Purpose: Mycophenolate has shown a strong synergistic effect when used in combination with oxaliplatin on inhibiting the growth of oral premalignant cells. We are developing novel mycophenolate patch formulations as a non-invasive therapeutic device for treating oral premalignant lesions. The purpose of this study is to develop a specific, sensitive and reproducible LC-MS/MS method capable of quantifying the levels of mycophenolate in rat tongue for tissue distribution studies.
Methods: Patch formulations were applied to the tongue of adult male Sprague Dawley rats for 4 hrs. Tongue tissues were then collected and washed with phosphate buffer. Mycophenolate was extracted from the tongue tissue by removing proteins using acetonitrile precipitation. Mycophenolate separations were carried out on an ACE Excel 2 Super C18 column (50 x 2.1 mm, 2 μm) with a mobile phase run in gradient elution of 0.1% FA in water (solvent A) and 0.1% FA in acetonitrile (solvent B) at a flow rate of 0.4 mL/min. Griseofulvin was used as internal standard. LC-MS/MS analysis was carried out on an API 4000 QTRAP LC-MS/MS system with a Turbo Ion Spray ion source. Tandem mass spectrometry was employed under positive electrospray ionization to detect the specific precursor to product ion transitions m/z 321.2 → 207.2 for mycophenolate and m/z 353.2 → 285.1 for the internal standard. Method validation was performed based on the FDA guideline for Bioanalytical Method Validation.
Results: The lower limit of detection (LOD) and the lower limit of quantitation (LLOQ) for mycophenolate were 2 ng/mL and 4 ng/mL in blank rat tongue, respectively. The method was linear from 4 ng/mL to 2000 ng/mL for rat tongue (r2 >0.999). The intra-batch and inter-batch precision for low (10 ng/mL), medium (800 ng/mL) and high (1500 ng/mL) quality control samples were between 2.94% - 13.5% and 3.21%-12.5%, respectively. The intra-batch and inter-batch accuracies were between 1.44% - 4.24% and 1.04% - 4.15%, respectively. The extraction recoveries were 96.92 ± 6.99% and the matrix effects were non-significant. Mycophenolate samples were stable in room temperature and autosampler for 16 hrs. The validated method was successfully applied to investigate the tissue distribution of mycophenolate following topical administration to male Sprague-Dawley rats.
Conclusion: We have established a specific, sensitive and reproducible LC-MS/MS method for the quantification of mycophenolate in rat tongue. This method was successfully applied to the analysis of mycophenolate levels in rat tongue for tissue distribution study.