Objective: This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify gentamicin residues in milk and to establish a withdrawal period for an intramuscular gentamicin product in dairy cows. Methods: Nine Holstein cows received a gentamicin product (50 mg gentamicin sulfate/ mL) at 0.1 mL/kg twice daily at 12 h intervals via intramuscular injection. Milk samples were collected before dosing and at 12 h intervals up to 168 h after the final administration and analysed with a validated LC-MS/MS method using heptafluorobutyric acid as an ion-pairing reagent. Results: The method showed recovery rates of 86%–104%, coefficients of variation of 3%–11%, and limits of detection and quantitation of 23 and 70 ng/mL, respectively. Mean gentamicin concentrations exceeded the maximum residue limit (MRL; 0.2 mg/kg) at 12 h during treatment and 12 h after the final administration, and were < MRL in all cows from 36 h after the last dose. Based on residue depletion data and a national non-statistical guideline, a withdrawal period of 40–48 h was derived, and the regulatory withdrawal period for this product was established as 3 days. Conclusions and Relevance: The validated LC-MS/MS method enables rapid quantitation of gentamicin residues in bovine milk. A 3-day regulatory withdrawal period for this product supports milk safety, regulatory compliance, and practical use in dairy herds.

Background/Aims: Tacrolimus has been used as an immunosuppressive agent in organ transplantation. Despite the therapeutic benefits, tacrolimus’s use is limited due to its nephrotoxicity. To reduce tacrolimus nephrotoxicity, effective humanized experimental models may be helpful. Here, we modeled tacrolimus nephrotoxicity using kidney organoids derived from human inducible pluripotent stem cells (iPSCs) in vitro. Methods: Kidney organoids were differentiated from the CMC11 iPSC cell line, re-seeded in 96-well plates, and treated with tacrolimus at doses of 0, 30, or 60 μM for 24 hours. This in vitro model was compared to a mouse model of tacrolimus nephrotoxicity and the associated mechanisms were investigated. Results: The size of the kidney organoids and cell viability decreased in dose-dependent manners after treatment with tacrolimus. The number of tubular cells decreased with a loss of polarity, similar to the effects seen in mouse tacrolimus nephrotoxicity. Ultrastructural analysis showed numerous vacuoles in the proximal tubular cells of the kidney organoids treated with tacrolimus. Tacrolimus treatment induced oxidative stress and mitochondrial dysfunction, and autophagic activity was enhanced in the kidney organoids. Rapamycin, an autophagy inducer, accelerated cell death in the kidney organoid model of tacrolimus nephrotoxicity, which was attenuated by treatment with 3-methyladenine, an autophagy inhibitor. These findings indicate that the augmentation of autophagy by rapamycin treatment accelerated tacrolimus nephrotoxicity. Conclusions Our data suggest that human kidney organoids are an effective in vitro model of tacrolimus nephrotoxicity and that autophagy plays a critical role in tacrolimus nephrotoxicity.