Determination Of Levamisole Research Articles

Simultaneous Determination of Levamisole, Mebendazole, and the Two Metabolite Residues of Mebendazole in Poultry Eggs by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

The quantitative determination of levamisole (LMS), mebendazole (MBZ), and the two metabolites of MBZ, 5-hydroxymebendazole (HMBZ) and 2-amino-5-benzoylbenzimidazole (AMBZ), in poultry eggs (hen, duck, and goose) was achieved with high-performance liquid chromatography–tandem triple quadrupole mass spectrometry (HPLC–MS/MS). Samples were pretreated by liquid–liquid extraction and solid-phase extraction (LLE–SPE) to extract the target compounds, and an Oasis MCX SPE column was used for purification. Determination was performed on an Xbridge C18 column with 0.1% formic acid aqueous solution and acetonitrile as mobile phases. LMS, MBZ, HMBZ, and AMBZ were detected in a triple-quadrupole mass spectrometer with ESI in positive mode and quantified with an external standard. In blank eggs, the target analyte concentrations were within the limits of quantification (LOQs)—25 μg/kg (LMS) and 150 μg/kg (MBZ, HMBZ, and AMBZ)—and the matrix-matched calibration curves had good linearity (R2 ≥ 0.9990). In the same concentration range, the average recoveries of the target analytes were 85.98–97.38% (n = 6); the relative standard deviation (RSD), intraday RSD, and interday RSD ranged from 2.06 to 4.22%, 1.40 to 5.85%, and 2.34 to 6.32%, respectively. The limits of detection (LODs) ranged from 0.03 to 0.33 µg/kg, and the LOQs ranged from 0.08 to 1.00 µg/kg. Experimental verification showed that the HPLC–MS/MS method exhibited high specificity and sensitivity for quantitative analyses of egg samples. This study provides a rapid, efficient, and sensitive method for the simultaneous detection of LMS, MBZ, HMBZ, and AMBZ residues in foods of animal origin.

Determination of Levamisole and Mebendazole and Its Two Metabolite Residues in Three Poultry Species by HPLC-MS/MS.

A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to simultaneously analyze levamisole (LMS) and mebendazole (MBZ) and its two metabolites, 5-hydroxymebendazole (HMBZ) and 2-amino-5-benzoylbenzimidazole (AMBZ), in poultry muscle (chicken, duck and goose). In the sample preparation process, basic ethyl acetate was used as the extraction agent, and the extracted samples were back-extracted with hydrochloric acid, purified by Oasis MCX solid-phase extraction (SPE) cartridges, and reconstituted in the initial mobile phase after being blown dry with nitrogen. Chromatographic separation was performed on an Xbridge C18 column (4.6 mm × 150 mm, 5 μm) with 0.1% formic acid in water and acetonitrile as the mobile phases, and gradient elution was performed at a flow rate of 0.6 mL/min and a column temperature of 35 °C. In blank poultry muscle samples, the spiked concentrations of LMS, MBZ, HMBZ, and AMBZ were within the range of the limit of quantitation (LOQ) to 25 μg/kg. The peak areas of the four target drugs had a good linear relationship with the concentration, and the determination coefficient (R2) values were higher than 0.9990. The average recoveries of LMS, MBZ, HMBZ, and AMBZ were 86.77–96.94%; the intraday relative standard deviations (RSDs) were 1.75–4.99% at LOQ, 0.5 maximum residue limit (MRL), 1.0 MRL, and 2.0 MRL; the interday RSDs were 2.54–5.52%; and the LODs and LOQs were 0.04–0.30 μg/kg and 0.12–0.80 μg/kg, respectively.

Determination of levamisole using ion-selective electrode

Current development of applied ionometry requires focusing of theoretical studies both on revealing the nature of the selectivity of electrode membranes and on the developing the new methods of membrane synthesis and their modification with the goal of obtaining more perfect structural units with improved functional properties. Elucidation of the relationship between the structural characteristics of the membranes and their effect on the electro-analytical properties is a rather important task. The interaction of organic cation levamisole with bromophenol blue was studied to meet the goals. Levamisole-selective sensor with a plasticized polyvinyl chloride (PVC) membrane has been developed. The electrode contains an ionic associate of levamisole with bromophenol blue. PVC was used as a matrix to model the membrane composition. Membranes plasticized with dibutyl phthalate (DBP), diethyl phthalate (DEP), dioctyl phthalate (DOP), dinonyl phthalate (DNP), dibutyl sebacate (DBP), tricresyl phosphate (TCP) were studied. It was shown that the nature of the plasticizer insignificantly affected the analytical characteristics of the electrode. The response is linear within the concentration range of 1 × 10–5 – 1 ×10–1 mole/liter with the slope of the electrode function 52.1 ± 1.0 mV/pC. The sensor has a quick response (10 sec) and can be used for at least 8 weeks without any divergence in the long run. The operation life of the membranes with a high plasticizer content is much longer. The electrode can be used within pH range of 3.0 – 8.0. Potentiometric selectivity coefficients of the electrode relative to a number of potentially interfering ions were determined. The developed electrode was tested in determination of levamisole in model solutions and pharmaceuticals («Decaris» and «Levamisole-Zdorovye»).

Determination of levamisole and tetramisole in seized cocaine samples by enantioselective high-performance liquid chromatography and circular dichroism detection

Levamisole, an anthelmintic drug, has been increasingly employed as an adulterant of illicit street cocaine over the last decade; recently, the use of tetramisole, the racemic mixture of levamisole and its enantiomer dexamisole, was also occasionally observed. A new enantioselective high-performance liquid chromatography (HPLC) method, performed on cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases in normal-phase mode, was validated to determine the enantiomeric composition of tetramisole enantiomers in seized cocaine samples. Furthermore, the hyphenation of the validated HPLC method with a circular dichroism (CD) detection system allowed the direct determination of elution order and a selective monitoring of levamisole and dexamisole in the presence of possible interferences. The method was applied to the identification and quantitation of the two enantiomers of tetramisole in seized street cocaine samples.

Determination of levamisole residue in animal livers by two liquid-liquid extraction steps-gas chromatography-mass spectrometry

A novel method wasdeveloped for the determination oflevamisole in animal livers by liquid-liquid extraction and gas chromatography-mass spectrometry. Levamisole hydrochloride was transferred to levamisole in alkalinesolution, andthen extracted intoethyl acetate. Two liquid-liquid extraction steps were carried out with HCI aqueous solution and potassium hydroxide-dichloromethane system in turn, and the liposoluble substances and the water-soluble substances were removed, respectively. The qualitative and quantitative analyses of levamisole were achieved by gas chromatography-mass spectrometry (GC-MS) system. The characteristic fragments m/z 148, 176 and 204 were selected and m/z 204 was used as the quantitative ion. A good linear relationship was obtained between the peak area and the mass concentration of levamisole from 0.25 mg/L to 3.0 mg/L with the correlation coefficient of 0.999. The limit of quantitation (LOQ) for levamisole was 5 microg/kg, which was much lower than the international maximum residue limit. The average recoveries of levamisole spiked in chicken liver, duck liver, rabbit liver and pig liver samples at three spiked levels ranged from 76% to 106% with the relative standard deviations less than 9%. The method is simple and stable without tedious sample preparation, and suitable for the determination of levamisole in animal livers.

Determination of levamisole in feeds by liquid chromatography coupled to electrospray mass spectrometry on an ion trap

Test methods have to be developed by laboratories for official control to monitor possible misuse of veterinary drugs in animal productions, also through feeding stuff. A novel method for identification and quantification of levamisole in feeds by liquid chromatography coupled to electrospray mass spectrometry in an ion trap (LC/ESI-MS/MS) is herein described; after a single-step cleanup by liquid-liquid extraction from the feed and separation by reversed-phase liquid chromatography, levamisole was determined and unambiguously confirmed by tandem mass spectrometry, on the basis of two product ions. The method was in-house validated, according to the Regulation 882/2004/EC, evaluating trueness, repeatability, within-laboratory reproducibility, ruggedness, specificity, and the limit of quantification (LOQ). The method is reliable and specific for complete and complementary feeds for pigs, cattle, rabbits and poultry; very good mean recoveries (higher than 92 %) and precision (RSD values < 15.2%) were attained. The LOQ at 2.0 mg/kg was verified. Moreover, we describe how the method was developed to support Italian Police investigations regarding illegal treatments of pigs; in this case, since the drug(s) added to the feed were unknown, a preliminary untargeted analysis was performed by full scan mass spectrometry on an ion trap, from 50 up to 2000 m/z; the presence of levamisole was hypothesised, on the basis of the most abundant ion and its fragmentation pattern. Then, levamisole was unambiguously confirmed by the ion trap LC/ESI-MS/MS method.

Determination of Levamisole in Urine by Gas Chromatography-Mass Spectrometry

The United States Public Health Service Substance Abuse and Mental Health Services Administration is alerting medical professionals that a substantial percentage of cocaine imported into the United States is adulterated with levamisole, a veterinary pharmaceutical that can cause blood cell disorders such as severe neutropenia and agranulocytosis. Levamisole HCl is the active ingredient in a number of veterinary drugs approved to treat worm infestations in animals. Levamisole HCl was also the active ingredient in a human drug for oral administration approved on June 18, 1990, as adjuvant treatment in combination with fluorouracil after surgical resection in patients with Duke's stage C colon cancer. This drug was withdrawn from the U.S. market around 2000, and it has not been marketed in the U.S. since then. The objective of this study was to develop a method to determine the amount of levamisole in urine samples. The procedure will be provided to state health laboratories as needed to be used in the evaluation of patients that have developed neutropenia or agranulocytosis in the setting of recent cocaine use. A gas chromatography-mass spectrometry method was validated and tested at two different laboratories, and the method limit of detection for levamisole is 1 ng/mL in urine when using a 5-mL sample. Confirmation of the stereoisomer of levamisole was done by high-performance liquid chromatography using a chiral column.

Development and validation of a simple stability-indicating TLC method for the determination of levamisole in pharmaceutical tablet formulation

Asimple, selective, precise, and stability-indicating thin-layer chromatographic (TLC) method has been established and validated for analysis of levamisole (LEV) in pharmaceutical dosage. Normal phase precoated aluminum TLC silica gel 60F254 plates were used as stationary phase and MeOH-toluene-chloroform (14:36:50 v/v/v) was used as mobile phase to determine LEV in pharmaceutical ingredients. The system was found to give compact spot for LEV (RF = 0.30). Determination was performed in absorbance mode at 223 nm. Regression analysis data for the calibration plots indicated good linear relationships between response and concentration over the range 60–2000 ng per spot and 0.998 correlation coefficient. The slope and intercept of the calibration plot were 4.946 and 267.6, respectively. The recovery of 88.44–102.57% with 2.02% inter-day and 1.91% intra-day relative standard deviations (RSDs) shows the validity of the method. Further, the limit of detection (LOD) and the limit of quantification (LOQ) were 2.2 ...

Spectrophotometric Simultaneous Determination of Levamisole and Triclabendazole in Tablets by Principal Component Regression and Partial Least Squares Chemometric Methods

Principal component regression (PCR) and partial least squares (PLS) chemometric methods were applied to the simultaneous quantitative analysis of levamisole (LVM) and triclabendazole (TCB) in tablets without using a preliminary separation, even in presence of the overlapping spectra of the above compounds. For both PCR and PLS, a concentration set containing 25 different mixtures of LVM and TCB in the linear concentration range was symmetrically prepared and then the absorbance values of the concentration set were measured at the wavelength set with Dl=0.1 nm in the spectral region of 225-322.3 nm. PCR and PLS calibrations were obtained by applying the PCR and PLS algorithms to the concentration set data (y-block) and their corresponding absorbance data (x-block). The validity of PCR and PLS chemometric methods was performed by using the independent synthetic mixtures and the standard addition technique. Then, these analytical methods were applied to the commercial tablets and a good agreement was obtained between experimental results provided by the application of the PCR and PLS to the synthetic and real samples.

Rapid, Simultaneous Determination of Levamisole and Abamectin in Liquid Formulations Using HPLC

The aim of the study was to develop a high performance liquid chromatography (HPLC) assay for the rapid, simultaneous determination of levamisole phosphate and abamectin in liquid formulations containing mixtures of solvents and medium chain mono‐ and diglycerides and triglycerides. The experimental procedure involved reversed‐phase‐HPLC with a Zorbax ODS column (7 µm particle size, 4.6 ID × 250 mm), acetonitrile‐water‐ and ammonia 1.0N volumetric solution (80:20:0.1, v/v/v) mobile phase, UV detection at 253 nm for both levamisole phosphate and abamectin. The flow rate of the mobile phase was 2 mL/min. The retention times of levamisole phosphate and abamectin varied from 2.45–2.46 min and 5.78–5.82 min, respectively. Formulation components did not give rise to any interfering peaks. Calibration curves were linear over the range 13.5–270 µg/mL for levamisole phosphate and 0.5–10 µg/mL for abamectin. The maximum intraday and interday coefficients of variation were 0.7% and 1% at 13.5 µg/mL for levamisole phosphate, and 2.2% and 5.8% at 0.5 µg/mL for abamectin, respectively. Accuracies were 98.9 ± 5.2% and 96.2 ± 3.1% (Mean ± SD, n = 14) for levamisole phosphate and abamectin, respectively, at concentrations of 27.3 µg/mL of levamisole phosphate and 1 µg/mL of abamectin. The assay was used to evaluate the stability of these drugs in the liquid formulations. A simple and accurate liquid chromatographic method was developed and validated for simultaneous determination of levamisole and abamectin in liquid formulations. Due to its simplicity and accuracy, the assay method is suitable for routine analysis of both drugs in liquid formulations.

Determination of levamisole in animal tissues using liquid chromatography with ultraviolet detection.

An efficient and sensitive liquid chromatographic method is described for the determination of the anthelminthic drug levamisole, in muscle, liver, kidney and fat of sheep, pigs and poultry, using thiabendazole as internal standard. Samples were extracted by homogenizing with chloroform, and were applied to Supelco Si solid-phase extraction columns and eluted with methanol. Chromatographic analysis was performed on a LiChrospher 60 RP-Select B column using methanol/ammonium acetate buffer 0.05 M (55/65, v/v) as mobile phase and reading at 220 nm. The quantification limit for the assay was 4 ng/g. Mean recoveries were about 84% for liver, 85% for kidney, 89% for muscle and 84% for fat. The assay has been used for statutory testing purposes.

A highly sensitive gas chromatographic determination of levamisole in milk

An efficient extraction and sensitive gas chromatographic method is described for the determination of the anthelminthic drug levamisole in milk. Levamisole was extracted from alkaline milk with ethyl acetate. Clean‐up of the extract was by a series of liquid‐liquid extraction steps. Levamisole residues in the extract were determined by gas chromatography with a nitrogen‐phosphorus detector. This method was satisfactory for determining levamisole residues in milk as low as 0.5 ng/g. Mean recoveries of 0.5–10.0 ng/g fortified milk samples ranged from 84.5 to 95.2%. Five replicate analyses performed on a milk containing incurred levamisole residues yielded a mean of 3.34 ng/g levamisole with a CV of 3.0%.

Determination of levamisole and thiabendazole in meat by HPLC and photodiode array detection

An HPLC method for the analysis of levamisole and thiabendazole has been developed with recoveries varying over 63-75%. The two anthelmintics are extracted from meat with ethyl acetate, purified by liquid/liquid extraction and analyzed quantitatively on a mu Bondapak C18 column. The optimum detection is achieved by means of a photodiode array detector at 240 nm for levamisole and at 300 nm for thiabendazole. The detection limits for both compounds in meat are 25 micrograms/kg and 5 micrograms/kg, respectively.

Determination of Levamisole by HPLC in Plasma Samples in the Presence of Heparin and Pentobarbital

Abstract A method which allows for the isolation of levamisole in plasma samples of rabbit which contained pentobarbital and heparin as well, used as an anaesthetic and an anticoagulant respectively, is described in this paper. The subsequent quantification was carried out using a ion-pair high-performance liquid chromatographic method in a reversed and isocratic phase at room temperature, using a UV detection at 225 nm. In our conditions the retention time for the levamisole was about 2.15 minutes and the average percentage of recuperation was that of 73.62%.

Gas-chromatographic determination of levamisole in human plasma-normalization and reliability of the method.

Levamisole has been used by many authors as an immunostimulant agent but the relationship between oral doses, plasma level and the therapeutic effects were not known. For such a study it was necessary to establish an accurate and reproducible method of measurement. We describe a modification of the gas chromatographic method of Wynants et al. (1975). The normalization of the extraction of the extraction process leads to greater reliability and accuracy (4 ng +/- 7.6%). Reproducibility was established using a series of 30 determinations from standard solutions and plasmas supplemented with Levamisole. The relative standard deviation in K evaluation was varied between 0.9 and 1.8%, 3.7 and 6.1% for standard solutions and plasmas respectively. Consequently this method was adopted for this pharmacokinetic study which was carried out on healthy and cancer patients.

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

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.