Rise of Pregabalin Poisoning and Abuse Cases in Serbia: A Ten-Year Retrospective Study.

Purpose: The aim of this study was to analyze data on gabapentinoid-related attendances to the National Poison Control Center of Serbia (NPCC), particularly abuse cases; to estimate its changes and to compare it with trends in national consumption rates of these drugs. We also aimed to analyze the main characteristics of the study population and to investigate the major clinical effects in poisoned patients. Patients and Methods: This is a retrospective study of patients admitted to the NPCC for acute poisoning involving gabapentinoids from 1 May 2012 to 1 October 2022. Results: There were 357 (95.5%) pregabalin-related and 17 (4.5%) gabapentin-related poisoning cases in 302 patients. Abuse of pregabalin was detected in 27.8% (84/302), while gabapentin abuse occurred in 0.7% (2/302) of all patients. A steady increase in rates of pregabalin poisoning and abuse cases strongly correlated with the increase in overall consumption of this drug, while there were no significant changes in rates of gabapentin consumption, poisoning and abuse rate during the study period. Most patients who abused pregabalin pregabalin were males (84.5%) and the median age was 26 years (range: 15-45 years). Almost 60% of patients who abused pregabalin (48/84) belonged to the migrant population. Co-ingestions occurred in 89.4% of pregabalin-related cases (319/357), resulting in more severe poisoning. The most often co-ingested drugs were benzodiazepines and among them clonazepam was detected in the largest number of cases. Conclusion: The poisoning and abuse cases involving pregabalin are on the rise in Serbia, which coincided with an increase in its overall consumption during the study period. Isolated pregabalin ingestions resulted in mild poisoning, although severe symptoms such as coma and bradycardia were recorded. When prescribing pregabalin to patients at risk of abuse caution is needed. Strengthening the measures for dispensing of pregabalin may reduce the risks associated with its abuse.

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.

Effects of memantine and its metabolite Mrz 2/373 on soman-induced inhibition of acetylcholinesterase in vitro.

Memantine is the non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, used in the treatment of Alzheimer's disease. It is also known that memantine pretreatment assured protection of skeletal muscles from poisoning with nerve agents and an interaction between memantine and AChE was proposed. In the study presented we examined interactions of memantine and its main metabolite (1-amino-3-hydroxymethyl-5-methyl adamantine, Mrz 2/373) with AChE in vitro as well as their effect on kinetics of the soman-induced AChE inhibition and aging. The results have shown that memantine and Mrz 2/373 exerted concentration-dependent inhibition of AChE, with Mrz 2/373 being a more potent inhibitor than the parent compound. Addition of soman 7.5 nmol/l induced gradual AChE inhibition that became almost complete after 20 min. Memantine (0.1, 0.5 and 1 mmol/l) and Mrz 2/373 (0.1, 0.5 and 1 mmol/l) concentration-dependently slowed down the AChE inhibition. After 30 min of incubation of AChE with soman, 5 min of aging and 20 min of reactivation by asoxime (HI-6 dichloride), AChE activity was 8.1% in control medium, 30.7% and 41.9% after addition of 1 and 10 mmol/l memantine, and 16.1% after addition of 1 mmol/l Mrz 2/373. It was concluded that it is possible that memantine and Mrz 2/373 can prevent AChE from inhibition by soman, which could, along with known memantine's neuroprotective activity, explain its potent antidotal effect in soman poisoning. The potential effect on aging of the soman-AChE complex warrants further studies.

Prophylactic potential of memantine against soman poisoning in rats.

BACKGROUND: Carbamates physostigmine and pyridostigmine have been used as a pretreatment against poisoning with nerve agents in order to reversibly inhibit and thus protect from irreversible inhibition a portion of acetylcholinesterase (AChE) in brain and respiratory muscles that is crucial for survival. Memantine, an adamantine derivative, has emerged as a promising alternative to carbamates, since it prevented the fasciculations and skeletal muscle necrosis induced by carbamates and organophosphates, including nerve agents. AIM: This experimental study was undertaken in order to investigate and compare the protective and behavioural effects of memantine and standard carbamates physostigmine and pyridostigmine in rats poisoned with soman and treated with atropine, oxime HI-6 and diazepam. Another goal was to elucidate the mechanisms of the antidotal effect of memantine and its potential synergism with standard antidotes against nerve agents. MATERIALS AND METHODS: Male Wistar rats were used throughout the experiments. In dose-finding experiments memantine was administered at dose interval 0-72 mg/kg sc 60 min before sc injection of soman. In time-finding experiments memantine was injected 18 mg/kg sc 0-1440 min before soman. Standard treatment antidotes - atropine 10 mg/kg, HI-6 50 mg/kg and diazepam 2.5 mg/kg - were administered im within 15 s post-exposure. Soman 0.75 LD50 was used to study its inhibitions of neuromuscular transmission on the phrenic nerve-diaphragm preparation in situ and of tissue AChE activity. Behavioural effects of the prophylactic antidotes were investigated by means of the rotarod test. Based on these data therapeutic index and therapeutic width was calculated for all three prophylactic agents. RESULTS: Memantine pretreatment (18 mg/kg sc) produced in rats poisoned with soman significantly better protective ratios (PRs) than the two carbamates - 1.25 when administered alone and 2.3 when combined with atropine pretreatment and 6.33 and 7.23 with atropine/HI-6 and atropine/HI-6/diazepam post-exposure therapy, respectively. The highest PR of 10.11 obtained in Atr/HI-6-treated rats was achieved after pretreatment with memantine 36 mg/kg. This additional protection lasted for 8 h. All three prophylactic regimens antagonised the soman-induced neuromuscular blockade, but the effect of memantine was fastest. Pretreatment with memantine assured higher AChE activity in brain and diaphragm than in unpretreated rats (46% vs 28% and 68% vs. 38%, respectively). All three prophylactic regimens affected the rotarod performance in rats, but the effect of memantine was relatively strongest. Memantine and pyridostigmine had lowest and highest therapeutic index and therapeutic width, respectively. CONCLUSIONS: Although memantine assures better and longer-lasting protection against soman poisoning in rats than the two carbamates, its small therapeutic index and narrow therapeutic width seriously limit its potential as a pretreatment agent. Despite its behavioural effects, memantine seems to be beneficial antidote when administered after soman, along with atropine/HI-6/diazepam therapy. Mechanism of the antidotal effect of memantine against soman poisoning appears to be a combination of AChE-protecting and NMDA receptor-blocking action.

Efficacy of antidotes and their combinations in the treatment of acute carbamate poisoning in rats.

BACKGROUND: Physostigmine and its analogues neostigmine, pyridostigmine and rivastigmine are carbamates nowadays used in many indications, including antidotal effects against antimuscarinic poisonings, reversal of competitive neuromuscular block, myasthenia gravis, Alzheimer's disease and prophylaxis against nerve agent intoxications. Use of these medicinal carbamates, but also of carbamate insecticides, created need for research into the potential and mechanisms of action of several antidotes against carbamate poisonings, including anticholinergics and oximes. AIM: The goal of this experimental study was to ascertain the life-preserving potential of anticholinergics atropine, hexamethonium and d-tubocurarine, oxime HI-6 and their combinations in rats poisoned with physostigmine or pyridostigmine. MATERIALS AND METHODS: Experiments were performed in Wistar rats. Carbamates were injected subcutaneously (sc) and antidotes intramuscularly (im). Median lethal dose (LD50) in animals treated with antidotes were compared to the ones in saline-treated rats and protective ratios (PRs) were calculated. Atropine (5, 10 and 20 mg/kg), hexamethonium (5, 10 and 20 mg/kg), d-tubocurarine (0.005, 0.010 and 0.020 mg/kg) and oxime HI-6 (25, 50 and 100 mg/kg) were used as monotherapies and in dual combinations, where atropine was the obligatory antidote. Biochemical experiments consisted in measuring of the cholinesterase activities in brain, whole blood and diaphragm in rats 5, 15, 30, 60, 120 and 240 min after poisoning with 0.8 LD50 of physostigmine or pyridostigmine. RESULTS: All the tested antidotes assured some degree of protection against the two carbamates. Atropine and hexamethonium produced better protection in physostigmine-poisoned rats, while d-tubocurarine and HI-6 were more efficacious in pyridostigmine-intoxicated animals. Oxime HI-6 50 mg/kg reactivated acetylcholinesterase (AChE) in brain inhibited by physostigmine and in diaphragm inhibited by pyridostigmine. CONCLUSIONS: Mechanism of physostigmine-induced lethal effect is predominantly central and it involves inhibition of brain AChE, while pyridostigmine produces the same effect exclusively outside the central nervous system, by inhibiting AChE in the respiratory muscles. As a consequence, increasing doses of atropine and their combination with hexamethonium assure excellent protection against physostigmine toxicity, while the best protection against pyridostigmine is provided by a strictly peripherally acting antinicotinic d-tubocurarine and bispyridinium oxime HI-6. The oxime acts as antidote against physostigmine and pyridostigmine poisoning by reactivating AChE in the brain and diaphragm, respectively.

Bioequivalence study of two formulations of itraconazole 100 mg capsules in healthy volunteers under fed conditions: a randomized, three-period, reference-replicated, crossover study.

BACKGROUND: The aim of the study was to evaluate the bioequivalence of two itraconazole 100 mg capsule formulations. RESEARCH DESIGN AND METHODS: The single-center, open-label, randomized, three-period, three-sequence, reference-replicated, cross-over study included 38 healthy subjects under fed conditions. In each study period (separated by a 14-day washout), a single oral dose of the test (T) or reference (R) product was administered. Blood samples were collected at pre-dose and up to 72.0 h after administration. The calculated pharmacokinetic parameters, based on the plasma concentrations of itraconazole and hydroxy itraconazole, were AUC0-72h, AUC0-∝, Cmax, Tmax, T1/2 and Kel. RESULTS: The 90% CI for the test/reference geometric means ratio for the parent compound, itraconazole, was in the range from 85.29% to 116.07% for AUC0-72h. Since the coefficient of variation (CV) for the reference product was 44.95% for Cmax, the 90% CI for this parameter for itraconazole was 93.49-133.78%, which was within the proposed limits of the EMA for bioequivalence of 72.15-138.59%. During the study, 4 subjects encountered a total of 14 mild adverse events. CONCLUSIONS: The use of the reference-scaling approach with 3-period design (TRR, RTR, and RRT) was an efficient way to demonstrate that two commercially available oral itraconazole formulations met the predetermined bioequivalence criteria.

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.

Determination of flumazenil in serum by liquid chromatography-mass spectrometry: Application to kinetics study in acute diazepam overdose.

BACKGOUND/AIM: Flumazenil is benzodiazepine receptor antagonist. It has been studied for a various indications, including reversal of sedation after surgery or diagnostic procedures, awakening of comatose patients in benzodiazepine overdose, or for symptomatic treatment of hepatic encephalopathy. Some drugs, like theophylline, may prolong its elimination half-life. Considering the long half-life of diazepam and its metabolites, concomitant use of theophylline may reduce the need for repeated dosing of flumazenil in patients with acute diazepam poisoning. The aim of this study was to introduce a reliable and accurate method for determining the concentration of flumazenil after therapeutic application in patients with acute poisoning, and using that method to assess whether the kinetics of flumazenil change in the presence of aminophylline (combination of theophylline and ethylenediamine in a 2:1 ratio) applied as concomitant therapy. METHODS: Blood samples from patients with acute diazepam poisoning that received flumazenil at the dose of 0.5 mg, or the same dose with 3 mg/kg of body weight of aminophylline, were collected 1, 3, 10, 30, 60, 120 and 240 min after its intravenous administration. Samples were prepared by solid-phase extraction on Oasis HLB cartridges with ethylacetate as extracting agens. Flumazenil was determined by liquid chromatography with mass spectrometry (LC-MS) in single ionmonitoring mode at m/z 304. Separation of flumazenil from matrix compound was performed on Lichrospher RP-8 column usingthe mixture of acidic acetonitrile and 20 mM of ammonium acetatein water (55 : 45) as a mobile phase. RESULTS: The applied analitycal method showed excellent recovery (94.65%). The obtained extracts were much cleaner than the extracts obtained by the sameextractant in the process of liquid-liquid extraction. The limit ofdetection of the LC-MS method described in this paper was 0.5 ng/mL and the limit of quantitation was 1 ng/mL. In the patientstreated with both flumazenil and aminophylline, the eliminationconstant for flumazenil was significantly lower and the elimination half-life was longer (p < 0.05) in comparison with the same parameters in.the patients who received flumazenil alone. CONCLUSION: The applied LC-MS method for the determination of flumazenil in serum samples of patients with acute diazepam poisoning is rapid, sensitive, precise and specific. Concomitant use with theophylline significantly prolonged elimination of flumazenil during the treatment of acute poisonings with diazepam.

Fatal cocaine intoxication in a body packer.

INTRODUCTION: 'Body packer' syndrome with severe intoxication or sudden death may happen in persons who smuggle drugs in their body cavities. In case of lethal outcome when carrying cocaine, it is important, but sometimes difficult to determine whether death was due to intoxication or due to other causes. Therefore, it is necessary not only to quantify cocaine and its metabolites in biological material, but also based on their distribution in body fluids and tissues to conclude whether it is acute intoxication. We described a well-documented case of fatal poisoning in a body packer and post mortem distribution of the drug in biological samples. CASE REPORT: A 26-year-old man was brought to hospital with no vital signs. Resuscitation measures started at once, but with no success. Autopsy revealed 66 packets of cocaine in his digestive tract, one of which was ruptured. Hyperemia of the most of all internal organs and pulmonary and brain edema were found. High concentrations of cocaine, its metabolites benzoylecgonine and ecgonine methyl ester, as well as cocaine adulteration levamisole were proven in the post mortem blood and tissues by liquid chromatography-mass spectrometry (LC-MC) method with selective-ion monitoring. CONCLUSION: The ratio of cocaine and its metabolites concentrations in the brain and blood obtained by LC-MS method can be used for forensic confirmation of acute intoxication with cocaine.

A randomized, open-label pharmacokinetic comparison of two oral formulations of fluconazole 150 mg in healthy adult volunteers.

BACKGROUND: Because of its systemic action, fluconazole is prescribed for a variety of fungal infections. However, therapeutic failure might result when a patient is switched between an innovator drug and a nonbioequivalent generic formulation. Pharmacokinetic (PK) studies investigating the bioequivalence of generic and innovator drugs can minimize such risks. OBJECTIVE: The aim of this study was to compare the PK profiles and relative bioavailabilities of 2 oral formulations of fluconazole: Diflucan (reference; Pfizer Corporation Austria GmbH, Wien, Austria) and Funzol (test; Bosnalijek d.d., Pharmaceutical and Chemical Industry, Sarajevo, Bosnia and Herzegovina), both prepared as capsules containing 150 mg of active drug. METHODS: A single oral dose of fluconazole was given under fasting conditions to healthy, white volunteers aged 18 to 55 years in this open-label, randomized, crossover study. A 3-week washout period was applied between each of the 2 doses. Serum samples were obtained before dosing and at various time points after dosing up to 144 hours and were analyzed for fluconazole concentration using a high-performance liquid chromatography-UV method. PK parameters representing the extent (AUC(0-infinity)) and rate (CmaX and T(max)) of absorption of fluconazole were obtained. An analysis of variance, a power analysis, 90% CI, and two 1-sided tests were used for statistical analysis of relative differences between the 2 drugs. Bioequivalence was concluded if the 90% CIs for the geometric mean ratios of AUC(0-infinity) and C(max) were between 0.80 and 1.25. A study investigator monitored the volunteers for adverse effects at 5 defined time points during the clinical part of the investigation. RESULTS: Thirteen men and 11 women (mean age, 33.3 years; mean weight, 73.6 kg) completed the study. The respective point estimates of the ratios of geometric means of log-transformed C(max) and AUC0(0-infinity) of fluconazole (test vs reference) were 0.985 and 1.047, with 90% CIs of 0.894 to 1.085 and 0.927 to 1.182, respectively. Differences in T(max) also did not reach statistical significance. No adverse effects were reported by the subjects or revealed by clinical or laboratory tests. CONCLUSIONS: The study failed to demonstrate any statistically significant differences in C(max) and AUCO(0-infinity) values between the test and reference formulations of oral fluconazole 150 mg in this small, select population of healthy volunteers. On that basis, and according to both the rate and extent of absorption, the test and reference formulations were considered bioequivalent.

Efficacy of trimedoxime in mice poisoned with dichlorvos, heptenophos or monocrotophos.

The aim of the study was to examine antidotal potency of trimedoxime in mice poisoned with three direct dimethoxy-substituted organophosphorus inhibitors. In order to assess the protective efficacy of trimedoxime against dichlorvos, heptenophos or monocrotophos, median effective doses and efficacy half-times were calculated. Trimedoxime (24 mg/kg intravenously) was injected 5 min. before 1.3 LD50 intravenously of poisons. Activities of brain, diaphragmal and erythrocyte acetylcholinesterase, as well as of plasma carboxylesterases were determined at different time intervals (10, 40 and 60 min.) after administration of the antidotes. Protective effect of trimedoxime decreased according to the following order: monocrotophos > heptenophos > dichlorvos. Administration of the oxime produced a significant reactivation of central and peripheral acetylcholinesterase inhibited with dichlorvos and heptenophos, with the exception of erythrocyte acetylcholinesterase inhibited by heptenophos. Surprisingly, trimedoxime did not induce reactivation of monocrotophos-inhibited acetylcholinesterase in any of the tissues tested. These organophosphorus compounds produced a significant inhibition of plasma carboxylesterase activity, while administration of trimedoxime led to regeneration of the enzyme activity. The same dose of trimedoxime assured survival of experimental animals poisoned by all three organophosphorus compounds, although the biochemical findings were quite different.

Biopharmaceutical characterization of sustained release matrix tablets based on novel carbomer polymers: formulation and in vivo investigation.

With the increased interest in modified release dosage forms and drug delivery systems, there is an increasing concern for biopharmaceutical characterization of the formulation in the early stages of drug product development. The main objectives of biopharmaceutical characterization are the in vitro and in vivo evaluation of the selected formulations in order to identify the factors influencing drug release; define the in vitro test methodology that would be predictive of drug products in vivo behavior and develop quantitative in vitro - in vivo correlation. The purpose of this study was to assess the potential of novel carbomer polymers, Carbopol 971P and Carbopol 71G, as a sustained release agents in matrix tablets containing high dosage drug substance. Although chemically identical, the two polymers exhibited substantially different drug release properties in vitro. Hypothetical in vivo drug release profiles were calculated by numerical deconvolution from cumulative urinary excretion data observed in vivo. The obtained results indicated that sound and reliable in vivo drug release profiles could be obtained from urinary excretion data and also, emphasized the need for in vitro testing under a range of experimental conditions in order to develop the biorelevant drug release methodology.

Serbia National Poison Control Centre: organization and current activities.

Ministry of Health of the former Federal Republic of Yugoslavia established the National Poison Control Centre in 1995. However, that was only the formally solution since clinical, analytical and experimental services in toxicology had worked independently for at least 40 years. Besides the Headquarters, NPCC has currently 2 main units, the Clinic of Emergency and Clinical Toxicology and Pharmacology and the Institute of Toxicology and Pharmacology. The latter is consisted of Toxicological Information Department, Department of Analytical Toxicology and Department of Experimental Toxicology and Pharmacology. The Mobile Toxicological Chemical Unit is a separate department that is activated from personnel of the NPCC in a case of chemical accidents and/or disasters. Clinical, information and analytical parts of NPCC have a 365-day/24-hour working service. The Clinic of Emergency and Clinical Toxicology and Pharmacology is a place where the intoxicated patients are treated, including those that need the intensive care measures. Toxicological Information Department uses the data from a self-made computer Database for different information purposes. Department of Analytical Toxicology is equipped with a lot of contemporary analytical equipment that is giving the opportunity of identification and quantification of chemicals/metabolites/degradation products in biological material, food, water, air and soil. Basic pharmacological and toxicological research of chemicals and pre-clinical investigations of antidotes are realized in the Department of Experimental Toxicology and Pharmacology. In terms of medical prevention and rational treatment of human poison exposures in Serbia, the current organization of NPCC has so far proven to be effective.

Relationship of hepatotoxicity and the target tissue dose of decabrominated diphenyl ether in subacutely exposed Wistar rats.

BACKGROUND/AIM: Based on numerous studies in animals, the most prominent toxic effects of decabrominated diphenyl ether (BDE-209) are observed in the liver, thyroid hormone homeostasis, reproductive and nervous systems. BDE-209 exhibits its toxic effects partly through the aryl hydrocarbon (Ah) receptor and consequent induction of hepatic microsomal enzymes. The aim of this study was to assess the hepatotoxic effect vs target tissue dose of BDE-209 in the subacutely orally exposed Wistar rats. METHODS: Effects were examined on male Wistar rats, weighing 200-240 g, exposed to doses of 1;000, 2,000 or 4,000 mg BDE-209/kg body weight (bw)/day by gavage during 28 days. Animals were treated according to the decision of the Ethics Committee of the Military Medical Academy, No 9667-1/2011. Evaluation of the hepatotoxic effect was based on: relative liver weight water and food intake, biochemical parameters of liver function [aspartate amino transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gama glutamyl transferase (γ-GT)], and oxidative stress parameters in liver homogenates [malondialdehiyde (MDA), superoxide dismutase (SOD), -SH] and morphological and pathohistological changes in the liver. For the assessment of internal dose-response relationship, lower confidence limit of Benchmark dose (BMDL) of 5% or 10% i.e. BMDL5 or BMDL10, were calculated using PROAST software. RESULTS: After the application of 1,000,2,000 or 4,000 mg BDE-209/kg bw/day, the concentrations of BDE-209 measured in liver were 0.269, 0.569 and 0.859 mg/kg of liver wet weight, (ww) respectively. Internal doses correlated with external (r = 0.972; p < 0.05) according to equation: internal dose (mg BDE-209/kg of liver ww) = 0.0002 x external dose (mg/kg bw/day) + 0.0622. Hepatotoxicity was demonstrated based on significant increase in AST and γ-GT activities and the degree of histopathological changes. The lowest BMDLs of 0.07228 mg BDE-209 /kg of liver ww, correlating to external dose of 39 mg/kg/day, indicated the increase of AST activity as the most sensitive biomarker of BDE-209 hepatotoxicity in subacutely exposed rats. CONCLUSION: The results of the present work add up to the issue ofBDE-209 toxicity profile with a focus on relationship between internal dose and hepatotoxicity. Critical internal dose for the effect on AST of 0.07 mg/kg of liver ww, corresponding to external dose of 39 mg/kg/ day, is the lowest dose ever observed among the studies on BDE-209 hepatotoxicity. For the persistent substances with low absorption rate such as BDE-209, critical effect based on internal dose in majority of cases is considered as more precisely deined than the effect established based on external dose, particularly.

Toxicokinetics and correlation of carbamazepine salivary and serum concentrations in acute poisonings.

BACKGROUND/AIM: Saliva is a body fluid which, like serum, can be used for determination of concentrations of certain drugs, both in pharmacotherapy as well as in acute poisonings. The aim of this study was to determine carbamazepine concentrations in both saliva and serum in acute poisoning in order to show if there is a correlation between the obtained values, as well as to monitor toxicokinetics of carbamazepine in body fluides. METHODS: Saliva and serum samples were obtained from 26 patients treated with carbamazepine and 20 patients acutely poisoned by the drug immediately after their admission in the Emergency Toxicology Unit. Determination of salivary and serum carbamazepine concentrations was performed by the validated high pressure liquid chromatography-ultraviolet (HPLC-UV) method. RESULTS: A significant correlation of salivary and serum carbamazepine concentrations in both therapeutic application and acute poisoning (r = 0.9481 and 0.9117, respectively) was confirmed. In acute poisonings the mean ratio between salivary and serum concentrations of carbamazepine (0.43) was similar to the mean ratio after its administration in therapeutic doses (0.39), but there were high inter-individual variations in carbamazepine concentrations in the acutely poisoned patients, as a consequence of different ingested doses of the drug. In acute poisoning the halftime of carbamazepine in saliva and serum was 12.57 h and 6.76 h, respectively. CONCLUSION: Our results suggest a possible use of saliva as an alternative biological material for determination of carbamazepine concentrations in therapeutic application and acute poisoning as well, and a possible extrapolation of the results obtained in saliva to serum concentrations of carbamazepine.

[Determination of morphine, codeine and 6-monoacetylmorphine in saliva of substance-abuse patients using HPLC/MS methods].

BACKGROUND/AIM: Saliva represents an alternative specimen for substances abuse determination in toxicology. Hence, the aim of this study was to optimize a method for saliva specimen preparation for heroin metabolites, morphine and 6-monoacetylmorphine (6-mam), and codeine determination by liquid chromatography-mass spectrometry (LC/MS), and to apply this method on saliva samples taken from the patients. METHODS: Saliva specimen was prepared using liqiud/liquid extraction of morphine, codeine and 6-mam by mixture of chloroform and isopropanol (9 : 1; v/v). Extracts were analysed by HPLC/MS technique: separation column Waters Spherisorb 5 microm, ODS2, 4.6 x 100 mm; mobile phase: ammonium acetate : acetonitile (80 : 20; v/v), mobile phase flow rate 0.3 mL/min; mass detection range: 100-400 m/z. Regression and correlation analyses were performed with the probalility level of 0.05. Concentrations of morphine, codeine and 6-mam were determined in saliva samples of the patients with "opiates" in urine identified by the test strips. RESULTS: Calibration for each analysed substance was done in the concentration range from 0.1 to 1 mg/L and the coefficient of correlation was R2 > 0.99. We obtained following calibration curves: y = 385531x + 14584; y = 398036x + 31542; and y = 524162x - 27105, for morphine, codeine and 6-mam, respectively. Recovery for morphine and codeine determination was 99%, while for 6-mam it was 94%. Limits of detection and quantification of a proposed method were 0.01 mg/L and 0.05 mg/L, respectively. Concentration of morphine in the saliva of the heroin users ranged between 0.54 and 5.82 mg/L, concentration of codeine between 0.05 and 5.33, and 6-mam between 0.01 and 0.68 mg/L. A statistically significant correlation between codeine and 6-mam concentrations was obtained. CONCLUSION: A proposed HPLC/MS method for morphine, codeine and 6-mam determination in saliva is accurate, simple, cheap and suitable for routine analysis and monitoring of heroin abuse.

[In vitro study of vitamins B1, B2 and B6 adsorption on zeolite].

BACKGROUND/AIM: Zeolites are the hydratised alumosilicates of alcali and earthalcali cations, which have a long three-dimensional crystal structure. Preparations on the basis of zeolites are used for adsorption of organic and nonorganic toxic substances and they, also, find more and more use in veterinary and human medicine and pharmacy. The aim of this study was to evaluate the possibilities of zeolite to adsorb vitamins B1, B2 and B6 in acid and neutral solutions, as well as the characteristics of the process (saturability, reversibility and competitiveness). METHODS: The specific and sensitive HPLC method with fluorescent detector was used for determination of vitamins B1, B2 and B6. Analyte separation and detection were carried out by applying the reverse-phase method on column C18. An in vitro experiment was done by testing the influence of pH value (2 and 7), concentration of vitamin solution (1, 2 and 5 mg/L), the length of contact with zeolite (10-180 min) and cation competitiveness on the exchange capacity, which is achieved by media and zeolite contact, as well as a possible vitamins desorption through changing pH value of the solution at 37 degrees C. Jon competitiveness was examined by adding commercial feed mixture (grower) with a defined content of the examined vitamins in zeolite solutions the pH = 2 and pH = 7. RESULTS: Vitamins B1, B2 and B6 were stable in both pH=2 and pH = 7 solutions at 37 degrees C, in the defined time intervals. In acid solution concentrations of vitamins significantly declined in the first 10 min, with no significant decline in further 30 min for all the three concentrations tests. In neutral solution, after the addition of 1% zeolite, decrease in vitamins concentrations was slightly lower than in acid solution, but also significant in the first 10 min of the contact with zeolite. It was found that zeolite, which adsorbed vitamins in acid solution, transferred in the neutral one released a significant quantity of adsorbed vitamins after 30 min of extraction on 37 degrees C. Vitamins B1, B2 and B6, from a commercial feed mixture in pH = 2 solution, at 37 degrees C, were significantly adsorbed on zeolite after 30 min of the contact (21.87%, 20.15% and 4.53%, respectively), while in neutral solution there was no statistically significant adsorption. Conclusion. Zeolite significantly adsorbs vitamins B1, B2 and B6 in acid and neutral solutions at 37 degrees C, already in the first 10 min of the contact. Adsorption was irreversible, but partly reversible after changing pH from acid to neutral. This is a significant ions competition for adsorption on zeolite in neutral solution, so no statistically significant vitamins B1, B2 and B6 adsorption occurs, while in acid solution competition is less, thus zeolite significantly adsorbs these vitamins, although in less degree than in conditions with no concurrent ions.

[Simultaneous determination of amoxicillin and clavulanic acid in the human plasma by high performance liquid chromatography-mass spectrometry (UPLC/MS)].

BACKGROUND/AIM: Quantitative analysis of amoxicillin and clavulanic acid in biological matrices requires sensitive and specific methods which allow determination of therapeutic concentration in 1 g/mL range. Analytical methods for determination of their concentrations in body fluids described in literature include high performance liquid chromatography coupled to UV detector (HPLC-UV) and liquid chromatography-mass spectrometry (LC-MS). The aim of this study was to develop sensitive and specific ultra performance liquid chromatography/mass spectrometry (UPLC/MS) method which could be used for the spectral identification and quantification of the low concentrations of amoxicillin and clavulanic acid in the human plasma. METHOD: A sensitive and specific UPLC/MS method for amoxicillin and clavulanic acid determination was developed in this study. The samples were taken from the adult healthy volunteers receiving per os one tablet of amoxicillin (875 mg) in combination with clavulanic acid (125 mg). RESULTS: Plasma samples were pretreated by direct deproteinization with perchloric acid. Quantification limit of 0.01 microg/ml for both amoxicillin and clavulanic acid was achieved. The method was reproducible day by day (RSD < 7%). Analytical recoveries for amoxicillin ranged from 98.82% to 100.9% (for concentrations of 1, 5 and 20 microg/mL), and recoveries for clavulanic acid were 99.89% to 100.1% (for concentrations of 1, 2 and 5 microg/mL). This assay was successfully applied to a pilot pharmacokinetic study in healthy volunteers after a single-oral administration of amoxicillin/clavulanic combination. The determined plasma concentrations of both amoxicillin and clavulanic acid were in the range of the expected values upon the literature data for HPLC-UV and LC-MS methods. CONCLUSION: The described method provided a few advantages comparing with LC/MS-MS method. The method is faster using running time of 5 minute, has lower limit of quantification (LOQ) and it could be used in pharmacokinetic studies of both amoxicillin and clavulanic acid.

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.