance of an adequate linking moiety design that allows controlled drug(s) release at the desired site of action is extensively studied for polymer-drug conjugates (PDCs). Redox-responsive self-immolative linkers bearing disulfide moieties (SS-SIL) represent a powerful strategy for intracellular drug delivery; however, the influence of drug structural features and linker-associated spacers on release kinetics remains relatively unexplored. The influence of drug/spacer chemical structure and the chemical group available for conjugation on drug release and the biological effect of resultant PDCs is evaluated. A "design of experiments" tool is implemented to develop a liquid chromatography-mass spectrometry method to perform the comprehensive characterization required for this systematic study. The obtained fit-for-purpose analytical protocol enables the quantification of low drug concentrations in drug release studies and the elucidation of metabolite presence. and provides the first data that clarifies how drug structural features influence the drug release from SS-SIL and demonstrates the non-universal nature of the SS-SIL. The importance of rigorous linker characterization in understanding structure-function correlations between linkers, drug chemical functionalities, and in vitro release kinetics from a rationally-designed polymer-drug nanoconjugate, a critical strategic crafting methodology that should remain under consideration when using a reductive environment as an endogenous drug release trigger.

Olanzapine Effects on Parvalbumin/GAD67 Cell Numbers in Layers/Subregions of Dorsal Hippocampus of Chronically Socially Isolated Rats.

Depression is linked to changes in GABAergic inhibitory neurons, especially parvalbumin (PV) interneurons, which are susceptible to redox dysregulation. Olanzapine (Olz) is an atypical antipsychotic whose mode of action remains unclear. We determined the effect of Olz on PV-positive (+) and glutamate decarboxylase 67 (GAD67) + cell numbers in the layers of dorsal hippocampus (dHIPP) cornu ammonis (CA1-CA3) and dentate gyrus (DG) subregions in rats exposed to chronic social isolation (CSIS), which is an animal model of depression. Antioxidative enzymes and proinflammatory cytokine levels were also examined. CSIS decreased the PV+ cell numbers in the Stratum Oriens (SO) and Stratum Pyramidale (SP) of dCA1 and dDG. It increased interleukin-6 (IL-6), suppressor of cytokine signaling 3 (SOCS3), and copper-zinc superoxide dismutase (CuZnSOD) levels, and it decreased catalase (CAT) protein levels. Olz in CSIS increased the number of GAD67+ cells in the SO and SP layers of dCA1 with no effect on PV+ cells. It reduced the PV+ and GAD67+ cell numbers in the Stratum Radiatum of dCA3 in CSIS. Olz antagonizes the CSIS-induced increase in CuZnSOD, CAT and SOCS3 protein levels with no effect on IL-6. Data suggest that the protective Olz effects in CSIS may be mediated by altering the number of PV+ and GAD67+ cells in dHIPP subregional layers.

Combined effect of cover crops and bio-fertilizer on sustainable popcorn maize production.

Cover crops play an important role in low-input cropping systems, increasing the use of agro-ecosystem services. Due to the lack of information about the impact of cover crops and bio-fertilizers on popcorn maize (Zea mays everta Sturt.) growth and yield quality, especially the popping volume and nutritive quality, such as concentrations of protein and mineral elements, this research aimed to provide essential information. The interrelation between popcorn maize productivity and quality with important groups of soil microorganisms presents additional novelty. The results demonstrated that field pea is a beneficial cover crop, especially when combined with a bio-fertilizer, supporting the accumulation of maize biomass, chlorophyll, yield potential, and the concentrations of protein, Ca, Mg, Fe, and Zn. In addition, field pea residues promoted N-fixing bacteria, and the number of total microorganisms, especially actinomycetes and decomposing bacteria, which could promote nutrient uptake and grain quality. Residues of cover crop mixtures, common vetch + winter oats and field pea + winter oats, promoted the total number of microorganisms in the soil, and up to the end of vegetation, a greater number of decomposition and ammonification microorganisms were found, especially when the bio-fertilizer was applied, which consequently could support greater maize biomass. Popping volume, as a main trait of popcorn maize, had the highest value in the common vetch + winter oats variant, supporting again the statement that quality traits could be enhanced in sustainable production. Unlike living cover crops, mulch mainly affected soil microbial communities and promoted the development of actinomycetes and cellulolytic microorganisms during the growing season. The results of this research could contribute to the development of sustainable popcorn maize production for improved grain quality. They could also serve as a basis for isolating beneficial soil microorganisms to develop new bio-fertilizers that could improve maize production in synergy with cover crops.

Transplantation of Human-Fetal-Spinal-Cord-Derived NPCs Primed with a Polyglutamate-Conjugated Rho/Rock Inhibitor in Acute Spinal Cord Injury.

Neural precursor cell (NPC) transplantation represents a promising therapy for treating spinal cord injuries (SCIs); however, despite successful results obtained in preclinical models, the clinical translation of this approach remains challenging due, in part, to the lack of consensus on an optimal cell source for human neuronal cells. Depending on the cell source, additional limitations to NPC-based therapies include high tumorigenic potential, alongside poor graft survival and engraftment into host spinal tissue. We previously demonstrated that NPCs derived from rat fetal spinal cords primed with a polyglutamate (PGA)-conjugated form of the Rho/Rock inhibitor fasudil (PGA-SS-FAS) displayed enhanced neuronal differentiation and graft survival when compared to non-primed NPCs. We now conducted a similar study of human-fetal-spinal-cord-derived NPCs (hfNPCs) from legal gestational interruptions at the late gestational stage, at 19-21.6 weeks. In vitro, expanded hfNPCs retained neural features, multipotency, and self-renewal, which supported the development of a cell banking strategy. Before transplantation, we established a simple procedure to prime hfNPCs by overnight incubation with PGA-SS-FAS (at 50 µM FAS equiv.), which improved neuronal differentiation and overcame neurite-like retraction after lysophosphatidic-acid-induced Rho/Rock activation. The transplantation of primed hfNPCs into immune-deficient mice (NU(NCr)-Foxn1nu) immediately after the eighth thoracic segment compression prompted enhanced migration of grafted cells from the dorsal to the ventral spinal cord, increased preservation of GABAergic inhibitory Lbx1-expressing and glutamatergic excitatory Tlx3-expressing somatosensory interneurons, and elevated the numbers of preserved, c-Fos-expressing, activated neurons surrounding the injury epicenter, all in a low percentage. Overall, the priming procedure using PGA-SS-FAS could represent an alternative methodology to improve the capabilities of the hfNPC lines for a translational approach for acute SCI treatment.

Olanzapine poisoning in patients treated at the National Poison Control Centre in Belgrade, Serbia in 2017 and 2018: a brief review of serum concentrations and clinical symptoms.

Olanzapine is a thienobenzodiazepine class antipsychotic that strongly antagonises the 5-HT2A serotonin receptor, but acute poisonings are reported rarely. Symptoms of an overdose include disorder of consciousness, hypersalivation, myosis, and coma. Serum concentration higher than 0.1 mg/L is toxic, while concentration above 1 mg/L can be fatal. Here we report key data about 61 patients admitted to the National Poison Control Centre in Belgrade, Serbia over olanzapine poisoning in 2017 and 2018. The ingested doses ranged from 35 to 1680 mg, and time from ingestion to determination from two to 24 hours. In 34 patients olanzapine serum concentrations were in the therapeutic range and in 27 in the toxic range. In five patients they were higher than fatal, but only one patient died. The most common symptoms of poisoning were depressed consciousness (fluctuating from somnolence to coma), tachycardia, hypersalivation, hypotension, myosis, and high creatine kinase. All patients but one recovered fully after nonspecific detoxification and symptomatic and supportive therapy.

Depletion of Mannose Receptor-Positive Tumor-associated Macrophages via a Peptide-targeted Star-shaped Polyglutamate Inhibits Breast Cancer Progression in Mice.

Although many studies have explored the depletion of tumor-associated macrophages (TAM) as a therapeutic strategy for solid tumors, currently available compounds suffer from poor efficacy and dose-limiting side effects. Here, we developed a novel TAM-depleting agent ("OximUNO") that specifically targets CD206+ TAMs and demonstrated efficacy in a triple-negative breast cancer (TNBC) mouse model. OximUNO comprises a star-shaped polyglutamate (St-PGA) decorated with the CD206-targeting peptide mUNO that carries the chemotherapeutic drug doxorubicin (DOX). In the TNBC model, a fluorescently labeled mUNO-decorated St-PGA homed to CD206+ TAMs within primary lesions and metastases. OximUNO exhibited no acute liver or kidney toxicity in vivo. Treatment with OximUNO reduced the progression of primary tumor lesions and pulmonary metastases, significantly diminished the number of CD206+ TAMs and increased the CD8/FOXP3 expression ratio (indicating immunomodulation). Our findings suggest the potential benefit of OximUNO as a TAM-depleting agent for TNBC treatment. Importantly, our studies also represent a novel design of a peptide-targeted St-PGA as a targeted therapeutic nanoconjugate. Significance: A peptide-targeted nanoformulation of DOX exclusively eliminates mannose receptor+ TAMs in breast cancer models, generating response without off-target effects (a drawback of many TAM-depleting agents under clinical study).

Current hurdles to the translation of nanomedicines from bench to the clinic.

The field of nanomedicine has significantly influenced research areas such as drug delivery, diagnostics, theranostics, and regenerative medicine; however, the further development of this field will face significant challenges at the regulatory level if related guidance remains unclear and unconsolidated. This review describes those features and pathways crucial to the clinical translation of nanomedicine and highlights considerations for early-stage product development. These include identifying those critical quality attributes of the drug product essential for activity and safety, appropriate analytical methods (physical, chemical, biological) for characterization, important process parameters, and adequate pre-clinical models. Additional concerns include the evaluation of batch-to-batch consistency and considerations regarding scaling up that will ensure a successful reproducible manufacturing process. Furthermore, we advise close collaboration with regulatory agencies from the early stages of development to assure an aligned position to accelerate the development of future nanomedicines.

Therapeutic potential of polypeptide-based conjugates: Rational design and analytical tools that can boost clinical translation.

The clinical success of polypeptides as polymeric drugs, covered by the umbrella term "polymer therapeutics," combined with related scientific and technological breakthroughs, explain their exponential growth in the development of polypeptide-drug conjugates as therapeutic agents. A deeper understanding of the biology at relevant pathological sites and the critical biological barriers faced, combined with advances regarding controlled polymerization techniques, material bioresponsiveness, analytical methods, and scale up-manufacture processes, have fostered the development of these nature-mimicking entities. Now, engineered polypeptides have the potential to combat current challenges in the advanced drug delivery field. In this review, we will discuss examples of polypeptide-drug conjugates as single or combination therapies in both preclinical and clinical studies as therapeutics and molecular imaging tools. Importantly, we will critically discuss relevant examples to highlight those parameters relevant to their rational design, such as linking chemistry, the analytical strategies employed, and their physicochemical and biological characterization, that will foster their rapid clinical translation.

Effect of urine adulterants on commercial drug abuse screening test strip results.

Immunochromatographic strips for urine drug screening tests (UDSTs) are common and very suitable for drug abuse monitoring, but are also highly susceptible to adulterants kept in the household, which can significantly alter test results. The aim of this study was to see how some of these common adulterants affect UDST results in practice and whether they can be detected by sample validity tests with pH and URIT 11G test strips. To this end we added household chemicals (acids, alkalis, oxidizing agents, surfactants, and miscellaneous substances) to urine samples positive for amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), tetrahydrocannabinol, heroin, cocaine, or benzodiazepines (diazepam or alprazolam) and tested them with one-component immunochromatographic UDST strips. The UDST for cocaine resisted adulteration the most, while the cannabis test produced the most false negative results. The most potent adulterant that barely changed the physiological properties of urine specimens and therefore escaped adulteration detection was vinegar. Besides lemon juice, it produced the most false negative test results. In conclusion, some urine adulterants, such as vinegar, could pass urine specimen validity test and remain undetected by laboratory testing. Our findings raise concern about this issue of preventing urine tampering and call for better control at sampling, privacy concerns notwithstanding, and better sample validity tests.

Clinical and analytical experience of the National Poison Control Centre with synthetic cannabinoids.

A rising number of patients are being treated for overdosing with new psychoactive substances (NPS) available at the illegal drug market in Serbia. The aim of this study was to report clinical and analytical experience of the National Poison Control Centre of Serbia (NPCC) with synthetic cannabinoids (SCs) and point to the NPS available at the illegal drug market in our country. From January 2013 to December 2016, 58 patients (aged between 14 and 25) were treated for the effects of synthetic cannabinoids at the NPCC. Tachycardia was established in 53, mydriasis in 31, somnolence, nausea, vomiting, and agitation in 16, dizziness in 10, disorientation in 9, dyspnoea and chest pain in 4, and loss of consciousness, pallor, paraesthesia, muscle twitches, and short-term memory impairment in 2 patients. After receiving symptomatic and supportive treatment in the emergency ward, all patients had fully recovered within 8 h and were discharged shortly afterwards. Another part of the study was focused on the analysis of the products known under their local street names as "Biljni tamjan" (herbal incense), "Beli slez", and "Rainbow Special" and the analysis of urine sampled from the patients with gas chromatography - mass spectrometry and high performance liquid chromatography. The detected synthetic cannabinoids were AB-PINACA, JWH-018, JWH-122, JWH-210, 5F-AKB48, and MDMB-CHMICA in herbal products and AB-FUBINACA, AB-CHMINACA, and MDMB-CHMICA in the urine samples. Our findings have shown the great capacity of NPCC to I) monitor NPS abuse in Serbia, II) reliably detect SCs in illicit products and biological samples, and III) clinically manage the adverse effects in their users. Future commitments of the NPCC will include systematic collection of relevant data on SCs and their adverse effects, detection of changes in purity and composition of the controlled NPS-based products, and raising the public awareness of NPS to improve the effectiveness of the national Early Warning System.

Influence of different seasons during late gestation on Holstein cows' colostrum and postnatal adaptive capability of their calves.

Season may affect calves' thermal comfort and behavior, but the data related to the overall influence of seasonal variations on dams' colostrum and postnatal adaptive capability of calves are limited. The aim of this study was to measure the effects of a 49-day-long low air temperature (LAT) season (5.20 ± 0.46 °C mean air temperature) and a 53-day-long high air temperature (HAT) season (27.40 ± 0.39 °C mean air temperature) on dams' colostrum quality and physiological, biochemical, hormonal, and oxidative stress parameters of their calves during the first 7 days of life. The dams' colostrum was sampled at 2, 14, and 26 h after calving, before feeding of their calves. Calves' blood samples were taken before the first colostrum intake and on days 1, 2, 3, and 7 of life. Calves' physiological parameters were measured on days 0 and 7. HAT season significantly reduced the quality of dams' colostrum. The ingestion of the low-quality colostrum, combined with the thermal discomfort during HAT season, probably provoked impaired physiological, biochemical, hormonal, and oxidative stress parameters in samples taken from the post-colostral calves. Additionally, intravenous glucose tolerance test was performed on day 7, which suggested an enhanced insulin response in HAT season calves. This study highlights the importance of adequate supporting strategies for the care of the late gestation cows and postnatal calves during the HAT season.

Cytotoxic and Antimicrobial Activity of Dehydrozingerone based Cyclopropyl Derivatives.

A small series of 1-acetyl-2-(4-alkoxy-3-methoxyphenyl)cyclopropanes was prepared, starting from dehydrozingerone (4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one) and its O-alkyl derivatives. Their microbiological activities toward some strains of bacteria and fungi were tested, as well as their in vitro cytotoxic activity against some cancer cell lines (HeLa, LS174 and A549). All synthesized compounds showed significant antimicrobial activity and expressed cytotoxic activity against tested carcinoma cell lines, but they showed no significant influence on normal cell line (MRC5). Butyl derivative is the most active on HeLa cells (IC50 = 8.63 µm), while benzyl one is active against LS174 and A549 cell lines (IC50 = 10.17 and 12.15 µm, respectively).

Intravenous lipid emulsion in treatment of cardiocirculatory disturbances caused by glyphosate-surfactant herbicide poisoning.

Introduction: Glyphosate is the first widely used herbicide against weed in genetically modified crops. Though glyphosate itself has a low toxicity, commercial products are more dangerous because of increased toxicity due to surfactants addition. There is no specific antidote for the poisoning with glyphosate-surfactant (Gly-SH). In recent times, the efficacy of intravenous lipid emulsion (ILE) administration for the treatment of acute poisoning caused by Gly- SH has been investigated. Case Report: A 50-year-old man was admitted 3 hours after self-poisoning with herbicide containing glyphosate and polyoxyethyleneamine, as a surfactant. On admission, the patient was in a coma, hypotensive (80/50 mmHg) and without spontaneous breathing. Electrocardiogram showed widecomplex tachycardia, and arterial blood gas (ABG) revealed acidosis (pH 7.07). Conventional treatment included mechanical ventilation, intravenous fluids, bicarbonate and dopamine. As there was no improvement, ILE was started. The patient received 100 mL of 20% Intralipid® bolus followed by infusion of 400 mL over 20 minutes. Prior to expiration of infusion, a gradual rise in blood pressure was noted, and within 2 hours sinus rhythm was restored. Conclusion: This case report suggests that the use of ILE may be an additional option for the treatment of cardiocirculatory disturbances caused by commercial products of glyphosate herbicide.

Comorbidity of harmful use of alcohol in population of schizophrenic patients.

BACKGROUND: Numerous studies carried out during the last twenty years point to an increase of co-morbidity of harmful use of alcohole caused disorders in the population of schizophrenic patients. The results show rate of this kind of co-morbidity between 35 and 80%. The aims of the investigation are: establishing frequency of harmful use of alcohol in the patients with diagnosis of schizophrenia, observed against the population statistics data; determination of possible predictors of harmful use of alcohol in the population of schizophrenic patients (adolsecent bihevioural problems, child neuroticism); determination of heritage role in the development of the both nosologic entities and the analysis of the outcomes of harmful use of alcohol in the population of schizophrenic patients (suicide attempts, cognitive impairment). SUBJECTS AND METHODS: The population included 50 inpatients between 20 and 50 years, with primary diagnosis of schizophrenia. Diagnostic procedure was carried out by application: Structured clinical interview ICD 10 (Mini International Neuropsychiatric Interview), Structured questionnaire for the assessment characteristics and effects of harmful use of alcohol in the population of schizophrenics - modified version, Mini mental state scale and Heteroanamnestic questionnaire. Comparison was made between the patients with schizophrenia and the patients with co-morbidity. RESULTS: The results revealed significantly higher rate harmful use of alcohol co-morbidity in the male population. There is a prominent significant difference in alcoholism heritage in co-morbidity group. A statistically significant difference between the sub-groups was found in the frequency of child neuroticism and adolescent behavioral problems. The results point to a higher suicidal risk and higher rate of cognitive impairment in the co-morbidity sub-group. CONCLUSIONS: Young male with schizophrenia and family history of alcoholism are especially susceptible by this type of co-morbidity. Presence of child neuroticism may represent "protective factor" for development of harmful use of alcohol. The study stressed some serious consequences of this type of co-morbidity: increase rate of suicide attempts, as well as more frequent development of organic brain tissue impairment.

Determination of carbamazepine in serum and saliva samples by high performance liquid chromatography with ultraviolet detection.

BACKGROUND/AIM: Carbamazepine is antiepileptic drug widely used for the treatment of epilepsy. Due to low therapeutic index of carbamazepine there is a need for routine measuring its concentrations in biological fluids. The aim of the study was to describe a method for concomitant determination of carbamazepine in the serum and saliva. METHODS: Separation of the drug from matrix is achieved by reversed-phase chromatography on a C18 column, with a mobile phase of methanol-water-acetic acid (65:34:1) at a flow-rate of 1.0 ml/min. Detection was effected by ultra-violet absorption at 285 nm. The total run time was 5 min. Samples were prepared by alkaline extraction (pH 10) using chlorophorm. RESULTS: Calibration curves were in the range 0.1-5 microg/mL for serum and saliva samples. Mean recoveries of spiked serum and saliva were 97.59 and 92.30%, respectively. Limits of detection (LOD) of carbamazepine in serum and saliva were 0.166 and 0.178 microg/mL, respectively. Limits of quantification (LOQ) in the serum and saliva were 0.237 and 0.226 microg/mL, respectively. The method precision was carried out with coefficient of variation of 2.10% and 4.03% for the serum and saliva, respectively. The obtained data showed that there was a strong correlation between saliva and serum concentrations (r = 0.9481, p < 0.001). CONCLUSION: The method described here is rapid, precise, accurate and simple, and can be used for quantitative determination of carbamazepine in human serum and saliva after therapy applying. Saliva samples could be used as an alternative matrix for therapeutic drug monitoring of this antiepileptic drug.

[Pharmacodynamic and pharmacokinetic effects of flumazenil and theophylline application in rats acutely intoxicated by diazepam].

BACKGROUND/AIM: The majority of symptoms and signs of acute diazepam poisoning are the consequence of its sedative effect on the CNS affecting selectively poli-synaptic routes by stimulating inhibitory action of GABA. The aim of the present study was to examine the effects of combined application of theophylline and flumazenil on sedation and impaired motor function activity in acute diazepam poisoning in rats. METHODS: Male Wistar rats were divided in four main groups and treated as follows: group I--with increasing doses of diazepam in order to produce the highest level of sedation and motor activity impairment; group II--diazepam + different doses of flumazenil; group III--diazepam + different doses of theophylline; group IV--diazepam + combined application of theophylline and flumazenil. Concentrations of diazepam and its metabolites were measured with LC-MS. The experiment was performed on a commercial apparatus for spontaneous motor-activity registration (LKB-Farad, Sweden). Assessment of diazepam-induced neurotoxic effects and effects after theophylline and flumazenil application was performed with rotarod test on a commercial apparatus (Automatic treadmill for rats, Ugo Basile, Italy). RESULTS: Diazepam in doses of 10 mg/kg and 15 mg/kg produced long-time and reproducible pharmacodynamic effects. Single application of flumazenil or theophylline antagonized effects of diazepam, but not completely. Combined application of flumazenile and theophylline resulted in best effects on diazepam-induced impairment of motoric activity and sedation. As a result of theopylline application there was better elimination of diazepam and its metabolites. CONCLUSION: Combined application of flumazenil and theophylline resulted in the best antidotal effects in the treatment of diazepam poisoned rats. These effects are a result of different mechanisms of their action, longer half-life of theophylline in relation to that of flumezenil and presumably the diuretic effect of theophylline.

[Analytical confirmation of lethal heroin overdose by the use of liquid chromatography methods].

BACKGROUND/AIM: Heroin is diacetylated morphine. Its ability to induce euphoria has led to its frequent abuse, giving rise to psychological and physical dependence. It has a short half-life, of approximately 2-6 min. In the brain, heroin undergoes deacetylation to 6-monoacetylmorphine (6-MAM) and morphine. Detection of 6-acetylmorphine in the urine is indicative of heroin use. The aim of this study was to compare sensitivity and reliability of two analytical methods, a multicolumn liquid chromatography system with UV scanning detector (HPLC-UV) and liquid chromatography-mass spectrometry detection (LC-MS) in opiate determining in post mortem material. METHODS: Post mortem samples (blood, urine and vitreous humor) were analyzed by liquid chromatography with UV and MS detection. The samples were prepared by liquid-liquid extraction with mixture chloroform-isopropanol (9:1). Separation was performed on C8 column with mobile phase composed of 55% acetonitrile-glacial acetic acid (99:1) and 45% 20 mM ammonium acetate. RESULTS: The analysis of blood samples, urine, and eye liquid by the use of multicolumn HPLC-UV method confirmed the presence of morphine in the samples of blood and urine, codeine only in urine, and 6-MAM in the samples of urine and eye liquid. Using LC-MS method morphine was confirmed in all of the samples, while codeine was confirmed in urine and in the sample of eye liquid. In the samples of eye liquid and urine 6-MAM was confirmed. CONCLUSION: For determination of opiates in post mortem material LC-MS technique is more sensitive and reliable as compared to multicolumn liquid chromatography.

[Determination of diazepam and its metabolites in serum by the use of liquid chromatography-mass spectrometry method].

BACKGROUND/AIM: Diazepam is a benzodiazepine anxyolitic. Metabolism of diazepam takes place in liver which generates pharmacologically active metabolites N-desmethyldiazepam, temazepam and oxazepam. The aim of this study was to develop and validate the method of liquid chromatographymass spectrometry (LC-MS) for separation and determination of diazepam and its active metabolites in the serum of rats samples after i.p. application of diazepam in a dose of 10 mg/kg. METHODS: The serum samples taken from Wistar rats, were used in LC-MS analysis after the application of 10 mg/kg of diazepam i.p. RESULTS: After alkaline extraction from the serum samples with diethylether and separation on a C18 reversed-phase column by using mobile phase methanolglacial acetic acid-water (50:1:49 v/v), diazepam and its metabolites were quantified. Determination was performed in a selective ion monitoring (SIM) mode, thereby the other exogenous and endogenous compounds did not interfere with this assay. Diazepam, N-desmethyldiazepam, oxazepam and temazepam were eluted in 14 minutes. The standard curve was linear in the range from 10-2 000 ng/ml. The limits of detection for diazepam, N-desmethyldiazepam, oxazepam and temazepam were 4.37, 3.13, 4.38 and 7.31 ng/ml, respectively. The limits of quantitation for diazepam, N-desmethyldiazepam, oxazepam and temazepam were 14.58, 10.41, 14.59 and 24.36 ng/ml, respectively. CONCLUSION: The described LC-MS is a simple, sensitive, specific and accurate method and could be used for routine identification and quantification of small concentrations of diazepam and its metabolites in biological fluids.

[Ecstasy tablets intoxication with lethal autcome].

BACKGROUND: Ecstasy, 3,4-methylenedioxymethamphetamine (MDMA), is a synthetic compound increasingly popular as a recreational drug. Tablets known as ecstasy contain MDMA, but may also contain caffeine, ephedrine, paramethoxyamphetamine, 3,4-methylenedioxyamphetamine (MDA), amphetamine, methamphetamine, and ketamine. After absorption MDMA is metabolized to MDA, 4-hydroxy-3-metoxymetamphetamine (HMMA) and 4-hydroxy-3-metoxyamphetamine (HMA). After that HMMA and HMA are conjugated and excreted by urine. The aim of this report was to confirm by toxicological post mortem analyses of poisoned person organs that ecstasy had been the cause of his death. CASE REPORT: We reported the death of a 17-year-old boy after the ingestion of ecstasy. MDMA and metabolites were determined by multicolumn high performance liquid chromatography with UV spectral detection (HPLC-UV). Toxicological tests showed the presence of MDMA in all samples. When examining post mortem material (the organs), the highest concentrations were measured in the stomach (835,97 microg/g) and kidney (801,14 microg/g). The minimal concentration was in the liver (22,26 microg/g). CONCLUSION: The obtained results of MDMA and its metabolites concentrations showed abuse of a high dose of ecstasy.