Cost-efficient production of uniformly 13C-labelled ochratoxin A using 13C-labelled yeast extract

Abstract Ochratoxin A (OTA) is a nephrotoxic and potentially carcinogenic mycotoxin produced by Aspergillus and Penicillium species, with frequent occurrence in food commodities such as cereals, coffee, and grapes. Stable isotope labelling is a powerful tool for both accurate quantification and metabolite identification, yet no published methodology exists for preparative-scale production of labelled OTA. This study presents a practical and cost-efficient approach for producing uniformly 13 C-labelled OTA using an in-house prepared 13 C-labelled yeast extract together with 13 C-glucose as carbon source for Aspergillus steynii on a modified liquid Czapek yeast autolysate (CYA) medium. Based on optimisation steps, fully labelled yeast extract was first produced by cultivating Saccharomyces cerevisiae on a modified yeast nitrogen base (YNB) containing 13 C-glucose. This extract was then used in OTA production, resulting in a high yield (28.2 mg/200 ml) of 13 C-OTA. The toxin was purified by liquid–liquid extraction, solid phase extraction and preparative HPLC, yielding 18 mg of OTA with near 100% purity and 99% 13 C enrichment, as confirmed by high-resolution mass spectrometry. A total of 16 g of 13 C-glucose was used for yeast extract and OTA production, corresponding to approximately 1.1 mg of purified 13 C-OTA per gram of labelled glucose. The overall recovery was comparable to that of non-labelled OTA produced under identical conditions. This work represents the first reported protocol for producing uniformly 13 C-labelled yeast extract and 13 C-labelled OTA. The resulting 13 C-OTA is suitable for use as an internal standard in liquid chromatography-mass spectrometry analyses and as a tracer for metabolic studies, which should enable accurate OTA measurements, as well as identifying its masked derivatives in complex biological systems.