Strategies for On-Line Sample Enrichment and Confirmation of Analytes in a Complex Food Matrix

Importance of the Topic

The monitoring of pesticide residues and flavonoid markers in citrus juices is critical to ensure food safety, regulatory compliance, and product authenticity. Carbendazim, a common fungicide, and naringin, a flavanone distinguishing orange from grapefruit juice, must be quantified at low parts-per-million levels in complex matrices such as orange juice.

Objectives and Study Overview

This study aimed to develop a streamlined UPLC–MS method for simultaneous detection and confirmation of carbendazim and naringin in orange juice. Key goals included enhancing sensitivity and selectivity, applying trap-and-elute with at-column dilution for sample enrichment, and demonstrating a user-friendly workflow suitable for routine QC laboratories.

Methodology and Instrumentation

• Sample Preparation: Orange juice was centrifuged to remove pulp. Solvent and matrix calibration standards (0.0137–10 ppm) were prepared by serial dilution. Samples were mixed with mobile phases, vortexed, and filtered prior to injection.
• Instrumentation Used:
• UPLC System: ACQUITY UPLC H-Class with CM-A or 2D configuration
• Trap Column: XBridge C18 Direct Connect HP (10 µm, 2.1 × 30 mm)
• Analytical Column: ACQUITY UPLC BEH C18 (1.7 µm, 2.1 × 150 mm)
• Detector: ACQUITY QDa single quadrupole mass detector with ESI+ for carbendazim and ESI– for naringin, coupled with PDA
• Software: Empower 3 CDS
• Chromatographic Conditions: 1D isocratic analysis (61:39 aqueous/methanol, 0.4 mL/min, 60 °C), and 2D trap-and-elute with at-column dilution (dilution pump 1.8 mL/min, elution pump 0.4 mL/min).
• MS Parameters: Single ion recording (SIR) for parent and in-source fragment ions (carbendazim m/z 579.07/159.97; naringin m/z 192.04/270.83) to confirm identity via ion ratios.

Main Results and Discussion

• Linearity and Sensitivity: Solvent calibration curves achieved R2 = 0.99 for carbendazim (3.33–10 ppm) and R2 = 1.00 for naringin (0.0412–10 ppm). With trap-and-elute and at-column dilution, detection down to 0.0137 ppm (naringin) and 0.37 ppm (carbendazim) was demonstrated, both with R2 = 1.00.
• Precision and Recovery: Precision at regulatory limits (0.123 ppm naringin, 10 ppm carbendazim) was ≤3.3% RSD in matrix. Recoveries were 95.7% for naringin and 89.7% for carbendazim with larger injection volumes (25 µL) under ACD conditions.
• Matrix Effects: Overlay of solvent vs. matrix chromatograms revealed co-eluting interferences. Ion ratio confirmation distinguished true analyte peaks (carbendazim ratio ~0.6; naringin ratio ~0.9) from matrix signals.
• At-Column Dilution Benefits: ACD mitigated solvent effects from high-organic diluents, enabled pure methanol injections without loss of peak shape or resolution, and supported larger injection volumes for enhanced sensitivity.

Benefits and Practical Applications

• Simplified Workflow: The ACQUITY QDa detector delivers mass confirmation with minimal tuning, facilitating deployment in QC labs.
• Enhanced Selectivity: SIR detection and ion ratio confirmation reduce false positives in complex food matrices.
• Improved Sensitivity: Multi-dimensional chromatography with ACD achieves lower detection limits and reliable quantification.
• Versatility: The approach supports different diluents and high-throughput analysis of pesticide residues and authenticity markers.

Future Trends and Potential Applications

• Extension to Other Analytes: Applying trap-and-elute with ACD to a broader range of pesticides and natural markers in diverse food matrices.
• Automation and Online Workflows: Integration with robotic sample preparation and real-time data processing for high-throughput testing.
• Advanced Detection: Coupling with high-resolution or tandem mass spectrometry for enhanced identification certainty.
• Sustainable Practices: Exploration of green solvents and reduced solvent consumption through optimized multi-dimensional setups.

Conclusion

The combined use of single-quadrupole mass detection with trap-and-elute and at-column dilution provides a robust, sensitive, and selective method for quantifying carbendazim and naringin in orange juice. This streamlined UPLC–MS workflow meets regulatory requirements, simplifies QC operations, and can be adapted for broader applications in food safety and authenticity testing.

References

1. US Food and Drug Administration. Orange Juice Products and Carbendazim: Addendum to FDA Letter to the Juice Products Association, Washington, DC, 2012.
2. Twohig M, Kruger DA, Gledhill A, Burgess J. Adulteration in Fruit Juices: A Solution to a Common Problem. Waters Application Note No. 720004173en, 2012.
3. Direct Injection of Orange Juice for the Rapid Detection of the Fungicide Carbendazim using ACQUITY UPLC I-Class with Xevo TQ-S. Waters Technology Brief No. 720004214en, 2012.