# Preparation of a Lyophilized Bovine Milk Reference Material for Quality Control of Amoxicillin Detection

## TL;DR

This paper describes the creation of a freeze-dried milk sample containing a precise amount of amoxicillin to help labs accurately test for antibiotic residues in milk.

## Contribution

The study introduces a new lyophilized bovine milk reference material with amoxicillin at the maximum residue limit for quality control in testing.

## Key findings

- A stable and homogeneous amoxicillin reference material was prepared with a certified value of (4.10 ± 0.13) μg/kg.
- The material meets the maximum residue limit requirements of the US, EU, and China.
- It provides a reliable tool for validating detection methods and ensuring milk safety.

## Abstract

Antibiotics such as amoxicillin are commonly used to treat bacterial infections in dairy cows, but improper use can leave drug traces in milk. These residues can harm consumer health and disrupt dairy production. To ensure milk safety, regulatory authorities set strict limits on how much of these drugs can remain in milk. Laboratories need reliable reference samples—called reference materials—to accurately check their testing methods for these residues. In this study, we developed a stable and uniform freeze-dried milk powder sample containing a known amount of amoxicillin, matching the maximum allowed limit of 4 μg/kg. We used a highly accurate measurement technique involving mass spectrometry to confirm the sample’s content. The final reference material was shown to be consistent across different batches and stable over time. This product provides laboratories with a trustworthy tool for validating their detection methods and improving the accuracy of milk safety monitoring, thereby helping to protect public health and support the dairy industry.

A bovine milk reference material was produced containing amoxicillin with a target concentration at the maximum residue limit. For the evaluation and characterization of the reference material, an isotope dilution–liquid chromatography/tandem mass spectrometry method was developed and validated. This material was prepared by collecting milk samples from cows treated with an appropriate amoxicillin injection, followed by freeze-drying, dilution, and homogenization of the authentic samples. The amoxicillin reference material, prepared in accordance with ISO Guide 35, was homogeneous and stable. It was assigned a certified value of (4.10 ± 0.13) μg/kg with a relative standard deviation between 2.93 and 5.29%, which meets the maximum residue limit (MRL) requirement of 4.0 μg/kg established by the United States, the European Union, and China. This reference material can be used for method validation and provides internal quality control assurance for the detection of amoxicillin.