LC–MS/MS with Postcolumn Ammonia Infusion Determines Ultratrace Enantiomers of Closantel in Bacteria

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Although closantel (CLO) is well-regarded as a curative antiparasitic drug, even low doses have been found to cause serious side effects in the human retina and central nervous system.

Researchers at South China Agricultural University in Guangzhou, China have been in search of an effective method to separate and determine enantiomers of the antiparasitic drug closantel (CLO) and its enantiomers in bacterial cells, as CLO has also been shown in recent studies to have potential for antibacterial applications (1). The research team developed an enhanced method of liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) for chiral separation, adding a postcolumn infusion of ammonia.

Published in the Journal of Chromatography A, the research addresses drug chirality as it relates to enantiomers, which are mirror-image pairs of compounds that cannot be superimposed onto one another. In a chiral drug, the researchers said (using thalidomide as an example), two enantiomers may have the same exact physicochemical properties, but they differ or could even be complete opposites in terms of metabolism and toxicity (1). While closantel (CLO) is known to have a curative effect when it comes to parasites, studies show it can cause nervous system or retinal damage in humans at low doses.

However, CLO doses as low as 0.5 μg/mL can also be significantly synergistic in their antibacterial effects when combined with colistin (CST) in treatment of bacterial cells. Since that concentration is extremely low, the researchers sought an ultrahigh-sensitivity method for detection of ultratrace enantiomers (1). An LC–MS/MS approach was devised that used a chiral column for separation of enantiomers and tested several chiral stationary phases (CSPs) for compatibility with MS, mobile phase and other modifiers, and varying column temperatures. A postcolumn T-piece, as the study illustrated, integrated an ammonia solution into the LC–MS/MS system at different concentrations and flow rates to obtain the optimum conditions for each. The addition of ammonia reversed the mobile phase pH, allowing optimal chromatographic separation of R-closantel and S-closantel with regard to alkaline.


The researchers said to their knowledge, there had been no prior reports of LC–MS/MS determining CLO enantiomers, although they cited two previous studies that attempted normal-phase high performance liquid chromatography (NP-HPLC) analysis. That approach fell short, they said, because of the NP mode’s incompatibility with MS in analyzing the bacterial cells (1).

This modified method was linear over a concentration range of 0.5 to 50 pg/mL (where R2 ≥ 0.99) for both R- and S-closantel, according to the results of the study (1). The detection limit was 0.15 pg/mL for all target analytes in the bacterial cells, and average recoveries of the two enantiomers were between 81.2% and 107.8% with relative standard deviations below 15%. These figures were adequate enough for the researchers to pronounce their experiment a success, and to suggest it as a reference point for future investigation into the biological behaviors of CLO enantiomers and their relationship to CST.


(1) Ding, T.; Liu, L.; Liu, Y.; et al. Chiral separation of racemic closantel and ultratrace detection of its enantiomers in bacteria by enhanced liquid chromatography–tandem mass spectrometry combined with postcolumn infusion of ammonia. J. Chromatogr. A 2023, 1698, 464001. DOI: 10.1016/j.chroma.2023.464001