Abstract: A method of high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for the determination of thyroid hormones in milk including 3,3′,5,5′-tetraiodothyronine (T4), 3,3′,5-triiodothyronine (T3) and 3,3′,5′-triiodothyronine (rT3). The sample was extracted with acetonitrile and centrifuged, then the up layer was alkalized with concentrated ammonia water and cleaned up with a Cleanert PAX cartridge. The chromatographic separation was carried out on a Zorbax Eclipse XDB-C18 column (150 mm×2.1 mm, 3.5 μm) with the mobile phase of 37% water containing 0.1% (v/v) acetic acid and 63% of methanol with an isocratic elution mode at a flow rate of 0.3 mL/min. The mass spectrometry detection was performed in positive electrospray ionization and monitored using multiple reaction monitoring (MRM) mode (m/z 652.0/605.5 and 652.0/478.6 for T3 and rT3; m/z 777.7/731.7 and 777.7/604.9 for T4; m/z 784.0/737.9 and 784.0/639.4 for T4-13C6). The analytes were identified by their retention times and relative abundance ratios of the characteristic product ions, and quantified by internal standard method. The method was linear with the correlation coefficients (r2) greater than 0.998 in the concentration ranges investigated. The limits of detection (LODs) were not more than 0.03 ng/g, and the limits of quantification (LOQ) were not more than 0.1 ng/g for the analytes. The recoveries were 80.61%-101.7% with the relative standard deviations (RSDs) of 1.48%-9.70%. The results of five real milk samples showed that the contents of T3 were 0.59-1.30 ng/g with the RSDs of 2.06%-7.70%, and T4 and rT3 weren’t found. The method presents many merits including simple sample pretreatment, high sensitivity, good reproducibility and unequivocal confirmation and quantification for the analytes. It could be used to monitor the levels of thyroid hormones in the milk for safety quality evaluation.

Key words: milk, thyroid hormone, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)