Analytical validation of a microplate reader-based method for the therapeutic drug monitoring of l-asparaginase in human serum
2002; Elsevier BV; Volume: 309; Issue: 1 Linguagem: Inglês
10.1016/s0003-2697(02)00232-4
ISSN1096-0309
AutoresClaudia Lanvers, João Paulo Vieira Pinheiro, Georg Hempel, Gudrun Wuerthwein, Joachim Boos,
Tópico(s)Childhood Cancer Survivors' Quality of Life
ResumoThe 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 β-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 2×10−5 U ASNASE in 20μl serum. Linearity was observed within 2.5–75 and 75–1250 U/L with coefficients of correlation of r2>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 r2=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.
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