Enzyme immobilized magnetic nanoparticles for in-line capillary electrophoresis and drug biotransformation studies: application to paracetamol.
Enzyme Immobilized Magnetic Nanoparticles (EMNPs) were injected and magnetically retained, as a microreactor, in the capillary of a capillary electrophoresis (CE) setup with UV detection. The enzyme horseradish peroxidase (HRP) was chemically immobilized onto commercially available magnetic 300 nm diameter nanoparticles. Paracetamol (acetaminophen: N-acetyl-p-aminophenol), a common analgesic drug, was used as model drug compound. The enzymatic reaction was studied in-line by CE in 12.5 mM phosphate buffer pH 7.4 containing 20 mg/ml sulfated- beta -cyclodextrin and 0.1 mM hydrogen peroxide. By means of the developed setup, the apparent Michaelis Menten constant between HRP and acetaminophen (APAP) was determined as K(m)(app) = 53+/-5 microM. This approach was found to be of interest for enzyme kinetics studies with short time resolution condition. Based on our results and from literature data, it was possible to infer that the in-line generated product was an APAP dimer. Higher enzyme immobilized beads loading in the CE setup generated the APAP dimer with two additional minor peaks likely attributed to APAP trimer and tetramer. N-acetyl-p-benzoquinone imine (NAPQI) was not generated during APAP short time migration through the in-line microreactor.
Yu
Donghui
D
Université Libre de Bruxelles, Pharmaceutical Institute, Campus Plaine CP 205/6, Boulevard du Triomphe, Brussels, Belgium.
Van Antwerpen
Pierre
P
Patris
Stéphanie
S
Blankert
Bertrand
B
Kauffmann
Jean-Michel
JM
D000082:Acetaminophen / Q000737:chemistry / Q000378:metabolism*; D001711:Biotransformation; D019075:Electrophoresis, Capillary; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D008280:Magnetics; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D013056:Spectrophotometry, Ultraviolet; D013499:Surface Properties
D004800:Enzymes, Immobilized; D000082:Acetaminophen; D006735:Horseradish Peroxidase