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Tutorials : Jun 1, 2013 ( )
Improving Compound Extraction Efficiency
Novel Method for Skin Tissues Allows Higher-Throughput Sample Processing!--h2>
Determining the skin penetration profile of active pharmaceutical ingredients present in creams, gels, lotions, and ointments is an important aspect of preclinical dermatological research, as it allows researchers to optimize their selection of formulation prototypes. The use of flow-through (or Bronaugh) diffusion cells using ex vivo human skin is a valuable in vitro model and suits the purpose of determining the skin penetration and distribution of compounds from topical formulations.
Flow-through cells have essentially two parts, a donor and a receiver compartment. The human skin membrane is placed in the donor compartment and a single dose of the formulation is applied to it at the beginning of the experiment. Beneath the skin is a constant flow of receiving fluid (phosphate buffer saline), which is continuously collected for analysis—this is the receiver compartment.
The amount of compound delivered into the epidermis and dermis, as well as in the receiving fluid, helps to select the best formulation during the optimization phase. In addition, results obtained may help to estimate the in vivo efficacy of the formulation selected. Therefore, it is important to ensure that the molecule of interest is fully extracted from the skin tissues.
The liquid extraction (LE) is a well-known technique that can be used for this purpose. Generally the skin sample is placed inside a 10 mL vial and the extraction solvent mixture is added, allowing for an overnight extraction process. While this is a straightforward approach, the possibility of using a faster method would allow high-throughput sample processing.
The Precellys®24 homogenizer (Bertin Technologies) can extract compounds from the skin in a more efficient manner, saving time and requiring less solvent (Figure 1). In addition, coupling the homogenization method with automation procedures could allow faster sample preparation before liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.
An in vitro skin penetration model was used to optimize three formulations (A, B, and C) containing the compound SB-275833. In addition, during method development, three different approaches were used to evaluate the extraction efficiency of the molecule from skin tissues (epidermis and dermis): LE for 15 hours, LE for 48 hours, and homogenization using Precellys 24 homogenizer and customized tubes with 2.8 mm stainless steel beads (Precellys lysing kit MK28-R). These tubes were developed to be used with a Biocap® decapper (Biosero) thus minimizing the time spent with manual tube handling.
The skin permeation study was set up using custom-made Bronaugh diffusion cells and dermatomed human abdominal skin (approximately 500 µm thickness) from plastic surgeries. The flow of PBS was set at 60 µL/min, with the time points collected hourly for six hours, for posterior analysis by LC-MS/MS.
After six hours, the skin samples were split into epidermis and dermis by placing them in an oven set at 60°C for two minutes. Each skin layer was placed in individual homogenization tubes and 500 µL of extraction solvent (1:1 methanol:water + 1% formic acid) was added. The Precellys 24 protocols for each layer were the following: epidermis—one cycle of 30 seconds at 6,500 rpm and dermis—two cycles of 60 seconds each at 6,500 rpm, with a 30-second interval in between.
Next, 1,000 µL of methanol was added to each tube, followed by a 10 second vortexing, sonication for 10 minutes, and finally a centrifugation step for five 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 samples (epidermis and dermis) used for LE were placed in 10 mL glass vials and 8 mL of a mixture of 1:1 methanol:water + 1% formic acid was added. The vials were incubated for 15 or 48 hrs at 5°C on a horizontal shaker. After this extraction step, the samples were sonicated for 10 minutes and then organized in a rack before being transferred and diluted in a 96-well plate using the liquid handler.
The compound was analyzed by LC-MS/MS using a Xevo TQ-MS (Waters) interface and multiple reaction monitoring in positive-ion mode for the transitions (m/z) 518.6>124.0 and 518.6>216.0. The chromatographic column was an UPLC BEH C18 50 × 2.1 mm, 1.7 μm particle size (Waters) with a mobile phase A containing 0.1% formic acid in water and a mobile phase B containing 0.1% formic acid in acetonitrile.
The flow rate was set at 0.4 mL/min with the following gradient 90:10 A:B from 0 to 0.4 min; 100:0 A:B from 0.6 to 1.3 min; and 90:10 A:B from 1.4 to 2 min. The injection volume was 5 µL, and the compound retention time was 1.1 minutes. The lower limit of quantification (LLOQ) was 20 pg/mL and the limit of detection, 5 pg/mL. The calibration curves were prepared over the range of 20 to 10,500 pg/mL, adequate for the detection and quantification of SB-275833 in the receiving fluid samples, dermis, and epidermis samples.
During the development of the extraction method, the number of replicates was minimized to a range of 15 to 27 for each of the formulations evaluated (replicates divided evenly across each extraction method). However, the automation methodology employed here (decapper and liquid handler) has proven suitable for a high-throughput screening of formulations using the in vitro skin penetration methodology, which generates about 400 samples per study (data not shown).
The dermis and epidermis values, considering the three different extraction methods, are shown on Figures 2A and 2B. The five-minute homogenization procedure using the lysing kit showed consistently increased extraction of the compound from the dermis samples, for the three formulations considered in this study.
For the epidermis samples, similarly higher extraction was observed only for formulations B and C. The LE for 15 and 48 hours was very comparable for dermis and epidermis samples, showing that a longer extraction did not improve its efficiency. This can be explained by the fact that the dermis is a thicker layer than the epidermis (350 µm compared to 150 µm), thus increasing the possibility of compound interaction and binding to the skin components, which can only be disrupted by a more intensive extraction method.
The skin penetration results are showed on Figure 3, where the cumulative amount over a period of six hours is displayed. As expected, the formulation ranking followed the amounts seen in the epidermis and dermis, namely B>A>C.
In conclusion, the extraction method using the Precellys homogenizer enhanced the high-throughput screening of formulations by decreasing the sample processing time (from 15 hours to five minutes), the volume of organic solvent used (from 8 mL to 1.5 mL), and by considerably increasing the extraction efficiency (up to 10 times for some samples).
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