Ultra-high sensitivity quantification of veterinary drug residues in products of animal origin

Methods

Standard preparation: A mixed standard solution was provided by the official food control authority and veterinary affairs of Geneva at a concentration of 1 µg/mL. A series of dilutions was performed to cover a range of concentrations from 0.005 to 100 ng/mL.

Sample preparation: Each sample was homogenized prior to weighing. A 5 g sample of pork, milk or chicken was combined with 0.1M EDTA-Mcllvaine buffer (4 mL for pork and chicken, 3 mL for milk). Pork and chicken samples were then homogenized for 75 seconds using a FASTH21, whereas milk samples were homogenized for 10 minutes using a mechanical shaker. Pork and chicken solutions were then combined with 16 mL acetonitrile and 5 g ammonium sulfate. Milk solutions were combined with 8 mL acetonitrile and 5 g ammonium sulfate. All samples were then further homogenized and centrifuged for 5 minutes at 4,700 rpm at 4°C. A 4 mL sample of the upper layer was removed and transferred into an evaporative vial with 400 µL of DMSO. Nitrogen was used to evaporate the sample at 40°C until a final weight of approximately 0.5 g was achieved. Then, 1 g of H₂O was added to each solution before vortexing for approximately 15 seconds, centrifuging for 5 minutes at 13,000 rpm and filtering through a 0.45 µm nylon filter prior to analysis.

Spiked sample preparation: 90 µL of prepared pork, chicken or milk matrix sample was added to 10 µL of a relevant standard solution. Three spike concentrations were prepared at 0.01, 0.1 and 1 ng/mL with standard solutions at 0.1, 1 and 10 ng/mL being used as spiking solutions.

Chromatography: An ExionLC AD system was used to perform the chromatographic separation with a Phenomenex Kinetex Polar C18 (2.6 µm, 100 Å, 100 x 2.1 mm) column.

Mass spectrometry: The analysis was performed using the SCIEX 7500 system. The system was operated using the time-scheduled multiple reaction monitoring (sMRM) mode (see Figure 2) using positive and negative electrospray ionization (ESI) switching. Q0D optimization was performed to enhance the signal of some compounds.⁵ Data were acquired using SCIEX OS software.

Data processing: Data were processed using SCIEX OS software with the AutoPeak algorithm for peak integration.⁶

Chromatographic separation

When analyzing many compounds in a single analytical method, chromatographic separation is an important factor to ensure that the number of compounds analyzed at a particular time is minimized. This allows the balance between the cycle time of the mass spectrometer and the dwell time for each analyte to be optimal. Figure 3 shows the chromatographic separation achieved in both positive and negative ion acquisition modes. It is important to note that baseline separation of compounds is not necessary in most cases, due to the specificity of a triple quadrupole mass spectrometer operated in MRM acquisition mode. Chromatography helps to minimize analyte concurrency and to reduce interferences that may occur from the sample matrix.

Sensitivity and specificity

Sensitivity and specificity are crucial for a successful analysis and triple quadrupole mass spectrometers are typically used to achieve high levels of these metrics. Figure 4 highlights the levels of sensitivity that can be achieved using the SCIEX 7500 system for 3 representative analytes at their respective LLOQs of 0.005 ng/mL in solution. The blank injection is free of interferences, therefore indicating that the peak observed at the LLOQ is genuine. The use of quantifier and qualifier MRM transitions enhances the specificity of the analysis in matrix by using ion ratios to better confirm the identity of the peak, relative to a single MRM transition. The ion ratio lines highlight the acceptable range within ±30% of the expected value for the qualifier peak.

Although sensitivity in solvent is an important factor for analysis, it is crucial to maintain the quality of analyses in complex matrices. The same 3 compounds analyzed in solvent in Figure 4 were spiked into the pork, milk and chicken matrices. The results of these analyses are shown in Figure 5. The XICs shown are for the lowest spike level analyzed (0.01 ng/mL), which equated to 0.01 µg/kg of compound in the pork or chicken samples and 0.005 µg/kg of compound in milk. These results demonstrate the ultra-high level of sensitivity that can be achieved in this assay.

Linear dynamic range

The linear range of an analyte is the span at which quantification is possible while achieving acceptable levels of precision and accuracy. Each calibration curve analyzed achieved an r value >0.99 with accuracy between ±30% of the expected value. The linear dynamic ranges are indicated in Tables 1 and 2, included at the end of this technical note, for compounds analyzed in positive and negative ion modes, respectively. Representative examples of 3 compounds with linear dynamic ranges spanning 4 orders of magnitude are shown in Figure 6.

Accurate and precise quantification

To ensure accurate and precise quantification, individual peaks must have enough data points. For the compounds analyzed, each peak consisted of more than 10 data points. Representative quantifier and qualifier ion MRM transitions are shown in Figure 7 for 3 analytes at a concentration of 10 ng/mL in solution.  

Accuracy was assessed at each point of the calibration curves. Accuracy values for all analytes were between ±30% of the expected value at each concentration.   

Precision is paramount to show the consistency of the instrument for analyses in solution and in sample. Table 3 outlines the %CV values achieved for the 3 compounds highlighted in Figure 7. Precision was assessed for compounds in solvent and spiked in matrix. Precision was calculated for the lowest concentrations tested: 0.01 ng/mL in solvent, 0.01 µg/kg in pork and chicken and 0.005 µg/kg in milk.

Minimum method performance requirements (MMPRs)

Many of the compounds analyzed in this study are prohibited for use in veterinary settings. For these compounds, it is paramount to achieve high levels of sensitivity to ensure that they are not used illegally. Prohibited compounds do not have MRL values, as their presence at any level is not permitted. Some of these prohibited compounds have been detailed in a specific EURL guidance that defines MMPR values, which correspond to the minimum compound concentrations that should be detected by official laboratories.³ Table 4 presents a compiled list of the prohibited compounds analyzed in this study that have MMPR values. The method developed was capable of detecting each of the prohibited compounds analyzed at concentrations lower or equal to the MMPR value.

Conclusions

• An ultra-sensitive and fast method for the quantification of more than 180 pharmacologically active compounds was developed
• Analysis was performed in 3 relevant food matrices, at concentrations as low as 0.01 µg/kg for pork and chicken and 0.005 µg/kg for milk
• Linear dynamic ranges spanned up to 4 orders of magnitude without the use of internal standard
• Accurate and precise quantification were achieved, and ion ratios were used to increase the specificity of the analysis
• The assigned MRL or MMPR values were achieved for all compounds analyzed.

References

1. Estelle Dubreil et al. (2017) Validation approach for a fast and simple targeted screening method for 75 antibiotics in meat and aquaculture products using LC-MS/MS, Food Additives & Contaminants: Part A, 34:4, 453-468.
2. Commission  regulation (EU) No 37/2010 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin, 22^nd December 2009.
3. EURL guidance on minimum method performance requirements (MMPRs) for specific pharmacologically active substances in specific animal matrices – September 2020.
4. EFSA - Scientific opinion on chloramphenicol in food and feed, 26^th November 2014.
5. Enabling new levels of quantification. SCIEX technical note, RUO-MKT-02-11886-A.
6. The Scheduled MRM algorithm Pro. SCIEX technical note, RUO-MKT-02-8539-A.
7. SCIEX OS software – Transforming your samples into meaningful analytical answers.
8. Commission regulation (EU) No 2019/1871, 7^th November 2019.
9. Commission implementing regulation (EU) No 2021/808, 22^nd March 2021.