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Tramadol is a centrally acting analgesic drug that has been used clinically for the last two decades to treat moderate to moderately severe pain in humans. The present study investigated tramadol administration in horses by intravenous, intramuscular, oral as immediate-release and oral as sustained-release dosage-form routes. Seven horses were used in a four-way crossover study design in which racemic tramadol was administered at 2 mg/kg by each route of administration. Altogether, 23 blood samples were collected between 0 and 2880 min. The concentration of tramadol and its M1 metabolite were determined in the obtained plasma samples by use of an LC/MS/MS method and were used for pharmacokinetic calculations. Tramadol clearance, apparent volume of distribution at steady-state, mean residence time ( MRT) and half-life after intravenous administration were 26 ± 3 mL/min/kg, 2.17 ± 0.52 L/kg, 83 ± 10 min, and 82 ± 10 min, respectively. The MRT and half-life after intramuscular administration were 155 ± 23 and 92 ± 14 min. The mean absorption time was 72 ± 22 min and the bioavailability 111 ± 39%. Tramadol was poorly absorbed after oral administration and only 3% of the administered dose was found in systemic circulation. The fate of the tramadol M1 metabolite was also investigated. M1 appeared to be a minor metabolite in horses, which could hardly be detected in plasma samples. The poor bioavailability after oral administration and the short half-life of tramadol may restrict its usefulness in clinical applications. [ABSTRACT FROM AUTHOR]

The article discusses the use of dependent-absorption apparent volume distribution at steady state (Vss/F) on pharmacokinetic analysis. It mentions that pharmacokinetic analysis is limited particularly those that are based on administration data from extravascular drug. It also states that the use of (Vss/F) in pharmacokinetic analysis determined by compartmental analysis is harmful and is strongly discouraged.

This study aimed to evaluate associations between prenatal and childhood exposure to phthalates and prenatal exposure to polychlorinated biphenyls (PCBs) and the development of 4-year-old children. Urinary metabolites of five phthalates were measured in women upon delivery, as well as serum concentrations of four PCB congeners. Postnatal phthalate metabolites were measured from children's urine obtained at the time of developmental assessment. The primary outcome was cognitive function as evaluated by the Wechsler Preschool and Primary Scale of Intelligence (WPPSI-III) administered at 4 years. Secondary outcomes were motor function and response to sensory stimuli as evaluated by the Developmental Coordination Disorder Questionnaire (DCDQ) and Short Sensory Profile (SSP) that the mothers filled out, respectively. The study included 57 mother-child pairs. Higher maternal phthalate metabolite concentrations were inversely associated with WPPSI-III scores among boys and not among girls. After using linear regression models and controlling for confounding variables, we found that higher levels of monobenzyl phthalate (MBzP) were the ones associated with lower WPPSI-III scores (p=0.004, 95 %CI [-14.18; -3.16]), lower DCDQ scores (p=0.007, 95 %CI [-6.08; -1.17] and lower SSP scores (p=0.004, 95 %CI [-7.47; -1.79]). No association was found between child urinary phthalate metabolite concentrations or maternal PCB blood concentrations and developmental function. These findings indicate that higher prenatal phthalate metabolite levels may be associated with deficits in neurologic development of young boys., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: In-utero phthalate exposure was shown to be associated with shortened anogenital distance (AGD) in male newborns, but findings among female are inconsistent. While phthalate exposure among pregnant women in Israel is widespread, no study has examined the association with offspring AGD. The objective of the current study was to investigate the association between maternal phthalates urinary concentration and offspring AGD at time of delivery among a birth cohort in Israel., Methods: We measured spot urinary concentration of monobutyl phthalate (MBP), monobenzyl phthalate (MBzP), mono-2-ethyl-5-carboxypentyl phthalate (MECPP), mono-2-ethyl-5-hydroxyhexylphthalate (MEHHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP) among women presenting to the delivery room at Shamir Medical Center in Israel. Birthweight, length and AGD were measured in all newborns using a standardized protocol. Each AGD measurement was adjusted to weight (ano-genital index). Confounders included socio-demographic characteristics, comorbidities and obstetrical history. Univariate and multivariate analyses assessed the associations between phthalates, confounders and AGD., Results: Overall, 193 mother and infant were analyzed. All newborns were born at term and had normal Apgar scores. Mean maternal age was 32 ± 4.7 years old. Mean birth weight and pregnancy week were 3183 ± 498 g and 39 ± 1.3, respectively. Median (IQR) urinary phthalate concentration adjusted to creatinine (ug/g) were 3.96 (2.2-6.6), 1.22 (0.7-2), 10.84 (7-20.4), 6.36 (3.3-11.2) and 0.64 (0.4-1.1) for MBP, MBzP, MECPP, MEHHP and MEOHP, respectively. Univariate comparison showed a significant association between higher than median MBzP concentration, higher Ano-Fourchetal index (AFI: 4.4 vs. 4.1, p = 0.037) and Ano-clitoral index (ACI: 11.5 vs. 10.4, p = 0.032) in infants. Total urinary phthalates concentration ≥26.25 μg/g was significantly associated with smaller penile width index (3.5 vs. 3.7, p = 0.022), higher ACI (11.6 vs. 10.3, p = 0.013) and a trend towards significance for higher AFI (4.3 vs. 4.1, p = 0.055). Following multivariate linear regression only PWI remained significantly associated with total phthalate urinary concentration., Conclusions: Maternal urinary phthalates concentration at delivery were not associated with female AGD, but total urinary phthalate concentration were inversely associated with penile width., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Matitiahu Berkovich reports financial support and equipment, drugs, or supplies were provided by Environment and Health Fund. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)

Antimicrobial drugs and coccidiostat compounds are commonly used in poultry farming. These compounds are subsequently excreted and released into the environment via broiler litter (BL) and can re-enter the food chain as fertilizer or animal feed. Such residue in animal feed can encourage the appearance of antibiotic-resistant bacteria as well as toxicity. Most analytical methods used to identify and quantitate these drug residues are traditional, and are specific to some antimicrobials and present limitations in assessing complex matrixes like BL. The aim of this study was to develop a multi-residue analytic method for assessing 30 antimicrobial drugs and coccidiostats associated with BL. We investigated the presence and the effects of biotic stack treatment on the degradation of drug residue in BL. Liquid-liquid extraction (LLE) and solid phase extraction (SPE) were replaced by Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) clean-up steps and detected by liquid chromatography mass spectrometry (LC/MS/MS). Results show that a wide spectrum of residues were detected from 0.4 to 8.9 mg kg -1 . Following lab-scale stacking treatment, tilmicosin and eight coccidiostats persisted in BL (26-100%). This research supports the need for better understanding, regulation, and management of the use of BL that might carry a high risk of residue drugs.

The use of antimicrobial drugs and coccidiostats in poultry farming is widespread, with a significant proportion of these drugs being excreted and released into the environment. The residues of such drugs in poultry litter (PL) can result in the development of antibiotic-resistant bacteria. The impact of different biotic treatments (aerobic, anaerobic, and stacking) on broiler litter (BL) before its use as animal feed has not been studied extensively, nor have the differences between antimicrobial-dependent and independent broiler farms been investigated. This study aimed to determine the resistant bacteria in BL used as ruminant feed before and after litter treatment. The results show that the most resistant bacteria before BL treatment were the Enterococcus species. This study also found that the quantity of amoxicillin-resistant Enterococcus detected in samples from antimicrobial-dependent farms was significantly higher than in those from antimicrobial-independent farms. Additionally, 14% of bacteria were multi-resistant to tetracycline, sulfafurazole, and erythromycin in antimicrobial-independent farm litters, significantly lower than those measured in antimicrobial-dependent broiler farm litter. This study highlights the importance of better understanding, regulating, managing, and using animal waste appropriately to reduce the number of antibiotic-resistant bacteria and minimize the use of antimicrobials that carry high risks for animals, humans, and the environment.

A study was conducted on six animal feed centers in Israel where fungal and mycotoxin presence was examined in maize and wheat silages. Fumonisin mycotoxins FB 1 and FB 2 were present in every maize silage sample analyzed. Interestingly, no correlation was found between the occurrence of specific mycotoxins and the presence of the fungal species that might produce them in maize and wheat silages. We further investigated the effect of pomegranate peel extract (PPE) on Fusarium infection and fumonisin biosynthesis in laboratory-prepared maize silage. PPE had an inhibitory effect on FB 1 and FB 2 biosynthesis by Fusarium proliferatum , which resulted in up to 90 % reduction of fumonisin production in silage samples compared to untreated controls. This finding was supported by qRT-PCR analysis, showing downregulation of key genes involved in the fumonisin-biosynthesis pathway under PPE treatment. Our results present promising new options for the use of natural compounds that may help reduce fungal and mycotoxin contamination in agricultural foodstuff, and potentially replace traditionally used synthetic chemicals., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Eight horses underwent IVRLP at two occasions through a 23-gauge 2 cm long butterfly catheter. Regional anaesthesia of the ulnar, median and medial cutaneous antebrachial nerves was performed prior, and an 8 cm rubber tourniquet was placed on the proximal radius for 30 minutes following the infusion. The first infusion consisted of 2 g of amikacin sulphate and 10 mg of dexamethasone phosphate diluted with 0.9% NaCl to a total volume of 100 ml. The second perfusion was performed after a 2-week washout period, the same protocol was used but without dexamethasone phosphate. Synovial fluid samples were collected from the metacarpophalangeal joint at T = 0, 0.5, 2, 12, 24 and 36 h post-infusion. Synovial fluid amikacin sulphate concentrations were determined by use of liquid chromatography/tandem mass-spectrometry. All horses (n = 8) remained healthy throughout the study, and no adverse effects associated with the study were encountered. No statistically significant differences were found in synovial fluid amikacin sulphate concentrations between the treatment and the control group at any of the time points. In conclusion, dexamethasone phosphate can be used in IVRLP concomitantly with amikacin sulphate in cases of distal limb inflammation and pain without decreasing the synovial fluid concentration of amikacin sulphate., (© 2022 John Wiley & Sons Ltd.)

Background: Polychlorinated biphenyls (PCBs) are persistent organic pollutants banned for use worldwide. Due to their biodegradation resistance, they accumulate along the food chain and in the environment. Maternal exposure to PCBs may affect the fetus and the infant. PCBs are immunotoxic and may damage the developing immune system. PCBs are associated with elevated IgE antibodies in cord blood and are considered to be predictive of atopic reactions. Several studies on the association between prenatal exposure to PCBs and atopic reactions were previously published, albeit with conflicting results. Objectives: To examine the association between maternal PCBs levels and atopic reactions in their offspring. Methods: During the years 2013-2015, a prospective birth cohort was recruited at the delivery rooms of Shamir Medical Center (Assaf Harofeh) and "Dana Dwek" Children's Hospital. Four PCBs congeners were investigated: PCBs 118, 138, 153, and 180. In 2019, when children reached the age of 4-6 years, mothers were interviewed using the ISAAC questionnaire to assess symptoms of atopic reactions, including asthma, allergic rhinitis, and atopic dermatitis. Results: One hundred and fifty mother-child dyads were analyzed. No significant differences were found in the median serum PCBs concentrations of each studied congener or total PCBs for asthma, allergic rhinitis, atopic dermatitis diagnosis, or parent-reported symptoms. No association was found between exposure to total PCBs and the risk for asthma symptoms or diagnosis, adjusted to maternal age and family member with atopic condition: aOR = 0.94, 95%CI: (0.88; 0.99). No association was observed between each studied PCB congener and asthma symptoms or diagnosis. The same results were found also for other studied outcomes-allergic rhinitis and atopic dermatitis. Conclusion: Our study joins a series of previous studies that attempt to shed light on environmental exposures in utero as influencing factors for atopic conditions in children. Our results reflect the complexity of the pathophysiology of these phenomena. No relationship between maternal serum PCBs levels was demonstrated for asthma, allergic rhinitis, or atopic dermatitis. However, additional multi-participant studies, with longer, spanning into later pediatric age follow up are needed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Berlin, Flor-Hirsch, Kohn, Brik, Keidar, Livne, Marom, Ovental, Mandel, Lubetzky, Factor-Litvak, Tovbin, Betser, Moskovich, Hazan, Britzi, Gueta, Berkovitch, Matok and Hamiel.)

Emergence of resistant bacteria during antimicrobial treatment is one of the most critical and universal health threats. It is known that several stress-induced mutagenesis and heteroresistance mechanisms can enhance microbial adaptation to antibiotics. Here, we demonstrate that the pathogen Bartonella can undergo stress-induced mutagenesis despite the fact it lacks error-prone polymerases, the rpoS gene and functional UV-induced mutagenesis. We demonstrate that Bartonella acquire de novo single mutations during rifampicin exposure at suprainhibitory concentrations at a much higher rate than expected from spontaneous fluctuations. This is while exhibiting a minimal heteroresistance capacity. The emerged resistant mutants acquired a single rpoB mutation, whereas no other mutations were found in their whole genome. Interestingly, the emergence of resistance in Bartonella occurred only during gradual exposure to the antibiotic, indicating that Bartonella sense and react to the changing environment. Using a mathematical model, we demonstrated that, to reproduce the experimental results, mutation rates should be transiently increased over 1,000-folds, and a larger population size or greater heteroresistance capacity is required. RNA expression analysis suggests that the increased mutation rate is due to downregulation of key DNA repair genes (mutS, mutY, and recA), associated with DNA breaks caused by massive prophage inductions. These results provide new evidence of the hazard of antibiotic overuse in medicine and agriculture., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants found in human tissues. PCBs can be transferred through the placenta and may disrupt the maternal thyroid homeostasis, and affect fetal thyroid hormone production. Several studies have shown that intrauterine exposure to PCBs might be associated with abnormal levels of thyroid hormones in mothers and their offspring. Objectives: To examine the associations between environmental exposure to PCBs and thyroid hormone levels in mothers and newborns. Methods: The EHF-Assaf-Harofeh-Ichilov cohort includes 263 mothers-newborns dyads. A total of 157 mother-newborn dyads had both PCBs and thyroid function measures. Regression models were used to estimate associations between maternal PCB exposure and maternal and newborn thyroid function, controlling for possible confounders. Results: Four PCBs congeners were analyzed: PCBs 118, 138, 153, and 180. ∑PCBs median (IQR) level was 14.65 (2.83-68.14) ng/g lipids. The median maternal thyroid-stimulating hormone (TSH) level was 2.66 (0.70-8.23) μIU/ml, the median maternal free thyroxine (FT4) level was 12.44 (11.27-13.53) μg/dL, the median maternal thyroid peroxidase antibodies (TPO Ab) level was 9.6 (7.36-12.51) IU/mL. Newborns' median total thyroxine (T4) level was 14.8 (7.6-24.9) μg/dL. No association was found between exposure to different congeners or to ∑PCBs and maternal TSH, FT4, thyroglobulin autoantibodies (Tg Ab), TPO Ab and newborn total T4 levels. In multivariable analysis a 1% change in ∑PCBs level was significantly associated with a 0.57% change in maternal TSH levels in women with body mass index (BMI) < 19. The same association was observed for each of the studied PCB congeners. Maternal TPO Ab levels statistically significantly increased by 0.53 and 0.46% for 1% increase in PCB 118 and 153 congeners, respectively. In women with BMI > 25, the association between the PCBs levels and maternal TSH levels was in the opposite direction. No association was found in women with normal BMI (19-24.9). Conclusions: Background exposure to environmentally relevant concentrations of some PCBs can alter thyroid hormone homeostasis in pregnant women and might be associated with abnormal TSH levels and TPO-Ab in women with low BMI. However, these findings require further investigation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Berlin, Barchel, Brik, Kohn, Livne, Keidar, Tovbin, Betser, Moskovich, Mandel, Lubetzky, Ovental, Factor-Litvak, Britzi, Ziv-Baran, Koren, Klieger, Berkovitch, Matok and Marom.)

The aim of this study was to investigate the safety and pharmacokinetics of trimethoprim-sulphadiazine administered via intravenous regional limb perfusion (IVRLP) into the cephalic vein. According to the hypothesis, the drug could be administered without adverse effects and the synovial concentrations would remain above the minimum inhibitory concentration (MIC) for trimethoprim-sulphadiazine (0.5 and 9.5 µg/mL) for 24 h. Ten ( n = 10) horses underwent cephalic vein IVRLP with an Esmarch tourniquet applied for 30 min. Four grams (4 g) of trimethoprim-sulphadiazine (TMP-SDZ) were diluted at 0.9% NaCl for a total volume of 100 mL. Synovial fluid and blood samples were obtained immediately before IVRLP and at 0.25, 0.5, 2, 6, 12 and 24 h after the initiation of IVRLP. Trimethoprim and sulphadiazine concentrations were determined using a method based on liquid chromatography/tandem mass spectrometry. The C max (peak drug concentration) values were 36 ± 31.1 and 275.3 ± 214.4 µg/mL (TMP and SDZ). The respective t max (time to reach C max ) values were 20 ± 7.8 and 26.4 ± 7.2 min. The initial synovial fluid concentrations were high but decreased quickly. No horse had synovial concentrations of trimethoprim-sulphadiazine above the MIC at 12 h. Severe vasculitis and pain shortly after IVRLP, lasting up to one week post-injection, occurred in five out of 10 horses. In conclusion, IVRLP with trimethoprim-sulphadiazine cannot be recommended due to the low concentrations of synovial fluid over time and the frequent severe adverse effects causing pain and discomfort in treated horses. Thus, in cases of septic synovitis with bacteria sensitive to trimethoprim-sulphadiazine, other routes of administration should be considered.

Objective:  The aim of this study was to determine the concentration of metronidazole in the distal interphalangeal joint (DIPJ) of the thoracic limb after administering metronidazole to standing horses by intravenous regional limb perfusion (IVRLP)., Methods:  Eleven healthy horses had a wide rubber tourniquet applied to the proximal aspect of the antebrachium for 0.5 hours and 500 mg of metronidazole diluted in physiologic saline solution to a total volume of 108 mL was administered by cephalic IVRLP. Synovial fluid samples were collected from the DIPJ before perfusion and at 0.25, 0.5, 2, 12 and 24 hours. Blood samples were obtained at the same time points for serum analysis. Concentrations of metronidazole were determined by liquid chromatography/tandem mass spectrometry., Results:  Four horses were excluded due to low synovial fluid concentrations and not completing the full tourniquet application time. The C max in the synovial fluid was 327 ± 208 µg/mL, and the t max was 26 ± 7 minutes. Only the concentrations of metronidazole at time points 0.25 and 0.5 hours were significantly different ( p  < 0.001) from synovial concentration before perfusion. The serum C max was 1.78 ± 0.93 µg/mL, and the t max was 76 ± 52min., Conclusion:  Metronidazole administered by IVRLP reached high concentrations in the synovial fluid at 0.5 hours. However, the concentrations rapidly decreased below the minimum inhibitory concentration of potential target pathogens. Effectiveness of metronidazole administered by IVRLP as a sole therapy against anaerobic infections of synovial structures of the distal limb cannot be determined by a pharmacokinetic study. However, the present study serves as the basis for future carefully planned clinical trials., Competing Interests: None declared., (Thieme. All rights reserved.)

Seafood is capable of bioaccumulating heavy metals (HM), making it a potentially major dietary source of HM for humans. Presently, little data exists on seafood from the eastern-most boundary of the Mediterranean Sea. This study aims to provide exposure insight of the Israeli population to HM through the consumption of locally caught seafood by assessing the levels of arsenic, mercury, cadmium, and lead in raw tissues of seafood. A wide survey of local fisheries was conducted providing 296 samples from 11 different species, including seven fish, two crustacean, and two cephalopod species. Total arsenic, cadmium, and lead were analyzed by graphite-furnace atomic absorption. Total mercury was measured by cold-vapor mercury analyzer. Arsenic speciation was performed by anion chromatography-inductively coupled plasma sector field mass spectrometry. Results suggested that the total arsenic concentrations were significantly higher in crustaceans and cephalopods than fish. Arsenic speciation revealed two samples that exceed 1 mg/kg of inorganic arsenic, whereas methylated arsenic was below the detection limit. Elevated mercury levels were detected in the commercial benthic species Mullus barbatus (red mullet), cadmium was detected in one-third of the samples, and lead detected in eight samples. Comparing the results to health guidelines, 99.4% of seafood tested in this study abide with acceptable levels of heavy metals in seafood, as defined by both Israeli and European Union guidelines., (© 2021 Institute of Food Technologists®.)

Objective:  The aim of this study was to determine the time (T max ) to the maximum concentration (C max ) of amikacin sulphate in synovial fluid of the radiocarpal joint (RCJ) following cephalic intravenous regional limb perfusion (IVRLP) using 2 g of amikacin sulphate., Methods:  Cephalic IVRLP was performed with 2 g of amikacin sulphate diluted in 0.9% NaCl to a total volume of 100 mL in six healthy adult mixed breed mares. An Esmarch's rubber tourniquet was applied for 30 minutes and the antibiotic solution was infused through a 23-gauge butterfly catheter. Synovial fluid was collected from the RCJ prior to the infusion and at 5, 10, 15, 20, 25 and 30 minutes after completion of IVRLP. The tourniquet was removed after the last arthrocentesis. Synovial fluid amikacin sulphate concentrations were determined by liquid chromatography/tandem mass spectrometry., Results:  The calculated mean T max occurred at 15 minutes (range: 10-20 minutes) post-perfusion. The highest synovial fluid amikacin sulphate concentration was noted at 10 minutes in 2 horses, 15 minutes in 2 horses and 20 minutes in 2 horses. The highest mean concentration was 1023 µg/mL and was noted at 20 minutes. Synovial mean concentrations were significantly different between 15 and 30 minutes., Clinical Significance:  In this study no T max occurred after 20 minutes; thus, 30 minutes of tourniquet application time appear to be excessive. The 20 minutes duration of tourniquet application appears sufficient for the treatment of the RCJ in cephalic IVRLP using 2 g amikacin sulphate in a total volume of 100 mL., Competing Interests: None declared., (Thieme. All rights reserved.)

Treated wastewater (TWW) constitutes a sustainable water resource and has been used for fish culture in some countries around the world, although there are no comprehensive data on the effect of TWW on fish growth and health in the context of aquaculture production. Our objectives were to examine how fish culture in TWW affected fish growth and fitness, as well as compliance with the international standards for safe consumption. Guppy (Poecilia reticulata) fingerlings were reared in 0%, 50% and 100% tertiary TWW (TTWW), from the age of five days, for a period of four months. In water analyses, 33 out of 67 tested organic micropollutants (OMPs) were detected in the TTWW samples at least once, at concentrations that are typically reported in domestic TTWW. Fish survival ranged between 77-80% and did not differ between treatment groups. Fish growth and mortality following challenge infection with Tetrahymena sp. (which ranged between 64-68%), were similar among treatment groups. Of tested immunological parameters, lysozyme and anti-protease was similar among treatments while complement activity was highest in the 50% TTWW-reared fish. No abnormalities were observed in the histopathological analysis. Levels of heavy metals, polychlorinated-biphenyls (PCBs) and organochlorines (OCs) in fish were below the detection limit and below the Food and Agriculture Organization of the United Nations (FAO) and the European Union EU maximal permitted levels in food fish. Results suggest that the yield of fish grown in TTWW is potentially similar to that in freshwater, and the produced fish comply with the standards of consumer safety. The results are in line with previous studies that examined the feasibility of TWW-fed aquaculture., Competing Interests: The authors have declared that no competing interests exist.

While the wheat-associated microbiome is of major agricultural importance, little is known about the alterations in wheat grain microbial community composition during storage. Characterization of the bacterial and fungal communities in stored wheat grains revealed the impact of phosphine fumigation, one of the most effective methods to eliminate insects in stored commodities, on the composition of the wheat grain microbiome. High-throughput amplicon sequencing of the bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) region was used to analyze the wheat grain microbiome at different times over as 6 months period of storage. Higher bacterial diversity was found across the samples during the first (immediately after harvest) and second (3 months later) time points, with a predominance of Proteobacteria , Firmicutes , Actinobacteria , Bacteroidetes and Planctomycetes . A two-fold decrease in the number of bacterial operational taxonomic units (OTUs) was observed in wheat grains at the last time point (6 months later), following phosphine treatment. In contrast to the effect of phosphine on bacteria, it did not affect fungal diversity in stored grains. The majority of fungal sequences were assigned to Ascomycota , followed by Basidiomycota , Glomeromycota , and unidentified fungi, which were evenly distributed throughout the storage period. Alpha and beta diversity analyses were confirmed by examination of the cultured microbial taxa obtained from the stored wheat grains. Mycotoxin analysis of wheat grains collected after phosphine fumigation revealed the presence of Fusarium toxins, primarily deoxynivalenol (DON). Several mycotoxigenic Fusarium spp. were also detected in the same samples. Results of the present study indicate that microbiome of stored, whole wheat grains was strongly affected by phosphine fumigation, which changed the structure of the microbial community leading to shifts in species composition toward mycotoxigenic strains. A better understanding of the complex interactions within the microbial communities of stored grains will assist in the development of novel biocontrol strategies to overcome mycotoxin contamination.

The objectives of this study were to evaluate the compatibility and the pharmacokinetic properties of combined amikacin and penicillin administration by intravenous regional limb perfusion (IVRLP) in horses. A tourniquet was applied proximal to the carpus of 7 clinically healthy adult horses and 2 g of amikacin and 10 × 10 6 IU of penicillin (100 mL total volume) were sequentially injected into the cephalic vein just distal to the tourniquet. Synovial samples were collected from the joint at several times after injection. All samples were analyzed for amikacin and penicillin concentration. The mean maximum concentration (C max ) of both amikacin and penicillin was over 10-fold the relevant minimal inhibitory concentration (MIC) for all horses and remained above those MICs for at least 24 hours. The results of this study indicate that combining amikacin with penicillin during IVRLP in normal horses delivers high therapeutic synovial concentrations of both drugs.

Objective: To determine the metacarpophalangeal joint fluid concentrations of ceftazidime administered via regional limb perfusion (RLP)., Animals: Eight healthy horses., Methods: RLP was performed by injecting 2 g of ceftazidime and 60 mL of perfusate volume in the cephalic vein of standing, sedated horses. Serum and synovial fluid from the metacarpophalangeal joint were collected before perfusion and at 0.5, 2, 6, 12, 24 hours postperfusion. Ceftazidime concentrations were measured via liquid chromatography. Maximal concentration (C max ), area under the curve (AUC), half-life of the drug (T ½), and the timing of C max (T max ) were determined to assess ceftazidime as a candidate drug for RLP. Continuous parameters were compared with the Mann-Whitney U test. P value ≤ .05 was considered statistically significant., Results: The C max of ceftazidime in synovial fluid (235 µg/mL) was 15 times higher than the minimal inhibitory concentration (MIC) for most bacteria involved in orthopedic infections, including resistant pathogens such as Pseudomonas aeruginosa (MIC = 16 µg/mL). However, synovial concentrations decreased quickly and remained above the MIC in only 1 horse by 6 hours postperfusion., Conclusion: RLP generated high synovial fluid concentrations of ceftazidime in the distal limb, but these concentrations decreased rapidly below the deliberately high MIC selected., Clinical Relevance: Once daily RLP, as applied in our study, with 2 g ceftazidime in standing horses, cannot be recommended for use in a clinical setting., (© 2017 The American College of Veterinary Surgeons.)

This study aimed to assess the occurrence of toxigenic fungi and mycotoxin contamination in stored wheat grains by using advanced molecular and analytical techniques. A multiplex polymerase chain reaction (PCR) strategy was established for rapid identification of mycotoxigenic fungi, and an improved analytical method was developed for simultaneous multi-mycotoxin determination in wheat grains by liquid chromatography-tandem mass spectrometry (LC/MS/MS) without the need for any clean-up. The optimized multiplex PCR method was highly specific in detecting fungal species containing species-specific and mycotoxin metabolic pathway genes. The method was applied for evaluation of 34 wheat grain samples collected from storage warehouses for the presence of mycotoxin-producing fungi, and a few samples were found positive for Fusarium and Aspergillus species. Further chemical analysis revealed that 17 samples contained mycotoxins above the level of detection, but only six samples were found to be contaminated over the EU regulatory limits with at least one mycotoxin. Aflatoxin B₁, fumonisins, and deoxynivalenol were the most common toxins found in these samples. The results showed a strong correlation between the presence of mycotoxin biosynthesis genes as analyzed by multiplex PCR and mycotoxin detection by LC/MS/MS. The present findings indicate that a combined approach might provide rapid, accurate, and sensitive detection of mycotoxigenic species and mycotoxins in wheat grains., Competing Interests: The authors declare no conflict of interest.

Objective: To determine the influence of 3 perfusate volumes on amikacin concentration in the metacarpophalangeal joint following cephalic regional limb perfusion (RLP) in standing horses., Animals: Seven healthy horses., Methods: Three perfusate volumes (100, 60, and 30 mL), containing 2 grams of amikacin, were tested during intravenous RLP at the cephalic vein, placing the tourniquet at mid antebrachium, in standing sedated horses. Synovial fluid was collected from the metacarpophalangeal joint before perfusion and at 30 and 120 minutes after perfusion. Serum samples were taken from the jugular vein at the same time points. Samples were analyzed for amikacin concentrations and a repeated measures ANOVA, followed by least squares difference pairwise comparisons to identify differences in amikacin concentration across perfusate volumes. Differences were considered significant at P<.05., Results: The mean amikacin concentration in synovial fluid at 30 minutes after perfusion was significantly higher following perfusate volume of 100 mL (579 μg/mL), compared to volumes of 60 mL (227 μg/mL) or 30 mL (282 μg/mL) (P<.05). When a threshold of 160 μg/mL was used, more horses reached the synovial therapeutic threshold following perfusate volume of 100 mL (100%), than horses receiving 60 mL (43%) and 30 mL (57%) at 30 minutes after injection., Conclusion: The use of 100 mL volume for RLP at the cephalic vein in standing horses resulted in higher concentration of amikacin in the synovial fluid and is recommended for use in clinical cases., (© Copyright 2016 by The American College of Veterinary Surgeons.)

Background: Drug administration as tablets to debilitated elderly patients in crushed form can modify the pharmacokinetic characteristics of the active components. Only scarce information is available on the pharmacokinetics when administered in such form. The aim of this study was to evaluate the pharmacokinetics of roxithromycin administered in crushed form and to compare it with the pharmacokinetics of a group of geriatric patients receiving it in the conventional tablet form., Methods: Twenty patients from the acute ward of the Shmuel Harofeh Geriatric Medical Center in stable, clinical, and hemodynamic condition were studied. Patients in group 1 (n = 10) received medications orally in tablet form. Group 2 (n = 10) included age- and disease-matched patients from the same department, who received oral roxithromycin in crushed tablet form. The mean daily dose was the same in both groups: 300 mg (150 mg twice daily). The patients received the drug for 3 days before the initiation of the study. Blood samples for determination of the roxithromycin concentration were taken at the baseline, 1 hour before the drug administration, and at 1, 3, 4, 6, 8, and 10 hours after drug administration. Roxithromycin concentration was measured by a liquid chromatography-tandem mass spectrometry method., Results: Pharmacokinetic parameters of roxithromycin were significantly different between the 2 groups: the Cmin and Cmax were significantly higher, the tmax significantly longer, AUC0-10 larger, and CL/F smaller in group 2., Conclusions: Roxithromycin pharmacokinetic parameters were significantly different between the 2 patient groups resulting in higher drug serum concentrations in the crushed tablets group. The impact of the increased drug exposure is unclear.

3-Methyl-methcathinone (3-MMC) is a novel, synthetic cathinone analog, recently linked to poisoning events among recreational users. The lack of pharmacological data on 3-MMC, prompted us to explore its pharmacokinetic profile as well as its effect on feeding behavior, weight gain, and serum biochemistry. 3-MMC was administered to male pigs (n=3, three months old) as a single intravenous dose (0.3 mg/kg), followed by a multiple oral dose administration (3 mg/kg) for five days and plasma and tissue concentrations determined. Concomitantly a control group consisting of two healthy male pigs received saline solution instead of 3-MMC according to the same administration schedule. 3-MMC effects on complete blood count, biochemistry, feed intake, and body weight were examined. The pigs were sacrificed and submitted to a pathological and histopathological examination. 3-MMC displayed rapid absorption with a peak concentration achieved within 5-10 min after oral ingestion and a plasma half-life of 0.8 h. The bioavailability was about 7%. 3-MMC tissue levels were below detectable levels 24 h after the last oral dosage. No treatment-related clinical signs were observed and no histopathological findings were detected. 3-MMC caused significant change in daily feed intake and weight gain over time. The animals treated with 3-MMC displayed a lower rate of increase in mean body weight. Caution needs to be practiced in terms of extrapolating the present data to human safety, due to the low sample size, low dosage, and the relatively short study duration as well as the lack of data on abuse potential of 3-MMC., (© The Author(s) 2015.)

Aim: To evaluate the association between cytochrome P450 2D6 (CYP2D6) phenotypes in paediatric patients with autistic spectrum disorders (ASD) treated with risperidone, adverse drug reactions (ADRs), and drug efficacy., Method: An observational cohort study of 40 children (34 males, six females; median age 7y range 3-18y) with autistic disorder, pervasive developmental disorder not otherwise specified, or Asperger syndrome diagnosed using the Autism Diagnostic Interview-Revised and treated with risperidone for at least 3 months. Charts were reviewed for demographic and clinical information, response to treatment was assessed by parents and the treating neurologist on a three-point scale, and information about ADRs was collected. Trough plasma levels of risperidone and its metabolites were determined and CYP2D6 genotyping was performed., Results: Twenty-six patients responded to therapy and 11 patients exhibited ADRs. CYP2D6 genotyping showed two patients to be poor metabolizers, two ultra-rapid metabolizers, seven intermediate metabolizers, and 29 extensive metabolizers. Both ultra-rapid metabolizer patients were non-responders and had no ADRs. In contrast, both poor metabolizer patients were responders but experienced ADRs. No correlation was found between risperidone dosage and either risperidone or drug metabolite plasma levels. There was no difference in risperidone or metabolite plasma levels when comparing responders to non-responders, or when comparing patients with or without ADRs., Interpretation: In patients with ASD treated with risperidone, a CYP2D6 phenotype may be associated with response to treatment and development of ADRs., (© 2014 Mac Keith Press.)

We aimed to determine the relationship between plasma and cerebrospinal fluid (CSF) concentrations of ibuprofen and the antipyretic effect in pediatric patients. A prospective cohort of infants and children aged 3 months to 15 years and treated with ibuprofen was studied. The patients received ibuprofen (via oral route, median dose of 10.0 mg/kg; 3.4-11.4 mg/kg range), samples of blood and CSF were collected, and body temperature was measured. Sequential analysis of the pharmacokinetic and pharmacodynamic data from 28 patients was performed using a population modeling approach. The observed concentration versus time data indicated substantial pharmacokinetic variability in absorption and distribution of ibuprofen between the patients. The pharmacokinetic modeling outcomes indicate that following a ∼25-minute lag time, ibuprofen is rapidly absorbed to the central compartment and rapidly equilibrates with the CSF, resulting in the total ibuprofen concentration in the CSF versus plasma (CCSF /Cplasma ) of 0.011 ± 0.007. The antipyretic effect of ibuprofen was best described by an indirect response PK-PD model incorporating patient baseline body temperature and ibuprofen concentration in the CSF. We conclude that the pharmacokinetic-pharmacodynamic modeling can be used to predict the time course of ibuprofen plasma and CSF concentrations and of the antipyretic effects in individual pediatric patients., (© 2014, The American College of Clinical Pharmacology.)

Ionophores are used as feed additives for the control of coccidiosis and growth promotion in farm animals. Reports of maduramicin toxicosis in farm animals are scarce. The present work describes an acute maduramicin toxicosis affecting 22 pregnant gilts, 2 pregnant sows and 2 boars, resulting in a total mortality of 65% within 2days. The clinical and histopathological findings observed shared similar characteristics to acute ionophore toxicosis in pigs, being characterized by severe myodegeneration in skeletal muscle and degenerative changes in the myocardium. Important clinical pathology indices found were elevated levels of CPK and ALT. In contrast to the pregnant gilts, the two pregnant sows completely recovered after 1month and farrowed 2months after the intoxication event healthy piglets. The lack of effect of maduramicin on the fetuses might be indicative of poor placental penetration of maduramicin. Moreover, the present work reports for the first time maduramicin levels in livers (0.5mg/kg) of gilts exposed to lethal concentrations of maduramicin (18.5mg/kg) in the feed. As the average feed intake of the gilts was estimated to be 3.5kg feed/day, the mean maduramicin intake leading to the observed high mortality rate was 0.4mg/kg body weight/day., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Serum and skin tissue azithromycin (AZM) concentrations were analysed in healthy and pyoderma affected dogs to determine AZM pharmacokinetics and to establish the effect of disease on AZM skin disposition. AZM was administered orally to two groups of healthy dogs: (1) at 7.02 mg/kg (n=7) and (2) at 11.2mg/kg (n=9). A crossover design was used on five of them. Seven dogs with pyoderma were treated with AZM at 10.7 mg/kg. The two groups of healthy dogs received AZM once daily over three consecutive days and dogs with pyoderma received the same treatment repeated twice with an interval of 1 week. AZM concentrations were determined by liquid chromatography-tandem mass spectrometry. AZM was rapidly absorbed and slowly excreted. In healthy dogs, maximum serum concentrations appeared 2h after administration and were (mean ± standard deviation) 0.60 ± 0.25 μg/mL and 1.03 ± 0.43 μg/mL, and the half-lives were 49.9 ± 5.10 and 51.9 ± 6.69 h for doses of 7.02 and 11.2mg/kg, respectively. Clearance (CL0-24/F) was similar in both dosing groups (1.24 ± 0.24 and 1.29 ± 0.24 L/h/kg) and the respective mean residence time (MRT0-24) was 11.1 ± 0.8 and 8.4 ± 2.2h. The skin concentration in healthy dogs was 3.5-6.5 and 5.0-12.0 times higher than the corresponding serum concentration after the two doses and increased after the cessation of AZM administration. The ratio increased significantly in inflamed tissue (9.5-26.2)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Anticoagulant rodenticides are frequently a cause of poisoning of domestic animals, wildlife, and human beings. A toxicosis in 6,000 laying hens caused by the malicious addition of unknown amounts of coumatetralyl bait as well as the insecticides aldicarb, methomyl, and imidacloprid in the drinking water, was investigated in the current study. In order to determine a possible carryover of coumatetralyl into eggs, a rapid and reliable analytical method was developed and fully validated for the simultaneous detection of 6 anticoagulant rodenticides (warfarin, coumatetralyl, coumachlor, bromadiolone, difenacoum, and brodifacoum) in yolk and albumen using high-performance liquid chromatography (HPLC) with fluorescence detection. The method developed was reproducible, sensitive, accurate, and linear within the range of 0.01-1 mg/kg, which is the concentration range of bromadiolone and warfarin found in yolk in previously reported studies. The coefficient of variations of within and between days was 1.0-8.5% for yolk and 0.6-3.8% for albumen, while recoveries from spiked albumen and yolk samples were all in the range of 79-99% and 51-95%, respectively. Limits of detection in yolk were 0.01 mg/kg for warfarin and 0.003 mg/kg for the remaining compounds; in albumen, the limit of detection was 0.003 mg/kg for warfarin, coumatetralyl, and coumachlor, and 0.0015 mg/kg for difenacoum and brodifacoum. The application of the validated method revealed the presence of coumatetralyl in the yolk only at levels of 0.0057 mg/kg and 0.0052 mg/kg on the second and fourth day of the poisoning. In conclusion, the HPLC method demonstrated suitability for application in official analysis of anticoagulants in hen eggs.

Objectives: Drug administration to debilitated elderly patients on enteral feeding through a nasogastric tube (NGT) can modify the pharmacokinetic characteristics of the drug and influence its therapeutic blood concentration. The aim of this study was to evaluate the pharmacokinetics of ciprofloxacin administered through an NGT and to compare it with those of a group of patients receiving the drug orally., Methods: Twenty patients in stable clinical and hemodynamic condition from the long-term care ward of a geriatric multilevel hospital were studied. Patients in group 1 (n = 10) had oropharyngeal dysphagia and received food and medications, including ciprofloxacin, by NGT. Group 2 (n = 10) included age- and disease-matched orally fed patients from the same department receiving ciprofloxacin orally. Blood samples for ciprofloxacin concentration were taken at steady state, before drug administration, time 0, and at 1, 2, 3, 4, 6, 8, and 10 hours after drug administration. Ciprofloxacin was measured using liquid chromatography with tandem mass spectrometric detection. The mean daily dose was the same in both the groups: 1000 mg (500 mg twice daily)., Results: Pharmacokinetic parameters of ciprofloxacin were not significantly different between the 2 groups: trough concentrations were 1.24 ± 0.95 μg/mL (0.25-3.67 μg/mL) versus 1.30 ± 0.61 μg/mL (0.21-2.36 μg/mL) (P = 0.76); Cmax 3.30 ± 2.16 μg/mL (1.54-8.62 μg/mL) versus 4.24 ± 1.99 μg/mL (2.24-9.02 μg/mL) (P = 0.356); tmax 2.8 ± 1.5 versus 3.1 ± 2.8 hours (P = 0.799); and AUC0-10 20.2 ± 12.1 μg·h·mL (9-51.07 μg·h·mL) versus 24.4 ± 13.0 μg·h·mL (5.57-52.48 μg·h·mL) (P = 0.493), in the oral fed versus NGT, respectively., Conclusions: Ciprofloxacin pharmacokinetic parameters are not significantly different between patients receiving the drug through NGT compared with those who received it orally, and therefore, in frail elderly patients, this route of administration can be considered.

The potent hepatotoxin and carcinogen aflatoxin B1 (AFB1) is a common mycotoxin contaminant of grains used in animal feeds. Aflatoxin M1 (AFM1) is the major metabolite of AFB1 in mammals, being partially excreted into milk, and is a possible human carcinogen. The maximum permitted concentration of AFM1 in cows' milk is 0.05 μg/kg in Israel and the European Union. Since milk yield and the carry-over of AFB1 in the feed to AFM1 in the milk are highly correlated, it was considered important to determine the AFM1 carry-over in Israeli-Holstein dairy cows, distinguished by world record high milk production. Twelve such cows were used to determine AFM1 carry-over following daily oral administration of feed containing ~86 μg AFB1 for 7 days. The mean carry-over rate at steady-state (Days 3-7) was 5.8% and 2.5% in mid-lactation and late-lactation groups, respectively. The carry-over appears to increase exponentially with milk yield and could be described by the equation: carry-over% = 0.5154 e(0.0521 × milk yield), with r(2) = 0.6224. If these data truly reflect the carry-over in the national Israeli dairy herd, the maximum level of AFB1 in feed should not exceed 1.4 μg/kg, a value 3.6 times lower than the maximum residue level currently applied in Israel.

The pharmacokinetics and metabolism of 2,2,3,3-tetramethylcyclopropanecarbonylurea (TMCU), a potent anticonvulsant compound, were studied in male Sprague-Dawley rats following i.v. (5 mg/kg), oral (20 mg/kg), and i.p. (20 mg/kg) administrations. Urine samples were analyzed by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry. Plasma samples were analyzed by GC/MS. TMCU absolute bioavailability was 83% and 90% following oral and i.p. dosing, respectively. Following i.p. administration, the peak plasma concentration (C(max)) obtained 45 min after dosing was 15.4 mg/l. Following oral dosing, C(max) was 6.5 mg/l, and it was reached after 4 h. The disposition kinetics of TMCU in rats was adequately described by a one-compartment open body model. TMCU is well distributed into the extravascular tissues with volume of distribution (V(ss)) of 0.87 l/kg and undergoes extensive metabolism. Only a small fraction of TMCU excreted unmetabolized in the urine (6.3 +/- 0.8%). trans-2-Hydroxymethyl-2,3,3-trimethylcyclopropanecarbonylurea (OH-TMCU) was a predominant metabolite of TMCU. Its structure was established by NMR and X-ray crystallography. Following i.p. administration of 5 and 20 mg/kg TMCU, the drug was excreted in the urine as OH-TMCU at an extent of 28.3 +/- 2.6% and 42.1 +/- 3.8%, respectively. A portion of OH-TMCU was excreted in the urine as TMCU sulfate and TMCU glucuronide.

Purpose: To compare the influence of enzyme-inducing comedication and valproic acid (VPA) on topiramate (TPM) pharmacokinetics and metabolism at steady state., Methods: Three groups were assessed: (a) patients receiving TPM mostly alone (control group, n =13); (b) patients receiving TPM with carbamazepine (CBZ; n = 13); and (c) patients receiving TPM with VPA (n = 12). TPM and its metabolites were assayed in plasma and urine by liquid chromatography-mass spectrometry (LC-MS)., Results: No significant differences were found in TPM oral (CL/F) and renal (CL(r)) clearance between the VPA group and the control group. Mean TPM CL/F and CL(r) were higher in the CBZ group than in controls (2.1 vs. 1.2 L/h and 1.1 vs. 0.6L/h, respectively; p < 0.05). In all groups, the urinary recovery of unchanged TPM was extensive and accounted for 42-52% of the dose (p > 0.05). Urinary recovery of 2,3-O-des-isopropylidene-TPM (2,3-diol-TPM) accounted for 3.5% of the dose in controls, 2.2% in the VPA group (p > 0.05), and 13% in the CBZ group (p < 0.05). The recovery of 10-hydroxy-TPM (10-OH-TPM) was twofold higher in the CBZ group than in controls, but it accounted for only <2% of the dose. The plasma concentrations of TPM metabolites were severalfold lower than those of the parent drug., Conclusions: Renal excretion remains a major route of TPM elimination, even in the presence of enzyme induction. The twofold increase in TPM-CL/F in patients taking CBZ can be ascribed, at least in part, to stimulation of the oxidative pathways leading to formation of 2,3-diol-TPM and 10-OH-TPM. VPA was not found to have any clinically significant influence on TPM pharmacokinetic and metabolic profiles.

Purpose: To characterize the metabolic profile of topiramate (TPM) in humans and to assess the influence of enzyme induction by carbamazepine (CBZ) on the pharmacokinetics and metabolic profile of TPM., Methods: Twelve healthy subjects received a single oral dose of TPM (200 mg) on two randomized occasions. On one occasion, TPM was administered alone, and on the other, it was given on day 18 of a 24-day treatment with CBZ (maintenance dosage, 600 mg/day). Blood and urine samples were collected for > or = 72 h after dosing. TPM and its metabolites were assayed in plasma and urine by a specific liquid chromatography-mass spectroscopy (LC-MS) method., Results: Mean TPM oral clearance (CL/F) increased from 1.2 L/h (control) to 2.2 L/h after CBZ treatment. Mean TPM half-life decreased from 29 h to 19 h. TPM was excreted extensively in urine both under noninduced (56%) and CBZ-induced conditions (40%). 2,3-O-Des-isopropylidene-TPM (2,3-diol-TPM) was identified as the most prominent urinary metabolite, with a recovery accounting for 3.2% and 7.9% of the TPM dose under noninduced and induced conditions, respectively. Corresponding recovery values for 10-hydroxy-TPM (10-OH-TPM) were 1.2% and 1.8%, respectively. The control AUC(metabolite)/AUC(drug) ratio for 2,3-diol-TPM and 10-OH-TPM were 1.5% and 0.6%, and they increased by threefold and twofold, respectively, after CBZ treatment., Conclusions: TPM remains appreciably excreted unchanged in urine (41%) under CBZ-induced conditions, even though TPM CL/F increased by twofold. Although 2,3-diol-TPM and 10-OH-TPM were measured in unconjugated form, the significant increases in their AUC and urinary excretion are consistent with the twofold increase in TPM clearance.

A novel liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for quantification of topiramate (TPM) and its metabolites 10-hydroxy topiramate (10-OH-TPM), 9-hydroxy topiramate (9-OH-TPM), and 4,5-O-desisopropylidene topiramate (4,5-diol-TPM) in plasma and urine. The method uses 0.5 mL of plasma or 1 mL of urine that is extracted with diethyl ether and analyzed by LC-MS. Positive ion mode detection enables tandem mass spectrometric (MS/MS) identification of the aforementioned four compounds. Calibration curves of TPM, 4,5-diol-TPM, 9-OH-TPM, and 10-OH-TPM in plasma and urine were prepared and validated over the concentration range of 0.625 to 40 microg/mL using TPM-d(12) as an internal standard. Calibration curves were linear over this concentration range for TPM and its metabolites. Accuracy and precision ranged in urine from 83% to 114% and 4% to 13% (%CV), respectively, and in plasma from 82% to 108% and 6% to 13%, respectively. The applicability of the assay was evaluated by analyzing plasma samples from a healthy subject who received a single oral dose of TPM (200 mg) and urine samples from 11 patients with epilepsy treated with TPM (daily dose between 100 to 600 mg) alone or with other antiepileptic drugs. Only TPM was detected and quantified in the plasma samples, and its concentration ranged between 0.7 and 4.3 microg/mL. The concentrations of TPM and 10-OH TPM were quantifiable in all urine samples and ranged from 20 to 300 microg/mL for TPM and from 1 to 50 microg/mL for 10-OH-TPM. The metabolites 4,5-diol-TPM and 9-OH-TPM were also detected in all urine samples, but their concentrations were quantifiable only in 4 patients. An unidentified peak in the chromatograms obtained from patients' urine was attributed to 2,3-O-desisopropylidene topiramate (2,3-diol-TPM). Due to a lack of reference material of 2,3-diol TPM and the similar MS/MS spectrum with 4,5-diol-TPM, the calibration curves of 4,5-diol-TPM were used for the quantification of its isomer 2,3-diol-TPM. Based on these determinations, the apparent 2,3-diol-TPM-to-TPM concentration ratio in patients' urine ranged from 0.05 to 0.51 and the 10-OH-TPM-to-TPM ratio ranged from 0.02 to 0.17. In conclusion, a novel LC-MS method for the assay of TPM and four of its metabolites in plasma and urine was developed. Its utilization for analysis of urine samples from patients with epilepsy showed that the method was suitable for analysis of TPM and its metabolites in clinical samples. Two quantitatively significant TPM metabolites (10-OH-TPM and 2,3-diol-TPM) and two quantitatively minor metabolites (9-OH-TPM and 4,5-diol-TPM) were detected and quantified in urine samples from patients with epilepsy.

Genetic polymorphism of the cytochrome P450 isoenzymes CYP2D6 and CYP2C19 was determined by phenotyping four ethnic groups of the Israeli population. The groups consisted of Ethiopian subjects, Yemenite subjects, and Russian subjects representing first-generation new immigrants and an Israeli Arab group. Dextromethorphan was used as the probe for CYP2D6 activity and mephenytoin was used for CYP2C19 activity. The two drugs were administered simultaneously and urine samples were collected over a period of 8 hours. The CYP2D6 phenotype was determined from the ratio of dextromethorphan conversion to dextrorphan and the CYP2C19 phenotype from the ratio of S-mephenytoin and R-mephenytoin. The used liquid chromatographic method was able to completely separate dextrorphan and dextromethorphan. Fluorescence detection allowed dextromethorphan quantification at 1 ng/mL. Mephenytoin enantiomers were completely separated in high-performance liquid chromatography and the respective fractions were collected and analyzed using a gas chromatography/mass spectrometry system with selective ion monitoring. The prevalence of poor metabolizer phenotype of dextromethorphan (CYP2D6) in the Yemenite (0%) and Ethiopian groups (0%) was significantly different from the prevalence in the Russian (17%) and Israeli Arab (9%) groups. A significant difference was also found in the distribution of the metabolic ratio of the extensive metabolizer phenotype between the Ethiopian group and the Russian and Yemenite groups. No significant difference was found in the prevalence of poor mephenytoin metabolizer phenotype (CYP2C19) between the Yemenite (8%), Ethiopian (6%), Russian (9%), and Israeli Arab (8%) groups. No difference was observed in the distribution of metabolic ratio within the extensive metabolizer phenotype subgroups of the four ethnic groups.