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
✍ Scribed by Christa E. Nath; Luciano Dallapozza; Adam E. Eslick; Ashish Misra; Deborah Carr; John W. Earl
- Publisher
- John Wiley and Sons
- Year
- 2009
- Tongue
- English
- Weight
- 358 KB
- Volume
- 23
- Category
- Article
- ISSN
- 0269-3879
- DOI
- 10.1002/bmc.1096
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✦ Synopsis
Abstract
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 C~18~ 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.