Ali Najafi, M.S., Research Scientist II at Emery Pharma presented this work at the 68th American Society for Mass Spectrometry (ASMS) Conference in June, 2020. Here, in this video, we presented the overview of the method for trace-level quantitative analysis of Poloxamers in biological samples by HPLC-ESI-QqQ MS. The developed method is based on a robust LC-MS/MS (MRM)-based approach and a simple protein precipitation-based sample preparation protocol to address challenges with quantification of trace amounts of this polymer in a variety of biological matrices.
The analytical performance of the method, including specificity, linearity and range, accuracy, precision, and recovery, was presented and shown per FDA and ICH recommendations. The linear dynamic range extended from 2 µg/mL to 100 µg/mL with an LLOQ of 2 µg/mL and LOD of 1 µg/mL. Various blank matrices showed endogenous interfering component levels ≤ 20% of the poloxamer signal at the LLOQ. The most accurate results were obtained when a quadratic model was applied. The response at the LLOQ showed a %CV ≤ 20%, and the accuracy was ≤ 20%. All other non-zero calibrators exhibited accuracy and %CV ≤15%.
The validated LC-MS/MS method was used to quantitate Poloxamer levels in human serum and gene therapy-based drug product to support clinical trials, gene therapy product release, and pharmacokinetic assays.
Abbreviation: ASMS: American Society for Mass Spectrometry, HPLC: High-Performance Liquid Chromatography, ESI-QqQ MS: Electrospray Ionization-Triple Quadrupole Mass Spectrometry, LC-MS/MS: Liquid Chromatography Tandem Mass Spectrometry, MRM: Multiple Reaction Monitoring, FDA: Food and Drug Administration, ICH: International Council for Harmonisation, LLOQ: Low Limit of Quantitation, LOD: Limit of Detection, CV: Coefficient of Variation.