Development and validation of an analytical method for the quantification of cytochrome c in skin transport studies

A simple isocratic HPLC method for the quantification of Cytochrome c in skin permeation samples was developed and validated. The mobile phase comprised a 41 : 59 mixture of an organic phase A (0.1% trifluoroacetic acid in a 90 : 10 mixture of MeCN–H₂O) and an aqueous phase B (0.1% trifluoroacetic acid in H₂O). The Cytochrome c retention and run times were 2.62 and 8.0 min, respectively—much shorter than those for existing gradient methods. The response was accurate, precise and linear from 2.5 to 25 μg/mL. The mean recoveries for intra‐day and inter‐day analysis ranged from 88.5 to 103.8% and the RSD varied from 0.05 to 1.55%. The assay was used to quantify transport of Cytochrome c across intact and laser‐microporated porcine skin in vitro. Cytochrome c permeation and the amount of protein retained within the membrane over 24 h were quantified as a function of the number of micropores. Although no Cytochrome c permeation was observed across intact skin, laser microporation enabled delivery of 22.9 ± 3.3 and 56.0 ± 15.9 μg/cm² of the protein across skin samples with 300 and 1800 micropores, respectively. In conclusion, the HPLC method provided a fast, efficient means to quantify Cytochrome c in samples from skin transport studies. Copyright © 2009 John Wiley & Sons, Ltd.