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Liquid chromatographic methods for biotransformation studies of ochratoxin A
Author(s) -
Schaut A.,
Saeger S. De,
Sergent T.,
Schneider Y.J.,
Larondelle Y.,
Pussemier L.,
Blank R.,
Peteghem C. Van
Publication year - 2008
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1021
Subject(s) - chromatography , ochratoxin a , chemistry , high performance liquid chromatography , ochratoxin , mass spectrometry , tandem mass spectrometry , liquid chromatography–mass spectrometry , mycotoxin , food science
Liquid chromatographic methods were used for the detection of ochratoxin A (OTA) and its metabolites ochratoxin alpha (OT α ), 10‐hydroxy OTA (10‐OHOTA), 4 R ‐hydroxy OTA (4 R ‐OHOTA) and the ethyl ester of OTA (OTC) in in vitro samples, obtained with Caco‐2 cell culture experiments and in in vivo urine samples from sheep. A high‐performance liquid chromatography method with fluorescence detection (HPLC‐FLD) and a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method were developed and validated for the detection of OTA and its metabolites OT α , 10‐OHOTA, 4 R ‐OHOTA and OTC, which was used as internal standard. The LOD/LOQ values for OT α , 4 R ‐OHOTA and OTA were 0.63/2.11, 0.99/3.31 and 0.84/2.81 µg/L, respectively, for the HPLC‐FLD method and 0.98/3.28, 1.11/3.72 and 0.88/2.96 µg/L, respectively for the LC‐MS/MS method. Within‐day and between‐day precision were both <12% for the HPLC‐FLD method, and <10% for the LC‐MS/MS method. The recovery of OTA and its metabolites ranged between 71 and 111% for the HPLC‐FLD method and between 79 and 110 % for the LC‐MS/MS method. In the first experiment only OTA was added to the Caco‐2 cells while in the second experiment 3‐methylcholanthrene (3MC) was also present in the cell culture systems. Besides OTA, which was recovered in all the samples, an unknown compound was also observed in the second experiment. When 3MC was added, the results showed that the OTA concentration in the basolateral samples was decreased by 50%. The methods were also implemented for the analysis of urine samples of sheep, fed increasing amounts of OTA. With the HPLC‐FLD method it could be concluded that the concentration of OTA and OT α increased according to ingested amounts of OTA, with OT α being the most abundant compound. The results obtained with the LC‐MS/MS method confirmed these results. Copyright © 2008 John Wiley & Sons, Ltd.