2023 FDA Science Forum
Simultaneous determination of tetracycline, oxytetracycline, and chlortetracycline using liquid chromatography tandem mass spectrometry
- Authors:
- Center:
-
Contributing OfficeCenter for Veterinary Medicine
Abstract
Tetracyclines are a group of broad-spectrum antibiotic compounds that have a common base structure. Tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC) are widely used in veterinary medicine due to their antimicrobial activity, low toxicity, and low cost. A novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed that identifies different forms of TC, OTC, and CTC standard solutions, including epimers and tautomers that may complicate the analysis. Epimer and tautomer products are isobaric compounds and cannot be distinguished by their respective mass. Therefore, optimization of the chromatographic conditions is desirable to separate and identify different forms of each drug. A solvent gradient using water and methanol containing 0.1% formic acid is used to elute all three compounds with a Waters Acquity HHS T3 column. Quantitation is performed by comparing each product quantifier ion peak area in the samples to that of an internal standard demeclocycline, which is structurally similar. The linearity of the assay is checked by injecting a series of standards from 100 ppb to 8.0 ppm into the LC-MS system. Epimerization is observed reversibly for TC, OTC, and CTC under different pH conditions. However, additional peaks are also observed for CTC. To clarify the identity of these peaks, CTC and epi-CTC solutions were analyzed on the mass spectrometer using multiple reaction monitoring (MRM) using m/z 479 → 462 and m/z 479 → 444 transitions. Further analysis of these two transitions over time, led to identification of different forms of CTC including keto-CTC, enol-CTC, iso-CTC along with their corresponding epimers. This method provides more accuracy and efficiency to detect TC, OTC, and CTC standard solution residues. Our results indicated that CTC goes through keto-enol tautomerization as well as an intra-molecular rearrangement resulting in formation of iso-CTC. The ability to test for different forms of these antibiotics can be applied by CVM to aid in its regulatory decision making.
