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.
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.
Nath
Christa E
CE
Department of Biochemistry, The Children's Hospital at Westmead, Westmead, NSW, Australia. christan@chw.edu.au
Dallapozza
Luciano
L
Eslick
Adam E
AE
Misra
Ashish
A
Carr
Deborah
D
Earl
John W
JW
D000293:Adolescent; D000970:Antineoplastic Agents / Q000097:blood / Q000378:metabolism* / Q000627:therapeutic use; D001215:Asparaginase / Q000097:blood / Q000378:metabolism* / Q000627:therapeutic use; D001216:Asparagine / Q000097:blood / Q000378:metabolism*; D001224:Aspartic Acid / Q000097:blood / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D029968:Escherichia coli Proteins / Q000627:therapeutic use; D005453:Fluorescence; D018698:Glutamic Acid / Q000097:blood / Q000378:metabolism; D005973:Glutamine / Q000032:analysis / Q000097:blood / Q000378:metabolism; D006714:Homoserine / Q000032:analysis; D006801:Humans; D016018:Least-Squares Analysis; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000970:Antineoplastic Agents; D029968:Escherichia coli Proteins; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D006714:Homoserine; D001216:Asparagine; D001215:Asparaginase