Determination of Ochratoxin A in Roasted Coffee According to DIN EN 14132

Determination of Ochratoxin A in Roasted Coffee According to DIN EN 14132

Significance of the Topic

Ochratoxin A is a potent nephrotoxic and carcinogenic mycotoxin frequently found in roasted coffee. Regulatory bodies enforce strict limits to protect consumer health. Implementing robust analytical methods ensures compliance and maintains product safety.

Objectives and Study Overview

This study validates a fluorescence-based HPLC method aligned with DIN EN 14132 to quantify ochratoxin A in roasted coffee. It assesses method performance across linearity, precision, accuracy, detection limits, and demonstrates applications on real samples. Additionally, system optimization explores solvent-saving columns and faster throughput.

Methodology

Coffee samples were extracted with methanol/sodium hydrogen carbonate, centrifuged, and subjected to cleanup using phenyl silane and immunoaffinity columns. The eluted extracts were concentrated, reconstituted in methanol/water/acetic acid, and injected into the HPLC system. Calibration standards ranged from 156.25 ng/L to 20 µg/L.

Instrumentation Used

• HPLC: Agilent 1260 Infinity LC with binary pump, degasser, thermostatted column compartment, and fluorescence detector
• Autosampler with temperature control
• Software: Agilent OpenLAB CDS ChemStation
• Columns: ZORBAX Eclipse Plus C18 (4.6×150 mm, 5 µm), Poroshell 120 EC-C18 (3.0×150 mm, 2.7 µm and 3.0×50 mm, 2.7 µm)
• Mobile phases: water and acetonitrile, each with 1% acetic acid

Results and Discussion

Calibration was highly linear (R2=1.00000). On the standard column, LOD and LOQ were 39 ng/L and 100 ng/L, with retention time RSD 0.27% and area RSD 0.39%. Accuracy was 96.5% at 8 µg/L and no carryover was detected. Use of the Poroshell column improved LOD to 14 ng/L, LOQ to 70 ng/L, enhanced peak shape, and reduced solvent use by 57%. Analysis of a commercial roast yielded 0.130 µg/kg, well below the 5 µg/kg limit. Shorter column formats and increased flow rates enabled run times as low as 1.5 minutes, increasing throughput three-fold.

Benefits and Practical Applications

The validated method offers high sensitivity and precision, meets regulatory requirements, and adapts to different throughput needs. Reduced solvent consumption and rapid analysis make it suitable for quality control laboratories in the coffee industry.

Future Trends and Applications

Advances may include coupling with mass spectrometry for confirmatory analysis, further miniaturization for ultra-fast chromatography, integration with automated sample preparation, and development of green analytical protocols.

Conclusion

The described HPLC-FLD method reliably quantifies ochratoxin A in roasted coffee according to DIN EN 14132. It combines excellent linearity, precision, and sensitivity with flexible throughput options, supporting regulatory compliance and efficient laboratory workflows.

References

1. Wikipedia contributors. Ochratoxin. Wikipedia.
2. Commission Regulation (EU) No 594/2012.
3. DIN EN 14132:2009.
4. DIN ISO 20481:2008.
5. Naegele E. Determination of Caffeine in Coffee Products According to DIN 20481.
6. DIN 10767:1992.
7. Naegele E. Determination of Chlorogenic Acid in Coffee Products According to DIN 10767.
8. DIN 10779:2011.
9. Naegele E. Determination of Methylcafestol in Roasted Coffee Products According to DIN 10779.