The paper method, also known as the PD (Paper Disc) method, is a commonly used technique for detecting β-lactam antibiotics in milk. Two widely applied versions of this method are the *Bacillus subtilis* paper test and the *Astragalus thermophilus* (or *Geobacillus thermophilus*) paper test. Both methods follow a similar operational procedure but use different bacterial strains to detect antibiotic residues. The *Bacillus subtilis* method is often prone to false positives, so a confirmatory step is typically performed by treating the milk sample with penicillinase to inactivate any penicillin present. This helps distinguish between true β-lactam antibiotics and other substances. The detection limit for this method can reach as low as 0.01 IU/mL.
On the other hand, the *Astragalus thermophilus* method not only detects β-lactam antibiotics but also identifies the presence of other bacteriostatic agents. It offers higher sensitivity, with a detection limit below 0.008 IU/mL, and results are usually available within 4 hours. Because of its accuracy and efficiency, the *Astragalus thermophilus* method is more widely used in practice than the *Bacillus subtilis* method.
In 1991, Shen Baosheng improved the paper method further, enhancing both the detection accuracy and the ability to identify specific types of antibiotics. The improved method increased the detection rates for various antibiotics, including chloramphenicol (0.01 mg/kg), oxytetracycline (0.05 mg/kg), streptomycin (1 mg/kg), erythromycin (0.05 mg/kg), and penicillin (0.0025 mg/kg).
In addition to the paper method, microbiological detection techniques such as the TTC (triphenyltetrazolium chloride) method and the swab method (STOP) are also widely used. These methods rely on the inhibition of microbial growth or color change due to antibiotic presence. While they are cost-effective and easy to perform in basic laboratories, they suffer from limitations such as long testing times, subjective visual interpretation, and complex procedures.
The TTC method, standardized in China’s GB5409-85, is particularly popular due to its simplicity and speed. It can provide results within 3–4 hours and is suitable for on-site testing in dairy farms and food hygiene departments. Its sensitivity varies depending on the antibiotic: 0.004 U/mL for penicillin, 0.5 U/mL for streptomycin, 0.4 U/mL for gentamicin, and 5 U/mL for kanamycin.
Rapid microbiological assays like the swab method and the CAST (Bovine Antibiotic and Sulfonamide Test) have gained popularity in countries like the United States and Canada. Recently, the Rapid Antibiotic Screening (FAST) method has been introduced, offering faster results. Although these methods may occasionally produce false positives, they are generally accepted with the possibility of follow-up confirmation tests.
Physical and chemical methods, such as high-performance liquid chromatography (HPLC), gas chromatography, and mass spectrometry, are more sensitive and accurate for detecting antibiotic residues. HPLC, in particular, is widely used due to its high resolution, automation, and versatility. It can separate and quantify even polar or ionic compounds. Reversed-phase HPLC, using C18 or C8 stationary phases, is especially effective for analyzing antibiotic residues in milk. UV detectors are commonly used, though pre-column derivatization is often necessary to enhance sensitivity and reduce background interference.
For example, Peng Li et al. successfully detected chloramphenicol in milk using HPLC with a UV detector at 278 nm after extraction with ethyl acetate. The method achieved an average recovery rate of 94.8% with a coefficient of variation less than 10%.
Overall, while microbiological methods remain practical and cost-effective, physical and chemical techniques offer greater precision and reliability, especially for trace-level detection. The choice of method depends on factors such as required sensitivity, available equipment, and the need for rapid or confirmatory analysis.
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