A survey of the occurrence of ochratoxin A in Madeira wines based on a modified QuEChERS extraction procedure combined with liquid chromatography–triple quadrupole tandem mass spectrometry

Abstract

Over the past years, to ensure food safety, the European Union (EU) has adopted specific legislation concerning the control of mycotoxins residue levels in different kinds of food. In this study, a fast, selective and sensitive reversed-phased liquid chromatography with tandem mass spectrometry (LC–MS/MS) methodology was developed and validated for quantification of ochratoxin A (OTA) in Madeira wines. Sample extraction and purification were performed with a modified QuEChERS-based (quick, easy, cheap, effective, rugged, and safe) procedure. Firstly, the homogenized samples are extracted and partitioned using an organic solvent and salt solution. Then, the supernatant is further extracted and cleaned using a dispersive solid phase extraction (dSPE) technique. Finally clear wine extracts were concentrated under vacuum to near dryness and taken up into initial mobile phase. Careful optimization of the LC–MS/MS parameters was achieved in order to attain a fast separation with the best sensitivity. The detection was carried out on a triple–quadrupole tandem mass spectrometer (MS/MS) by electrospray ionization in positive ion mode (ESI+) with multiple reaction monitoring (MRM). MS/MS conditions were optimized in order to increase selectivity, selecting the corresponding product ions (precursor-to-fragment m/z 404→239; m/z 404→m/z 358) for quantification and identification. The performance of the method was assessed and compared to the European Commission (EC) Regulations, by studying the selectivity and specificity, limit of detection (LOD), limit of quantification (LOQ), linear dynamic range, matrix effect, accuracy, precision, robustness/ruggedness and uncertainty. The validation was performed by analyzing recovery samples at four different spiked concentrations, 0.5, 1.0, 5.0 and 10.0μg/kg, with four replicates (n=4) at each concentration. Recoveries ranged from 87.2% to 102.6%, with relative standard deviations below 9% in all cases. The intra-day precision and inter-day precision, expressed as relative standard deviation, were lower than 7% and 14%, respectively. Matrix effects were observed by comparing the slope of matrix-matched standard calibration with that of solvent. Good linearity was achieved at the concentration levels of 0.50–22.5μg/L. The LOQ and LOD, 0.4 and 0.1μg/kg, respectively, at the signal-to-noise ratio (S/N) of 10 and 3, were lower than the concentration usually permitted by legislation in wines. The method was successfully applied to evaluate the occurrence of OTA in 30 Madeira wine samples. None of the analyzed samples exceeded the maximum permissible limit for wines (2.0μg/kg) set by the EC, which confirms that the risk of OTA occurrence in Madeira wines is, as expected, is very low.