(T1030-09-59) Development of a Fast and Robust LC-MS/MS Method for Simultaneous Analysis of a Wide Range of Antibiotics: A Cost-Effective Solution for Pharmaceuticals

Purpose: A large group of different antibiotics have been used in pharmaceuticals for multiple purposes such as cell culture, upstream cell selection, monitoring, etc. Nevertheless, exposure to antibiotics even at low levels can lead to detrimental effects on human health, such as allergic reactions, disruption of the digestive system’s functionality, and chronic toxic effects, among others^1-3. This concern is particularly crucial for patients who are already immunocompromised and are undergoing multiple therapies simultaneously⁴. Consequently, it is essential to control the presence of unexpected antibiotic residues at low levels in biologics products before releasing each batch. Therefore, a quick and robust LC-MS/MS method for simultaneous analysis of a wide range of different antibiotics used, is developed to be a fast, accurate, and low-cost solution.

Methods: In this method, multiple reaction monitoring (MRM) is used which is a mass spectrometry technique to select and quantify specific compounds and ions within complex mixtures by using two mass stages. More than one product ion can be selected and measured by the second mass analyzer. Due to the molecule of interest being effectively filtered twice, the background noise can be minimized, resulting in high sensitivity (parts per billion, ppb). Furthermore, antibiotics with similar retention times (RT) can be effectively separated by exploiting the ion difference.

Results: Four model antibiotics, including Ampicillin, Sulfamethoxazole, Trimethoprim, and Benzylpenicillin (penicillin G), were successfully analyzed simultaneously at ppb levels (ranging from 0.1ppb to 1000ppb), yielding an impressive correction coefficient of R=0.9998.

Conclusion: Overall, the development of a quick and robust LC-MS/MS method for the simultaneous analysis of various antibiotics serves as a valuable solution to ensure the controlled presence of antibiotics at low levels in biologics products. Furthermore, it has the flexibility to adapt to other antibiotics (e.g., Streptomycin, Amikacin, Kanamycin, etc.) by modifying the LC or MS/MS condition parameters accordingly.

References: 1. Chen, J., Ying, G. G., & Deng, W. J. (2019). Antibiotic residues in food: extraction, analysis, and human health concerns. Journal of Agricultural and Food Chemistry, 67(27), 7569-7586.
2. Bedford, M. (2000). Removal of antibiotic growth promoters from poultry diets: implications and strategies to minimize subsequent problems. World’s Poultry Science Journal,56, 236-243.
3. Sarmah, A. K., Meyer, M. T., & Boxall, A. B. (2006). A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere, 65(5), 725-759.
4. El‐Najjar, N., Hösl, J., Holzmann, T., Jantsch, J., & Gessner, A. (2018). UPLC–MS/MS method for therapeutic drug monitoring of 10 antibiotics used in intensive care units. Drug Testing and Analysis, 10(3), 584-591.

Figure1 Chromatograph of Mixture Standard (Blue: Ampicillin; Grey: Trimethoprim; Green: Sulfamethoxazole; Red: Penicillin G)

Figure 2 Calibration Curve of Four Antibiotics