Determination of levamisole in plasma and animal tissues by gas chromatography with thermionic specific detection

doi:10.1016/S0378-4347(00)80134-6

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 224, Issue 1, 12 June 1981, Pages 25-32

https://doi.org/10.1016/S0378-4347(00)80134-6 Get rights and content

Abstract

A rapid and sensitive method has been developed for the determination of the anthelmintic levamisole in plasma and tissues from man and animals. The procedure involves the extraction of the drug and its internal standard from the biological material at alkaline pH, back-extraction into sulphuric acid and re-extraction into the organic phase (heptane—isoamyl alcohol). Several extraction steps can be omitted, however, whenever the gas chromatographic background permits and some operations can be simplified using Clin Elut^TM extraction tubes.

The analyses were carried out by gas chromatography using a nitrogen-selective thermionic specific detector. The detection limit was 5 ng, contained in 1 ml of plasma or in 1 g of the various tissues, and recoveries were sufficiently high (79–86%).

The method was applied to human plasma samples in a comprehensive bioavailability study of levamisole in healthy volunteers, and to plasma and tissues in a residue trial in cattle. The effect of the blood collection technique on the plasma levels was also studied and pointed to decreased plasma concentrations when Vacutainer^® tubes were used.

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Cited by (43)

Estimation of the withdrawal time of levamisole in eggs after oral administration to laying hens
2018, Journal of Food Protection
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.
A sensitive LC-MS/MS method for determination of levamisole in human plasma: Application to pharmacokinetic study
2011, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Citation Excerpt :
Various methods have been published for the determination of levamisole in biological fluids. They included gas chromatography with nitrogen-phosphorus detection (GC-NPD) [2,3], flame ionization detection (GC-FID) [4], liquid chromatography (LC) with ultraviolet (UV) detection [5–7], LC–MS [8,9] or LC–MS/MS [10]. The detection limit of levamisole of the GC-NPD method was 5 ng/mL in plasma.
A sensitive and rapid LC–MS/MS method was developed and validated for the determination of levamisole in human plasma. The assay was based on liquid–liquid extraction of analytes from human plasma with ethyl ether. Chromatographic separation was carried on an Agilent HC-C₈ column (150 mm × 4.6 mm, 5 μm) at 40 °C, with a mobile phase consisting of acetonitrile—10 mM ammonium acetate (70:30, v/v), a flow rate of 0.5 mL/min and a total run time of 6 min. Detection and quantification were performed by mass spectrometry in the multiple reaction monitoring mode with positive electrospray ionization m/z at 205.1 → 178.2 for levamisole, and m/z 296.1 → 264.1 for mebendazole (internal standard). The assay was linear over a concentration range of 0.1–30 ng/mL with a lower limit of quantification of 0.1 ng/mL. The coefficient of variation of the assay precision was less than 8.5%. The assay was successfully used to analyze human plasma samples in a pharmacokinetic study where levamisole was administered as a liniment.
A fluorescence enhancement-based sensor using glycosylated metalloporphyrin as a recognition element for levamisole assay
2006, Biosensors and Bioelectronics
A fluorescence sensor based on the supermolecular recognition by glycosylated metalloporphyrin for levamisole (LEV) assay is reported. For the preparation of a LEV-sensitive active material, 5, 10, 15, 20-tetrakis[2-(2, 3, 4, 6-tetraacetyl-β-D-glucopyranosyl)-1-O-phenyl] porphyrin and its metal complexes were synthesized and used in an optode membrane prepared by including glycosylated metalloporphyrin in chitosan matrice. The immobilized glycosylated metalloporphyrin is shown to be weakly fluorescent as a result of the inhibiting of the electron tansfer by central metal. The fluorescence enhancement of the metalloporphyrin modified optode membrane by LEV is based on the complexation with the central metal moiety of metalloporphyrin and weakening the inhibiting of the electron tansfer for metalloporphyrin. The glycosylated metalloporphyrin/chitosan optode membrane showed excellent selectivity toward LEV with respect to a number of interferents and exhibited stable response. The calibration graph obtained with the proposed sensor was linear over the range of 1.3 × 10⁻⁵–3.5 × 10⁻⁷ M L⁻¹, with a detection limit of 3.5 × 10⁻⁷ M L⁻¹ for LEV. The prepared sensor is applied for the determination of LEV in pharmaceutical preparations and the results agreed with the values obtained by the pharmacopoeia method.
Pharmacokinetics and brain distribution of unbound levamisole in the anesthetized rats using microdialysis and microbore column liquid chromatography
2006, Analytica Chimica Acta
A sensitive microbore liquid chromatographic method was developed for analysis of protein-unbound levamisole in blood and brain of anesthetized rats. Microdialysis probes were simultaneously inserted into the jugular vein toward right atrium and the striatum of brain of the male anesthetized Sprague–Dawley rats for biological fluid sampling after the administration of levamisole (3 mg/kg, i.v.) through the femoral vein. Assay of unbound levamisole was carried out using a microbore Phenyl-Hexyl column (150 mm × 1 mm i.d.; particle size 5 μm). The mobile phase consisted of methanol–10 mM 1-octanesulfonic acid which was adjusted to pH 3.0 with orthophosphoric acid (52:48, v/v) and the flow-rate is 0.05 ml/min. The assay was performed by monitoring the wavelength of maximum UV absorbance at 213 nm. The retention time of levamisole in rat blood and brain dialysates were found to be 10.5 min. The present assay enhanced the detection sensitivity and enabled the determination of levamisole at 50 ng/ml. Average in vivo recoveries of blood and brain dialysate were 46 ± 4 and 12 ± 4%, respectively, at concentration of 1, 5, 10 μg/ml. The result indicates that this method was suitable to investigate the disposition of levamisole and its penetration into blood–brain barrier (BBB).
Liquid chromatographic method with ultraviolet absorbance detection for measurement of levamisole in chicken tissues, eggs and plasma
2003, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
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 μm C₁₈ 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 μg/g for all tissues and 0.003 μg/ml for plasma. Limit of detection was 0.001 μg/g for tissues and plasma.
Liquid chromatographic determination of levamisole in animal plasma: Ultraviolet versus tandem mass spectrometric detection
2003, Analytica Chimica Acta
Citation Excerpt :
Other specificity experiments (interference of analogous compounds) were not performed because levamisole is the only compound belonging to the group of imidazolthiazoles which is used in veterinary medicine [10]. To extract levamisole (and the IS) from the animal plasma, a liquid–liquid extraction based on the procedure described by Woestenborghs et al. [1], was evaluated during preliminary experiments. A mixture of hexane:isoamyl alcohol (95:5, v/v) was used instead of heptane:isoamyl alcohol.
This work presents the optimization and validation of a liquid chromatography (LC)–ultraviolet (UV) detection method and a tandem mass spectrometry (LC–MS/MS) method with positive electrospray ionization for the determination of levamisole, an anthelmintic for veterinary use, in animal plasma. A liquid–liquid extraction procedure in alkaline medium, using hexane-isoamyl alcohol (95:5, v/v) as extraction solvent, was performed to clean-up the plasma samples prior to LC-UV analysis. The sample preparation prior to LC–MS/MS analysis consisted of a protein precipitation step, using acetonitrile. Methyllevamisole was used as the internal standard. Chromatographic separation was achieved on a LiChrospher^® 60 RP-select B (5 μm) column using a mixture of 0.1 M ammonia acetate in water and acetonitrile as the mobile phase. A gradient (flow rate 1 ml min⁻¹) or isocratic (flow rate 0.2 ml min⁻¹) elution was performed in case of the LC-UV and LC–MS/MS methods, respectively. UV detection was at 235 nm. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode.
The methods were validated (linearity, precision, trueness, limit of quantification, limit of detection, specificity) according to the requirements defined by the European Community. Good linearity was achieved over the concentration ranges tested (0–0.5 μg ml⁻¹ and 0–4.0 μg ml⁻¹, r>0.99, goodness-of-fit <10%). Limits of quantification of 0.025 μg ml⁻¹ and 0.1 μg ml⁻¹ were achieved for the LC–MS/MS and LC-UV methods, respectively. The limits of detection were 0.009 and 0.077 μg ml⁻¹, respectively. The results for precision (within-day and between-day) fell within the ranges specified.
The methods have been successfully used for the determination of levamisole in plasma samples from two pigs medicated via drinking water. A good correlation was observed between the results of both methods (r²=0.9831, slope=1.13, intercept=0.005 μg ml⁻¹), proving their usefulness for the application in the field of pharmacokinetic and residue analysis.

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Determination of levamisole in plasma and animal tissues by gas chromatography with thermionic specific detection

Abstract

Anal. Biochem.

J. Med. Chem.

Amer. J. Trop. Med. Hyg.

Ann. Soc. Belg. Med. Trop.

Fortschr. Arzneimittelforsch.