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.

Reliable analysis of ultratrace antibiotics in bacterial cells may become a new means to elucidate the antibacterial mechanism, drug resistance and environmental fate. In this work, an ultrahigh-sensitive, accurate and enhanced liquid chromatography-tandem mass spectrometric method was first developed for chiral separation and detection of racemic closantel, as an antibacterial adjuvant. Optimizing acetonitrile-water-formic acid system that is compatible with mass spectrometry as a mobile phase, the baseline separation of two enantiomers was achieved by using EnantioPak® Y1-R chiral column, and the resolution of the two analytes was more than 1.95. Further adopt the strategy of postcolumn infusion of ammonia, the mobile phase pH was reversed from acidic condition suitable for the optimal chromatographic separation of R- and S-closantel to alkaline, so that closantel could realize efficient electrospray ionization under the preferred negative ion mode. The bacterial cells were subjected to be frozen-cracked, and the analytes were extracted with acetonitrile after clipping the pointed bottom of the Eppendorf tube into a new tube. The method was linear over concentration ranges of 0.5-50 pg/mL (r2≥0.99) for R- and S-closantel. The detection limits of target analytes were all 0.15 pg/mL in bacterial cells. The average recoveries of two enantiomers ranged from 81.2% to 107.8% with relative standard deviations below 15%. The method proposed might be important support for the deep research of the stereoselectivity of biological activity, toxicity and metabolism of closantel enantiomers.

Determination of closantel enantiomers in black goat plasma and their pharmacokinetic characteristics.

Closantel is a broad-spectrum antiparasitic drug widely used in livestock. It is a chiral drug consisting of a pair of enantiomers. An accurate, selective, and validated method was established for separating and detecting closantel enantiomers in black goat plasma. Separation of enantiomers was achieved in Chiralpak AD-3 based on amylose tris (3,5-dimethyl phenyl carbamates) under a gradient mobile phase composed of n-hexane-TFA (100:0.1, v/v) and IPA-MeOH-TFA (99:1:0.1, v/v/v). Mean recoveries of the two enantiomers at three spiking levels ranged from 97.4 % to 102.0 % with a relative standard deviation ≤ 4.5 %. The limit of quantification was 1.35 µg/mL for each enantiomer in plasma. The proposed method was successfully applied to investigate the enantioselective pharmacokinetics of closantel enantiomers (E1 and E2) in black goats following an intramuscular closantel sodium at 5 mg/kg b.w. The results revealed that peak plasma concentration and area under the curve were significantly different between the two enantiomers (p < 0.05). Cmax and AUC0-∞ for closantel E1 were approximately 3 times greater than closantel E2. These findings would help in further evaluation of pharmacokinetic, pharmacodynamic, and toxicity of the individual enantiomers of closantel in food animals.