Detecting Antibiotic Residues Using High-Performance Liquid Chromatography

Researchers from Addis Ababa University in Addis Ababa, Ethiopia tested new methods for separating and simultaneously determining antibiotic residues in food and environmental samples. Publishing their work into the Journal of Chromatography A, the researchers used high-performance liquid chromatography with diode-array detection (HPLC-DAD) was used to detect residues in samples.

Antibiotics function by killing harmful bacteria or stopping bacteria from multiplying. They can be classified into various groups, such as penicillins, cephalosporins, and more, that can each treat different conditions. However, a significant global health challenge is an uptick in antimicrobial resistance (AMR). According to the World Health Organization, AMR occurs when bacteria, viruses, fungi, and parasites no longer respond to antimicrobial medicines, rendering them ineffective (2). This can make certain infections difficult or impossible to treat, increasing the risk of disease spread, severe illness, disability, and death. According to the Centers for Disease Control and Prevention, 1.27 million deaths in 2019 could be attributed to bacterial AMR (3).

white round medicine tablet antibiotic pills | Image Credit: © Kadmy - stock.adobe.com

Antibiotics are expelled from the body and entering the environment, rendering them organic micropollutants. These pollutants can dissolve in water or attach to solid materials, with most of them persisting in the environment for a long time, with current cleaning procedures being unable to entirely remove metabolites from surface waters. Various techniques have been tested for determining pharmaceutical residues in environmental samples, with LC being the most used technique for this purpose.

For this study, the scientists used a high-performance liquid chromatography with diode-array detection (HPLC-DAD) method for separating and simultaneously determining ten multiclass antibiotics residues in various environmental samples. Sample sources include river water, irrigation dam water, hospital wastewater, cow urine, milk and sediment samples. The antimicrobials and anthelminthic agents selected for this study were amoxicillin (Amoxa), metronidazole (Met), tinidazole (Tin), ciprofloxacin (Cipro), chloramphenicol (CAF), cephalexin (Ceph), cefuroxime (Cefu), cefpodoxime (Cefp), albendazole (Albe), and cloxacilln (Cloxa). They were chosen due to their constant use in Ethiopia for treating humans and animals.

In addition to the HPLC-DAD method, the scientists optimized a surface floating organic droplets-air assisted liquid-liquid micro extraction (SFOD-AALLME) method for extracting the residues from various real samples. A combination of liquid-liquid microextraction (LLME), air-assisted mass transfer, and surface floating organic droplets the technique brings these concepts together to increase extraction efficiency and decrease solvent consumption. A small quantity of an organic solvent is added to an aqueous phase, causing floating droplets to develop on the surface. Afterwards, air flow improves mass transfer between organic droplets and the aqueous phase, making it easier for analytes to partition into the organic phase, where they are concentrated. This proves advantageous for analyzing biological, culinary, and environmental samples, as the scientists claim, since the technique is very sensitive, requires minimal solvent use, and yield quick extraction periods (1).

For each antibiotic, the HPLC-DAD method yielded strong linearity, having a coefficient of determination falling between 0.9982 and 0.9999. The ranges for the limits of detection and quantification were 0.07–0.15 and 0.26–0.49 μg/L, respectively. The method also achieved a good recovery range of 87.3–100.8%. The SFOD-AALLME method proved its linearity, accuracy, and precision as well, at least regarding the multiclass antibiotic residues under study. Overall, when compared to traditional extraction methods, the suggested approaches within this study proved themselves more time-efficient and environmentally friendly due to the small amount of less harmful extraction solvent used during procedures.

References

(1) Abera, S.; Yaya, E. E.; Chandravanshi, B. S. Development of HPLC-DAD Method for the Separation and Simultaneous Determination of Ten Antibiotic Residues in Food and Environmental Samples Using Surface Floating Organic Droplet-Based Air-Assisted Liquid-Liquid Micro-Extraction. J. Chromatogr. A 2025, 1748, 465817. DOI: 10.1016/j.chroma.2025.465817

(2) Antimicrobial Resistance. World Health Organization 2023. https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance (accessed 2025-3-13)

(3) Antibiotic Resistance Threats in the United States, 2019. Centers for Disease Control and Prevention 2019. http://dx.doi.org/10.15620/cdc:82532 (accessed 2025-3-13)