DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY IMAGING OF TAPE-STRIPPED STRATUM-CORNEUM SAMPLES
DESI, which was proposed by Takáts et al. in 2004,12) is the first ambient ionization method18) to allow the direct analysis of solid-state samples by mass spectrometry (MS) in an open environment. DESI generates gas-phase ions derived from analytes by using electrospray ionization. In addition to providing fast profiling with minimal or no sample pretreatment, DESI has already been used as a MSI method in a variety of applications.13)
To date, applications of MSI to skin samples have mostly used time-of-flight secondary-ion mass spectroscopy (TOF-SIMS) and matrix-assisted laser desorption-ionization MSI (MALDI-MSI), presumably because of the broader interests in applications that provide high spatial resolution.19–21) In fact, TOF-SIMS has been used for MSI analysis with submicron resolution, whereas, due primarily to the intrinsic characteristics of the ionization method used, the targets are limited to low-molecular-weight compounds.19) Conversely, MALDI-MSI offers relatively modest spatial resolution (~micron level) but permits the analysis of mid- to high-molecular-weight macromolecules (i.e., proteins and polymers) and gives more options for advanced analysis by MS such as MS/MS and/or high-resolution m/z measurements. To the best of our knowledge, only few studies have applied 2D-DESI MSI to the analysis of substances in skin samples,22) excluding the direct or indirect (offline) profiling (non-imaging) of the surface of skin in forensic,23) security,24) and healthcare studies.25) Note that most of the skin samples used in previous MSI studies consist of slice tissues or biopsies, which involve invasive procedures and are thus not suited for the daily evaluation of cosmetic products.
The tape-stripping technique16,17) is also used in cosmetic science as a convenient method to rapidly and noninvasively collect stratum-corneum samples. The technique involves using an adhesive tape to collect a sheet of the stratum corneum from the skin surface. Repeating the process at the same site allows literally “layer-by-layer” sampling for rapid depth profiling. By combining 2D-DESI MSI with tape stripping, we developed a rapid method for analyzing the distribution of quasi-drugs26) and endogenous molecules27) in the stratum corneum.
Figure 3 shows the general workflow for the proposed ex vivo 2D-DESI MSI method.26) Briefly, after applying a topical cream on the skin, the sampling site is gently washed with soap and water. After allowing the skin sample to equilibrate for 15 min at room temperature, the stratum-corneum samples are collected by tape stripping with an adhesive tape under an established protocol (gentle pressing at about 0.22 MPa followed by abrupt stripping, etc.). By repeating the process at the same site, deeper layers of the stratum corneum can be obtained. The collected stratum-corneum samples were directly analyzed by 2D-DESI MSI.
|Fig. 3. Workflow of 2D-DESI MSI for tape-stripped stratum-corneum samples. (1) Stratum-corneum samples are collected from cosmetic-applied regions of the skin by stripping with adhesive tape. (2) Consecutive samplings allow depth profiling. The collected stratum-corneum samples are analyzed from their backsides by 2D-DESI MSI. (3) Line scanning the region of interest generates datasets containing location (X–Y), m/z, and signal intensities, which may be converted to visual distributions.|
To validate and optimize the method, we visually inspected the stratum-corneum samples collected by tape stripping. Figure 4(a) shows microscopy images of stratum-corneum samples collected by repeated tape stripping from the inner forearm of a healthy male volunteer (aged mid 40s). As expected, the stratum corneum collected on the first strip consists of a “sheet” of flattened cells, whereas that collected from the fifth and tenth strips appear more scattered and less dense. The cells from the deeper samples have smaller diameters (10–20 μm) because the cells are immature (in terms of keratinization).
|Fig. 4. (a) Microscopic images of tape-stripped stratum-corneum cells collected consecutively from the same site by using dedicated adhesive tape (SkinChecker; Promotool, Tokyo). Light absorbance as a function of strip number demonstrating (a) the difference in cell appearance and (b) the gradual decrease in the quantity of collected cells upon stripping from the deeper part of the stratum corneum.|
To compare the spatial resolution of 2D-DESI MSI to the size of stratum-corneum cells, in Fig. 4(a), yellow circles are drawn to show the typical resolution of 2D-DESI MSI (500-μm diameter). The results clearly show that the proposed 2D-DESI MSI method collects information on stratum-corneum cells averaged over the targeted area of the skin. TOF-SIMS or MALDI-MSI is clearly the preferred choice when a five- to ten-fold greater spatial resolution is required. We thus use 2D-DESI MSI as a complementary technique for the rapid acquisition of molecular distributions of larger areas (e.g., the human face).
For quantitative discussions, light absorbance is often used to estimate how much of the stratum-corneum protein is collected by tape stripping.28) Figure 4(b) shows the absorbance at 430 nm for tape-stripped stratum-corneum samples that were taken repeatedly from the three vicinal sites from the inner forearm of the male volunteer. The results reveal that the absorbance decreases for the deeper strips, which is consistent with the population of the cells, as determined by visual inspection [(Fig. 4(a)]. To convert absorbance into the quantity of protein, the samples are dispersed into a 0.1% RapiGest® (Waters, Inc.) aqueous detergent solution and the protein content quantified by using the Lawry method. The protein quantity thus obtained is used to correct quantitative data on target molecules from each strip.
Figure 5 shows an example of the evaluation of transdermal drug delivery by 2D-DESI MSI. Stratum-corneum samples were collected after the application of a topical cream containing 4-methoxy salicylic acid potassium salt [4MS(K)], which is a registered mild whitening drug, onto the inner forearm of a healthy male volunteer. For demonstration purposes, a test formulation containing 1% 4MS(K) was applied at a rate of ~50 mg/cm2, and the applied site was left exposed (i.e., not occluded by a cotton mask or a wrap film) for 1 h. After gently cleaning the skin with soap and warm water, the skin was allowed to equilibrate for 10 min at room temperature, the stratum-corneum samples were then collected by tape stripping. Small pieces of adhesive tape containing stratum corneum were directly analyzed by 2D-DESI MSI. The DESI experimental conditions were similar to the standard settings suggested by the manufacturer for samples on a glass surface, except that the distance between the DESI spray nozzle and the MS inlet was increased slightly (0.5–1 mm) in order to obtain a stable signal.
|Fig. 5. Transdermal drug-delivery evaluation by 2D-DESI MSI for stratum-corneum samples collected after application of topical cream containing 4-methoxy salicylic acid potassium salt [4MS(K)]. Depth and planar profile information are obtained from the direct analysis of samples collected consecutively by using adhesive tape from the inner forearm of a healthy male volunteer. The molecules are identified by tandem mass spectrometry. In this case, 4MS(K) accumulates mostly on the second and third strips. The method is useful for rapid screening of penetration enhancers formulated for topical cosmetic products.|
The data show that most of the 4MS(K) molecules accumulate on the second and third stratum-corneum layers, which is typical for compounds having moderate partition coefficients (P) [log P=2.10±0.26 for the free acid form of 4MS(K), calculated by ACD/Labs from Advanced Chemistry Development, Inc.]. The low concentration of 4MS(K) molecules in the first layer is attributed to the washing with soap, which removes any topical cream remaining on the surface. The identity of the analyte molecule is confirmed based on the presence of the major MS/MS product ion (m/z 123, CO2 eliminated ion). The middle panel on the right of Fig. 5 shows the depth profile of a putative endogenous molecule (m/z 187); the amount of this molecule decays gradually upon approaching the skin surface. Although its identity is currently unknown even after high-resolution MS and MS/MS measurements, circumstantial evidence suggests that the molecule originates from the body. We have been using this technology in our laboratory to evaluate the early performance of penetration enhancers, which play a crucial role in the transdermal delivery of hydrophilic (low log P) quasi-drugs. Note that the planar distribution is also very valuable for analyzing the penetration route, which will be discussed elsewhere.
The 2D-DESI MSI method was also applied to an analysis of the distribution of NMF amino acids across the human face.27) As mentioned above, NMF amino acids are produced through the enzymatic degradation of filaggrin protein in the skin.29–31) In the process of keratinization, some amino acids are converted by heat, UV light, and/or enzymes, and act as natural humectants in the stratum corneum. We are interested in understanding the relationship between the molecular composition of NMF amino acids and skin conditions. The moderate spatial resolution of 2D-DESI MSI is advantageous for obtaining macroscopic molecular-distribution information from a human face within a reasonable time (∼8 h for half the face at 0.5 mm resolution).
Figure 6 shows a representative facial NMF distribution for a healthy female subject. Stratum-corneum samples were collected from eight sites over half of her face. Three samples were collected consecutively from each site by using the adhesive tape originally developed for gentle “layer-by-layer” sampling, and the results for the second sheets are shown. After estimating the protein quantity by near-infrared absorbance (850 nm), the stratum-corneum samples were directly analyzed by 2D-DESI MSI (i.e., without sample preparation).
|Fig. 6. Molecular distribution of endogenous amino acids over a human face by 2D-DESI MSI coupled with tape-stripping stratum-corneum sampling technique. Polyacrylate-based adhesive tape (25×70 mm2) was used to collect the stratum corneum from eight sites of the face of a healthy female subject. Three samples were collected consecutively from each site, and the results of the second samples are shown. The quantity of stratum-corneum protein is estimated from the absorbance at 850 nm. The figure shows the different facial distributions of serine (Ser) and pyrrolidone carboxylic acid (PCA), which are representative of humectant molecules called natural moisturizing factors.|
As an example, Fig. 6 shows the difference in distribution between serine (Ser) and pyrrolidone carboxylic acid (PCA). Serine is the most abundant NMF amino acid and is not converted in the stratum corneum during turnover. The distribution of serine is uniform across the (half-) face and similar to that of the stratum-corneum protein, which suggests that serine could serve as an index of the basal NMF-generation activities. Conversely, PCA, which is a conversion product of glutamine (Gln), exhibits a unique distribution pattern, indicating the differences in biological activity in the stratum corneum for different parts of the face. Unfortunately, the significance of these molecular conversions in the stratum corneum remains unknown. To find answers to this question, the relationship between NMF composition and skin parameters, such as bleomycin-hydrase activity,31) skin water content, and trans-epidermal water loss, are currently being investigated in detail.
The extractive nature of DESI is useful for analyzing skin samples because the selectivity of analytes can be easily controlled by changing the polarity of the spray solvent. In our experience, an acetonitrile–water (1 : 1, v/v) mixture containing 0.1% formic acid is the best balance for sensitivity and detection coverage for analyzing NMF amino acids. Seventeen out of twenty-one typical NMF amino acids were observed from the stratum corneum of healthy male and female subjects.27) For the analysis of skin lipids, such as fatty acids, glycerides, cholesterol, and ceramides, MeOH including 0.1% formic acid works best for the same reasons.27)
Absolute quantitation is a challenging task for most MSI techniques that desorb analytes from condensed-phase samples without the help of liquid dissolution or the use of chromatographic separation. In addition to the matrix effect, which alters signal intensity, the recovery of the analyte from the region of interest affects the quantitative accuracy. To estimate the recovery (or extraction efficiency) of NMF from stratum-corneum samples by using 2D-DESI MSI, we measured (data not shown) the residual amounts of serine in the samples that were formerly analyzed by 2D-DESI MSI. As a result, the recovery of serine by 2D-DESI MSI was approximately 50% in comparison with the recovery obtained by LC-MS/MS measurements of a solvent extract of the same sample. Briefly, the quantification by 2D-DESI MSI was based on an external calibration established for serine that was deposited onto a tape-stripped stratum corneum, and the peak volume obtained for m/z 104 under the negative-ion detection mode was plotted against the amounts that were spotted (11–280 pmol/mm2, the standard addition method). For LC-MS/MS quantification, serine was quantified in the form of a 3-aminopyridyl-N-hydroxysuccinimidyl carbamate derivative32) by reversed-phase liquid chromatography based on the internal standard method using serine-13C3, 15N as an internal standard. Because the extraction efficiency of 2D-DESI MSI depends on the chemical properties of the analytes, the sampling location, and DESI MSI parameters, quantitative data of 2D-DESI MSI need to be carefully considered, as is the case for other MSI methods.
The other important factor that may impact the quantitative performance of 2D-DESI MSI is possible variations in the quantity of stratum corneum collected after the application of a cosmetic.33,34) This is influenced by the degree of hydration and other ingredient-associated factors, which may influence the adhesive strength of stratum-corneum cells.34) The only possible countermeasure against this effect is to do a carefully controlled experiment each time. For example, to determine if the stratum corneum is completely removed under the given conditions, all stratum-corneum layers are removed by repeated tape stripping 20 to 30 times from the same site until all the stratum-corneum cells are collected. The number of strippings needed varies depending on the thickness of the stratum-corneum layer (body-site dependent) and on the adhesive strength of the stripping tape. By plotting the estimated amount of stratum-corneum protein versus strip number, the influence of the applied product can be estimated. Unfortunately, this process is invasive and tedious and so cannot be performed on a regular basis. In practice, our laboratory uses up to five strippings with a dedicated mildly adhesive tape (in-house product) to avoid potential skin troubles such as irritation and pigmentation. To ensure quantitative accuracy, the amount of protein on each strip is always measured by near-infrared or visible-light absorbance.
Thus, the proposed technique of applying 2D-DESI MSI to stratum-corneum samples obtained by tape stripping is a practical method that allows molecular events on human skin to be rapidly analyzed in a nearly noninvasive manner. Because DESI ionizes lipids, proteins, and even nonpolar compounds (by a technique known as Reactive-DESI35)), the technique is expected to gain widespread use in advancing skin research in cosmetic science.