Determination and Confirmation of Priority Pesticide Residues in Baby Food

James Morphet and Peter Hancock, Waters Corporation, Manchester, UK

AIM

To utilize the power of the Waters® UltraPerformance Liquid Chromatography (UPLC®) System combined with the fast MS acquisition rates of the Xevo™ TQ Mass Spectrometer for rapid determination and confirmation of pesticide residues in baby food.

INTRODUCTION

As the population of the world grows, it becomes increasingly important to produce enough food to satisfy the needs of its inhabitants. This escalation in consumer demand has led farmers to use increasing amounts of pesticides to improve their yields and make their operations more cost-effective. This growth in the use of pesticides, coupled with poor agricultural practices and illegal use, can pose significant risks to human health through the presence of pesticide and metabolite residues in food products.

Most countries have strict regulations that govern pesticides. Legislation imposes Maximum Residue Limits¹ (MRLs) for pesticide residues in food products requiring analytical techniques that are sensitive, selective, and robust. The EU baby food directive, 2003/13/EC² covers a list of both GC amenable and LC amenable pesticides. These prohibited pesticides have a maximum level in baby food that should not exceed 0.003 mg/kg, or not greater than a limit between 0.004-0.008 mg/kg. This is considered to be among the strictest legislation in the world.

The need to meet mandated detection limits, develop generic sample preparation techniques for complex matrices, and the desire to increase sample throughput are the main challenges that face food safety testing laboratories today. The use of a single multi-residue method per instrument dramatically improves return on investment in a laboratory by removing the need to change method parameters. This is often the case in labs that analyze a wide variety of commodities with various lists of legislated pesticides.

The following application note describes a solution for the rapid analysis of pesticides in fruit- and meat-based baby food extracts that is able to exceed both current European and worldwide legislation.

EXPERIMENTAL

A DisQuE™ (QuEChERS style dispersive solid-phase extraction) extraction was utilized for this multi-residue method as described below:

Extraction procedure³

1. Add 15 g of homogenized baby food to the 50 mL DisQuE extraction tube. Add 15 mL of 1% acetic acid in acetonitrile.

2. Shake vigorously for 1 minute and centrifuge > 1500 rcf for 1 minute.

3. Transfer 1 mL of the acetonitrile extract into the 2 mL DisQuE cleanup tube.

4. Shake for 30 seconds and centrifuge >1500 rcf for 1 minute.

5. Transfer 100 μL of final extract into an autosampler vial. Dilute with 900 μL water, mix, and inject.

Refer to Appendix 1 at the end of this document for further MS parameters.

Appendix 1. Xevo TQ MS parameters

Quanpedia: Database and method creation tool

Method creation has been streamlined with the use of Quanpedia,™ a searchable database for quantitative LC/MS method information that can be updated with user information.

Each entry in Quanpedia is populated with information that associates the compound name with details of optimal SIR/MRM acquisition methods, acceptable confirmatory ion ratios, appropriate LC methods, and expected peak retention times. For fast-paced, multi-user environments that require quality results the first time, Quanpedia offers a simple and convenient way to rapidly create complete LC/MS data and acquisition methods.

Acquisition and processing methods

These data were acquired using Waters MassLynx™ Software v. 4.1. Incorporated into MassLynx, the IntelliStart™ Software automates optimization of MS parameters for the sample and also monitors the health of the MS system, which reduces the time for operator-intensive troubleshooting and upkeep.

Figure 1. Quanpedia is a simple and easy-to-use method creation tool and MS database.

RESULTS AND DISCUSSION

The analysis of the priority pesticide residues in baby food was achieved by combining the ACQUITY UPLC System with the Xevo TQ MS System – UltraPerformance LC with tandem quadrupole mass spectrometry (UPLC/MS/MS) operated in MRM mode. This tandem quadrupole mass spectrometer offers a highly specific and selective detection technique that has become the technique of choice within the laboratory.⁴

The selectivity given using a tandem quadrupole mass spectrometer (Xevo TQ MS System) shows an advantage over a single quadrupole instrument as it allows co-eluting compounds to be identified and quantified with confidence. Figure 2 shows fensulfothion sulfone and terbufos sulfone that co-elute at 3.32 minutes. All dwell times were optimized to give approximately 12 data points across each peak.

Figure 2. Chromatogram showing all 17 pesticide residues in one injection at 1 ng/mL in water.

These data were processed using TargetLynx™ Application Manager. This quantification package from MassLynx Software enables automated data processing and reporting for quantitative data, which incorporate a range of confirmatory checks that identify samples that fall outside user-specified or regulatory thresholds.

QCMonitor: Automating your quality control

The QCMonitor™ is an automated tool that provides real-time quantitative data quality monitoring to determine whether injections meet tolerances specified by the user. QCMonitor will automatically decide if subsequent samples should be injected, or if more detailed checks are required to ensure the best use of valuable laboratory resources. Injections that fail to meet the acceptance criteria set for calibration curves, QC samples, and blanks can be re-injected or, in worst case scenarios, the batch can be skipped over, allowing for other revenue generating batches to be injected. This is especially important during overnight slots when the instrumentation is left unattended. An email facility is also available to send messages if and when deviations occur, alerting chemists to problems as soon as they arise.

Diagnosis of whether co-extracted interferants are the source of QCMonitor flags is also made easier by the ability to acquire high-sensitivity full scan data simultaneously with MRM, adding a new dimension to QC for LC/MS/MS analyses. QCMonitor can be found in the TargetLynx Software Method Editor, as shown in Figure 3.

Figure 3. The features of the TargetLynx Software Method Editor detail the parameters that can be modified within QCMonitor. Highlighted are the settings for the calibration standards, the limits that must be obeyed, and the type of actions that can be applied if the injection fails to meet the set criteria.

A calibration curve was prepared in matrix matched standards and injected. Excellent linearity was achieved using a weighting factor of 1/x with a high coefficient of determination. This is shown in Figure 4.

Figure 4. TargetLynx Software browser view showing cadusafos in fruit-based baby food extract at 0.001 mg/kg. Also highlighted are the red boxes which show ion ratios that are out of tolerance.

With TargetLynx Software as standard, the process of quantification is simpler than ever. The results browser and report generator clearly indicate when samples contain residues that are above minimum reporting levels.

The advantage of using the ACQUITY UPLC System with the Xevo TQ MS System is that ion ratio confirmation is also possible. Figures 4 and 5 show an ion ratio for disulfoton sulfone that fails to meet the criteria required. The TargetLynx Software Method Editor can be easily manipulated by the user to set pass and fail criteria for each compound with regard to ion ratio. The ability of each injection to meet these criteria is then shown with a red or a white box. This injection shows a concentration of 0.7 ng/mL (0.007 mg/kg for the sample), but the ratio between the areas of the primary and secondary MRM transition traces do not lie within the limits set by the chemist. Within the EU, ion ratio confirmation is important for pesticide analysis as documented in SANCO/2007/3131.⁵ When the mouse is positioned over this red box, a further message is displayed detailing the problem. In this instance the actual ion ratio does not lie within the limits specified by the chemist. This feature automatically performs these calculations, which allows chemists to use their time more cost-effectively and improve laboratory workflow.

Figure 5. Closeup view showing the type of user message TargetLynx Software produces to flag an out-of-range ion ratio result.

TrendPlot: Monitor long-term system health

The TrendPlot™ tool provides confirmation that the Xevo TQ MS System results generated by your laboratory are consistently of the highest quality. It is possible to choose specific injections by charting both short-term intra-batch and long-term inter-batch trends in your analytical performance. In this example, ethoprophos samples have been plotted with the outlier easily seen in Figure 6.

Figure 6. TrendPlot Tool shows outliers within a batch or performance of the instrument when data from several batches is added together.

CONCLUSION

A rapid multi-residue method was developed for the determination and confirmation of LC amenable priority pesticides. The analysis of pesticides in fruit- and meat-based baby food extracts exceeds current worldwide legislated limits.

Improved efficiency and increased sample throughput was realized through the combination of powerful UPLC and fast MS acquisition technologies. ACQUITY UPLC combined with the Xevo TQ MS offers:

• Enhanced chromatographic resolution and short analysis times.

• Incorporation of confirmatory MRM traces.

• Complies with legislative regulations such as SANCO.

• IntelliStart technology that is designed to reduce the burden of complicated operation, training new users, time-intensive troubleshooting, and upkeep.

• The compact features of the ACQUITY UPLC and Xevo TQ MS Systems will give any laboratory an advantage as it gives high-end performance with a benchtop footprint.

• Automated system setup and quality control system checks for simple access.

The benefits of this Waters UPLC/MS/MS solution for a revenue conscious laboratory can be realized through increased efficiency by analytical time savings, a decreased need for sample retesting, and increased lab productivity. Cost savings can be made by lowering the use of lab consumables with the environmental impact of solvent usage also being reduced.

The sensitivity achieved for a large number of pesticide residues in real food matrices indicates this UPLC/MS/MS method is the ideal basis for rapid analysis of pesticides in a wide range of food samples.

References

1. Commission of the European Communities EC 396/2005, OJ 2005; L70:1.

2. Commission of the European Communities EC 2003/13, L41:33-36.

3. DisQuE Dispersive Sample Preparation Kit Care and Use Manual, P/N 715001888.

4. Leandro CC, Hancock P, Fussell RJ, Keely BJ. J. Chrom A. 2007; 1144:161.

5. Method Validation and Quality Control Procedures for Pesticide Residues in Food and Feed. Document No SANCO/2007/3131.