Development of a Solid Phase Extraction Cartridge‐Based Analytical Method for Determination of Free Drug in Nanoliposomal Oncology Drug Formulations

A reliable SPE-free drug testing method was developed for active loading nanoliposome formulations through a systematic development approach. Ultrafiltration (UF), nanoparticle exclusion chromatography (HPLC-nPEC), solid phase extraction (SPE), and size exclusion chromatography (SEC) were screened for liposome-free drug separation. Full-scale method development was then conducted on the selected SPE technique. HLB 3cc (60 mg) SPE cartridges were used for developing a liposome-free drug testing procedure. SPE retention capacity for the Mirati drug was determined (1028 µg per cartridge). SPE testing conditions were studied to achieve a good separation between drug-loaded liposomes and free drug, which include cartridge conditioning, buffer wash steps for liposomal drug elution, organic media wash steps for free drug elution, and sample size. Qualification of this newly developed SPE method has demonstrated sufficient specificity/selectivity, excellent detection linearity (correlation coefficient (R) > 0.999) over a study concentration range (1.0 to 46.9 µg/mL), acceptable LOQ (0.52 µg/mL equivalent to 1.7% of free drug in a liposome formulation at 2.5 mg/mL), good accuracy/recovery (within 90% to 94%) for free drug in spiked samples at 4.5%, 9%, and 18% levels, satisfactory method precision (RSD (n = 6) of <2.0% for free drug analysis), and 2-day solution stability for standard and sample solutions (2-8°C). In comparative sample testing, SPE free drug results were in good agreement with SEC results for testing active loading formulations. A significant difference in free drug detection was observed among SPE, SEC, and HPLC-nPEC methods for testing passive loading formulations.