Determination of Erythromycin Residues in Antibiotic Fermentation Residues Using the Wayeal LCMS-TQ9200 Liquid Chromatography- Mass Spectrometry System

Erythromycin is a macrolide antibiotic produced by the fermentation of Streptomyces erythreus. It exerts its antibacterial effect primarily by inhibiting bacterial protein synthesis. Erythromycin production primarily relies on bio-fermentation technology, involving steps such as strain selection, seed culture, large-scale fermentation in tanks, extraction, and purification. After fermentation, erythromycin is extracted with organic solvents (such as butyl acetate), followed by purification through crystallization or ion-exchange resin separation, ultimately yielding clinical-grade salts (e.g., erythromycin ethylsuccinate). However, the erythromycin production process generates a significant amount of mycelial dregs (fermentation residue), which may contain residual antibiotics, microbial proteins, and metabolic by-products. If not managed properly, these residues may enter the ecological chain through environmental discharge or use as feed additives, leading to multiple hazards: Residual erythromycin can promote the spread of antibiotic resistance genes in soil and water bodies, disrupting microbial community balance; amplified through the food chain, it may induce the emergence of resistant bacterial strains in animals or humans, undermining antibiotic efficacy; furthermore, the underutilized organic matter within the residues can cause environmental pollution. Therefore, strict regulation of mycelial residue is a critical measure for controlling pollution from erythromycin production.

This experiment was conducted with reference to the standard "T/PIAC 00003—2021 Group Standard of China Pharmaceutical Industry Association - Method for the determination of erythromycin in antibiotic residue, organic fertilizer base materials, crops, and environmental media," utilizing the Wayeal LCMS-TQ9200 liquid chromatography-mass spectrometry system to determine the erythromycin content in the mycelial dregs. The experimental results indicate that the system suitability test demonstrated good peak shape and good linearity, meeting the experimental requirements.

1. Instrument and Reagents

1.1 Configuration List of LCMS

Table 1 List of Instrument Configuration

1.2 Reagents and Standards

Table 2 List of Reagents and Standards

1.3 Experiment Material and Auxiliary Equipment

Ultrasonic cleaner

Vortex mixer

High-Speed centrifuge

2. Experiment Method

2.1 Sample Pretreatment

Weigh 0.5g of the sample (accurate to 0.001 g) into a glass-stoppered test tube. Add 50mL of acetonitrile, vortex for 1 minute to homogenize the mixture, and perform ultrasonic-assisted extraction for 20 minutes. Then transfer the mixture to a 50mL centrifuge tube and centrifuge at 4000rpm for 10 minutes. Collect an appropriate amount of the supernatant and pass it through a 0.22μm filter membrane. Discard at least 1mL of the initial filtrate, then transfer the remaining filtrate into an amber LC vial for analysis.

2.2 Experiment Conditions

2.2.1 Liquid Chromatography Method Conditions

Chromatography column: C18 1.7μm 2.1x50mm

Mobile phase: A: Acetonitrile, B: 0.1% Formic acid in water

Flow rate: 0.3mL/min

Column temperature: 30℃

Injection volume: 2μL

2.2.2 Mass Spectrometer Method Conditions

Table 3 Mass Spectrometry Ion Source Parameters

3. Experiment Result

3.1 System Suitability Test

The system suitability test results showed well-defined target peaks without interference from extraneous peaks, meeting all experimental requirements.

Fig 1 Chromatogram of Erythromycin A Standard Working Solution (0.5ng/mL)

3.2 Linear Range

The calibration curve for erythromycin A was prepared by serially diluting the standard solution using intermediate concentration working solutions. The curve demonstrated excellent linearity across the range of 0.5-500ng/mL, with a correlation coefficient (R²) greater than 0.99.

Table 4 Linear Range of Erythromycin A

Fig 2 Calibration Curve for Erythromycin A

3.3 Limit of Detection (LOD) and Limit of Quantification (LOQ)

In this method, the limit of detection (LOD) and limit of quantification (LOQ) for the erythromycin A standard solution were determined to be 0.2ng/mL and 0.5ng/mL, respectively. The corresponding signal-to-noise ratios (S/N) were 110.02 and 292.20, substantially exceeding the minimum requirements of 3 and 10, thereby fulfilling the experimental sensitivity criteria.

Figure 3. Extracted Ion Chromatograms for the LOD and LOQ of Erythromycin A

3.4 Precision Test

The erythromycin A solution was injected consecutively seven times, with the results shown in the figure below. The retention time deviation for erythromycin A was 0.19%, and the peak area deviation was 0.96%, both of which are below 5%, meeting the experimental requirements.

Figure 4. Precision Chromatograms of Erythromycin A Sample (7 Injections)

3.5 Sample Test

Following the aforementioned pretreatment method, the target peak area in the solid sample was 7.4E6. Calculated via the external standard method, the erythromycin A content in the sample solution was determined to be 400 ng/mL.

Fig 5 Chromatogram of the Erythromycin A Sample Test

4. Conclusion

This method utilizes the Wayeal LCMS-TQ9200 liquid chromatography-mass spectrometry system for the determination of erythromycin A content in antibiotic fermentation residues. The data indicate that the method demonstrates excellent performance: all chromatographic peaks exhibit optimal shape without tailing, and the sensitivity meets experimental requirements. The linear correlation coefficient (R²) exceeds 0.99. The retention time and peak area deviations for all compounds across seven consecutive injections are within 1%, indicating high precision. The sample chromatograms show no interference from extraneous peaks, and the measured sample content is 400ng/mL. This method equipped with the Wayeal LC-MS/MS system, fulfills the requirements for routine qualitative and quantitative analysis of the test samples.