Hydrophilic interaction liquid chromatography–tandem mass spectrometry quantitative method for the cellular analysis of varying structures of gemini surfactants designed as nanomaterial drug carriers

• An LC–MS/MS method for the quantification of 18 surfactants with varying structures was developed.
• Tested compounds belong to diquaternary ammonium gemini surfactant molecules.
• HILIC chromatography was employed allowing for a short simple isocratic run of only 4 min.
• The method was applied to assess the cellular uptake within PAM 212 cells for two lead compounds.

Diquaternary gemini surfactants have successfully been used to form lipid-based nanoparticles that are able to compact, protect, and deliver genetic materials into cells. However, what happens to the gemini surfactants after they have released their therapeutic cargo is unknown. Such knowledge is critical to assess the quality, safety, and efficacy of gemini surfactant nanoparticles. We have developed a simple and rapid liquid chromatography electrospray ionization-tandem mass spectrometry (LC–ESI–MS/MS) method for the quantitative determination of various structures of gemini surfactants in cells. Hydrophilic interaction liquid chromatography (HILIC) was employed allowing for a short simple isocratic run of only 4 min. The lower limit of detection (LLOD) was 3 ng/mL. The method was valid to 18 structures of gemini surfactants belonging to two different structural families. A full method validation was performed for two lead compounds according to USFDA guidelines. The HILIC–MS/MS method was compatible with the physicochemical properties of gemini surfactants that bear a permanent positive charge with both hydrophilic and hydrophobic elements within their molecular structure. In addition, an effective liquid–liquid extraction method (98% recovery) was employed surpassing previously used extraction methods. The analysis of nanoparticle-treated cells showed an initial rise in the analyte intracellular concentration followed by a maximum and a somewhat more gradual decrease of the intracellular concentration. The observed intracellular depletion of the gemini surfactants may be attributable to their bio-transformation into metabolites and exocytosis from the host cells. Obtained cellular data showed a pattern that grants additional investigations, evaluating metabolite formation and assessing the subcellular distribution of tested compounds.