Concentrations Of Levamisole Research Articles

COMPARATIVE EFFICACY OF ORAL FORMULATIONS OF IVERMECTIN AND LEVAMISOLE UNDER IN VITRO CONDITIONS AGAINST HAEMONCHUS CONTORTUS

Anthelmintic resistance (AR) is the major constraint towards controlling parasitism of livestock. This study was executed to evaluate relative efficacy of oral formulation consisting of ivermectin (IVM) and levamisole (LEV) towards Haemonchus contortus under in-vitro condition. The worms were isolated from abomasa (n=384), collected from nearby slaughter house. The egg hatch test (EHT), adult motility test (AMT), and larval development test (LDT) were performed for estimation of lethal concentration (Lc) 90. A typical sigmoid dose response was converted to linear function by using Probit transformations. Different concentrations of LEV (0.172, 0.086, 0.043, 0.021, 0.010, 0.005 μl/mL), and IVM (0.002, 0.001, 0.0005, 0.00025, 0.000125, 0.0000625 μl/mL) were incubated with 100 eggs / concentration. The LC 90 values of LEV and IVM were found higher than values suggested by World Association for the Advancement of Veterinary Parasitology (WAAVP), which is (0.1µg/ml). The dose-dependency stipulates development of (AR) anthelmintic towards H. contortus.

Pharmacokinetics and therapeutic efficacies of fenbendazole in comparison with levamisole in helminth-infected Caspian turtles (Mauremys caspica).

The pharmacokinetics and bioavailability of fenbendazole and levamisole were determined in Caspian turtles after a single intravenous (i.v.) and subcutaneous (s.c.) administration. Thirty turtles diagnosed as naturally infected with Serpinema microcephalus and Falcaustra armenica nematodes received fenbendazole (50 mg/kg) or levamisole (10mg/kg) by i.v. and s.c. administrations. Blood samples were collected at time 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h after drug administration. Plasma drug concentrations were determined by a validated high-performance liquid chromatography method. Data were analyzed by noncompartmental methods. The mean elimination half-life of levamisole was 5.16 h and 12.03 h for i.v. and s.c. routes, respectively, and for fenbendazole, the mean elimination half-life was 25.38 h (i.v.) and 29.77 h (s.c.). The total clearance and volume of distribution at steady-state for levamisole and fenbendazole following i.v. administration were 0.22, 0.44 ml/g/h, and 1.06 and 7.35 ml/g, respectively. For the s.c. route, the peak plasma concentration of levamisole and fenbendazole was 10.53 and 5.24 μg/mL, respectively. The s.c. bioavailability of levamisole and fenbendazole was complete. Considering high anthelmintic efficacy and bioavailability after s.c. administration of levamisole and fenbendazole, and the absence of adverse effects, this route of administration is an easy and efficacious way of treating nematodes in Caspian turtles.

Deleterious effects of levamisole, a cocaine adulterant, in rabbit aorta

Levamisole, a veterinary anthelmintic drug, is one of the most widely used and dangerous cocaine adulterants. Like cocaine, levamisole acutely blocks noradrenaline reuptake but with much less potency, although its vascular effects are not well known. In this study, we evaluated the vascular effects of levamisole and cocaine in rabbit aortic rings used for isometric recording of tension in organ baths and protein expression by western blot. Our results indicated that levamisole (10−5–10−3 M) induced a concentration-dependent relaxation in rings precontracted with noradrenaline (10−7–3 × 10−7 M). Furthermore, it reduced the contractile response to phenylephrine (10−9–3 × 10−5 M) that was not modified by cocaine (10−5–10−4 M), and reduced α1-adrenergic receptor expression. Levamisole (10−6–10−4 M) produced a potentiation of the electrical field stimulation that was not further enhanced by the combination of both drugs. However, high concentrations of levamisole (10−3 M) abolished adrenergic neurotransmission whether administered alone or with cocaine (10−4 M). In addition, levamisole (10−5–10−3 M) also decreased endothelium-dependent relaxation to acetylcholine that was not further impaired by cocaine (10−4 M), and that was partially reversed by superoxide dismutase (SOD, 200 U/ml). These results demonstrate that levamisole has a dual effect on the adrenergic system, and its effects are independent of the presence of cocaine. At lower concentrations, it enhances the contractile sympathetic response by blocking presynaptic α2-adrenergic receptors, while at high concentrations, the effect of the antagonism of α1-adrenergic receptor prevails. In addition, levamisole induces endothelial dysfunction by reducing NO bioavailability, and this effect could be in part mediated by oxidative stress.

Evaluation of in vitro methods of anthelmintic efficacy testing against Ascaridia galli.

To investigate methods for in vitro assessment of anthelmintic efficacy against the chicken nematode Ascaridia galli this study firstly evaluated sample preparation methods including recovery of eggs from excreta using different flotation fluids and induced larval hatching by the deshelling-centrifugation method and the glass-bead method with or without bile. It then evaluated two in vitro assays, the in-ovo larval development assay (LDA) and larval migration inhibition assay (LMIA), for anthelmintic efficacy testing against A. galli using fresh eggs and artificially hatched larvae, respectively. Four anthelmintics, thiabendazole (TBZ), fenbendazole (FBZ), levamisole (LEV) and piperazine (PIP) were employed using an A. galli isolate of known susceptibility. The results suggested that the LDA and LMIA could successfully be used to generate concentration response curves for the tested drugs. The LDA provided EC50 values for inhibition of egg embryonation of 0.084 and 0.071 μg/ml for TBZ and FBZ, respectively. In the LMIA, the values of effective concentration (EC50) of TBZ, FBZ, LEV and PIP were 105.9, 6.32, 349.9 and 6.78 × 107 nM, respectively. For such in vitro studies, a saturated sugar solution showed high egg recovery efficiency (67.8%) and yielded eggs of the highest morphological quality (98.1%) and subsequent developmental ability (93.3%). The larval hatching assays evaluated did not differ in hatching efficiency but the deshelling-centrifugation method yielded larvae that had slightly better survival rates. For final standardization of these tests and establishment of EC50 reference values, tests using isolates of A. galli of defined resistance status need to be performed.

Agricultural contaminants in amphibian breeding ponds: Occurrence, risk and correlation with agricultural land use

Anthropogenic pressure such as agricultural pollution globally affects amphibian populations. In this study, a total of 178 different compounds from five agrochemical groups (i.e. antimicrobial drugs residues (ADRs), coccidiostats and anthelmintics, heavy metals, mycotoxins and pesticides) were determined monthly, from March until June 2019 in 26 amphibian breeding ponds in Flanders, Belgium. Furthermore, a possible correlation between the number and concentration of selected contaminants that were found and the percentage of arable land within a 200 m radius was studied. Within each group, the highest detected concentrations were obtained for 4-epioxytetracycline (0.422 μg L-1), levamisole (0.550 μg L-1), zinc (333.1 μg L-1), 3-acetyldeoxynivalenol (0.013 μg L-1), and terbuthylazine (38.7 μg L-1), respectively, with detection frequencies ranging from 1 (i.e. 3-acetyldeoxynivalenol) to 26 (i.e. zinc) out of 26 ponds. Based on reported acute and chronic ecotoxicological endpoints, detected concentrations of bifenthrin, cadmium, copper, cypermethrin, hexachlorobenzene, mercury, terbuthylazine, and zinc pose a substantial ecological risk to aquatic invertebrates such as Daphnia magna and Ceriodaphnia dubia, which both play a role in the food web and potentially in amphibian disease dynamics. Additionally, the detected concentrations of copper were high enough to exert chronic toxicity in the gray treefrog (Hyla versicolor). The number of detected compounds per pond ranged between 0 and 5 (ADRs), 0 - 2 (coccidiostats and anthelmintics), 1 - 7 (heavy metals), 0 - 4 (mycotoxins), and 0 - 12 (pesticides) across the four months. Furthermore, no significant correlation was demonstrated between the number of detected compounds per pond, as well as the detected concentrations of 4-epioxytetracycline, levamisole, copper, zinc, enniatin B and terbuthylazine, and the percentage of arable land within a 200 m radius. For heavy metals and pesticides, the number of compounds per pond varied significantly between months. Conclusively, amphibian breeding ponds in Flanders were frequently contaminated with agrochemicals, yielding concentrations up to the high μg per liter level, regardless of the percentage surrounding arable land, however showing temporal variation for heavy metals and pesticides. This research also identifies potential hazardous substances which may be added to the European watch list (CD 2018/408/EC) in the future.

Weak Binding of Levamisole by the Cocaine-Binding Aptamer Does Not Interfere with an Aptamer-Based Detection Assay.

Levamisole is a common and harmful adulterant of street samples of cocaine and can cause electrochemical tests for cocaine to give false negative results. To see if levamisole would interfere with aptamer-based bioassays, we analyzed the binding of levamisole to the cocaine-binding DNA aptamer. At low aptamer concentrations (0.5 to 20 μM) using isothermal titration calorimetry methods and thermal stability measurements, no binding of levamisole to the cocaine-binding aptamer was observed. At higher levamisole concentrations (500 μM), weak binding to the cocaine-binding aptamer was detected using nuclear magnetic resonance (NMR) spectroscopy chemical shift perturbations. NMR-detected titrations show that levamisole binding is competitive with cocaine binding, indicating that both ligands share a common binding site. Finally, we show that the presence of levamisole does not interfere with the photochrome aptamer switch binding assay for cocaine. We conclude that assays using low concentrations of cocaine, and consequently low concentration of levamisole as an adulterant, should be unaffected by the weak binding of levamisole.

IN VIVO AND IN VITRO EVALUATION OF PHARMACOLOGICAL ACTIVITIES OF Hedychium coccineum RHIZOMES EXTRACT

The study reports the in vivo antidiarrheal and in vitro anthelmintic, cytotoxic, and thrombolytic activity of methanol extract of Hedychium coccineum rhizomes (MEHC). The antidiarrheal activity was determined using Castor oil-induced diarrhea and Gastrointestinal motility test in mice at the doses of 200 and 400 mg/kg body weight, whereas an aquarium worm, Tubifex tubifex, was used to determine the anthelmintic activity. The cytotoxic and thrombolytic activity of MEHC was performed by Brine shrimp lethality bioassay and clot lysis method respectively. In antidiarrheal, castor oil-induced diarrhea and gastrointestinal motility exhibited a significant reduction in diarrhea and defecation and an extremely significant inhibition in intestinal motility and peristalsis index by 200 and 400 mg/kg of MEHC. The MEHC (5, 10, and 20 mg/mL) showed a significant dose-dependent manner paralysis time and times to death in multiple comparisons to the different levamisole concentrations (0.5, 0.8, and 1 mg/mL) at in vitro anthelmintic activity. The brine shrimp lethality bioassay exhibited a weak LC50 (681.95 µg/mL; R² = 0.951) while in thrombolytic a significant percentage of clot lysis (32.70%, P < 0.05) demonstrated. The findings demonstrate that H. coccineum rhizomes could be potential sources for biological activity.

Use of levamisole-adulterated cocaine is associated with increased load of white matter lesions.

BackgroundCocaine use has been associated with vascular pathologies, including cerebral white matter hyperintensities. Street cocaine is most often adulterated with levamisole, an anthelminthic drug that may also be associated with vascular toxicity. However, whether levamisole exposure from cocaine consumption further accelerates the development of white matter lesions remains unknown.MethodsWe investigated the association of cocaine and levamisole exposure with white matter hyperintensities in 35 chronic cocaine users and 34 healthy controls. We measured cocaine and levamisole concentrations in hair samples, which reflected exposure up to 6 months previously. We assessed the number and total surface area of the white matter hyperintensities using structural MRI (FLAIR sequence). Using generalized linear models, we analyzed the contributions of cocaine and levamisole to the number and area of white matter hyperintensities, accounting for several confounding factors.ResultsAnalysis using generalized linear models revealed that cocaine users had more white matter hyperintensities in terms of total surface area, but not in terms of number. Further generalized linear models that included cocaine and levamisole hair concentrations (instead of group) as predictors indicated that levamisole exposure was strongly associated with more and larger white matter hyperintensities, suggesting that the elevated white matter hyperintensities in cocaine users were driven mainly by levamisole exposure. Finally, white matter hyperintensities in levamisole-exposed cocaine users were located primarily in the periventricular and juxtacortical white matter.LimitationsThe sample size was moderate, and blood pressure was not systematically assessed.ConclusionAs an adulterant of cocaine, levamisole appears to increase the risk of white matter injury.

Digenean Transversotrema haasi (Witenberg, 1944) infection in cultured Dicentrarchus labrax, and in vitro anthelmintic assays

Abstract A little knowledge still available for fish digenean, although some of them have pathogenic and economic importance. Fish morbidity and mortalities were reported in two fish farms culturing D. labrax fingerling with the primarily isolated parasites belonged to fish digenean trematodes. Therefore, this study was directed mainly to investigate the main causative digenean parasites. Additionally, the ecological water parameters were reported, and the anthelmintic efficacy using levamisole was evaluated. The prevalent digenean parasite was identified Digenean, Transversotrema haasi, an extra-intestinal digenean that was mainly isolated from gill and skin. The infected D. labrax exhibited abnormal external, and internal lesions indicative of deteriorating health status. The recorded infection rates were 48.67 and 38.67%, parasitic intensities, 6.14 and 2.4 and mortalities rates 28 and 19.33% in Manzala, and Wadi-mariot respectively. The recoded ecological parameters were ranged from 14.22 - 29.7 oC temperature, 7.49 - 8.36 pH, and 13.54 - 34.36 ppt for salinity. Besides, the in vitro therapeutic baths with levamisole were dose-dependent, with the highest anthelmintic efficacy at higher doses effective against digenean T. hassi. Higher anthelmintic efficacy was obtained at 70 µg/l levamisole concentration, the lowest mean viability 0.33, the highest parasitic mortality rate 81.81% occurred at the lowest exposure time. Although the anthelmintic efficacy of the levamisole bath has been proven, other friendly environmental agents of plant origin should be taken under consideration to avoiding potential environmental and human hazards.

Development and Validation of a Highly Sensitive Liquid Chromatography-Tandem Mass Spectrometry Technique to Determine Levamisole in Plasma and Saliva.

Levamisole is used as a steroid-sparing drug for the treatment of frequently relapsing or steroid-dependent idiopathic nephrotic syndrome in children. As part of a large multicentre randomized controlled trial with levamisole, pharmacokinetic and pharmacodynamic parameters of levamisole in children with idiopathic nephrotic syndrome were investigated, as well as the feasibility of using saliva as an alternative and patient-friendly matrix for determining levamisole concentrations. In this study, the authors presented the development and validation of a highly sensitive method for determining levamisole in plasma and saliva using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In 100 μL samples, proteins were precipitated with 750 μL acetonitrile/methanol 420:80 (v/v) with levamisole-D5 as an internal standard. Calibration standards were prepared over a range of 0.1 ng/mL-50 ng/mL. To determine ultrafiltration efficiency, the ultrafiltrate was obtained by centrifuging blank plasma samples over the filter. Both filtered and nonfiltered samples were analyzed. For plasma, accuracy and within-run and between-run imprecision were between 95.0% and 100% and <14.5%, respectively, and for saliva, between 100.9% and 107.5%, and <13.3%. No significant matrix effects were observed. Samples were stable at benchtop for 24 hours and -80°C, for at least 14 months (stability experiments ongoing). The ultrafiltration efficiency of unbound concentrations in plasma was lower than 85% (58.9%) but stable, and, therefore, the observed concentration should be corrected. Based on observations, the developed measure can determine levamisole concentrations in participant saliva samples.

Pharmacokinetics of levamisole after intramuscular and oral administrations to Caspian salmon (Salmo trutta caspius).

The pharmacokinetic parameters of levamisole were determined in the Caspian salmon after intramuscular (IM), oral by gavage, and oral by feed administrations. Eighty-one healthy fish in three different groups received levamisole at the dose of 25mg/fish by each route. Blood samples were collected at time points of 0, 0.5, 1, 2, 4, 6, 12, 14, and 24hr after administrations. Plasma levamisole concentrations were measured by a validated high-performance liquid chromatography (HPLC) assay and were analyzed using a noncompartmental approach. The mean terminal half-life was 4.56, 3.95, and 2.91hr for IM, gavage and feed routes, respectively. The peak plasma concentration for IM, gavage, and feed routes of levamisole were 35.53, 4.63, and 8.36µg/ml, respectively, at the time of 0.25 for IM, and 1hr for gavage and feed. The relative bioavailability for gavage and feed routes was 54.80 and 69.30. The similar bioavailability for gavage and feed might be indicative of similar efficacy for these routes of administrations. Further studies are warranted to evaluate the absolute oral bioavailability and the effective dose in Caspian salmon.

Ascaridia Galli Infection in Laying Hens and the Results of in Vitro Efficacy of Levamisole, Piperazine and Carvacrol, Whether is Necessary to Change the Deworming Protocols?

Abstract We present a case of Ascaridia galli infection in laying hens on a farm in central Serbia. During the rearing period on litter, ascaridosis was diagnosed at 15 weeks of age by routine parasitological fecal examination. Pullets were treated with flubendazole for one week, and two weeks later the hens were moved to battery cages. The production results were within technological standards until the 23rd week and the medical health status was without any clinical symptoms. After that period weight loss began, the egg production dropped to 70% and eggs were of poor quality. Subsequently, severe feather pecking and an increase of mortality were reported. The postmortal examination showed severe anemia and several gross lesions in the liver, intestines, lungs, and kidneys. Different sizes of A. galli were found in the lumen of the duodenum and jejunum. Gross changes of the intestinal mucosa were present, such as a thickened intestinal wall with hemorrhagic spots, inflammation and necrotic patches. Histopathological examination showed marked changes in the intestines, liver and kidneys. All visible live parasites were collected and stored in Earle’s balanced salts, and females were used for in vitro susceptibility testing. Median lethal concentration (LC50) of piperazine, levamisole and carvacrol for A. galli was 119.7μM, 2.71μM and 3.26μM, were applied, respectively. Based on our results, it is likely that reinfection occurred after completed dehelmintization. In relation to the new circumstances and the regulation for laying hen welfare the deworming protocol should be changed in order to ensure successful dehelmintization. In order to prevent reinfection the treatment must be done at the end of the rearing period and thus be maximally effective.

High concentration of levamisole in the diet of Colossoma macropomum (Pisces: Serrasalmidae) is effective for controlling monogenean parasites.

This study investigated the effects of diets supplemented with levamisole on monogeneans on the gills of Colossoma macropomum. Fish were fed with diets containing levamisole at concentrations of 0, 300, 600, 900 and 1200 mg kg-1 for 24, 96 and 240 h and the infection by Anacanthorus spatulatus, Notozothecium janauachensis and Mymarothecium boegeri were evaluated. None of the levamisole concentrations caused either mortality or behavioral alterations in fishes during 240 h of feeding. After 24 h of feeding with 1200 mg kg-1 of levamisole, the abundance of N. janauachensis decreased in comparison with treatments of 0, 300, 600 and 900 mg kg-1, as did the abundance of M. boegeri after 240 h of feeding with 1200 mg kg-1 of levamisole. The efficacy of 900 mg kg-1 of levamisole was only 55.7% after 96 h of feeding, but it was 84.6% after 240 h of feeding with 1200 mg kg-1. Our results show that 1200 mg kg-1 of levamisole for 10 days has good anthelmintic efficacy against monogeneans of C. macropomum. Since monogeneans elicit some of the worst problems in C. macropomum, this study has provided evidence of an effective control method that may be used in fish farms.

Toxicological, hematological and immunological effects of levamisole and ivermectin diet supplementation on Colossoma macropomum (Serrasalmidae).

This study investigated the toxicological, hematological and immunological effects of diets supplemented with levamisole and ivermectin on Colossoma macropomum. Fish were fed for 24, 96 and 240 h with diets containing 300, 600, 900 and 1200 mg of levamisole kg-1 of feed. None of the levamisole concentrations caused either mortality or behavioral alterations among the fish over 10 d of feeding. In comparison, a single feeding of ivermectin at 4500, 9000, 13500 or 18000 mg kg-1 caused 100% mortality among the fish within 10 h. There were no alterations in erythrocyte parameters or albumin in any treatments with levamisole after 24, 96 and 240 h of feeding. At concentrations of 900 and 1200 mg kg-1, levamisole caused decreases in the albumin-to-globulin ratio compared to the control group and the 300 and 600 mg kg-1 diet treatment groups. Levels of glucose and total plasma protein were higher in the fish fed with 600, 900 and 1200 mg kg-1 than in the controls and the 300 mg kg-1 diet treatment group. After 10 d of receiving feed supplemented with levamisole, globulin levels were higher in the 600 mg kg-1 group than in the controls. Respiratory burst activity of leukocytes also increased in the fish supplemented with the 900 and 1200 mg kg-1 diets compared to the controls and other treatments. This is the first study to investigate how diets supplemented with these drugs affect C. macropomum. Our research indicates that all the levamisole concentrations tested can be used in the diet of C. macropomum for antiparasitic treatments against helminth species, and that dietary treatments with levamisole can stimulate components of the innate immune system.

Distribution and elimination of levamisole in eggs and tissues after oral administration to laying hens, determined by LC-MS/MS

ABSTRACTLevamisole was administered to laying hens, and concentrations in eggs and tissues (thigh muscle, breast muscle, liver and kidney) were determined by a newly developed liquid chromatography tandem mass spectrometry method, which allowed trace level quantification of levamisole. The adopted analytical method showed good sensitivity, repeatability and percentage of recovery from spiked matrices. Maximum concentrations of levamisole were found on the first day after the administration (531.1 μg/kg in liver, 164.3 μg/kg in egg yolk, 130.7 μg/kg in kidney, 78.0 μg/kg in breast muscle, 70.7 μg/kg in thigh muscle and 64.0 μg/kg in egg white), after which there is a decline. The compound was rapidly eliminated from eggs, with a half-life of 1.3 days. Elimination appeared to be slower in thigh muscle (3.5 days), breast muscle (3.4 days) and liver (3.3 days). According to this experiment, the levamisole withdrawal periods calculated for eggs, liver, kidney, breast muscle and thigh muscle in laying hens were 14.1, 6.1, >4.0, 14.5 and 13.0 days, respectively. The longest time for levamisole residues to be completely released from tissues was seen in liver samples (37.4 days), followed by thigh muscle, breast muscle and kidney. Elimination from eggs was fastest (16.4 days for levamisole residues to drop below the method quantification limit).

Pharmacokinetics of levamisole in the red-eared slider turtles (Trachemys scripta elegans).

The pharmacokinetics and bioavailability of levamisole were determined in red-eared slider turtles after single intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration. Nine turtles received levamisole (10mg/kg) by each route in a three-way crossover design with a washout period of 30days. Blood samples were collected at time 0 (pretreatment), and at 0.25, 0.5, 1, 1.5, 3, 6, 9, 12, 18, 24, 36, and 48hr after drug administration. Plasma levamisole concentrations were determined by a high-performance liquid chromatography assay. Data were analyzed by noncompartmental methods. The mean elimination half-life was 5.00, 7.88, and 9.43hr for IV, IM, and SC routes, respectively. The total clearance and volume of distribution at steady state for the IV route were 0.14Lhr-1 kg-1 and 0.81L/kg, respectively. For the IM and SC routes, the peak plasma concentration was 9.63 and 10.51μg/ml, respectively, with 0.5hr of Tmax . The bioavailability was 93.03 and 115.25% for the IM and SC routes, respectively. The IM and SC route of levamisole, which showed the high bioavailability and long t1/2ʎz , can be recommended as an effective way for treating nematodes in turtles.

Sorption and leaching behavior of bithionol and levamisole in soils

The batch experiments were conducted to understand sorption process of bithionol (BIT) in yellow soil (YS) and red soil (RS), while column leaching experiments were performed to evaluate the leaching behavior of BIT and levamisole (LEV) in the tested soils. The adsorption and desorption data fitted well with the Freundlich isotherms (R2 ≥ 0.94). The distribution coefficient of BIT in the YS and RS were 104 and 98.3 L/kg, respectively. Hysteresis was observed for bithionol desorption in the YS and RS, with hysteresis coefficient of 0.917 and 0.928, respectively. Dissolved organic matter (DOM) addition and acid condition enhanced the adsorption of BIT in the soil. Both BIT and LEV showed poor leaching potential in the tested soils. More than 80% of BIT and LEV remained in the surface soil layer and the amount of the two target compounds in the leachates accounted for less than 1% of overall recovery. DOM showed little influence on the concentration of BIT and LEV in the leachates collected at different time. The results could fill the gap on the behavior of BIT and LEV in soil under laboratory conditions.

Cocaine adulteration with the anthelminthic tetramisole (levamisole/dexamisole): Long-term monitoring of its intake by chiral LC-MS/MS analysis of cocaine-positive hair samples.

Recent studies indicate that not only the anthelminthic levamisole but also the racemate tetramisole (R-/S-phenyltetraimidazothiazole, PTHIT) was found as an adulterant for cocaine. We herein report on the investigation of the prevalence of PTHIT among cocaine-positive hair samples and the discrimination of the presence of its stereoisomers levamisole and dexamisole. Cocaine-positive hair samples were collected in a forensic context in 2015 and mainly 2017 (n=724). Cocaine and PTHIT concentrations have been determined by achiral liquid chromatography-tandem mass spectrometry (LC-MS/MS). For distinction of levamisole/dexamisole chiral LC-MS/MS was performed. Cocaine hair concentrations ranged from 500 (cut-off) to approximately 800 000pg/mg. The study demonstrates a strong prevalence of PTHIT in cocaine users' hair (87%, n=627). PTHIT hair concentrations ranged from below LLOQ 3.5 to approximately 61 000pg/mg (median: 260pg/mg). Surprisingly, enantiomeric ratios of levamisole/dexamisole ranged from 0.17 to 1.34 (median: 0.63). Therefore, PTHIT-adulterated street cocaine samples (n=24) seized between 2013 and 2016 were tested. Samples mainly contained racemic tetramisole (87.5%), only one sample contained levamisole only and two samples contained non-racemic PTHIT. Our experiments suggest that the presence of tetramisole in biological samples may have hitherto been underestimated. Most probably higher dexamisole than levamisole concentrations in hair specimens arise from stereoselective metabolism and/or elimination. This is particularly important in light of the different pharmacological activities of the two enantiomers and potentially different adverse effects. Toxicological interpretations in intoxication cases with adulterated cocaine should not only consider levamisole but also tetramisole and terminology in scientific contributions should be used accordingly.

Estimation of the Withdrawal Time of Levamisole in Eggs after Oral Administration to Laying Hens

This study was conducted to evaluate withdrawal time of levamisole in eggs after oral administration in laying hens at different doses. Sampling of eggs was conducted for 37 days after the end of treatment, and levamisole concentrations were measured by liquid chromatography-tandem mass spectrometry validated according to the Commission Decision 2002/657/EC. Estimated validation parameters were as follows: decision limit, 0.54 μg/kg; detection capability, 0.56 μg/kg; limit of detection, 0.04 μg/kg; limit of quantification, 0.15 μg/kg; accuracy (recovery), between 92.9 and 102.3%; precision (relative standard deviation), ≤4.62%; and within-laboratory precision (relative standard deviation), ≤5.19%. Levamisole residue levels were significantly higher in egg yolks than in egg whites. The highest levels of levamisole were detected on day 2 posttreatment in groups receiving 50 mg/kg of body weight (556.2 μg/kg in egg yolks and 166.5 μg/kg in egg whites). Significant elimination occurred within 5 days after the cessation of treatment in all groups, with an elimination half-life of 1.3 days. Levamisole was still detectable on day 30 after the end of treatment in egg whites (0.06 μg/kg) and on day 37 in egg yolks (0.06 μg/kg). The longest withdrawal time for levamisole in eggs (14.9 days) was determined in a group treated with 25 mg of levamisole per kg of body weight for two consecutive days. According to the results, oral treatment of laying hens with levamisole may result in noncompliant egg samples even 14 days after treatment.

Development and validation of a high-performance thin-layer chromatographic method for the simultaneous determination of levamisole and cocaine in seized cocaine sample

Cocaine is the second most illicit drug worldwide. Levamisole, an anthelminthic drug, has been identified as a cocaine adulterant in the United States since the last decade. Now, its presence has increasingly been discovered in street cocaine samples seized in India. Recently, it has been observed in many street cocaine samples seized by investigation agencies in India. Due to the incidence of toxicity caused by this contamination which has been increasing rapidly since 2002, it is important to understand the chemical separation, identification, and quantification of this drug. In the present study, an attempt has been made to develop a new high-performance thin-layer chromatographic (HPTLC) method for the simultaneous discrimination and quantification of levamisole in seized cocaine samples. Chromatography was performed using a pre-coated silica gel aluminum plate as the stationary phase, and the mobile phase cyclohexane—toluene—diethyl amine (75:15:10) was able to discriminate between cocaine hydrochloride and levamisole hydrochloride significantly. Densitometric analysis was performed in the absorbance mode at 230 nm. The linear regression analysis of data for the calibration curve showed good linearity over a concentration range of 100–1200 ng per band and 200–2400 ng per band with a regression value of 0.997 and 0.995 for levamisole hydrochloride and cocaine hydrochloride, respectively. The developed method is used to quantify cocaine content as well as to estimate the concentration of adulterant levamisole. The method was validated according to the International Conference on Harmonization (ICH) guidelines and can be used in the identification and quantification of levamisole and cocaine in seized cocaine samples. The present method is simple, reproducible, and repeatable, which can easily be performed in any laboratory.