A Sensitive And Rapid LC-MS/MS Method For The Determination Of Zanamivir In Human Plasma: Method Development And Validation

Abstract

A sensitive and rapid LC-MS/MS method was developed and validated for the quantification of Zanamivir in human plasma using Remdesivir as an internal standard (IS). The method employed a Waters Alliance HPLC system coupled with a SCIEX QTRAP 5500 mass spectrometer using an electrospray ionization interface in positive ion mode. Chromatographic separation was achieved on an isocratic system with a mobile phase of MeOH and ammonium formate (30:70 v/v) at a 1.0 mL/min flow rate. The method demonstrated excellent linearity in the 4–80 ng/mL concentration range with a correlation coefficient (R²) of 0.99941. The retention times of Zanamivir and Remdesivir were 2.600 min and 3.956 min, respectively, with a total run time of 6 minutes. Validation studies were performed by FDA guidelines, assessing system suitability, sensitivity, accuracy, precision, recovery, and stability. The % recovery for Zanamivir across all QC levels was above 99%, and the matrix effect was negligible with a %CV of 0.83. Stability studies showed Zanamivir remained stable under varied storage conditions, including freeze-thaw cycles, bench-top, and long-term storage at −80 °C. The developed method was precise, accurate, and robust, making it suitable for pharmacokinetic studies and therapeutic drug monitoring of Zanamivir.