Separation and Determination of the Enantiomeric Levamisole and Dexamisole in Equine Plasma Samples Using Chiral Polysaccharide Column/ LC-MS/MS

Author(s): Abdul Khader Karakka Kal*, Tajudheen K. Karatt, Moses Philip, Samir Meissir, Jahfar Nalakath

Journal Name: Current Analytical Chemistry

Volume 16 , Issue 6 , 2020


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Graphical Abstract:


Abstract:

Background: Drug Enforcement Administration confirmed that many manufacturers began adding tetramisole or its individual isomers to cocaine as an adulterant, and believed that tetramisole may augment cocaine’s effects. In recent times, there is an increasing trend in the usage of tetramisole and its individual enantiomer in race sports especially in horse and camel races. So it’s is very much required to confirm the stereochemistry of this illicit drug in the routine race day samples coming to the anti-doping labs in order to avoid legal arguments and challenges to the analytical findings.

Methods: The aim of the study was to develop a simple, rapid and accurate method for the chiral separation and determination of enantiomeric mixtures of levamisole and dexamisole using Thermo Q-Exactive High-Resolution Mass Spectrometer. In order to evaluate the suitability of the method for determining the enantiomeric purity of tetramisole, validation studies were also carried out by using equine plasma.

Results: The enantio-separation was achieved using the Lux i-cellulose-5 column. Isocratic flow was used with a 1:1 mixture of mobile phase A (10 mM ammonium acetate in water) and mobile phase B (acetonitrile), at a flow rate of 0.6 mL/min. The run time was 8.0 min, and the column temperature was 50°C. Dexamisole eluted at 5.94 min, and levamisole eluted at 6.62 min, giving the R-value of 1.50. The obtained inter-day precisions of dexamisole, levamisole were 3.16% and 2.85%, respectively. The accuracy of dexamisole was in the range of 97.78 to 102.44%, and that for levamisole was 99.16 to 102.82%. The limit of quantification value for both isomers in this method was 0.1 ng/ mL. The method was linear in the range of 0 to 50 ng/mL.

Conclusion: Chromatographic separation was achieved using the polysaccharide cellulose chiral column, and the reverse-phase separation approach was found to have the highest potential for successful chiral resolution in LC-MS. Linearity, precision, accuracy, detection limit, recovery, and the matrix effect in equine plasma were determined. Under the optimized conditions, the validated method can be applied for the identification and detection of the tetramisole enantiomers in different sources of illicit drugs of abuse.

Keywords: Chirality, dexamisole, equine plasma, HRMS, levamisole, reverse phase chiral, tetramisole.

[1]
Tracy, T.S.; Korzekwa, K.R.; Gonzalez, F.J.; Wainer, I.W. Cytochrome P450 isoforms involved in metabolism of the enantiomers of verapamil and norverapamil. Br. J. Clin. Pharmacol., 1999, 47(5), 545-552.
[http://dx.doi.org/10.1046/j.1365-2125.1999.00923.x ] [PMID: 10336579]
[2]
Takahashi, H.; Echizen, H. Pharmacogenetics of warfarin elimination and its clinical implications. Clin. Pharmacokinet., 2001, 40(8), 587-603.
[http://dx.doi.org/10.2165/00003088-200140080-00003 ] [PMID: 11523725]
[3]
Islam, M.R.; Mahdi, J.G.; Bowen, I.D. Pharmacological importance of stereochemical resolution of enantiomeric drugs. Drug Saf., 1997, 17(3), 149-165.
[http://dx.doi.org/10.2165/00002018-199717030-00002 ] [PMID: 9306051]
[4]
Mehvar, R.; Reynolds, J.M.; Robinson, M.A.; Longstreth, J.A. Enantioselective kinetics of verapamil and norverapamil in isolated perfused rat livers. Pharm. Res., 1994, 11(12), 1815-1819.
[http://dx.doi.org/10.1023/A:1018935921473] [PMID: 7899248]
[5]
Thienpont, D.; Vanparijs, O.F.; Raeymaekers, A.H.; Vandenberk, J.; Demoen, J.A.; Allewijn, F.T.; Marsboom, R.P.; Niemegeers, C.J.; Schellekens, K.H.; Janssen, P.A. Tetramisole (R 8299), a new, potent broad spectrum anthelmintic. Nature, 1966, 209(5028), 1084-1086.
[http://dx.doi.org/10.1038/2091084a0] [PMID: 5925183]
[6]
Vanhoutte, P.M.; Van Nueten, J.M.; Verbeuren, T.J.; Laduron, P.M. Differential effects of the isomers of tetramisole on adrenergic neurotransmission in cutaneous veins of dog. J. Pharmacol. Exp. Ther., 1977, 200(1), 127-140.
[PMID: 189006]
[7]
Keiser, J.; Utzinger, J. Efficacy of current drugs against soil-transmitted helminth infections: Systematic review and meta-analysis. JAMA, 2008, 299(16), 1937-1948.
[http://dx.doi.org/10.1001/jama.299.16.1937] [PMID: 18430913]
[8]
Nowak, L.G.; Rosay, B.; Czégé, D.; Fonta, C. Tetramisole and levamisole suppress neuronal activity independently from their inhibitory action on tissue non-specific alkaline phosphatase in mouse cortex. Subcell. Biochem., 2015, 76, 239-281.
[http://dx.doi.org/10.1007/978-94-017-7197-9_12] [PMID: 26219715]
[9]
Przegaliński, E.; Bigajska, K.; Siwanowicz, J. Psychopharmacological profile of dexamisole. Pol. J. Pharmacol. Pharm., 1980, 32(1), 21-29.
[PMID: 7454609]
[10]
Broséus, J.; Gentile, N.; Esseiva, P. The cutting of cocaine and heroin: A critical review. Forensic Sci. Int., 2016, 262, 73-83.
[http://dx.doi.org/10.1016/j.forsciint.2016.02.033] [PMID: 26974713]
[11]
Martello, S.; Pieri, M.; Ialongo, C.; Pignalosa, S.; Noce, G.; Vernich, F.; Russo, C.; Mineo, F.; Bernardini, S.; Marsella, L.T. Levamisole in illicit trafficking cocaine seized: A One-Year Study. J. Psychoactive Drugs, 2017, 49(5), 408-412.
[http://dx.doi.org/10.1080/02791072.2017.1361558 ] [PMID: 28813206]
[12]
Raymon, L.P.; Isenschmid, D.S. Letter to the editor: The possible role of levamisole in illicit cocaine preparations J. Anal. Toxicol., 2009, 33(9), 620-622..
[http://dx.doi.org/10.1093/jat/33.9.620] [PMID: 20040138]
[13]
European Community. Council Directive 96/22/EC concerning the prohibition on the use in stock farming of certain substances having a hormonal or thyrostatic action and of ß-agonists. Eur. Commun.Off. J., 1996, 125, 3-9.https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A31996L0022
[14]
Davis, E.; Loiacono, R.; Summers, R.J. The rush to adrenaline: Drugs in sport acting on the beta-adrenergic system. Br. J. Pharmacol., 2008, 154(3), 584-597.
[http://dx.doi.org/10.1038/bjp.2008.164] [PMID: 18500380]
[15]
The World Anti-Doping Code. International Standard, the Prohibited List. 2015.https://www.wada-ama.org/sites/default/files/resources/files/wada-2015-prohibited-list-en.pdf
[16]
Dolezˇalova´. M.; Tkaczykova´, M. LC determination of the enantiomeric purity of levamisole using stationary phase with bonded naphthylethylcarbamoylated-beta-cyclodextrin. J. Pharm. Biomed. Anal., 2001, 25, 407-415.
[17]
Madry, M.M.; Kraemer, T.; Baumgartner, M.R. Cocaine adulteration with the anthelminthic tetramisole (levamisole/dexamisole): Long-term monitoring of its intake by chiral LC-MS/MS analysis of cocaine-positive hair samples. Drug Test. Anal., 2019, 11(3), 472-478.
[http://dx.doi.org/10.1002/dta.2505] [PMID: 30239147]
[18]
Armstrong, D.W.; Chang, C.D.; Lee, S.H. (R)- and (S)-Naphthylethylcarbamate-substituted β-cyclo-dextrin bonded stationary phases for the reversed-phase liquid chromatographic separation of enantiomers. J. Chromatogr. A, 1991, 539, 83-90.
[http://dx.doi.org/10.1016/S0021-9673(01)95362-2]
[19]
Hermansson, J.; Grahn, A. Resolution of racemic drugs on a new chiral column based on silica-immobilized cellobiohydrolase Characterization of the basic properties of the column. J. Chromatogr. A, 1994, 687, 45-59.
[http://dx.doi.org/10.1016/0021-9673(94)00886-8]
[20]
Stalcup, A.M.; Agyei, N.M. Heparin: A chiral mobile-phase additive for capillary zone electrophoresis. Anal. Chem., 1994, 66(19), 3054-3059.
[http://dx.doi.org/10.1021/ac00091a011] [PMID: 7978302]
[21]
Carlo, B.; Daniele, T.; Edoardo, F.; Anna, M.D.P.; Francesca, R.; Marco, G.; Elia, D.B. Determination of levamisole and tetramisole in seized cocaine samples by enantioselective high-performance liquid chromatography and circular dichroism detection. J. Chromatogr. A, 2014, 1363, 150-154.
[http://dx.doi.org/10.1016/j.chroma.2014.07.069] [PMID: 25124228]
[22]
John, F.C.; Valerie, L.C.; Donald, F.L. 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. J. Anal. Toxicol., 2012, 36, 130-135.
[http://dx.doi.org/10.1093/jat/bkr025] [PMID: 22337783]
[23]
Tachibana, K.; Ohnishi, A. Reversed-phase liquid chromatographic separation of enantiomers on polysaccharide type chiral stationary phases. J. Chromatogr. A, 2001, 906(1-2), 127-154.
[http://dx.doi.org/10.1016/S0021-9673(00)00955-9 ] [PMID: 11215884]
[24]
Tajudheen, K.K.; Jahfar, N.; Zubair, P.; Peter, H.A.; Abdul, K.K.K.; Syed, M.A.P.; Laya, S. Mass spectrometric method for distinguishing isomers of dexamethasone via fragment mass ratio: An HRMS approach. J. Mass Spectrom., 2018, 53, 1046-1058.
[http://dx.doi.org/10.1002/jms.4279] [PMID: 30098588]
[25]
Peng, T.; Farkas, L. Poster Communication at Chirality. Geneva, Switzerland, , 2008.
[26]
Maier, N.M.; Cox, G. Poster Communication P-220 at Chirality., 2009.
[27]
Tajudheen, K.K.; Ramy, S.; Jahfar, N.; Zubair, P.; Peter, H.A.; Abdul, K.K.K. Separation and identification of the epimeric doping agents – Dexamethasone and betamethasone in equine urine and plasma: A reversed phase chiral chromatographic approach. Steroids, 2018, 140, 77-82.
[http://dx.doi.org/10.1016/j.steroids.2018.10.003] [PMID: 30296548]
[28]
Liming, P.; Swapna, J.; Chankvetadzeb, B.; Farkasa, T. Reversed-phase chiral HPLC and LC/MS analysis with tris(chloromethyl-[phenylcarbamate) derivatives of cellulose and amylose as chiral stationary phases. J. Chromatogr. A, 2010, 1217, 6942-6955.
[http://dx.doi.org/10.1016/j.chroma.2010.08.075] [PMID: 20863505]
[29]
Tajudheen, K.K.; Abdul, K.K.K.; Ramy, S.; Jahfar, N.; Zubair, P. Use of polysaccharide-based chiral columns: Enantiomeric separation of seven pairs of abused drugs by high-performance liquid chromatography–mass spectrometry. Forensic Toxicol., 2019, 37, 254-260.
[http://dx.doi.org/10.1007/s11419-018-0453-6]
[30]
Ikeda, K.; Hamasaki, T.; Kohno, H.; Ogawa, T.; Matsumoto, T. Direct separation of enantiomers by reversed-phase high performance liquid chromatography on cellulose tris(3,5-dimethylphenyl-carbamate). Chem. Lett., 1989, 18(6), 1089-1090.
[31]
Peng, L.; Jayapalan, S.; Chankvetadze, B.; Farkas, T. Reversedphase chiral HPLC and LC/MS analysis with tris(chloromethylphenylcarbamate) derivatives of cellulose and amylose as chiral stationary phases J. Chromatogr. A, 2010, 1217, 6942-6955..
[http://dx.doi.org/10.1016/j.chroma.2010.08.075] [PMID: 20863505]


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VOLUME: 16
ISSUE: 6
Year: 2020
Page: [761 - 767]
Pages: 7
DOI: 10.2174/1573411015666190808103143
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