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Tetracycline antibiotic (TC) is one of the most commonly used antibiotics in the animal production sector. They are used to avoid animal infections, treat diseases and protect animal growth.
Excessive use of antibiotics in animal-derived foods (such as milk and meat) may lead to residual violations, thereby posing health risks to consumers. The presence of tetracycline residues in food can cause a series of problems, including:
In order to reduce risks and protect consumer health, regulatory agencies around the world have formulated guidelines for acceptable levels of veterinary drugs in foods. In the United States, these levels are called "tolerable levels", while China, Canada, and the European Union refer to them as "maximum residue levels" (MRL). 1,2
According to the regulatory requirements, the use of highly sensitive and selective technology to provide accurate monitoring of tetracycline levels is a key factor in protecting consumer health.
This article outlines a UHPLC/MS/MS method that provides the speed and sensitivity required for the quantitative analysis of tetracycline in meat products.
In order to improve the efficiency of the test laboratory, the QS-Works high-throughput autosampler is used for the following reasons:
The outlined method shows the excellent sensitivity, recovery, precision, linearity, and selectivity required for the analysis of tetracycline in meat products at low tolerance levels specified by various regulatory agencies around the world.
A series of hardware is used for analysis. Chromatographic separation was performed on the PerkinElmer QSight® LX50 UHPLC system. The detection process is completed with PerkinElmer QSight® 420 MS/MS detector.
The QS Works autosampler with high capacity is used to increase the productivity of the laboratory. The Simplicity 3Q™ software platform is used to perform data acquisition and processing as well as all instrument control.
The MS source parameters and LC method are shown in Table 1. See Table 2 for the multiple reaction monitoring mode (MRM) transitions of tetracycline.
Table 1. LC method and MS ion source parameters. Source: PerkinElmer Food Safety and Quality
Table 2. MRM conversion. Source: PerkinElmer Food Safety and Quality
At least two MRM transitions are monitored for each analyte. This is to reduce the number of false positives and negatives in the overview method.
The optimization of MS/MS parameters, including the selection of precursor ions and product ions, inlet voltage (EV/CCL2) and collision energy (CE), is accomplished by the injection of standard solutions.
The optimization of source conditions is achieved by injecting pure standard solution and LC flow, and the acquisition MS method is based on these optimized conditions.
All reagents, solvents and diluents used are LC/MS grade. All tetracycline standards are from Sigma-Aldrich® Inc. These standards are stored in a refrigerator at -20 °C to prevent their degradation.
All stock and mixed drug solutions used in all tetracycline spike and calibration procedures are prepared in methanol. In order to reduce the risk of degradation of standard products, all working standard products and stock standard products are stored in the refrigerator until needed.
The homogeneous sample used for this procedure is created as follows:
This article aims to evaluate the performance of the high-volume QS-Works autosampler through a validated sample preparation procedure for the determination of tetracycline in meat samples.
The use of high-throughput autosamplers (such as QS-Works) can increase the productivity and efficiency of the test laboratory.
QS-Works allows food testing laboratories with a large number of samples to process samples more quickly, while at the same time improving data quality and consistency due to its robotic sampling unit (Figure 1).
Figure 1. QS-Works autosampler. Image source: PerkinElmer Food Safety and Quality
The reference target level is determined by using the maximum residue level (MRL) of tetracycline in food determined by Health Canada or the allowable amount of the US FDA (X in Table 3).
Table 3. Results of retention time, matrix effect (ME), linearity, recovery, and reproducibility (%RSD). Source: PerkinElmer Food Safety and Quality
The detection threshold or "yes/no" screening level should be equal to or lower than 0.5X. The chromatograms (XIC) of the four tetracyclines showed good peak resolution (Figure 2).
Figure 2. XIC chromatograms of a) oxytetracycline, b) tetracycline, c) chlortetracycline, and d) doxycycline. Image source: PerkinElmer Food Safety and Quality
For confirmation and identification purposes, two ion transitions were observed, thereby improving the selectivity and specificity of identification. The ratio of each ion is determined by calculating the peak area of the ion with a lower intensity and the peak area of the ion with a higher intensity to generate the ion ratio.
One of the main problems in LC/MS/MS method development is the sample matrix effect (ME). This is especially true when analyzing food due to the complexity and diversity of food sample matrices.
In the presence of charge competing matrix components, ESI is susceptible to ionization inhibition by the analyte. Matrix-induced enhancement effects may appear in ESI, which may lead to large deviations in quantification.
In order to overcome the sample matrix effect, a variety of methods have been used, including:
The sample matrix effect is calculated by comparing the slope of the calibration curve obtained from the meat sample matrix with the slope obtained from the pure reagent (RO).
The sample matrix effect (expressed as a percentage) for each analyte is then determined by comparing the percentage difference between the slopes.
The matrix effect of most compounds is less than 20% (Table 3).
Matrix matching calibration is used in this analysis to quantify all analytes. This is to control the matrix effect and reduce the level of variation in the analysis results.
Use pure reagents (RO) and matrix matching (MM) standards for calibration.
Each calibration curve constructed from RO and meat sample matrix showed good linearity, with a correlation coefficient (R2) greater than 0.99. See Figure 3 and Figure 4 for typical examples of calibration curves.
Figure 3. Tetracycline calibration curve obtained using standards prepared only with reagents. Image source: PerkinElmer Food Safety and Quality
Figure 4. Calibration curve of tetracycline obtained from a standard prepared from a meat sample matrix. Image source: PerkinElmer Food Safety and Quality
Evaluate the degree of carryover by injecting a reagent blank after the high-concentration standard. No residue was found in any test.
Add oxalic acid to the aqueous mobile phase 3 to prevent the tetracycline from being chelated by the metal ions of the LC component or column. This also helps to maintain good peak shape and reproducibility throughout the run.
This method shows good precision, with RSD less than 5% for five repetitions. See Table 3 for the effect of using oxalic acid and reconstituted solvent in the mobile phase on reproducibility.
By adding oxalic acid to the mobile phase and reconstitution solvent, the reproducibility and peak shape of all analytes are improved. The limit of quantification (LOQ) of each tetracycline analyzed was lower than the allowable amount of meat samples.
This indicates that the technology meets the sensitivity level required for the analysis of tetracycline tolerance levels in meat.
The LOQ of tetracycline is estimated based on a signal-to-noise ratio (S/N) of 10. The LOQ of each drug under study is 1.5 ± 1.0 µg/L.
These results indicate that the method can be used for rapid screening and quantification of tetracycline in meat samples, and the absolute recovery rate of tetracycline is in the range of 70-120% (Table 3). The use of deuterated tetracycline can compensate for the loss of tetracycline during this extraction process.
PerkinElmer high-throughput QS-Works autosampler and LX 50 UHPLC coupled with QSight 420 MS/MS system are used to demonstrate UHPLC/MS/MS procedures.
The instrument shows good sensitivity in identifying and quantifying tetracycline in homogeneous meat samples. The low detection level obtained by this method helps support low regulatory limits for routine screening and quantitative analysis.
Made from materials originally written by Saba Hariri, Abir Khaled, Avinash Dalmia and Feng Qin from PerkinElmer, Inc.
This information is derived from materials provided by PerkinElmer Food Safety and Quality and has been reviewed and adapted.
For more information on this source, please visit PerkinElmer Food Safety and Quality.
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