We report the improvement of a method for the detection of ochratoxin A (OTA) and its thermal degradation product 2'R-ochratoxin A in dried blood spots (DBS) by high performance liquid chromatographic (HPLC) tandem mass spectrometry (MS/MS). The DBS technique was advanced for the analysis of these two compounds in DBS with unknown amounts of blood as well as varying hematocrit values. Furthermore the comparability of venous vs. capillary blood was investigated. Human whole blood samples were spotted, dried, and extracted with a solvent consisting of acetone, acetonitrile and water for analysis by HPLC-MS/MS. Quantification was carried out by stable isotope labelled internal standards. Blood samples of volunteers (n=50) were used to further optimize and simplify the procedure. Ochratoxin A and 2'R-ochratoxin A concentrations found in the entire spots (approx. 100 μL blood) were compared with punched DBS discs of 8.8mm size containing approximately 20 μL blood. As a result the amounts of both toxins in a punched 8.8mm disc correlate well with the entire DBS. Also the use of capillary blood from finger-pricks versus venous blood was evaluated. The analyte levels correlate as well indicating that the less invasive finger-prick sampling gives also reliable results. The influence of hematocrit was investigated in a range of 25-55% according to the hematocrit in the used real blood samples (34-46% hematocrit). However no significant hematocrit effect was observed for the utilized real blood samples. Moreover different blood volumes were spotted and punched as a minimal spot size is usually recommended for accurate analysis. In this experiment finger-prick samples typically consist of about 90 μL blood. Therefore spots of 75, 100 and 125 μL blood were prepared and analyzed. Similar to the hematocrit effect, no considerable influence was observed.
Keywords: Biomonitoring; Dried blood spot; Hematocrit; Metabolism; Mycotoxin; Ochratoxin.
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