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Development of a Chiral Method for Levamisole and its Metabolite, Aminorex, for Monitoring Abuse in Horse Racing and Cocaine Adulteration

By: David S Bell, Denise Wallworth, Reporter US Volume 33.4

Background

Concern over the abuse of amphetamines and other stimulants goes beyond its prevalence in the human population. Unscrupulous and unethical animal handlers administer drugs to their charges to enhance performance through stimulation, cardiovascular augmentation, reduction of pain or inflammation, increasing muscle mass, and other means. Improvements in analytical technologies have facilitated the detection of illicit drugs and other substances and continue to be a significant underpinning to criminal investigations into animal abuse1.

Illicit Uses of Levamisole

Levamisole, a tetramisole enantiomer, is sometimes given to horses as an anthelmintic treatment. Concern has arisen because levamisole is metabolized to the amphetamine-like drug aminorex (Figure 1). Investigation into race horses that tested positive for aminorex suggested that its source was from the administration of levamisole2. Because of the potential for abuse, the therapeutic use of levamisole is banned in some countries. The presence of aminorex in the animal may be from the administration of levamisole or from synthetic, racemic aminorex. Studies have shown that these two administration routes result in significantly different ratios of the aminorex enantiomers3. Additionally, levamisole is thought to play a role in the in vivo production of a significant performance enhancing drug, pemoline. Levamisole is also used as an adulterant in cocaine; its addition is likely intended to extend the effect of cocaine by metabolizing to aminorex just as the effect of cocaine diminishes4.

Analyte Chemical Structures

Figure 1. Analyte Chemical Structures

Importance of Chiral Discrimination

Chiral HPLC analysis can play a significant role in understanding whether detected levamisole or aminorex is due to legal administration of drugs or illicit use to enhance performance.
A simple chiral HPLC method for levamisole and related compounds may therefore be of utility in both the determination of illicit use within the horse racing industry as well as in investigations into cocaine abuse and tracing studies.

Objectives of the Study

The aim of this study was to develop a method to differentiate the enantiomers of tetramisole (levamisole and dexamisole) for drug product analysis and performance enhancing activities in horse racing. An added bonus would be to have a method that could simultaneously separate aminorex and cocaine from the tetramisole enantiomers which would be useful in cocaine-related studies.

Experimental

In previous work, Astec® CYCLOBOND® cyclodextrin-based chiral stationary phases (CSP) were shown to be effective in separating the enantiomers of ‘-conazole’ based compounds that are structurally similar to tetramisole. For that reason, a selection of Astec CYCLOBOND phases based on derivatized ß-cyclodextrin was chosen for initial column screening to resolve the tetramisole enantiomers using an MS-compatible mobile phase system comprising 100 mM ammonium acetate and acetonitrile. Five Astec CYCLOBOND columns were screened (Table 1). Each column was 25 cm × 4.6 mm I.D. packed with 5 µm particles. Since levamisole is related to both the metabolic production of aminorex as well as a common cocaine adulterant, the developed separation conditions were investigated for their simultaneous separation.

 

Table 1. Technical Specifications

Column Cyclodextrin type Cyclodextrin derivative
(2- and 3-position hydroxyls)
Astec CYCLOBOND I 2000 HP-RSP Beta (ß)* R,S-Hydroxypropyl ether
Astec CYCLOBOND I 2000 DMP Beta (ß) 3,5-Dimethylphenylcarbamate
Astec CYCLOBOND I 2000 DNP Beta (ß) 2,6-Dinitro-4-trifluoromethyl phenyl ether
Astec CYCLOBOND I 2000 DM Beta (ß) Dimethyl
Astec CYCLOBOND I 2000 AC Beta (ß) Acetyl
*Cycloheptylamylose, glucose units: 7, stereogenic centers: 35, cavity size: 0.78 nm.

Results

Of the five ß-cyclodextrin phases screened, only the dimethylphenylcarbamate derivative (Astec CYCLOBOND I 2000 DMP) showed significant selectivity toward the tetramisole enantiomers. (A detailed explanation of the separation mechanism behind the cyclodextrin-based CSPs can be found in Reference 5.) Method optimization resulted in the chromatographic separation shown in Figure 2. Confirmation of elution order was determined by injection of a standard levamisole preparation (Figure 3).

Under the initial mobile phase conditions (20% acetonitrile) aminorex eluted very early. Retention was increased by reducing the mobile phase to 10% acetonitrile which permitted the simultaneous resolution of all target analytes in less than 10 minutes (Figure 4).

Chiral Separation of Tetramisole Enantiomers on Astec CYCLOBOND I 2000 DMP

Figure 2. Chiral Separation of Tetramisole Enantiomers on Astec CYCLOBOND I 2000 DMP

Conditions
column:
Astec CYCLOBOND I 2000 DMP, 25 cm × 4.6 mm I.D., 5 µm (Product No. 20724AST); mobile phase: [A] 100 mM ammonium acetate, pH 5 with acetic acid; [B] acetonitrile; (80:20, A:B); flow rate: 1.0 mL/min; column temp.: 35 °C; detector: UV, 230 nm; injection: 10 µL; sample: 200 µg/mL tetramisole in mobile phase A:methanol (80:20, v/v)

 

Identification of Elution Order of Tetramisole Enantiomers on Astec CYCLOBOND I 2000 DMP

Figure 3. Identification of Elution Order of Tetramisole Enantiomers on Astec CYCLOBOND I 2000 DMP

Conditions as in Figure 2 except
sample: 100 µg/mL levamisole in 80:20 (A:B)

 

Separation of Tetramisole Enantiomers and Related Compounds on Astec CYCLOBOND I 2000 DMP

Figure 4. Separation of Tetramisole Enantiomers and Related Compounds on Astec CYCLOBOND I 2000 DMP

Conditions
column:
Astec CYCLOBOND I 2000 DMP, 10 cm × 2.1 mm I.D., 5 µm (custom); mobile phase: [A] 100 mM ammonium acetate, pH 5 with acetic acid; [B] acetonitrile; (90:10, A:B); flow rate: 0.2 mL/min; column temp.: 35 °C; detector: UV, 220 nm; injection: 2 µL; sample: 100 µg/mL tetramisole, 50 µg/mL each aminorex and cocaine in water:acetonitrile (20:80, v/v)

Conclusions and Observations

Astec CYCLOBOND I 2000 DMP was shown to be effective for the chiral resolution of tetramisole enantiomers, levamisole and dexamisole. The conditions are also shown to separate a known metabolite of levamisole (aminorex) and thus may be used in certain studies aimed at understanding the origins the aminorex found in horse racing monitoring procedures. In addition, cocaine, which is often laced with levamisole, also separates within a reasonable time window, allowing the conditions to be useful in drug abuse investigations. Future work will focus on the ability of the method to resolve the aminorex enantiomers once standards of the individual enantiomers or an authenticated racemic mixture can be obtained.

Legal Information

Astec and CYCLOBOND are registered trademarks of Sigma-Aldrich Co. LLC

Materials

     

 References

  1. Wong, J. K. Y.; Wan, T. S. M. Doping control analyses in Horse racing: A clinician's guide. Veterinary Journal 2014, 200(1), 8-16.
  2. Ho, E. N. M.; Leung, D. K. K.; Leung, G. N. W.; Wan, T. S. M.; Wong, A. S. Y.; Wong, C. H. F.; Soma, L. R.; Rudy, J. A.; Uboh, C.; Sams, R. Aminorex and rexamino as metabolites of levamisole in the horse. Analytica Chimica Acta 2009, 638(1), 58-68.
  3. Barker, S. A. 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. J. Vet. Pharmacol. Ther. 2009, 32(2), 160-166.
  4. Hofmaier, T.; Luf, A.; Seddik, A.; Stockner, T.; Holy, M.; Freissmuth, M.; Ecker, G. F.; Schmid, R.; Sitte, H. H.; Kudlacek,O.. Aminorex, a metabolite of the cocaine adulterant levamisole, exerts amphetamine like actions at monoamine transporters. Neurochemistry International 2014, 73, 32-41.
  5. Armstrong, D. W.; Chang, C. D.; Lee, S. H. (R)- and (S)-naphthylethylcarbamatesubstituted ß-cyclodextrin bonded stationary phases for the reversed-phase liquid chromatographic separation of enantiomers. J. Chromatogr. 1991, 539(1), 83-90.

 

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