Direct monitoring of ochratoxin A in cheese with solid-phase microextraction coupled to liquid chromatography-tandem mass spectrometry.

An in situ application of solid-phase microextraction (SPME) as a sampling and sample preparation method coupled to HPLC-MS/MS for direct monitoring of ochratoxin A (OTA) distribution at different locations in a single cheese piece is proposed. To be suited to the acidic analyte, the extraction phase (carbon-tape SPME fiber) was acidified with aqueous solution of HCl at pH 2, instead of the traditional sample pre-treatment with acids before SPME sampling. For calibration, kinetic on-fiber-standardization was used, which allowed the use of short sampling time (20 min) and accurate quantification of the OTA in the semi-solid cheese sample. In addition, the traditional kinetic calibration that used deuterated compounds as standards was extended to use a non-deuterated analogue ochratoxin B (OTB) as the standard of the analyte OTA, which was supported by both theoretical discussion and experimental verification. Finally, the miniaturized SPME fiber was adopted so that the concentration distribution of OTA in a small-sized cheese piece could be directly probed. The detection limit of the resulting SPME method in semi-solid gel was 1.5 ng/mL and the linear range was 3.5-500 ng/mL. The SPME-LC-MS/MS method showed good precision (RSD: approximately 10%) and accuracy (relative recovery: 93%) in the gel model. The direct cheese analysis showed comparable accuracy and precision to the established liquid extraction. As a result, the developed in situ SPME-LC-MS/MS method was sensitive, simple, accurate and applicable for the analysis of complicated lipid-rich samples such as cheese.