title: Development of a new LC-MS/MS based enzyme activity assay for recombinant urate oxidase in plasma and its application to pharmacokinetics in human.
abstract: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of recombinant urate oxidase in human plasma. This assay was based on the determination of enzyme reaction product, (15)N-allantoin, and phenacetin was used as an internal standard (IS). Separation was achieved on a C18 column by the mobile phase of 30% water (containing 0.5% formic acid) and 70% methanol. Quantification was done using multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 161 → m/z 118 for (15)N-allantoin and m/z 180 → m/z 110.1 for IS at positive ionization mode. The calibration curve was established over the range of 2.077-42.06 U/l and the correlation coefficient was larger than 0.99. The intra-day and inter-day relative standard deviations were less than 10.6%. Accuracy determined at three concentrations ranged between 98.6% and 109.2%. This method was successfully applied to a pharmacokinetic study of intravenous recombinant urate oxidase produced from Escherichia coli in Chinese healthy volunteers.
authors.affiliation: Research Institute of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Central South University, 410013 Changsha, Hunan, PR China.
authors.forename: Xi
authors.initials: X
authors.lastname: Luo
authors.forename: Ning Fang
authors.initials: NF
authors.lastname: Cai
authors.forename: Ze Neng
authors.initials: ZN
authors.lastname: Cheng
mesh_terms: D002138:Calibration; D002681:China; D002853:Chromatography, Liquid / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D006801:Humans; D007262:Infusions, Intravenous; D008297:Male; D011994:Recombinant Proteins / Q000097:blood; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014503:Urate Oxidase / Q000097:blood* / Q000302:isolation & purification; D055815:Young Adult
chemical_list: D011994:Recombinant Proteins; D014503:Urate Oxidase