Combining SWATH^® Acquisition with MRM^HR for the analysis of veterinary drugs in tissue

Using the SCIEX X500R QTOF System

Jasmin Meltretter
SCIEX, Germany

Introduction

Veterinary drugs are commonly used in livestock breeding to prevent or treat infections of the animals and to ensure their optimal growth. Legal regulations define waiting periods between the application of active pharmaceutical ingredients and the release of the animals for food manufacturing. Veterinary drugs which still find their way into human nutrition represent a potential risk to human health, e.g. in terms of possible allergenic reactions or reproductive dysfunctions. Furthermore, abuse of antibiotics in animals may also contribute to the development of antimicrobial resistance.

Therefore, European guidelines require careful control of veterinary drugs residues in animal products.¹ However, the resulting sample matrix is often complex making sensitivity and selective detection of these residues more difficult.

Here a versatile and sensitive workflow is presented on the SCIEX X500R QTOF System which combines a non-targeted screening workflow using SWATH Acquisition looped with highly selective MRM^HR acquisition. Confident identification of veterinary drug residues according legal requirements² is achieved by accurate precursor and fragment mass measurement and their compound specific ion ratios, as reported in the SCIEX OS Software.

Key takeaways

• The X500R QTOF System is a flexible high resolution mass spectrometer allowing the collection of high mass accuracy, high resolution data for MS and MS/MS data with high acquisition rates (up to 100 Hz)
• Multiple scan types can be combined into a single experiment to create a very powerful workflow
  • TOF MS experiment for high resolution quantitation on most compounds
    
  • SWATH acquisition full scan MS/MS experiments for confident identification and targeted quantitation
    
  • Targeted MRM^HR experiment for high selectivity quantitation on specific compounds
• SCIEX OS Software, a robust platform for the confident detection and quantification of veterinary drugs from a tissue sample is demonstrated

Methods

Sample preparation: Liver tissue was mixed with extraction solution (acetonitrile, water, formic acid) and homogenized. Following centrifugation for 5 minutes, a 5 mL aliquot from the supernatant was concentrated under nitrogen flow. After addition of 2.5 mL of solvent A, the extract was vortexed, centrifuged and filtered prior to injection. Aliquots of the extracts were spiked with a standard solution yielding final concentrations of 0.2, 1, 5, 10, and 50 ng/mL (corresponding to 0.08, 0.4, 2, 4, and 20 µg/kg liver).

Chromatography: Veterinary drugs were chromatographically separated on a SCIEX ExionLC™ AD UHPLC System, using a Phenomenex Kinetex C18 column (150 x 2.1 mm, 2.6 μm). Chromatographic separation was achieved using the gradient in Table 1. Oven temperature was set to 30 °C. Injection volume was 5 µL.

Mass spectrometry: The SCIEX X500R QTOF system was operated in positive mode with electrospray ionization. Data acquisition was performed using TOF MS mode looped with eight SWATH MS/MS experiments and Scheduled MRM^HR experiment. Variable Q1 SWATH Acquisition was used, calculated with the SWATH Variable Window Calculator. MRM^HR experiments were acquired in fragment mode with a TOF scan window of 20 Da. Figure 2 shows the MS method as displayed in SCIEX OS Software.

Data processing: Data processing was done in SCIEX OS software 1.3.

Quantitative Results

On the SCIEX X500R QTOF System, TOF MS mode is the typical acquisition mode for quantitation, providing non-targeted data collection which can be subsequently processed in SCIEX OS Software using a list of target compounds. For the 27 analytes of interest, TOF MS mode provides excellent sensitivity in the standard solution at 1 ng/mL (Figure 3).

However, in very complex matrices such as liver extracts, interferences may hamper the sensitive detection of certain analytes. For example, the signal for azithromycin in matrix spiked at 0.2 ng/mL shows a shoulder from a matrix interference which is not chromatographically resolved, and which makes an accurate integration and thus quantitation difficult (Figure 1, left). In such a case, quantitation can be alternatively performed using the comprehensive MS/MS traces from SWATH Acquisition experiments within the same method. Using the higher selectivity provided by the fragment ion XICs, the interference observed in the TOF MS trace can be removed (Figure 1, middle). If even higher selectivity and sensitivity is needed, a true MRM^HR experiment can be included within the same experimental method which can provide even better signal-to-noise ratios (Figure 1, right). The increase of signal-to-noise performance in MRMHR is due to the use of a more narrow Q1 isolation window as well as a collision energy and declustering potential specifically tuned for the compounds of interest. This can provide both increased signal and decreased noise. In the non-targeted SWATH Acquisition experiments, more generic parameters must be used.

Qualitative Results

SCIEX OS Software displays several key parameters that allow for the confident identification of a detected compound, meeting the European Union criteria of identification points.² First, it calculates mass error of the precursor ion as well as of the fragment ions. Second, the ion ratio measured in unknown samples is compared to the one calculated from standards. Both the mass error and the ion ratio confidences are clearly displayed with a traffic light system in SCIEX OS Software results table, using a green checkmark for signals which meet the identification criteria. This allows the user to easily review large data sets and filter for positively detected compounds (Figure 4).

Typically, the ion ratio can be calculated from the area of the precursor ion and the area of one fragment. Alternatively, if the TOF MS trace is confounded by interferences, two MS/MS fragments can be used. MS/MS fragments can be taken either from the SWATH Acquisition experiment or, if higher selectivity is needed, from an MRM^HR experiment.

Conclusions

The SCIEX X500R QTOF System is a powerful instrument for the sensitive analysis of veterinary drugs in complex matrices, with a unique combination of versatile acquisition modes for different requirements:

• TOF MS data as standard trace used for quantitation.
• Concurrent acquisition of SWATH Acquisition MS/MS data, used for identification with the help of accurate fragment masses and compound specific ion ratios as required by official guidelines. Furthermore, MS/MS fragments from the SWATH Acquisition experiments can be used for quantitation, if the TOF MS traces shows interferences.
  
• Concurrent acquisition of targeted MRM^HR data in the same experiment increased selectivity for analytes which show interferences both in TOF MS and SWATH acquisition MS/MS data.
  

References

1. European Commission, Commission Decision 37/2010/EU of 22 December 2002. ABl. :L15/1-72 (2002)
2. European Commission, Commission Decision 2002/657/EC of 12 August 2002. ABl. L221, 8-36 (2002)
   

Acknowledgements

We would like to thank Anton Kaufmann from the Cantonal Laboratory of Zurich, Switzerland, who provided the liver extracts and the chromatographic method