# Depletion of Amoxicillin and Its Major Metabolites in Anatolian Water Buffalo Milk After Intramuscular Administration

## TL;DR

This study tracks how amoxicillin and its metabolites leave Anatolian water buffalo milk after treatment, helping determine safe waiting times before milk can be sold.

## Contribution

The study provides the first detailed depletion data for amoxicillin and its metabolites in Anatolian water buffalo milk.

## Key findings

- Amoxicillin concentrations in buffalo milk fell below EU MRL by the sixth milking and were undetectable by the tenth.
- Two metabolites persisted longer than amoxicillin, remaining detectable up to the thirteenth milking.
- A practical withdrawal period of 96 hours (≈8 milkings) was estimated using the EMA 95/95 tolerance limit approach.

## Abstract

Antibiotics can be necessary to treat infections in dairy animals, but traces of these drugs can pass into milk if it is collected too soon after treatment. Water buffalo are an important milk species worldwide, yet there is limited information on how quickly some antibiotics leave buffalo milk. In this study, five lactating Anatolian water buffaloes received one intramuscular injection of amoxicillin, and milk was sampled at every milking for seven days. We measured amoxicillin and two major breakdown products using a highly sensitive laboratory method. Amoxicillin was highest at the second milking and then decreased; mean concentrations fell below the European Union MRL by around the sixth milking and were below the LOD after the tenth milking. Because one individual animal was slightly above the MRL at the sixth milking (4.2 µg/kg), all individual samples were below the MRL from the seventh milking onward. The two breakdown products persisted longer than AMOX, remaining detectable up to about the thirteenth milking, whereas AMOX was no longer detectable from the tenth milking onward. Based on the EMA-recommended 95/95 tolerance limit approach, the withdrawal time corresponded to 84.7 h after dosing; when rounded up to the next 12 h milking interval, this gives a practical withdrawal period of 96 h (≈8 milkings). This evidence can help farmers, veterinarians, and regulators set practical waiting times for buffalo milk and suggests that breakdown products may also be useful to track in safety testing.

Antibiotic residues in water buffalo milk are a food-safety concern, yet depletion data are scarce. The purpose of this study was to characterize the depletion profiles of amoxicillin (AMOX) and its two major metabolites, amoxicilloic acid (AMA) and amoxicillin diketopiperazine-2′,5′-dione (2,5-DKP), in Anatolian water buffalo milk after a single intramuscular administration and to estimate a milk withdrawal time relative to the EU MRL. We tested the hypothesis that AMOX concentrations would decrease below the EU MRL over successive milkings and that AMA and 2,5-DKP would exhibit depletion kinetics distinct from the parent compound. Five lactating Anatolian water buffaloes received a single intramuscular injection of amoxicillin (15 milligrams per kilogram). Milk was collected at each milking (twice daily) for seven days and analyzed by liquid chromatography–tandem mass spectrometry with quantification limits below the European Union maximum residue limit for amoxicillin in milk (4 micrograms per kilogram). Amoxicillin peaked at the second milking (mean 13.65 micrograms per kilogram), mean concentrations fell below the maximum residue limit from the sixth milking, and they became non-quantifiable from the tenth milking onward. Two major metabolites, amoxicillinic acid and amoxicillin diketopiperazine-2′,5′-dione, peaked earlier (2,5-DKP Tmax 12 h) or at higher concentrations (AMA Cmax 32.64 µg/kg vs. AMOX 13.65 µg/kg) and remained detectable up to the thirteenth milking, with longer apparent terminal half-lives (32.0 and 52.8 h) than amoxicillin (23.5 h); the mixed-effects model confirmed different depletion rates among analytes (milking × analyte interaction p = 4.63 × 10−5). A log-linear withdrawal model applying the EMA 95/95 tolerance limit indicated that the first time point at which the upper tolerance limit fell below the EU MRL was 84.7 h after dosing; rounded up to the next 12 h milking interval, this corresponds to a reported withdrawal period of 96 h (≈8 milkings). These results provide species-specific residue kinetics for amoxicillin in Anatolian buffalo milk and support considering metabolites in monitoring and withdrawal-time decisions.