Coupling immobilized enzymes flow reactors with supercritical fluid extraction for analytical purposes

Two immobilized enzyme reactors (IMERs), working under supercritical fluid extraction (SFE) conditions, are described for the determination of hydrogen peroxide levels in oil samples and cholesterol concentration in food samples. The first enzymatic system is based on the oxidative coupling of 4-aminoantypirine–2-chlorophenol in the presence of horseradish peroxidase (HRP) and hydrogen peroxides to form a quinoid dye. The second enzymatic system consists of a mixture of enzymes, HRP and cholesterol oxidase (COD), which in the presence of oxygen transforms cholesterol to 4-cholesten-3-one. Both enzymes did not show significant differences in working lifetime when they were used in the overall analytical procedure for nine days. For hydrogen peroxide in oil samples, the limit of detection was about 0.78 μg ml−1, whereas a value of 1.04 μg ml−1 was obtained for cholesterol in fat samples. This reaction-extraction combination was developed at 172 bar and 35 °C over 25 min for the HRP system and at 210 bar and 35 °C over 30 min for the HRP–COD-reactor. The HRP enzymatic reaction-SFE method exhibits a good relationship for the hydrogen peroxide determination up to 200 μg ml−1, whereas this range was 150 μg ml−1 for the determination of cholesterol by the HRP–COD method. These methods were applied for the determination of the corresponding analytes in edible oils and fat-containing samples.