title: Analytical validation of a microplate reader-based method for the therapeutic drug monitoring of L-asparaginase in human serum.
abstract: The enzyme L-asparaginase (ASNASE), which hydrolyzes L-asparagine (L-Asn) to ammonia and L-aspartic acid (L-Asp), is commonly used for remission induction in acute lymphoblastic leukemia. To correlate ASNASE activity with L-Asn reduction in human serum, sensitive methods for the determination of ASNASE activity are required. Using L-aspartic beta-hydroxamate (AHA) as substrate we developed a sensitive plate reader-based method for the quantification of ASNASE derived from Escherichia coli and Erwinia chrysanthemi and of pegylated E. coli ASNASE in human serum. ASNASE hydrolyzed AHA to L-Asp and hydroxylamine, which was determined at 710 nm after condensation with 8-hydroxyquinoline and oxidation to indooxine. Measuring the indooxine formation allowed the detection of 2 x 10(-5)U ASNASE in 20 microl serum. Linearity was observed within 2.5-75 and 75-1,250 U/L with coefficients of correlation of r(2)>0.99. The coefficients of variation for intra- and interday variability for the three different ASNASE enzymes were 1.98 to 8.77 and 1.73 to 11.0%. The overall recovery was 101+/-9.92%. The coefficient of correlation for dilution linearity was determined as r(2)=0.986 for dilutions up to 1:20. This method combined with sensitive methods for the quantification of L-Asn will allow bioequivalence studies and individualized therapeutic drug monitoring of different ASNASE preparations.
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster, Albert-Schweitzer-Str. 33, 48149 Muenster, Germany. lanvers@uni-muenster.de
authors.forename: Claudia
authors.initials: C
authors.lastname: Lanvers
authors.forename: Joao Paulo
authors.initials: JP
authors.lastname: Vieira Pinheiro
authors.forename: Georg
authors.initials: G
authors.lastname: Hempel
authors.forename: Gudrun
authors.initials: G
authors.lastname: Wuerthwein
authors.forename: Joachim
authors.initials: J
authors.lastname: Boos
mesh_terms: D001215:Asparaginase / Q000097:blood* / Q000737:chemistry; D001216:Asparagine / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D002138:Calibration; D016974:Dickeya chrysanthemi / Q000201:enzymology; D016903:Drug Monitoring / Q000379:methods*; D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D016014:Linear Models; D008832:Microchemistry / Q000379:methods; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: C024848:beta-aspartylhydroxamic acid; D001216:Asparagine; D001215:Asparaginase