Determination of ochratoxins in nuts and grain samples by in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry.

A simple and sensitive method for the determination of ochratoxins A and B in nuts and grain samples was developed using an automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS). Ochratoxins were separated within 5 min by high-performance liquid chromatography using an Inertsil ODS-3 column with 5mM anmonium acetate/acetonitrile (65/35, v/v) as the mobile phase. Electrospray ionization conditions in the positive ion mode were optimized for mass spectrometric detection of ochratoxins. The pseudo molecular ion M+H was used to detect ochratoxins with selected ion monitoring (SIM) mode. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 μL of sample using a Carboxen-1006 PLOT capillary column as an extraction device. The extracted ochratoxins were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME/LC-MS with SIM method, good linearities of the calibration curves (r=0.9993 for ochratoxin A and r=0.9989 for ochratoxin B) were obtained in the concentration range from 0.5 to 20 ng/mL. The detection limits (S/N=3) for ochratoxins A and B were 92 and 89 pg/mL, respectively. The in-tube SPME method showed above 15-19-fold greater sensitivity than the direct injection method (10 μL injection). The within-day and between-day precisions (relative standard deviations) were below 5.1% and 7.7% (n=6), respectively. This method was applied successfully to analysis of nuts and grain samples without interference peaks. The recoveries of ochratoxins spiked into extraction solution from nut samples were above 88%. Ochratoxins were detected at 0.7-8.8 ng/g levels in various nuts and grain samples.