A Novel Liquid Chromatography-Tandem Mass Spectrometry Method for Monitoring Plasma l-Asparaginase Activity in Pediatric Patients With Acute Lymphoblastic Leukemia Receiving Pegaspargase Treatment.

Background: Acute lymphoblastic leukemia (ALL) is the predominant malignancy in pediatric patients. As a crucial constituent of ALL chemotherapy, l-asparaginase is recognized as an integral element of treatment with a threshold concentration of 0.1 IU/mL used in treatment protocols. This study presents a novel liquid chromatography-tandem mass spectrometry method for evaluating plasma l-asparaginase activity in pediatric patients with ALL undergoing pegaspargase therapy.

Methods: Initially, an enzyme incubation was conducted using 20 μL of plasma and 100 μL of l-asparagine (0.1 mol/L) at 37°C for 15 minutes. The reaction was stopped by adding sulfosalicylic acid and methanol. After the addition of isotope internal standard l-aspartic 13C4 acid, subsequent centrifugation, and dilution, the plasma samples underwent analysis by liquid chromatography-tandem mass spectrometry on a hydrophilic interaction liquid chromatography analytical column. A flow rate of 0.3 mL/min was used during isocratic elution using a mobile phase consisting of methanol-water (95:5, vol/vol) with 0.2% formic acid within a 5-minute run.

Results: The calibration curves exhibited excellent linearity ranging from 0.1 to 15 IU/mL, with determination coefficients (r2) exceeding 0.99. The precision and accuracy ranged from 1% to 7% and from 93% to 110%, respectively. The relative recovery fell within the range of 98%-100%, and the internal standard-normalized matrix effect ranged from 95% to 101%. The stability was satisfactory across various conditions.

Conclusions: This method was fully validated and successfully applied to quantify l-asparaginase activity in plasma samples of 15 children with ALL, enabling the monitoring of l-asparaginase activity with mass spectrometry.