Determination of Amphenicol Antibiotic Residues in Chicken Meat by Wayeal LCMS-TQ9200

Keywords: LCMS, amphenicol, food safety, veterinary drug residues.

1. Instrument and Reagents

1.1 Configuration List of LCMS

Table 1 Instrument Configuration List

1.3 Experiment Materials and Auxiliary Equipment

Ultrasonic cleaner

Vortex Mixer

Water bath nitrogen evaporator

High-speed centrifuge

2. Experiment Methods

2.1 Solution Preparation

2.1.1 2% Ammoniated ethyl acetate solution: Take 4mL of ammonia water and dilute it to 200mL with ethyl acetate.

2.1.2 4% Sodium chloride solution: Take 4g of sodium chloride, dissolve it in water, and dilute to 100mL.

2.1.3 4% Sodium chloride-saturated n-Hexane solution: Take a suitable amount of 4% sodium chloride solution, add an excess of n-hexane, mix, allow to stand for layer separation, and take the upper n-hexane layer.

2.1.4 Mixed internal standard working solution: Take an appropriate amount of the mixed internal standard stock solution, dilute with 20% methanol to achieve chloramphenicol-D5 at 10ng/mL and other internal standards (florfenicol-D3, thiamphenicol-D3, and florfenicol amine-D3) at 50ng/mL each.

2.2 Sample Pretreatment

2.2.1 Sample Extraction: Weigh 2g of sample (accurate to ±0.01g), add 100µL of mixed internal standard working solution, vortex for 1 min, add 10mL of 2% ammoniated ethyl acetate solution, vortex for 10 min, centrifuge at 8000rpm for 5 min, and collect the supernatant. Add another 10mL of 2% ammoniated ethyl acetate solution to the residue, and repeat the extraction procedure. Combine the two supernatants, evaporate to dryness under a stream of nitrogen at 50°C, and reserve for use.

2.2.2 Sample Purification: Add 3mL of 4% sodium chloride solution to the purification residue, vortex to dissolve, then add 5mL of 4% sodium chloride-saturated n-hexane solution, vortex for 30s, centrifuge at 8000 rpm for 5min, and discard the upper n-hexane layer. Repeat the degreasing procedure once. Add 5mL of 2% ammoniated ethyl acetate solution, vortex for 10min, centrifuge at 8000 rpm for 5min, and collect the supernatant. Add another 5mL of 2% ammoniated ethyl acetate solution to the residue and repeat the extraction procedure. Combine the supernatants, evaporate to dryness under a stream of nitrogen at 50°C, add 1mL of 20% methanol solution, vortex for 30s, filter through a membrane, and await instrumental analysis.

2.3 Experiment Conditions

2.3.1 Liquid Chromatography Conditions

Chromatography column: C18 1.7μm 2.1x50mm

Mobile phase: A: Acetonitrile; B: 2 mM Ammonium Formate in Water

Flow rate: 0.3mL/min

Column temperature: 40 ℃

Injection volume: 5µL

2.3.2 Mass Spectrometry Conditions

Table 3 Ion Source Parameters

3. Experiment Result

3.1 Standard Chromatogram

The determination of four amide antibiotics and their internal standards was completed within 6 minutes. All peaks showed good peak shapes without tailing, and each compound demonstrated satisfactory response, meeting the experimental requirements.

Fig 1 Chromatogram of 4 Amphenicol Antibiotics and Internal Standard (Chloramphenicol 0.2ng/mL, Other Antibiotics 1ng/mL)

3.2 Linear Range

Take an appropriate amount of the amide alcohol mixed standard solution and dilute it stepwise to a series of concentrations to prepare a standard curve. Isotope internal standard method was used for quantitative analysis. The linear range was 0.2-10 ng/mL for chloramphenicol and 1-50 ng/mL for florfenicol amine, thiamphenicol, and florfenicol. The deviation between the linear detection results and the known concentrations was less than the maximum allowable deviation, with R² values ranging from 0.9958 to 0.9998, indicating excellent linearity for all components.

Table 4 Linear Range of Compounds

Fig 2 Calibration Curve Data of the Four Compounds

3.3 LOD and LOQ

The Chinese National Food Safety Standard GB 31658.20-2022 specifies that for this method, the limit of detection (LOD) for chloramphenicol is 0.1µg/kg and the limit of quantification (LOQ) is 0.2µg/kg; while for thiamphenicol, florfenicol, and florfenicol amine, the LOD is 0.5µg/kg and the LOQ is 1 µg/kg. The signal-to-noise ratios for all target compounds at the specified concentrations of the limit of detection (LOD) and limit of quantification (LOQ) are significantly greater than 3 and 10, which complies with the sensitivity requirements specified in the national standard.

Table 5 Limits of Detection and Quantification for Each Compound

Fig 3 Chromatograms of Detection Limits and Quantification Limits for Four Compounds

3.4 Precision Testing

Take low, medium, and high concentrations of amphenicol standard mixture solutions, and perform six consecutive injections to compare the deviations in retention time and peak area. The results are shown in the table below. The retention time deviations of all amphenicol compounds were less than 1%, and the peak area deviations were less than 5%, meeting the precision requirement of RSD ≤ 15% specified by the national standard.

Table 6 Precision Test for Each Compound

Fig 4 Precision Chromatograms of Four Amide Alcohol Compounds

3.5 Matrix Spiking Recovery Test

The accuracy of this detection method was evaluated using a spike recovery test. Add known concentrations of standard samples to the blank matrix sample at different ratios for pretreatment, preparing matrix-spiked samples at high, medium, and low concentrations. Each concentration level of the spiked samples was independently analyzed six times. The recovery rate was calculated by comparing the detected concentration in the matrix before and after spiking, thereby evaluating the accuracy of the method. The results are shown as follows: in chicken matrix, the spike recovery rates of all compounds ranged from 94.5% to 107.2%, with a CV within 5%, indicating that the accuracy meets the method requirements.

Table 6 Recovery Rates of Compounds in Spiked Chicken Matrix

3.6 Sample Test

Randomly purchased chicken breast samples from supermarkets were extracted and purified using the aforementioned sample pretreatment method. After testing the samples on the instrument, none of the four amide antibiotics were detected.

Fig 5 Chromatogram of Four Amide Alcohol Compounds in Supermarket Chicken Breast Samples

4. Conclusion

This method utilizes the Wayeal LCMS-TQ9200 liquid chromatography mass spectrometry system for the determination of amphenicol antibiotics in animal-derived foods, allowing for simultaneous acquisition of compounds in both positive and negative ion modes. The data indicates that the method delivers well-shaped chromatographic peaks with no tailing, and its sensitivity meets national standard requirements. The linear correlation coefficients are all greater than 0.99. The retention time deviation for all compounds across six consecutive injections is within 1%, and the peak area deviation is within 5%, demonstrating good precision. The spiked recovery rates in chicken matrix range from 94.5% to 107.2%, with a CV within 5%, indicating that the accuracy meets methodological requirements. Randomly purchased chicken breast samples from supermarkets were analyzed, and none of the four amphenicol antibiotics were detected. This demonstrates that the method, equipped with Wayeal's liquid chromatography- mass spectrometry system, meets the requirements for routine qualitative and quantitative detection of the target samples.