ABSTRACT
An electrochemical method based on differential pulse voltammetry is presented for the determination of AZT in whole blood of fasted subjects. A protein-free supernatant of whole blood is prepared using HClO4 precipitation followed by neutralization with phosphate buffer. The AZT is reduced at a hanging mercury drop electrode. The linear dynamic range of standards in buffer is from the detection limit of 4.1 nM to 206.5 microM (1.1 to 55,200 ng/ml). However, in spiked blood samples the linear dynamic range is from 0.029 to 0.29 microM (7.75 to 77.5 ng/ml). The whole blood assay yields a recovery of 92.30 +/- 5.92% compared to the standard solution assay. After a 30-min preparation time, each sample can be analyzed in 10 min by a manual procedure.
Subject(s)
Electrochemistry/methods , Zidovudine/blood , Chemical Precipitation , Humans , Oxidation-Reduction , Perchlorates , Sensitivity and SpecificityABSTRACT
An amperometric method for alkaline phosphatase is described and compared to the most widely used spectrophotometric method. Catalytic hydrogenation of 4-nitrophenylphosphate (the substrate in the spectrophotometric method) gives 4-aminophenylphosphate (the substrate in the amperometric method). The latter substrate has the formula C6H6NO4PNa2.5H2O and a Mr of 323. The Michaelis constant for 4-aminophenylphosphate in 0.10 M, pH 9.0. Tris buffer is 56 microM, while it is 82 microM for 4-nitrophenyl phosphate. The amperometric method has a detection limit of 7 nM for the product of the enzyme reaction, which is almost 20 times better than the spectrophotometric method. Similarly, with a 15-min reaction at room temperature and in a reaction volume of 1.1 ml, 0.05 microgram/l alkaline phosphatase can be detected by electrochemistry, almost an order of magnitude better than by absorption spectrophotometry. Amperometric detection is ideally suited for small-volume and trace immunoassay.
