An isocratic fluorescence HPLC assay for the monitoring of l-asparaginase activity and l-asparagine depletion in children receiving E. colil-asparaginase for the treatment of acute lymphoblastic leukaemia

• Published in: Biomedical chromatography Vol. 23; no. 2; pp. 152 - 159
• Main Authors: Nath, Christa E., Dallapozza, Luciano, Eslick, Adam E., Misra, Ashish, Carr, Deborah, Earl, John W.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.02.2009
• Subjects: Adolescent; Antineoplastic Agents - blood; Antineoplastic Agents - metabolism; Antineoplastic Agents - therapeutic use; Asparaginase - blood; Asparaginase - metabolism; Asparaginase - therapeutic use; Asparagine - blood; Asparagine - metabolism; Aspartic Acid - blood; Aspartic Acid - metabolism; Child; Child, Preschool; Chromatography, High Pressure Liquid - methods; Drug Stability; Escherichia coli; Escherichia coli Proteins - therapeutic use; Fluorescence; Glutamic Acid - blood; Glutamic Acid - metabolism; Glutamine - analysis; Glutamine - blood; Glutamine - metabolism; Homoserine - analysis; HPLC; Humans; l-asparaginase activity; l-asparagine depletion; Least-Squares Analysis; Precursor Cell Lymphoblastic Leukemia-Lymphoma - drug therapy; Reference Standards; Reproducibility of Results; Sensitivity and Specificity
• ISSN: 0269-3879; 1099-0801
• DOI: 10.1002/bmc.1096

A novel assay for the determination of l‐asparaginase activity in human plasma is described that is based on the HPLC quantitation of l‐aspartic acid produced during enzyme incubation. Methods for monitoring l‐asparagine depletion are also described. Chromatography of l‐aspartic acid, l‐asparagine and l‐homoserine (the internal standard) involved derivatization with o‐pthaldialdehyde, then separation from other amino acids on a Phenomenex Luna C18 column using a 1 mL/min flow rate and a mobile phase consisting of di‐potassium hydrogen orthophosphate propionate buffer, pH 6, with 10% methanol and 10% acetonitrile. Fluoresence detection was at excitation/emission wavelengths of 357/455 nm. Under these conditions l‐aspartic acid, l‐asparagine and l‐homoserine had retention times of 3.5, 9.8 and 17.7 min, respectively. The l‐asparaginase assay was linear from 0.1 to 10 U/mL activity and interday precision and accuracy were less than 13%. The limit of quantitation was approximately 0.03 U/mL. The assay utility was established in 12 children who received E. coli l‐asparaginase as treatment for acute lymphoblastic leukaemia. Copyright © 2008 John Wiley & Sons, Ltd.