Analytical validation of a microplate reader-based method for the therapeutic drug monitoring of L-asparaginase in human serum

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, sensitise 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 Eseherichia 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 mul serum. Linearity was observed within 2.5-75 and 75-1250 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 Lis r(2) = 0.986 for dilutions up to 1:20. This method combined with sensitive methods for the quantification Of L-Asn will allots bioequivalence studies and individualized therapeutic drug monitoring of different ASNASE preparations. (C) 2002 Elsevier Science (USA). All rights reserved.