HPLC‐MS/MS Method for Monitoring of L‐Asparaginase Activity by Using Asparagine and Aspartic Acid Plasma Levels

The effectiveness of L-asparaginase and therefore the effectiveness of acute lymphoblastic leukemia treatment will be understood by determining the substrates of the enzyme, L-asparagine and L-glutamine. For this purpose, the high-performance liquid chromatography-tandem mass spectrometry method was developed and validated by analyzing the L-asparaginase substrates asparagine and glutamine and its products aspartic acid and glutamic acid from plasma. Acetonitrile and ammonium acetate were used at 0.4 mL/min in gradient mobile phase flow using a HILIC column for chromatographic separations. The linear amino acid range was found to be 500-5000 ng/mL for asparagine, aspartic acid, and glutamic acid, and 5-50 μg/mL for glutamine, respectively. Detection limit and quantitation limit were found to be 100-500 ng/mL for asparagine, aspartic acid, and glutamic acid, and 1-5 μg/mL for glutamine, respectively. The validated method has been successfully applied to plasma samples. The method was found to be selective and reproducible.