Differentiation of tetracycline isomers by cyclodextrin-enhanced ion mobility-mass spectrometry with computational insights

Since tetracycline (TC) and doxycycline (DC) differ only in the position of a single hydroxyl group, this nearly identical core structure makes their chromatographic separation extremely difficult. This study employed ion mobility mass spectrometry (IM-MS) combined with computational modeling to investigate the identification of TC and DC via non-covalent interactions with cyclodextrins (CD).

The TC/DC complexes with α-, β-, and γ-CD were examined. Baseline separation was achieved for α-CD (R_p-p = 1.175) and β-CD (R_p-p = 1.079), while γ-CD showed poor resolution (R_p-p = 0.098). The effect of metal ion additives was further investigated, and the results showed that the addition of most metal ions did not significantly improve the resolution but instead led to a deterioration in separation performance. Theoretical calculations were performed to obtain the optimized structures of the three [TC/DC+α/β/γ-CD + H]⁺ complexes. As the CD cavity size increased, the TC/DC molecules were embedded more deeply, resulting in a greater number of interaction sites. Meanwhile, the DC-CD complexes exhibited a more loose structure compared to TC-CD, with correspondingly larger mobility values. For the quantitative analysis of TC/DC using the β-CD-based IM-MS system, revealing good linear relationships, with a limit of detection of 1.32 nmol L^-1 and RSD ≤ 3.16%. The method was applied to the determination of DC/TC in hair, urine, and chicken meat, yielding recoveries ≥ 80.42% and RSD ≤ 4.69%.

The results demonstrate that the proposed method based on IM-MS combined with computational modeling can serves as a viable alternative for the isomer separation of TC and DC, or analogous isomers in real samples.