Rapid, sensitive, and easy UHPLC-MS/MS analysis of fungicides in fruit juices with QuEChERS

Significance of the Topic

Fungicide residues such as carbendazim, thiabendazole, imazalil, fenbuconazole, and difenoconazole may persist in commercial fruit juices and occasionally exceed regulatory maximum residue limits. Rapid, sensitive, and cost-effective analytical methods are essential for routine monitoring to ensure food safety, comply with international standards, and protect consumers.

Goals and Overview of the Study

This study aimed to develop a rapid UHPLC-MS/MS assay for quantitation of key fungicides in orange and apple juices. Objectives included:

• Achieve a separation gradient of under four minutes
• Attain a lower limit of quantification (LLOQ) of 1 ng/mL without solid-phase extraction (SPE)
• Ensure recoveries between 70 % and 120 % and relative standard deviations (RSD) below 20 % in line with SANTE guidelines
• Simplify sample preparation using only a QuEChERS protocol

Methodology and Instrumentation

Sample Preparation:

• QuEChERS extraction: 15 mL juice + 15 mL acetonitrile with salting-out reagents
• Dispersive SPE cleanup in 15 mL tubes (magnesium sulfate, PSA, C18) to remove matrix interferences
• Dilution of extract with 95:5:0.1 water/methanol/ammonia to optimize chromatographic peak shape

Instrumentation:

• UHPLC: Vanquish Flex Binary system with Hypersil GOLD VANQUISH C18 column (50 × 2.1 mm, 1.9 µm)
• MS/MS: TSQ Endura triple quadrupole mass spectrometer with heated electrospray ionization (HESI) in positive mode, ultrafast selected reaction monitoring (500 SRM/s)
• Software: Chromeleon 7.2 for instrument control, data acquisition, and compliance-ready reporting

Main Results and Discussion

Method Optimization:

• Injection volume reduced to 20 µL to avoid peak tailing of early-eluting analytes
• Sub-4 minute gradient achieved (90:10 to 5:95 water–formic acid/methanol–formic acid)
• Retention times reproducible (RSD 0.05–0.16 %) across 22 injections

Calibration and Linearity:

• Matrix-matched calibration from 1 to 100 ng/mL displayed linearity coefficients (R²) > 0.997
• Accuracy within 80–120 % of true values across the range

Quality Control and Recovery:

• QC levels at 3, 40, and 80 ng/mL: accuracy 86.9–108.7 %, precision RSD 1.2–9.4 %
• Recovery in orange and apple juices ranged 80–92 % with RSD < 12 %

Matrix Effects:

• Orange juice exhibited up to 32 % signal suppression for thiabendazole; apple juice suppression < 12 %
• Dilution strategy minimized matrix impact on quantitation

Detection Limits:

• Method detection limits below 0.5 ng/mL for all fungicides

Benefits and Practical Applications

Key advantages of the developed approach include:

• High throughput: sub-4 minute runs and minimal sample preparation
• Cost efficiency: elimination of SPE and evaporative concentration steps
• Regulatory compliance: meets SANTE 11945/2015 guidelines for pesticide residues
• Wide applicability: suitable for routine monitoring in food safety laboratories and QA/QC environments

Future Trends and Potential Applications

Further developments may involve:

• Extension to additional pesticide classes and more complex matrices
• Automation of QuEChERS workflows and on-line sample cleanup
• Integration with high-resolution MS for non-targeted screening
• Miniaturization and ambient ionization techniques for field-based testing

Conclusion

The described UHPLC-MS/MS method using a simplified QuEChERS protocol provides rapid, sensitive, and reliable quantitation of multiple fungicides in fruit juices at low-nanogram-per-milliliter levels. It streamlines sample preparation, reduces cost per sample, and adheres to strict regulatory performance criteria, making it an effective tool for food safety monitoring.

References

1. European Commission SANTE/11945/2015. Guidance Document on Analytical Quality Control and Method Validation Procedures for Pesticide Residue Analysis in Food and Feed, 2015.
2. Hollosi L., Bousova K., Ates E., Mittendorf K. Determination of Carbendazim and Benomyl Residues in Oranges and Orange Juice by Automated Online Sample Preparation Using TLX-LC-MS/MS. Thermo Fisher Scientific Application Note AN52252, 2012.
3. Standardization Administration of China. GB 14870-1994: Maximum Residue Limits of Carbendazim in Foods, 1994.
4. Fernandes V.C., Domingues V.F., Mateus N. Determination of Pesticides in Fruit and Fruit Juices by Chromatographic Methods. J. Chromatogr. Sci. 2011, 49, 715–730.
5. Xiongfeng H., Qun X., Rohrer J. Determination of Carbendazim in Orange Juice. Thermo Fisher Scientific Application Note AN1067, 2016.
6. Dioumaeva I. LC/MS/MS of Fungicides and Metabolites in Orange Juice with Agilent Bond Elut™ Plexa™ and Poroshell™ 120. Agilent Application Note 5991-0051EN, 2012.
7. Young M.S., van Tran K., Shia J.C., Burgess J.A., Mullen L., Fountain K.J. QuEChERS Sample Preparation for LC/MS and LC/UV Determination of Carbendazim and Other Conazole Fungicides in Orange Juice. Waters Application Note 720004457EN, 2012.