Liquid chromatographic methods for biotransformation studies of ochratoxin A

Abstract

Liquid chromatographic methods were used for the detection of ochratoxin A (OTA) and its metabolites ochratoxin alpha (OTalpha), 10-hydroxy OTA (10-OHOTA), 4R-hydroxy OTA (4R-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 OTalpha, 10-OHOTA, 4R-OHOTA and OTC, which was used as internal standard. The LOD/LOQ values for OTalpha, 4R-OHOTA and OTA were 0.63/2.11, 0.99/3.31 and 0.84/2.81 microg/L, respectively, for the HPLC-FLD method and 0.98/3.28, 1.11/3.72 and 0.88/2.96 microg/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 OTalpha increased according to ingested amounts of OTA, with OTalpha being the most abundant compound. The results obtained with the LC-MS/MS method confirmed these results.

Comparative Toxicity of Mycotoxins to Navel Orangeworm (Amyelois transitella) and Corn Earworm (Helicoverpa zea)

“…In contrast with AFB1, there are no reports of insect detoxification of OTA. Although we failed to detect metabolites of OTA produced by midgut proteins of either H. zea or A. transitella, bioactivation is involved in its genotoxicity to other organisms (Manderville 2005;Schaut et al 2008), and the absence of major pathways that produce bioactivated metabolites also may reflect adaptation of these insects to the presence of this mycotoxin in their diet.…”

Teratogenicity of Ochratoxin A and the Degradation Product, Ochratoxin α, in the Zebrafish (Danio rerio) Embryo Model of Vertebrate Development

“…The hydrolysis reaction was followed by normal-phase thin-layer chromatography (TLC) using toluene/EtOAc/90% formic acid (5:4:1) as a solvent system [ 25 ], and fluorescence under long-wave UV ( i.e. , 366 nm) for detection; the identity of OTα was subsequently confirmed by previously described HPLC-FL and LC-MS methods [ 26 , 27 ] using a commercially available analytical standard of OTα (Romer Labs, Union, MO, USA). All other chemicals and reagents were purchased from either VWR Scientific (Radnor, PA, USA), or Sigma-Aldrich (St. Louis, MO, USA).…”

Mycotoxin Biomarkers of Exposure: A Comprehensive Review

“…(), however, analyzed urine and feces of rats and chickens, and they were able to find a similar pattern of OTA metabolites after liver microsomal incubation (Yang et al., ). Hydroxylated compounds found after liver cell incubations, especially 7‐OH‐OTA, (4R)‐OH‐OTA, and (4S)‐OH‐OTA, have been scarcely studied in biological fluids (Schaut et al., ). The analyses of these hydroxylated OTA‐forms and OTB in human urine could increase the knowledge, and obtain a reliable pattern, of OTA excretion.…”