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

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: Based on observations, the developed measure can determine levamisole concentrations in participant saliva samples.

Pharmacokinetics and pharmacodynamics of single and multiple-dose levamisole in belugas (Huso huso): Main focus on immunity responses.

The pharmacokinetics of levamisole were determined in the belugas after single intravascular (IV), and single and multiple-dose oral by feed administrations. Also, the effect of levamisole (LVM) on the stress and immune responses of belugas were assessed. One hundred-fourteen healthy belugas in 4 different groups received single LVM administration at the doses of 50 and 100 mg/kg via IV and oral routes. A separate group of 24 belugas were administered oral LVM at the dose of 100 mg/kg for 5 days. Blood samples were collected at different time points after administrations to measure plasma concentrations of LVM by a validated high-performance liquid chromatography (HPLC) assay. For immunological evaluations, a total of 126 belugas received 50 and 100 mg/kg LVM via medicated feed for 5 days or served as the control without any medication; blood samples were recovered on day 0, 1, 3, 5, 7, 10, and 14 to measure hemolytic activity of the complement system (HAC50), serum lysozyme activity, serum antibacterial activity, glucose, cortisol, total protein, albumin and C3 contents. In the single-dose administration, quantified LVM concentrations were dose-dependent and the oral bioavailability was in the range of 43.2-49.6%. In the multiple-dose administration, the peak plasma concentration at the steady state was 45.2 mg/ml, and accumulation ratio was calculated as 3.6. In the immunological study, LVM especially at the dose of 100 mg/kg increased HAC50, lysozyme and antibacterial activity in the sera of treated fish. No significant effect of LVM on glucose and albumin content was observed, but cortisol levels decreased and C3 content was increased, more significantly by LVM at the dose of 100 mg/kg. Our results indicate that LVM is well absorbed after oral administration and reached to concentrations that can affect stress indicators and improve immune responses in belugas.

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 (10 mg/kg) by each route in a three-way crossover design with a washout period of 30 days. 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 48 hr 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 9.43 hr for IV, IM, and SC routes, respectively. The total clearance and volume of distribution at steady state for the IV route were 0.14 L hr-1  kg-1 and 0.81 L/kg, respectively. For the IM and SC routes, the peak plasma concentration was 9.63 and 10.51 µg/ml, respectively, with 0.5 hr 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.

Pharmaco-parasitological evaluation of the ricobendazole plus levamisole nematodicidal combination in cattle.

The goals of the current study were to evaluate the potential pharmacokinetic (PK) interactions and the clinical efficacy occurring after the subcutaneous (s.c.) administration of ricobendazole (RBZ) and levamisole (LEV) given both separately and co-administered to calves naturally infected with susceptible gastrointestinal nematodes. The clinical efficacy was shown in two seasons, winter and spring, with predominance of different nematode populations. Groups of 15 calves were treated with RBZ alone, LEV alone and RBZ + LEV combination, and an untreated group was kept as a Control. RBZ and LEV plasma concentrations were quantified by HPLC. The clinical efficacy was determined by the faecal egg count reduction test. RBZ and LEV have similar plasma persistence, being detected in plasma over 24 hr post-treatment. No PK interactions were observed after the combined treatment, with similar PK parameters (p > .05) obtained for the single-drug and the combination-based strategy. In winter, the observed clinical efficacies were 96%, 99% and 100% for groups treated with RBZ, LEV and RBZ + LEV, respectively; however, in spring, the efficacies were 95%, 93% and 96% for the same groups. Remarkably, the combination was the only treatment that achieved 100% clinical efficacy against both Haemonchus spp and Ostertagia spp in winter; but the increased presence of Ostertagia spp. in spring (28% in untreated group) determined a tendency to reduced efficacies compared to winter time (only 10% of Ostertagia spp. in untreated group), even for the combined treatment. Overall, in a scenario where the nematode population is susceptible, the RBZ + LEV treatment may be a valid combination in cattle to delay the development of resistance, especially in winter when this combination achieved 100% of efficacy. Thus, selection of anthelmintic resistance will never occur. In fact, this is one of the greatest challenges for the whole cattle production system: to be one step ahead of anthelmintic resistance.

Population pharmacokinetics of levamisole in children with steroid-sensitive nephrotic syndrome.

AIM: The aim was to investigate the population pharmacokinetics of levamisole in children with steroid-sensitive nephrotic syndrome. METHODS: Non-linear mixed effects modelling was performed on samples collected during a randomized controlled trial. Samples were collected from children who were receiving 2.5 mg kg(-1) levamisole (or placebo) orally once every other day. One hundred and thirty-six plasma samples were collected from 38 children from India and Europe and included in the analysis. A one compartment model described the data well. RESULTS: The apparent clearance rate (CL/F) and distribution volume (V/F) were 44 l h(-1) 70 kg(-1) and 236 l 70 kg(-1) , respectively; estimated interindividual variability was 32-42%. In addition to allometric scaling of CL/F and V/F to body weight, we identified a significant proportional effect of age on CL/F (-10.1% per year). The pharmacokinetics parameters were not affected by gender, tablet strength or study centre. The median (interquartile range) maximum plasma concentration of levamisole was 438.3 (316.5-621.8) ng ml(-1) , and the median area under the concentration-time curve was 2847 (2267-3761) ng ml(-1) h. Median tmax and t½ values were 1.65 (1.32-2.0) h and 2.60 (2.06-3.65) h, respectively. CONCLUSIONS: Here, we present the first pharmacokinetic data regarding levamisole in children with steroid-sensitive nephrotic syndrome. The pharmacokinetic profile of levamisole in children was similar to findings reported in adults, although the elimination rate was slightly higher in children.

A sensitive method for the determination of levamisole in serum by electrochemiluminescence.

A novel method was developed for the determination of levamisole by electrochemiluminescence. The method was based on electrochemiluminescence signal enhancement produced by Ru(bpy)(3)(2+), which reacted with the tertiary amine group of levamisole on a platinum electrode in 12 mmol/L borate buffer (pH 9). A linear relationship between the luminous intensity and concentration of levamisole in the range 0-1 × 10(-7) mol/L was obtained and the detection limit was 1.76 × 10(-11) mol/L. The method is sensitive, selective, simple and convenient. The method has been successfully applied to the analysis of levamisole in serum.

A green, fluorescent probe employing erythrosine-B for tracing the accidental administration of levamisole in milk and plasma samples.

A simple and sensitive fluorescent probe has been developed and optimized to detect the non-intentional administration of levamisole (LVM). LVM is used as an anthelmintic therapy in cows, and hence, its residues appear in the drained milk until 60 hours after administering the drug. Meanwhile, levamisole is known to be an adulterant to cocaine and could be detected in addicts' plasma samples. Owing to its severe side effects, including agranulocytosis, which is lethal in many cases, detection and quantification of LVM in milk and plasma samples are of utmost importance. Therefore, a sensitive and selective analytical method is required for this purpose. This work develops a highly fluorescent probe obtained through the reaction between LVM and erythrosine-B in an acidic medium, where the produced ion pair complex has been measured at 553 nm after excitation at 528 nm. The proposed method provides linearity over the concentration range of 0.5-2.0 µg mL-1 for LVM, with a corresponding detection and quantitation limit of 0.5 and 0.3 µg mL-1. Full validation was performed, permitting the application of the suggested method to perform simple extraction steps. All the applied procedures followed the guidelines offered by green analytical chemistry, where the Green Analytical Procedure Index (GAPI) assessed the greenness of the proposed tool, and the yielded pictograms proved the eco-friendliness of the offered tool.

Bioanalytical methods for quantitation of levamisole, a widespread cocaine adulterant.

Abstract Levamisole is an anthelminthic that was first used as a de-worming agent in humans and animals. It has also been used to treat inflammatory conditions as well as certain types of cancer. Levamisole was discontinued for human use in the early 21st century due to toxic side effects including agranulocytosis and vasculitis. Recently, levamisole was discovered as a cocaine adulterant after reports emerged of drug users with the above disorders. As the prevalence of cocaine usage has grown in the last 15 years, measurement of levamisole in human samples has become increasingly important. This review focuses on the various bioanalytical methods available for the determination of levamisole in human plasma and urine. Earlier methods employed gas chromatography coupled with nitrogen-selective thermionic specific detection and nitrogen-phosphorus detection, as well as high performance liquid chromatography coupled with ultraviolet detection. In addition, gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) have also been described. Currently, GC-MS appears to be the method of choice however recent developments in the area of LC-MS/MS make this technology an attractive alternative. The merits of both GC-MS and LC-MS/MS for the determination of levamisole are evaluated on the basis of sample preparation, chromatographic separation conditions, run time, and analytical performance. In addition, emerging methods in this area are also reviewed.

Determination of levamisole in the plasma of patients with falciparum malaria using high-performance liquid chromatography.

BACKGROUND: The oral antihelmintic drug levamisole reduces sequestration of late stage parasites in falciparum malaria. Levamisole has been also identified as a cocaine adulterant. In the present study, authors developed a sensitive and selective HPLC-assay for the determination of levamisole in the plasma from patients with falciparum malaria. METHODS: Chromatographic separation was achieved by using a C18 column and with an isocratic elution system comprising phosphate buffer and acetonitrile. The eluate was monitored at 235 nm by diode array detection. RESULTS: The calibration curve for levamisole was linear in the range from 50 to 2000 ng/mL (r2 > 0.999). The limit of quantification was 28 ng/mL and the inter- and intraday coefficients of variation were less than 7%. No interference from commonly prescribed malaria treatments was observed. CONCLUSIONS: The HPLC method is simple, rapid, and robust and is suited for monitoring levamisole patients in routine or toxicological studies.

Electromembrane extraction of levamisole from human biological fluids.

Electromembrane extraction coupled with high-performance liquid chromatography (HPLC) and ultraviolet (UV) detection was developed for the determination of levamisole in some human biological fluids. Levamisole migrated from 4 mL of different acidized biological matrices, through a thin layer of 2-nitrophenyl octyl ether containing 5% tris-(2-ethylhexyl) phosphate immobilized in the pores of a porous hollow fiber, into a 20-µL acidic aqueous acceptor solution present inside the lumen of the fiber. The parameters influencing electromigration were investigated and optimized. Within 15 min of operation at 200 V, levamisole was extracted from different biological fluid samples with recoveries in the range of 59-65%, which corresponded to preconcentration factors in the range of 118-130. The calibration curves showed linearity in the range of 0.5-10, 0.2-10 and 0.1-10 µg/mL for plasma, urine and saliva, respectively. Limits of detection of 0.1, 0.07 and 0.05 µg/mL and limits of quantification of 0.5, 0.2 and 0.1 µg/mL were obtained for plasma, urine and saliva, respectively. The relative standard deviations of the analysis were found to be in the range of 5.6-9.7% (n = 3). Electromembrane extraction was successfully processed for determination of levamisole in plasma, urine and saliva samples.

Plasma and Urine Levamisole in Clinical Samples Containing Benzoylecgonine: Absence of Aminorex.

Aminorex has been reported as a metabolite of levamisole in man, but data on the aminorex concentrations in clinical samples are scant. We thus measured levamisole, aminorex and benzoylecgonine in urine, and levamisole and aminorex in plasma using achiral liquid chromatography-high resolution mass spectrometry. Centrifuged urine (50 µL) was diluted with LC eluent containing internal standard (benzoylecgonine-D3, 25 µg/L) (450 µL). For plasma, sample (200 µL) and Tris solution (2 mol/L, pH 10.6, 100 µL) were added to a 60.5 × 7.5 mm i.d. glass test tube. Internal standard solution (ketamine-D4, 200 µg/L) (10 µL) was added and the tube contents vortex-mixed (5 s). Butyl acetate:butanol (9 + 1, v/v; 200 µL) was added and after vortex-mixing (30 s) and centrifugation (13,680 × g, 4 min), the extract was evaporated to dryness and reconstituted in 10 mmol/L aqueous ammonium formate containing 0.1% (v/v) formic acid (150 µL). Prepared samples and extracts (100 µL) were analyzed using an AccucoreTM Phenyl-Hexyl column (2.6 mm a.p.s., 100 × 2.1 mm i.d.) maintained at 40°C. MS detection was in positive mode using heated electrospray ionization (ThermoFisher Q-ExactiveTM). Intra- and inter-assay accuracy and precision were ±20%, and ≤11%, respectively, for all analytes in both matrices. Lower limits of quantitation were 0.1 and 1 µg/L (all analytes) in plasma and urine, respectively. Of 100 consecutive urine samples submitted for drugs of abuse screening containing benzoylecgonine, levamisole was detected in 72 (median 565, range 4-72,970 µg/L). Levamisole was also measured in eight plasma samples (median 10.6, range 0.9-64.1 µg/L). A number of metabolites of levamisole (4-hydroxylevamisole, levamisole sulfoxide, levamisole glucuronide, and hydroxylevamisole glucuronide) were tentatively identified in urine. Neither aminorex, nor any of its reported metabolites were detected in any sample.

Monitoring of Levamisole Concentration in Serum of Traffic Participants after Cocaine Consumption.

This study highlights the problem of levamisole-adulterated cocaine in context of active traffic participation. For the purposes of levamisole concentration monitoring in human serum, an analytical method based on LC-MS/MS and solid-phase extraction was applied. A Luna 5 µm C18 (2) 100 A, 150 mm × 2 mm column and a mobile phase consisting of A (H2 O/methanol = 95/5, v/v) and B (H2 O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and with 0.1% acetic acid (pH = 3.2), were used. The validation experiments demonstrated that the method applied was appropriate for levamisole quantification in human serum. For 23% of levamisole-positive samples, the concentrations exceeded 20 ng/mL. Therefore, the interaction of this drug with cocaine has to be considered as important for active traffic participation. As a consequence, monitoring of levamisole concentration in human serum is recommended, as long as it is used as cocaine adulterant.

Pharmacokinetics of levamisole in cancer patients treated with 5-fluorouracil.

PURPOSE: To investigate the pharmacokinetics of levamisole and a metabolite, p-hydroxylevamisole in patients with colorectal cancer treated with 5-fluorouracil (5-FU). METHODS: Following an intravenous bolus dose of 5-FU, 20 patients with colorectal cancer received oral doses of 50 mg levamisole every 8 h for 3 days. Immediately after the last dose, blood and urine samples were collected over at least an 8-h period. Samples were assayed for levamisole and p-hydroxylevamisole by GC/MS. The levamisole plasma and urine data were subjected to pharmacokinetic analysis using NONMEM software. RESULTS: Substantial interpatient variability was observed in the levamisole plasma concentration-time curves. Patients with cardiovascular or gastrointestinal complications demonstrated altered absorption of levamisole. Pharmacokinetic parameter values for levamisole were similar to those obtained previously in healthy subjects and other cancer patients. CONCLUSIONS: There is no evidence that the pharmacokinetics of levamisole are altered by 5-FU administered immediately prior to levamisole administration. The relationship between the substantial intersubject variability in levamisole plasma concentration-time curves and clinical outcome following 5-FU/levamisole adjuvant chemotherapy should be examined.

Liquid chromatographic method with ultraviolet absorbance detection for measurement of levamisole in chicken tissues, eggs and plasma.

The development and validation of a high-performance liquid chromatographic and UV detection method was accomplished for quantitative determination of levamisole in chicken tissues, eggs and plasma. The chromatographic separation was achieved on Luna 5 microm C(18) column using a mobile phase of 0.2% acetic acid in water:methanol (50:50 (v/v)) and Pic B-7 low UV reagent and the pH was adjusted to 7.31 with ammonium hydroxide and UV wavelength was 225 nm. Limits of quantification were 0.025 microg/g for all tissues and 0.003 microg/ml for plasma. Limit of detection was 0.001 microg/g for tissues and plasma.

Comparison of three routes of administration of a water soluble anthelmintic: levamisole.

The plasma concentration of levamisole was determined by liquid chromatography in three groups of 20 heifers treated with levamisole by intramuscular administration, drenching and in drinking water respectively. Maximum concentrations were observed at three hours (1.10 +/- 0.16 microgram/ml) and six hours (0.64 +/- 0.06 microgram/ml) after intramuscular administration and drenching respectively. Large individual variations in plasma concentrations were observed following treatment in drinking water with four animals showing maximum concentrations below 0.1 microgram/ml. Variation in water intake therefore makes the latter method less reliable.

Pharmacokinetics of levamisole in sheep.

Levamisole, at a dose rate of 7.5 mg/kg, produced mean peak plasma concentrations of 3.1, 0.7 and 0.8 microgram/ml in four sheep after administrations by the subcutaneous, oral and intraruminal routes respectively. The mean peak concentrations in abomasal fluid were 33, 164 and 21 micrograms/ml respectively. The bioavailability of levamisole to the systemic compartment was less after oral and intraruminal administration than after subcutaneous administration. In six sheep there were no significant differences in the plasma concentrations obtained after subcutaneous administration in the thoracic, neck or gluteal regions. Dividing the dose between five sites in the gluteal region produced higher peak plasma concentrations than when injected into a single site.

Plasma protein binding of levamisole in several species.

The binding of levamisole to total plasma proteins of 6 animal species was determined in vitro by equilibrium dialysis. The percentage of bound drug protein was independent of levamisole concentration within the range studied, 5-50 micrograms/ml (ANOVA). Levamisole was bound to a low extent to plasma proteins of each animal species (19.40-25.91%). There were significant differences in the extent of levamisole binding among species (ANOVA). Owing to the low degree of protein binding and the high volume of distribution of levamisole, the variations in protein binding due to different factors would not be of major clinical importance in its therapeutic application.

Lymphocytes T and pharmacokinetics after a single oral dose of levamisole in healthy and cancer subjects.

Levamisole, an anthelmintic drug with immunopotentiating activity, is shown to have variable effects on cell-mediated immunity. We have studied the effect of a single oral dose (2.5 mg/kg or 5 mg/kg) on early and spontaneous E rosettes percentages in healthy and cancer patients. Pharmacokinetic study of this compound was conducted in parallel. The results indicated that single administration of levamisole (2.5 mg/kg) in healthy men can promote an increase of early E rosettes with mean peak plasma level of 0.8 micrograms. ml-1. On the other hand, there was no change in the proportion of early E rosettes in cancer patients and in the proportion of spontaneous E rosettes in healthy and cancer subjects.

Effect of first-pass hepatic metabolism on the disposition of levamisole after intravenous administration in rabbits.

OBJECTIVE: To evaluate the contribution of first-pass hepatic metabolism of levamisole on levamisole disposition in rabbits. ANIMALS: 30 male New Zealand White rabbits. PROCEDURES: Rabbits were randomly placed into 2 groups. Rabbits in the first group received levamisole via the marginal ear vein at the following 3 doses: 12.5, 16, and 20 mg/kg (5 rabbits for each dose). Rabbits of the second group received levamisole via the jejunal vein at the same doses (5 rabbits each). During the following 240-minute period, plasma samples were obtained and quantified for levamisole concentrations by reversed-phase high-performance liquid chromatography. RESULTS: No significant differences were found between pharmacokinetic parameters calculated by compartmental or noncompartmental analysis. Mean hepatic extraction ratio ranged from -0.044 to 0.017 and from 0.020 to 0.081 when area under the plasma concentration-time curve values were obtained after compartmental or noncompartmental analysis, respectively. After compartmental analysis, plasma concentration decreased bi-exponentially. Mean pharmacokinetic parameter values were as follows for each dose (12.5, 16, and 20 mg/kg, respectively): after levamisole administration via the marginal ear vein, volume of distribution at steady state (Vss) = 4.26, 4.33, and 3.20 L/kg; total body clearance (CI) = 49.04, 43.77, and 39.26 mL/kg x min; and half-life associated with beta-phase (t1/2beta) = 77.93, 85.39, and 69.79 minutes. After levamisole administration via the jejunal vein, Vss = 4.38, 2.85, and 2.97 L/kg; CI = 48.14, 42.40, and 39.69 mL/kg x min; and t1/2b = 101.9, 76.71, and 76.13 minutes. CONCLUSIONS: Levamisole has a low degree of hepatic extraction in rabbits.

Anthelmintic efficacy, safety, and residue evaluation of levamisole gel formulation in cattle.

Anthelmintic efficacy, safety, and residue studies were conducted in beef calves using a levamisole gel formulation. The effectiveness of levamisole gel and tablet formulations was studied in 30 calves with experimental infection of Ostertagia ostertagi. Removal of 33- to 34-day-old O ostertagi was 99.6% and 97.7%, for the gel and tablet formulation, respectively, when administered orally to supply 8 mg of levamisole HCl equivalent/kg of body weight. A comparative blood concentration study was also used to demonstrate bioequivalence of the levamisole gel to the tablet formulation. In a cross-over-designed test, 5 cattle/treatment group were dosed orally with levamisole 11.5% gel or levamisole tablets at the dosage rate of 8 mg of levamisole HCl/kg. Blood levamisole values were similar with the levamisole gel and tablet formulations. In a safety study, 3 groups of 6 calves each were given levamisole gel orally to provide 8, 24, or 40 mg of levamisole HCl/kg. A 4th group served as controls. Adverse clinical signs were not observed in cattle treated with the recommended dosage level of 8 mg/kg. Transient salivation was noticed in 1 placebo control calf, 2 calves given 24 mg/kg, and 4 calves given 40 mg/kg. Edible tissues from cattle given a single oral dose of levamisole gel (8 mg/kg) were analyzed for drug residues 2 hours and 2, 3, 5, and 7 days after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)