Hydroxy levamisole and its phase II conjugates as potential indicators of levamisole doping in thoroughbred horses.

RATIONALE: According to previous research, aminorex is the major metabolite of levamisole; however, in the screening of levamisole-positive racehorse urine and plasma samples, aminorex could only be detected in trace amounts or not at all. In forensic laboratories, hydroxy levamisole and its phase II conjugates make it easier to confirm levamisole misuse and to differentiate between the abuse of levamisole and aminorex. This study aimed to identify the major levamisole metabolites that can be detected along with the parent drug. METHODS: The study describes levamisole and its metabolites in thoroughbred horses following oral administration and in vitro with equine liver microsomes. The plausible structures of the detected metabolites were postulated using liquid chromatography combined with high-resolution mass spectrometry. RESULTS: Under the experimental conditions 26 metabolites (17 phase I, 2 phase II, and 7 conjugates of phase I metabolites) were detected (M1-M26). The major phase I metabolites identified were formed by hydroxylation. In phase II, the glucuronic acid conjugates of levamisole and hydroxy levamisole were detected as the major metabolites. In plasma, the parent drug and major metabolites are detectable for up to eight days, while in urine, they are detectable for up to twenty days. Levamisole levels rapidly increased at 45 min following administration, then declined gradually until detectable levels were reached approximately 8 days after administration, according to a pharmacokinetics study. CONCLUSIONS: A prolonged elimination profile and relatively high concentration of hydroxy metabolites suggest that the detection of hydroxy metabolites is imperative for investigating levamisole doping in horses.

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.

The cocaine cutting agent levamisole is frequently detected in cocaine users.

Cocaine use in Australia is increasing, with approximately 2.5% of the surveyed population having used cocaine. In the USA, levamisole, a widely used anti-helminthic veterinary drug has been increasingly detected as a cutting agent in cocaine seizures. Levamisole is known to cause agranulocytosis in humans. We ascertained the prevalence of levamisole-adulterated cocaine, detectable in the urine from patients that had undergone a pathology request for a urine drug screen. We assayed routinely requested urines that were positive for cocaine on immunoassay with liquid chromatography high resolution quadrupole time of flight mass spectrometry (LC-QToF). We investigated available urine samples from a period of 2 years that had a positive result for cocaine. In addition, we examined samples that were below the cut-off for cocaine on immunoassay. Specimens were analysed for the presence of levamisole and other 'unknown' drugs. In the period under investigation the laboratory examined 3665 urine samples for cocaine: 1.4% (n = 51) of the samples were positive for cocaine by immunoassay and half of these (n = 26, 51%) were further examined by LC-QToF. In addition, we examined 10 samples that were negative by immunoassay (as defined by AS/NZS 4308:2008). Levamisole was detected in the urine of cocaine users in approximately 75% of cases. Other illicit drugs were also frequently found in this cohort. The most common illicit drugs detected were methamphetamine, ecstasy and cannabis. Australian cocaine is widely adulterated with levamisole. Cocaine users are at risk of levamisole related health problems in addition to the problems related to cocaine.

Confirmed case of levamisole-associated multifocal inflammatory leukoencephalopathy in a cocaine user.

Levamisole is a common adulterant in cocaine and has previously been associated with a variety of serious complications including multifocal inflammatory leukoencephalopathy (MIL). There have been several reports of MIL in patients taking cocaine and, though suspected, the presence of levamisole was not confirmed. We present a case of a 63-year-old woman presenting with stupor and spastic quadraparesis found to have urine positive for cocaine and levamisole. An MRI brain revealed innumerable FLAIR hyperintensities with restricted diffusion and incomplete ring-enhancement. This is the first case to confirm the presence of levamisole in a patient with MIL associated with cocaine use.

Purpura and leukopenia in a cocaine user.

A previously healthy 42-year-old woman presented to the emergency department (ED) for arthralgias and painful lesions on her ears, feet, and knee (Figures 1 and 2) that had developed over the last month. She had no significant past medical history and was not taking any prescribed medications. The rash was purpuric with violaceous borders and hemorrhagic bullae. While she had mild pain with movement, her joint examination was otherwise normal and without signs of infection. ED laboratory testing revealed leukopenia (2500/mm(3)) and cocaine metabolites in her urine.

Prevalence of levamisole and aminorex in patients tested positive for cocaine in a French University Hospital.

CONTEXT: The prevalence of levamisole in urine samples of subjects positive for cocaine in the US was estimated at 78% (95%confidence interval or CI: 73%-83%). However, levamisole was not quantified, and at the time of this report, aminorex was not known to be a possible metabolite of levamisole in human. Moreover no data are available in Europe. OBJECTIVE: The aim of this study was to determine the prevalence and concentration of levamisole and aminorex in positive cocaine urine toxicology tests, and in serum samples of cocaine-positive subjects driving under the influence of drugs or forensic autopsies. MATERIALS AND METHODS: Consecutive urine toxicology samples tested positive for cocaine by immunoassay (EMIT, Siemens) from April to May 2014 at the toxicology laboratory of a French University Hospital, and blood samples of cocaine-positive subjects driving under the influence of drugs or forensic autopsies from April to December 2014 were analyzed by liquid chromatography-tandem mass spectrometry or LC-MS/MS (3200 QTrap, AB Sciex) to detect and quantify the presence of levamisole and aminorex. RESULTS: Forty-two urine samples tested positive for cocaine in 39 patients (79.5% males) with a median age of 43 [range: 20-51] years. Toxicological analyses were mainly required by addictions care centers (n = 17; 40%) in the context of the routine management of addict patients. Cocaine concentrations were above the limit of quantification for 33 patients, with a median value of 228 [0-645,000] ng/ml. Levamisole was detected in 32 urine samples (76%) (median concentration: 1,430 ng/ml, range: 30-258,000). Aminorex was never detected. During the study period, levamisole was detected in 87.5% of cocaine-positive blood samples of the subjects driving under the influence of drugs or forensic autopsies. DISCUSSION: In this prospective study, the prevalence of levamisole in cocaine-positive samples was 76%. Over this period, although clinical complications related to cocaine use were reported (agitation, road accident, and cardiac arrest), no complication specifically related to levamisole or aminorex was reported. CONCLUSION: Our results show a high prevalence of levamisole in samples of subjects positive for cocaine, close to the prevalence found in the US. This high prevalence raises issues with respect to well-identified health risks associated with regular consumption of levamisole.

Metabolism of levamisole and kinetics of levamisole and aminorex in urine by means of LC-QTOF-HRMS and LC-QqQ-MS.

The antihelminthic drug Levamisole can enhance cocaine effects by conversion into the amphetamine-like drug aminorex. We describe an LC-MS method for the determination of levamisole and its metabolite aminorex in human urine. Selectivity is given, calibration curves were linear within the calibration range 2.5-250 ng/mL; limits of the method were LoD 0.51 ng/mL, LoQ 1.02 ng/mL for levamisole and LoD 0.65 ng/mL, LoQ 0.76 ng/mL for aminorex. Precision data was in accordance with the guidelines (intraday precision for aminorex ranged between 5.75 and 11.0 % for levamisole between 8.36 and 10.9 %; interday precision for levamisole 10.9-16.9 % and for aminorex 7.64-12.7 %; accuracy data for levamisole -1.96 to -14.3 % and for aminorex-11.9 to-18.5 %). The validated method was successfully applied to study the urinary excretion of levamisole after the administration of 100 mg of levamisole orally. Levamisole and aminorex could be detected in post-administration urine samples. Levamisole could be detected up to 39 h after ingestion, while aminorex was detectable up to 54 h. Maximum aminorex concentrations were 45 ng/mL urine. Further metabolites of levamisole after oral ingestion by means of liquid chromatography hybrid quadrupole time-of-flight high-resolution mass spectrometry (LC-QTOF-HRMS) were identified. Only 0.5 % of the ingested drug was quantified as unchanged levamisole in urine. Besides aminorex, five isomers of aminorex and 4 hydroxy-metabolites of aminorex or its isomers were found. Furthermore, levamisole is also hydroxylated and eliminated free or conjugated with sulfate or glucuronide into urine.

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 phenyltetrahydroimidazothiazole enantiomers (Levamisole/Dexamisole) in illicit cocaine seizures and in the urine of cocaine abusers via chiral capillary gas chromatography-flame-ionization detection: clinical and forensic perspectives.

Illicit cocaine laboratories in South America have been adding phenyltetrahydroimidazothiazole enantiomers (levamisole and/or tetramisole) to refined illicit cocaine for over 8 years. A chiral capillary gas chromatographic methodology is presented for phenyltetrahydroimidazothiazole enantiomer determination in illicit cocaine samples and in the urine of cocaine abusers. Illicit cocaine samples (N = 752) and urine specimens from cocaine abusers (N = 50) that contained phenyltetrahydroimidazothiazole were analyzed for enantiomeric composition. Legitimate commercial preparations of phenyltetrahydroimidazothiazole are either 100% levamisole or a 50:50 mixture of levamisole and dexamisole (tetramisole). Specimens that contain phenyltetrahydroimidazothiazole mixtures that are other than 50:50 preparations will be enhanced in one isomer over the other, and they are referred to as either "levamisole-enhanced" or "dexamisole-enhanced". Cocaine samples were found to contain levamisole (N = 495, 66%), tetramisole (N = 143, 19%), and levamisole-enhanced enrichment (N = 114, 15%); urine specimens contained levamisole (N = 23, 46%), levamisole-enhanced enrichment (N = 10, 20%), and dexamisole-enhanced enrichment (N = 13, 26%). The toxicological and forensic aspects of these findings are discussed.

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.

Determination of aminorex in human urine samples by GC-MS after use of levamisole.

The Drug Enforcement Administration (DEA) reports that as of October 2010, 79% of all cocaine seized in the United States contained levamisole. The equine conversion of levamisole to aminorex has been demonstrated. However, the metabolic fate of levamisole in humans is unknown. Nevertheless, as aminorex is amphetamine-like and hallucinogenic, it may be used as an adulterant to increase the effects of cocaine. We report here the results of in vivo studies demonstrating for the first time that not only equine, but also canine and human metabolism all result in aminorex formation. Levamisole and aminorex were extracted from real urine samples by liquid-liquid extraction and identified and quantified by GC-MS (identification by 3 ions per substance, LLOQ at 0.15ng/ml for both).

Detection of levamisole exposure in cocaine users by liquid chromatography-tandem mass spectrometry.

Levamisole, a veterinary antihelminthic, was recently recognized as an adulterant in cocaine and is known to cause severe adverse reactions in some cocaine users. Because of the health concerns involving levamisole-adulterated cocaine, we developed a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of levamisole in urine. This method was used to determine the prevalence of levamisole in cocaine-positive patient samples. All cocaine-positive urine samples that were sent to the San Francisco General Hospital Clinical Laboratory were tested for levamisole for one month. For LC, an Agilent 1200 series was used with a C(18) column and a gradient of mobile phase A (0.05% formic acid) and B (acetonitrile/methanol). Detection was carried out with an Applied Biosystems QTRAP(®) LC-MS-MS. The levamisole LC-MS-MS method was linear over the range of 5-2500 ng/mL (r > 0.996). Interassay and intraassay CVs were < 6%. The lower limit of detection for levamisole was 0.5 ng/mL. Out of 949 total urine drug screens, 20% were positive for benzoylecgonine, and of those, 88% were positive for levamisole. The high prevalence of levamisole-adulterated cocaine and potential toxicity in cocaine users is a serious public health concern. These findings validate the utility of an LC-MS-MS method for the detection of levamisole.

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.

Levamisole-induced occlusive necrotizing vasculitis of the ears after use of cocaine contaminated with levamisole.

Based on the best available data, approximately 2.1 million Americans use illicit cocaine each month; for the last several months, 30% of that cocaine has been "cut" with a veterinary pharmaceutical, levamisole. Levamisole can cause agranulocytosis, leaving patients susceptible to fulminate and opportunistic infections and also can cause a debilitating cutaneous necrotizing vasculitis. In this manuscript, we describe a case and provide an image of levamisole-induced necrotizing vasculitis of the ears.

The formation of aminorex in racehorses following levamisole administration. A quantitative and chiral analysis following synthetic aminorex or levamisole administration vs. aminorex-positive samples from the field: a preliminary report.

Beginning in 2004, the horseracing industry experienced an epidemic of drug positives for the amphetamine-like drug aminorex. Investigation of the therapeutic treatment of the horses called positive for this drug suggested that its source was from the administration of the anthelmintic levamisole. This study examines the urine concentrations of aminorex as a function of time following administration of synthetic, racemic aminorex. Confirmation of the presence of aminorex in urine samples from the horses known to be treated with levamisole is also presented as are data concerning the concentrations of aminorex in positives called from the field and the corresponding concentrations of levamisole found in the same samples. Furthermore, this study illustrates that the chiral isomer distribution of aminorex found in samples from the field is significantly different from that arising from the administration of synthetic, racemic aminorex and is similar to that observed from aminorex arising from levamisole administration. An examination of the chiral isomer distribution of aminorex and a determination of the presence of levamisole in a sample may be used to assess the source of an aminorex positive, distinguishing it from an intentional synthetic, racemic aminorex administration. The role of levamisole in aminorex formation is also discussed.

Levamisole for corticosteroid-dependent nephrotic syndrome in childhood. British Association for Paediatric Nephrology.

In children with corticosteroid-responsive nephrotic syndrome who are dependent on high-dose prednisolone, alkylating therapy often fails to maintain a remission, and long-term immunosuppression may be hazardous. An alternative approach to treatment is to use an immunostimulant such as levamisole. 61 children with frequently relapsing corticosteroid sensitive and dependent nephrotic syndrome were randomly allocated to receive levamisole, 2.5 mg/kg on alternate days (31 patients) or placebo (30 patients) for a maximum of 112 days. After entry to the trial, prednisolone was progressively reduced and was stopped by 56 days. The two groups were well matched for age and sex distribution, indices of corticosteroid toxicity, and previous alkylating therapy. 14 patients in the levamisole group and 4 in the placebo group remained in remission at 112 days (log rank analysis p less than 0.01). No significant adverse events were recorded. Levamisole is effective in maintaining a steroid-free remission in this condition and has few side-effects.