May 15, 2015 (Vol. 35, No. 10)
Leandro Santos Investigator Center for Skin Biology, Stiefel
Vanessa Yu Ph.D. Field Scientist Bertin
The extraction of compounds from ex vivo human skin is a critical aspect of the in vitro penetration assay, which is used for the selection of molecules in early stages of preclinical dermal development, as well as during the formulation optimization process. While the liquid extraction procedure is commonly used for this purpose, the tissue homogenization technique provides a faster and more efficient option, characteristics that are critical in a high-throughput setting.
The Precellys® Evolution homogenizer (Bertin Technologies) in Figure 1 is capable of homogenizing human skin in a fast (24 samples in 7 minutes) and efficient manner (recovery levels generally above 75%), with significantly less solvent (1.5 mL) than the liquid extraction approach, which is demonstrated in this study.
The first step of the tissue extraction method development is the assessment of the extraction efficiency, which sometimes involves the evaluation of different solvent mixtures containing water, acetonitrile, and methanol, as well as pH modifiers (formic or acetic acid and sodium carbonate), to increase the compound recovery. The validation step starts with spiking known concentrations of the compound of interest on skin sections, which are incubated for a set period of time and then homogenized, extracted, and analyzed by UPLC-MS/MS. Additional steps are also included during the method development and intended to evaluate compound stability in the receiving media and extraction solvent mixture, as well as precision, accuracy, and linearity of the analytical method.
In this study a proprietary GSK compound was used as a model molecule for the evaluation of the Precellys Evolution in the homogenization of skin samples. Skin sections from abdominoplasties were defatted and dermatomed at 500 µm, cut in a rectangular shape with an area of 1 cm2 (average mass of 150 mg) and placed inside reinforced tubes with 2.8 mm stainless steel beads (Precellys lysing kit MK28-R, Bertin Technologies). The skin was then spiked with 10 µL of Low, Mid and High concentrations (four replicates of each) of the compound of interest (dissolved in 100% acetonitrile) and incubated at 37°C for 4 hours. Controls were prepared in triplicate per concentration, spiking the solutions in tubes containing only the extraction solvent, without the incubation step. After incubation of the skin samples, 500 µL of the solvent extraction mixture (1:1 methanol:water + 0.1% formic acid) were added, and the samples homogenized using the Precellys Evolution set at four cycles of 9000 rpm (30 seconds/cycle) with a pause of 90 seconds between cycles. After the samples cooled down, 1,000 µL of methanol were added to each tube, followed by sonication for 10 minutes, and then centrifuged for 5 minutes at 10,000 g and 5°C. The tubes were placed in a rack, and samples were transferred and diluted in a 96-well plate using an EVO® 200 liquid handler (Tecan).
The GSK compound was analyzed by UPLC-MS/MS using a Xevo TQ-MS (Waters Instruments) interface and multiple reaction monitoring in positive-ion mode. The chromatographic column was an UPLC BEH C8 30 × 2.1 mm, 1.7 μm particle size (Waters Instruments) with a mobile phase A containing 0.1% of formic acid in water and a mobile phase B containing 0.1% of formic acid in acetonitrile. The flow rate was set at 0.4 mL/min with the following gradient: 70:30 A:B from 0 to 0.1 min; 1:99 A:B from 0.2 to 1.0 min; and 70:30 A:B from 1.1 to 1.3 min. The injection volume was 5 µL and the compound retention time was 0.73 minutes. The Lower Limit of Quantification (LLOQ) was 0.20 ng/mL and the calibration curve was prepared over a range of 0.235 to 146 ng/mL, with a coefficient of determination of at least 0.99 and residuals below 15% for the 8 concentrations used in the calibration curve.
The results are summarized in Figure 2, where the averaged amounts of compound extracted from the skin sections, using the Precellys Evolution, are compared to the respective controls for each of the concentrations. The extraction efficiency ranged from 75.6 to 81.8% (low and high concentrations, respectively), which is suitable for the evaluation of compounds in the in vitro human skin penetration assay. Additionally, the analytical variability (measured as standard deviation) ranged from 5.7 to 14%, demonstrating that the extraction, as well as the analytical method, generated consistent values and can be reliably used for their intended purpose. It was also observed that the spiked concentrations spanning from 6.58 to 355 ng of compound, which comprises a 50-fold range, accounts for the intrinsic experimental variability seen when using different skin donors. It is well known that ethnicity, gender, and age influence the delivery of compounds to the skin, which is therefore an important factor to be considered when developing the extraction method.
In conclusion, the extraction method using the Precellys Evolution homogenizer demonstrated a high compound recovery efficiency (above 75%), low variability for three different concentrations (below 15%), and decreased processing time when compared to the usual liquid extraction procedure (24 samples in 7 minutes in comparison to an overnight procedure).