Application of ACQUITY TQD for the Analysis of Pesticide Residues in Baby Food

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OVERVIEW
This application note assesses the suitability of the Waters® ACQUITY™ TQD for tandem quadrupole-based analysis of pesticide residue in baby food. Polarity switching, differing dwell times and ion ratio robustness will also be assessed.

INTRODUCTION
The European Union residue monitoring program, 2005-2007, establishes the need to cover 55 active ingredients in various foods, including baby foods1,2. Twenty of these pesticides are suitable for multi-residue LC/MS analysis; only one has a negative polarity in electrospray mode, normally requiring two injections (one in each polarity ion mode). Consequently compounds with negative polarity are often excluded from monitoring programs.

Ideally, these should be determined in a single analysis with polarity switching.
Chemists analyzing pesticide residues are under increasing pressure to broaden the range of pesticides determined in a single analysis: to improve limits of detection, precision and quantitation; to increase confidence in the validity of residue data; to provide faster methods and to reduce usage of hazardous solvents while maintaining or reducing costs. In order to meet these demanding requirements, the scope, sensitivity, efficiency and speed of multiresidue methods of analysis must be improved.

The introduction of the ACQUITY TQD featuring the TQ detector (Figure 1) allows scientists to perform UPLC® analysis of pesticides while harnessing all the benefits that this new tandem quadrupole instrument brings to the laboratory. With IntelliStart™ technology, the instrument is designed to remove the burden of complicated operation, time-intensive troubleshooting and upkeep. Its small footprint will give any laboratory an advantage as this powerful tool removes the need for larger instrumentation.

EXPERIMENTAL
The sample extraction method has been previously reported3. Extracts of blank baby food matrix in acetonitrile were provided along with mixtures of the compounds in acetonitrile by the Central
Science Laboratory (CSL), York, UK. Extracts for injection were prepared by spiking the compounds into the baby food matrix. The supernatant was analyzed on the ACQUITY TQD following dilution with water (1:9) v/v.

UPLC conditions

LC system:     Waters ACQUITY UPLC® system
Column:      ACQUITY UPLC BEH C18 column
     2.1 x 50 mm, 1.7 ?m
     Column temp.: 40 °C
Flow rate:      600 mL/min
Mobile phase A:      Water
Mobile phase B:      Methanol
Gradient:      Time 0 min 90% A
     Time 4 min 100% B
     Time 5 min 100% B
Total run time:      7 min
Injection volume:      20 mL

MS conditions

MS system:      Waters TQ detector
Ionization mode:      ESI positive and negative
Switching time:      0.02 s
Capillary voltage:      2500 V
Cone voltage:      35 V
Desolvation gas:      Nitrogen, 800 L/Hr, 400 ?C
Cone gas:     Nitrogen, 50 L/Hr
Source temp.:      140 °C
Acquisition:      Multiple Reaction Monitoring (MRM)
Collision gas:      Argon at 4.0 x 10-3 mBar

The ACQUITY TQD was tuned so that the precursor and product ions were resolved with a peak width at half height of less than 0.7 Da.

Acquisition and processing methods
Waters MassLynx™ software v4.1 was used for acquisition and its TargetLynx™ application manager used for data processing.

RESULTS AND DISCUSSION
All compounds were separated successfully. Figure 2 shows the Total Ion Chromatogram (TIC) for all compounds.

The polarity switching capability of the ACQUITY TQD is demonstrated when comparing two co-eluting compounds, lenacil (negative ion compound) and phorate sulfone (positive ion compound). Figure 3 shows the chromatography achieved with 12 data points over each peak. Both compounds show good linearity and give good correlation coefficients (r2), illustrated in Figure 4, while using the 20 millisecond inter-scan delay for polarity switching.

The ACQUITY TQD has a greater dynamic range than 0.0005 to 0.5 mg/mL, a good practical working range for quantitation that is equivalent to 3 orders of magnitude, as illustrated in Figure 5.
Here, the range is extended from 0.0005 mg/mL to 2.5 mg/mL, equivalent to 4.5 orders of magnitude. This wider range shows the instrument can perform beyond the usual range of quantification.

The ACQUITY TQD was tested over a 100 injection sequence to assess its robustness to matrix. This 12-hour batch analysis contained a series of five matrix-matched standards, between 0.005 and 0.250 mg/mL, delivering approximately 200 mg of matrix. The results for three compounds are illustrated in Figure 6, where the peak area/concentration ratio is plotted against injection number.

All three compounds display excellent percent relative standard deviations (%RSD) over the injection sequence indicating good instrument robustness.

The ACQUITY TQD's travelling wave collision cell was tested for use at short dwell times for the multi residue method. Figure 7 shows methiocarb in fruit-based baby food at a concentration of 0.05 mg/mL. The sample was injected seven times in succession using the same MRM transition, at differing dwell times from 5 to 500 milliseconds. The data shows that the signal intensity remains consistent over the range of dwell times. As the dwell time decreases the number of points per peak increases from eight points for the dwell time of 500 milliseconds to 220 points for the shortest dwell time of five milliseconds.

TargetLynx was used to provide automatic quantification and confirmation with two MRM transitions processed for each residue.
The browser produced by TargetLynx for thiabendazole at a spiked concentration of 0.01 mg/mL in fruit-based baby food is illustrated in Figure 8.

All residues could be screened and confirmed to a concentration of 0.01 mg/mL.
Two MRM transitions were monitored for each compound. The primary transition is used for quantification and the secondary transition is used for conformation purposes.
Confirmation is achieved by calculating the ion ratio between the primary and secondary transition. All other injections must have a ratio that lies within 20 percent of the standard for them to be positively confirmed by this technique. Figure 9 shows the ion ratios of three example compounds at concentrations between 0.005 and 0.250 ?g/mL in baby food over 100 injections. All injections lie within the ±20% boundary required for confirmation with the average difference shown in the header of the graph.

CONCLUSION
A fast and simple UPLC method involving polarity switching of MRM transitions has been successfully transferred to the ACQUITY TQD for the determination of 52 pesticides. Of these, 21 pesticides and seven metabolites are included in the EU residue monitoring program, 2005-2007.

The ACQUITY TQD was capable of very fast polarity switching, allowing the analysis of positive and negative compounds in a single injection.

The use of very short dwell times of 5 ms was found to have no effect on signal intensity, indicating that sensitivity can be maintained as the number of residues is increased.

Robustness of the ACQUITY TQD was proven with a 100 injection sequence after delivery of approximately 200 mg of matrix to the instrument.

Confirmation was achieved using a secondary MRM transition over a 100 injection sequence, where the variance was less than 20 percent in all cases.

Acknowledgements
The authors would like to thank Richard Fussell at the Central Science Laboratory (CSL), Sand Hutton, York, UK for kindly supplying the sample extracts that were analyzed in this project.

References
1.     AGROW Magazine 468 (2005) 9.
2.      C.C. Leandro, P. Hancock, R.J. Fussell, B.J. Keely, J. Chromatogr. A 1144 (2007) 161-169. doi: 10.1016/j.chroma.2007.01.030.
3.      Determination of Pesticides in Food using UPLC with Polarity Switching Tandem Quadrupole LC/MS/MS, Waters Application Note 720001995EN.