Abstract: A comprehensive method for simultaneous identification and detection of 16 anabolic steroid hormones (ASs, including andorgens, gestagens and their esters)in muscle samples was developed with liquid chromatography coupled to quadrupole/linear ion trap mass spectrometry (LC-Q/Trap-MS). The ASs in muscle samples were extracted with acetonitrile under ultrasonic assistance. The extract was defatted by n-hexane with liquid-liquid partitioning and followed by clean-up with NH2 solid phase extraction (SPE) cartridge. The separation of analytes was carried out on a CAPCELL PAK C18 MGIII column (150 mm×2.0 mm, 5.0 μm) using mobile phases of 0.1% (v/v) formic acid in acetonitrile and 0.1% (v/v) formic acid-5 mmol/L ammonium formate aqueous solution with gradient elution. A scheduled multiple reaction monitoring (sMRM) in positive mode as survey scan and an enhanced product ion (EPI) scan as dependent scan in an information-dependent acquisition (IDA) experiment was adopted in mass spectrometry acquisition. On-line lab-built MS/MS library and internal standards were employed for the identification and quantification. As a result, the 16 ASs showed good linearity with all correlation coefficients (r) no less than 0.9990 within the linear ranges. The limits of quantification (LOQs, S/N≥10) for the 16 ASs were in the range of 0.029~0.36 μg/kg. At the three spiked levels (0.5, 2.0 and 20 μg/kg), the overall recoveries ranged from 89.9% to 118% with the relative standard deviations (RSDs) no more than 16.2% under within-laboratory reproducibility conditions. The proposed method can be used to identify and detect the 16 ASs in a single run, which makes it effective in residue surveillance of anabolic hormones in muscle samples.

Key words: anabolic steroid hormones (ASs), muscle, quadruple/linear ion trap mass spectrometry (Q/Trap-MS), residues, liquid chromatography (LC)