Determination of drugs in biosamples at picomolar concentrations using competitive ELISA with electrochemical detection: application to steroids.
A competitive ELISA with electrochemical detection in a flow injection system (FIA) has been developed for determinations of the steroid drug budesonide in biological samples. Plasma samples were cleaned from interfering and cross-reacting compounds by two pretreatment steps consisting of a solid-phase extraction and a liquid chromatography fractionation. The enzyme label was alkaline phosphatase, which was used with p-aminophenyl phosphate (PAPP) as a substrate. The product, p-aminophenol, was detected electrochemically at a glassy carbon electrode at 250 mV (vs Ag/AgCl). The limited stability of both the substrate and the product influenced the performance of the method and had to be taken into account in the procedure by a normalization with time. Budesonide could be quantified in plasma samples down to 10 pM. The major sensitivity-limiting factor was the amperometric background response, probably due to spontaneous hydrolysis of PAPP to p-aminophenol.
Department of Analytical Chemistry, University of Lund, Sweden.
K
K
Kronkvist
U
U
Lövgren
L E
LE
Edholm
G
G
Johansson
D000469:Alkaline Phosphatase / Q000378:metabolism; D000627:Aminophenols / Q000032:analysis; D000814:Aniline Compounds / Q000378:metabolism; D019819:Budesonide; D002853:Chromatography, Liquid; D003429:Cross Reactions; D004563:Electrochemistry; D004566:Electrodes; D004797:Enzyme-Linked Immunosorbent Assay; D017022:Flow Injection Analysis; D005938:Glucocorticoids / Q000097:blood*; D006801:Humans; D009943:Organophosphorus Compounds / Q000378:metabolism; D011282:Pregnenediones / Q000097:blood*; D012680:Sensitivity and Specificity
D000627:Aminophenols; D000814:Aniline Compounds; D005938:Glucocorticoids; D009943:Organophosphorus Compounds; D011282:Pregnenediones; D019819:Budesonide; C068798:4-aminophenylphosphate; D000469:Alkaline Phosphatase