Determination of salicylate in blood serum by flow injection with immobilized salicylate hydroxylase

A flow injection (FI) enzymatic system, based on the use of immobilized salicylate hydroxylase in glass beads, was developed for the determination of salicylate. Salicylate hydroxylase and nicotinamide adenine dinucleotide (NADH) are used to convert salicylate to catechol. The reaction of catechol w...

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Bibliographic Details
Published in:Journal of AOAC International 2001-09, Vol.84 (5), p.1363-1369
Main Authors: VILA, Marta M. D. C, TUBINO, Matthieu, DE OLIVEIRA, Graciliano NETO
Format: Article
Language:English
Subjects:
Analysis of complex biological substances
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Bioreactors
Biotechnology
Blood and biological fluids
Calibration
Colorimetry
Enzymes, Immobilized - chemistry
Flow Injection Analysis
Fundamental and applied biological sciences. Psychology
Humans
Hydrogen-Ion Concentration
Indicators and Reagents
Methods. Procedures. Technologies
Mixed Function Oxygenases - chemistry
NAD - analysis
Salicylates - blood
Temperature
Various methods and equipments
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Summary:A flow injection (FI) enzymatic system, based on the use of immobilized salicylate hydroxylase in glass beads, was developed for the determination of salicylate. Salicylate hydroxylase and nicotinamide adenine dinucleotide (NADH) are used to convert salicylate to catechol. The reaction of catechol with 4-aminophenol at high pH yields a colored product which is detected spectrophotometrically at 565 nm. Ten samples of human serum containing from 5.0 x 10(-4) to 5.0 x 10(-3) mol/L added salicylate were analyzed and the recovery was determined. Eight additional serum samples containing salicylate were analyzed by the Trinder test and the proposed method. The results obtained with the 2 methods showed good agreement by the statistical Student's t-test. The relative precision of the method is about 3.4% (RSD of the mean recovery). Considering the lowest concentration analyzed, the quantitative limit of detection is about 0.2 x 10(-5) mol/L (3 x SD). The volume of the sample used was 150 microL. The proposed method was also used to analyze medicines containing acetylsalicylic acid. The results were statistically compared with those obtained through the U.S. Pharmacopoeia procedure and showed excellent agreement.
ISSN:1060-3271
1944-7922
DOI:10.1093/jaoac/84.5.1363