<b>Validation method for determining the concentration of norfloxacin-tylosin combination in broiler chicken tissue using high-performance liquid chromatography</b>

Background: The combination of norfloxacin and tylosin is an alternative to prevent antibiotic residue and resistance. Measurement of the levels of the combination of norfloxacin-tylosin in tissue using high-performance liquid chromatography (HPLC) to obtain parameters for further research, such as pharmacokinetics and residue analysis. When broiler chickens are given norfloxacin-tylosin antibiotics, they make antibiotics more effective against bacterial illnesses. These results indicate that the right mix of antibiotics can be used as a single medicine to achieve synergistic effects. Also, reach the SDGs for no hunger, good health, and well-being. Aim: This study aimed to ascertain the correct validation method for the combination antibiotic norfloxacin-tylosin in broiler chicken tissues, specifically the liver, kidney, and muscle. Methods: HPLC consistently detected norfloxacin-tylosin in broiler chicken tissue. Mobile phase methanol/acetonitrile was 50:50 (v/v). The chromatographic equipment utilized a 250 mm Shimpack ODS C18 column with a four μm diameter. Solution detection happened at 1 mL/min flow rate, 30°C oven temperature, and 20 μL injection volume. Light had a wavelength of 279 nm. In Bogor, Indonesia, PT Mensana Aneka Satwa manufactured liquid antibiotics and combined them with broiler chicken tissue from 28-day-old antibiotic-free-fed chicks. Results: The tissue, liver, kidney, and muscle showed linearity values of R2=0.9999, R2=0.9992, and R2=0.9939 for norfloxacin and R2=0.9969, R2=0.9964, and R2=0.9938 for tylosin. Norfloxacin had liver, kidney, and muscle accuracy scores of 100.57%, 101.85%, and 105.41%, respectively, while tylosin had 103.89%, 95.83%, and 105.49%. Norfloxacin showed intraday precision values (%CV) of 0.77%, 4.18%, and 4% in the liver, kidney, and muscle, while tylosin had 0.74%, 1.84%, and 1%. Tylosin retained 3.325-3.358 minutes and norfloxacin 3.083-3.092. The LOD values for norfloxacin in the liver, kidney, and muscle were 1.57, 5.10, and 1.4 μg/mL, respectively. Tylosin levels in liver, kidney, and muscle were 1.72, 1.15, and 1.51 μg/mL, while norfloxacin had LOQ values of 5.24, 17.01, and 4.98 μg/mL. Conclusion: According to the European Medicines Agency Guideline, the mixed solution of norfloxacin and tylosin demonstrated favourable outcomes regarding sensitivity, specificity, linearity, accuracy, and precision.