Chromatographic Isotope Effect: Retention Time Changes for Polydeuterated Laquinimod in Reverse Phase HPLC

Vladimir Ioffe, Analytical Development, Global R&D, Teva Pharmaceutical Industries, Kfar Saba, Israel (Vladimir.Ioffe@teva.co.il)
Shiri Zigman, Analytical Development, Global R&D, Teva Pharmaceutical Industries, Kfar Saba, Israel

Laquinimod is a medicine developed by Active Biotech and Teva for the treatment of multiple sclerosis (MS) and additional indications of other autoimmune and neurodegenerative diseases.

As a part of development, several polydeuterated analogues of Laquinimod were prepared.

During analytical method development we observed noticeable reduction of retention time with increase of deuterium atoms in the molecule.D5-Laquinimod (five hydrogen atoms replaced with deuterium) elutes about 0.2 min earlier than the non-deuterated (RT is about 15.7 min), while D16-Laquinimod (sixteen hydrogen atoms replaced with deuterium) elutes more than 1.0 min earlier.

The difference of chromatographic behavior of deuterated compounds, as compared to nondeuterated prototypes, is discussed in relation with the differences between C-H and C-D bonds.

Deuterium atom is smaller than hydrogen; consequently, C-D bonds are shorter than C-H, which results in smaller "molar volume" and reduced lipophilicity of polydeuterated molecules. RP chromatography is based, mainly, on hydrophobic interactions. Therefore retention of compounds with lower lipophilicity should be weaker. As a result, in RP LC, molecules containing heavier isotopes of hydrogen elute earlier. This was suggested as a major contribution to the observed "Isotope effect".