A LC-MS/MS method for determination of clenbuterol enantiomers in animal tissues and its application to the enantioselective distribution in Bama mini-pigs

Abstract

Objectives

To establish and validate a simple and reliable analytical method for separation and determination of clenbuterol enantiomers (R-(-)-clenbuterol & S-(+)-clenbuterol) in animal tissues, and apply it to the enantioselective distribution of clenbuterol in Bama mini-pigs.

Methods

A LC-MS/MS analytical method was developed and validated in positive multiple reaction monitoring mode with electrospray ionization. After perchloric acid deproteinization, samples were pretreated only by one step liquid–liquid extraction using tert-butyl methyl ether under strong alkaline condition. Teicoplanin was used as chiral selector and 10 mM ammonium formate methanol solution was used as mobile phase. The optimized chromatographic separation conditions were completed in 8 min. Two chiral isomers in 11 edible tissues from Bama mini-pigs were investigated.

Results

R-(-)-clenbuterol and S-(+)-clenbuterol can be baseline separated and accurately analyzed with a linear range of 5–500 ng/g. Accuracies ranged from −11.9–13.0% for R-(-)-clenbuterol and −10.2–13.2% for S-(+)-clenbuterol, intra-day and inter-day precisions were between 0.7 and 6.1% for R-(-)-clenbuterol and 1.6–5.9% for S-(+)-clenbuterol. R/S ratios in edible tissues of pigs were all significantly lower than 1.

Conclusions

The analytical method has good specificity and robustness in determination of R-(-)-clenbuterol and S-(+)-clenbuterol in animal tissues, and can be used as a routine analysis method for food safety and doping control. There is a significant difference in R/S ratio between pig feeding tissues and pharmaceutical preparations (racemate with R/S ratio of 1), which makes it possible to identify the source of clenbuterol in doping control and investigation.