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An analytical method for the simultaneous determination of chiral thyroxine and the related iodinated chiral compounds using LC-MS/MS is introduced in this study. D-Thyroid hormones (THs), which are not commercially available, were produced through the racemization reaction of the L-THs in acetic acid solution containing salicylaldehyde. The solution containing D- and L-THs after the reaction was used for optimizing the chiral separation. The D- and L-THs were well separated enantiomerically under isocratic conditions in 70 % acetonitril