A general liquid chromatography tandem mass spectrometry method for the quantitative determination of diquaternary ammonium gemini surfactant drug delivery agents in mouse keratinocytes’ cellular lysate

• An LC–MS/MS method for the quantification of 29 gemini surfactants was developed.
• Tested compounds belong to diquaternary ammonium gemini surfactant molecules.
• Full validation according to the USFDA was performed on a candidate gemini surfactant.
• The method was applied to assess the cellular uptake within PAM 212 cells.

Development of a simple and rapid liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the quantification of diquaternary ammonium gemini surfactants, utilized as gene deliver agents, is required for the evaluation of their post transfection fate. The continued development of efficient and safe gene delivery agents will benefit directly from an understanding of their rate of uptake, metabolism and excretion. A method is reported that is specific for the quantification of twenty-nine individual diquaternary ammonium gemini surfactant molecules and was validated for N,N-bis(dimethylhexadecyl)-1,3-propane-diammonium dibromide (G16-3) within PAM212 cell lysate according to USFDA bioanalytical method validation guidelines. The 10 min chromatographic separation procedure utilized an Agilent Zorbax CN column (100 mm × 2.1 mm with 3 μ particles) with LC–MS grade water and acetonitrile, both containing 0.3% (v:v) formic acid and 1 mM triethylamine. Extraction of the gemini surfactant from PAM212 keratinocyte cell lysate was performed using octanol and 10 μL aliquots were injected onto the column. The standard curve was linear from 0.30 μg/mL to 220 μg/mL (r2 ≥ 0.999) for G16-3 and precision and accuracy were within USFDA specified limits. G16-3 analyte was assessed as stable during storage in the auto-injector, bench-top, freeze–thaw cycling and long-term (60 days) storage at −20 °C. Evaluation of the cellular uptake and fate of G16-3, during both the incubation and post incubation periods, has established the potential of the application of the LC–MS/MS quantification method for monitoring diquaternary ammonium gemini surfactants in transfection studies.