Optimisation and validation of a new analytical method for the determination of four natural and synthetic hormones using LC-ESI-MS/MS

A rapid liquid chromatographic-tandem mass spectrometric method was developed for the simultaneous determination of four natural and synthetic hormone residues (progesterone, testosterone, trenbolone acetate and zeranol) in animal tissue samples. Sample preparation was optimised to minimise time and solvent consumption. Meat samples were mechanically homogenised and digested in a procedure that gave similar recoveries to those enzymatically hydrolysed by Helix pomatia. Efficient extraction was achieved using acidified acetonitrile (1% acetic acid). Chromatographic conditions were optimised to minimise matrix effects. Analytes were separated using a C18 column with gradient elution using ammonium formate solution in methanol (MeOH)/water (1:9) and MeOH mobile phases. Finally, residues were qualitatively and quantitatively determined by electrospray ionisation tandem mass spectrometry in multiple reaction monitoring mode. Different parameters for LC-MS/MS (e.g., declustering potential and collision energy) were optimised using API 6500QT; all analytes were measured using positive-mode electrospray ionisation (ESI+) except zeranol which was measured in negative mode (ESI-). Due to LC-MS/MS signal enhancement/suppression, the determination of hormones was based on matrix-matched standard calculations. The method was validated for the four hormones on meat samples at different fortification levels and showed accepted performance criteria according to European Commission Decision 2002/657/EC. Decision limits and detection capabilities were estimated for all analytes.