(W0930-09-51) LCMS-MS Method Development and Detection Challenges of low Molecular Weight Hydrophobic Topical Dermatological Products in Support of In Vitro Permeation Testing (IVPT)

Purpose: In Vitro permeation testing (IVPT) examines the penetration profile of API (active pharmaceutical ingredient) across a skin tissue membrane (fresh, flash frozen or cadaver) using Franz-type diffusion cell systems containing typically aqueous receiving media. Using liquid chromatography mass spectrometry (LCMS) detection accompanied by noted and newly developed extraction techniques to enhance sensitivity of the targeted compounds for both qualitative and quantitative analysis. The current study examines several procedures (e.g., Protein precipitation, liquid-liquid extraction (LLE), solid-phase extraction (SPE) to enhance the sensitivity of reference molecule TER001 in the presence of a solubilizer and the associated suppression of signal using LCMS detection.

Methods: LCMS-MS method development for compound TER-001 using the instrument detector and organic solvent to isolate the associated masses and parameters were developed and the optimized parameters were integrated into a detection method along with the selection of a gradient, retention time, column, peak shape, and instrument solvents. Method Extraction-1 was prepared as a dilution of standards prepared in phosphate buffer solution (1xPBS), typical IVPT receiving medium into an organic solvent. Method Extraction-2 was prepared using a LLE procedure to isolate and enhance sensitivity. Standard samples were prepared in receiving media containing 0.5% solubilizer material and extracted using ethyl acetate. Samples were evaporated until dry and reconstituted with 80:20 Methanol: Water containing 0.1% Acetic Acid. Method Extraction-3 examined SPE procedures. A solid-phase 96-well plate was conditioned/rinsed with organic and aqueous solutions. Samples were added to the 96-well plate and treated with wash steps prior to elution into a collection plate. Samples were evaporated until dry and reconstituted with Acetonitrile. All extraction methods were analyzed for API content using the above developed LCMS-MS method. Solubility studies were then conducted with TER001 in various percentages of a solubilizer material added to the 1xPBS receiving media.

Results: The addition of the necessary solubilizer into matrix samples showed varying levels of analytical impact within the three extraction procedures. The preliminary data on matrix samples resulted in peak sensitivity at the higher end of linearity curves with Method Extraction-3 showing the most sensitive with a generated upper limit of quantitation (500 ng/mL) at a peak height of >4.0e⁵ cps. The lower limit of quantitation suffered from low sensitivity, background noise, and possible suppression due to the presence of solubilizer content in all extraction methods. At lower end of the curve the signal to noise ratio in support of bioanalytical method validation requirements is not demonstrated across the different extraction methods evaluated. Additionally, samples analyzed with the various extraction methods with higher solubilizer content in the receiving media presented additional suppression and increased loss of signal on mass spectrometry detection.

Conclusion: In conclusion, our study experiments presented the analytical challenges involved in the detection of the hydrophobic molecule in an aqueous based media required for IVPT studies. Further investigation into LCMS-MS method development is warranted to provide suitable conditions for specific extraction procedures and isolation of target molecules in the presence of solubilizers to support IVPT methodology. Such processes and procedure performances will assist in developing higher sensitive analytical detection in studies with low concentration of topical dermatological products.