Method Validation Report for the Estimation of Nelarabine in K3edta-Human Plasma by Using Lc-Esi-Ms/Ms

Abstract

Author want to define the procedure for robust and sensitive method utilized in bio-analytical research to establish a simple technique for determining the level of nelarabine in K₃EDTA plasma from human beings, is a combination of liquid chromatography, electro spray ionization and mass spectro-photometry (LC-MS/MS) parameters were systematically optimized, by using methanol: 5mM ammonium acetate in water (80:20%v/v) as mobile phase, flow rate of 1.2mL/minute, Zorbax SB-C18; 2.1*50mm, 5µm Agilent Technologies column. Solid-phase extraction (SPE) was further optimized to improve recoveries and minimize matrix effects. The final method achieved instrument detection limits as low as 0.01fg (femto-grams) on-column, retention time for nelarabine observed at 2.65 ± 0.03minutes with run time 4.0 minutes. Calibration linearity concentration range of 2.00 to 1000ng/mL with a correlation coefficient (r²) of ≥ 0.9997, %Mean ISTD recovery with correction factor for Nelarabine = 83.79; %CV of ISTD recovery (Extracted) for Nelarabine = 6.15, recoveries within 70–130%, and intra / inter-day precision (RSD ≤20%) confirmed the robustness and reproducibility of the protocol. The LC-MS/MS technique that was created to quantify the amount of Nelarabin in the biological matrix worked well for routine blood sample analysis from patients for pharmacokinetics research and medication monitoring.