Methods for analysis of citrinin in human blood and urine

Citrinin (CIT), produced by several Penicillium, Aspergillus, and Monascus species, has been detected as contaminant in feeds, grains, and other food commodities. CIT can co-occur with ochratoxin A (OTA), a mycotoxin also known for its nephrotoxicity, and this raises concern regarding possible combined effects. But, in contrast to OTA, data on CIT contamination in foods for human consumption are scarce, and CIT biomonitoring has not been conducted so far due a lack of suitable methods for human specimen. Thus, it was the aim of the present study to develop sensitive methods for the analysis of CIT in human blood and urine to investigate human exposure. To this end, we assessed different methods of sample preparation and instrumental analysis for these matrices. Clean-up of blood plasma by protein precipitation followed by LC-MS/MS-based analysis allowed robust detection of CIT (LOD 0.07 ng/mL, LOQ 0.15 ng/mL). For urine, sample clean-up by an immunoaffinity column (CitriTest(®)) proved to be clearly superior to SPE with RP(18) material for subsequent analysis by LC-MS/MS. For CIT and its metabolite dihydrocitrinone (HO-CIT), the LOD and LOQ determined by external calibration curves in matrix were 0.02 and 0.05 ng/mL for CIT, and those for HO-CIT were 0.05 and 0.1 ng/mL urine. The newly developed method was applied in a small pilot study: CIT was present in all plasma samples from 8 German adults, at concentrations ranging from 0.11 to 0.26 ng/mL. The molar (nM) concentrations of CIT are similar to those measured for OTA in these samples as a result of dietary mycotoxin intake. CIT was detected in 8/10 urines (from 4 adults and 6 infants) in a range of 0.16-0.79 ng/mL, and HO-CIT was present in 5/10 samples at similar concentrations. Thus, CIT is excreted in urine as parent compound and also as metabolite. These first results in humans point to the need for further studies on CIT exposure.