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 % acetonitrile containing 0.1 % formic acid on a CROWNPAK® CR-I (+) column, but some peaks, such as those of diiodo-D-tyrosine (D-DIT)/monoiodo-L-tyrosine, diiodo-D-thyronine/diiodo-L-tyrosine and D-thyroxine/triiodo-L-thyronine, overlapped chromatographically, causing misinterpretation in impurity analysis. This was overcome by using the gradient condition providing the best chiral selectivity (α) and resolution (Rs) ranging from 1.14 to 1.37 and from 2.39 to 4.52, respectively. The linearity was above 0.999 and the detection limits ranged from 8.2 to 57.7 ng/mL by the separation method. This method was applied to identify and quantify chiral impurities in authentic standards and pharmaceuticals. As a result, D-enantiomers corresponding to the L-THs standards as well as L-DIT were commonly observed as impurities. In the stability test of DL-thyroxine under acidic conditions for identifying the distribution of chiral products, it was observed that the formation of DIT by hydrolysis increased over time. Additional products formed through esterification, including thyroxine methyl ester and diiodo-tyrosine methyl ester, were newly separated and identified using a C18 column.
Keywords: Chiral stationary phase; Enantiomer separation; Racemization; Thyroid hormone.
Copyright © 2021 Elsevier B.V. All rights reserved.