Patulin and Hydroxymethylfurfural in Apple Juice Using In-Line SPE Cleanup and Concentration

Significance of the Topic

The accurate measurement of patulin and Hydroxymethylfurfural (HMF) in apple products is critical for food safety and quality control. Patulin, a mycotoxin produced by mold, serves as an indicator of contamination, while HMF forms during thermal processing of sugars and reflects heat-induced damage.

Objectives and Study Overview

This study aims to develop a reliable, inline solid-phase extraction (SPE) coupled with high-performance liquid chromatography (HPLC) method for simultaneous cleanup, concentration, and separation of patulin and HMF in complex apple juice and sauce matrices.

Methodology and Instrumentation

Sample Preparation:

• Apple sauce and unfiltered juice treated with pectinase, centrifuged, and filtered to remove solids.
• Inline SPE cleanup and concentration using mixed-mode sorbent for reproducible analyte retention and matrix removal.

Chromatographic Conditions:

• Analytical column: Thermo Scientific Acclaim C30, 3 µm, 2.1 × 150 mm.
• SPE column: Thermo Scientific Acclaim Mixed-Mode WCX-1, 3.0 × 50 mm.
• HPLC system: Dionex UltiMate 3000 RSLC with dual-gradient pumps and 10-port auxiliary valve for forward elution.
• Mobile phases: phosphate buffer pH 6.5/acetonitrile on left pump; water, acetonitrile, ammonium formate–formic acid buffer pH 3.1 on right pump.
• Gradient program optimized to wash out organic acids and phenolics, then elute patulin and HMF.
• Flow rate: 0.40 mL/min per pump; injection volume: 60 µL; column temperature: 25 °C.
• Detection: diode array at 276 nm, 5 Hz, 1.0 s response time.

Main Results and Discussion

The inline SPE approach effectively excluded early-eluting matrix interferences, concentrating patulin and HMF for clear baseline separation on the C30 column. Two distinct peaks corresponding to HMF and patulin were resolved within a nine-minute run time. Recovery rates exceeded 90 % for both analytes, demonstrating superior reproducibility over traditional off-line ethyl acetate extraction which underperforms for HMF.

Benefits and Practical Applications

This method offers:

• Reduced manual handling and sample preparation time.
• Improved precision and recovery, especially for HMF.
• High throughput with automated SPE–HPLC integration.
• Applicability to routine quality control in juice manufacturing and research laboratories.

Future Trends and Potential Applications

Advancements may include adaptation to additional mycotoxins or heat‐damage markers in diverse food matrices. Integration with mass spectrometry detection could enhance selectivity for multi-residue screening. Miniaturized or high-throughput SPE cartridges and rapid UHPLC gradients are expected to further reduce analysis time and solvent consumption.

Conclusion

The inline SPE–HPLC–DAD method provides a robust, reproducible, and efficient solution for simultaneous determination of patulin and HMF in apple products, meeting stringent requirements for food safety monitoring and quality assessment.