2.1.

Volunteers

The study was undertaken on nine healthy volunteers with skin phototypes 2 and 3,¹⁰ aged between 24 and 45 years. Approval was obtained for these experiments from the Ethics Committee of the University Hospital Charite´. The volunteers provided written informed consent prior to entry into the study.

2.2.

Topical Application of the Emulsions

An oil/water (o/w) emulsion provided by Beiersdorf AG (Hamburg, Germany) was used containing 4% 4-methylbenzylidene camphor. The investigation of the fluorescence was performed using an o/w emulsion containing 0.2% of the dye curcumin (MERCK-Schuchardt, Hohenbrunn, Germany). This fluorescent dye was chosen because this fluorophore is permitted for use in foods. Furthermore, a comparable behavior of the time-dependent spectroscopic changes was found for curcumin and the UV filter substance.

The emulsions were topically applied to a skin area on the flexor forearms of the volunteers as described previously.⁷ A skin area of 10×8 cm marked with a permanent marker was treated with 2 mg/cm² emulsion. The emulsion was distributed homogeneously inside the marked area using a gloved finger (pressure in the range of 60 g/cm²) saturated with the emulsion.

2.3.

Tape Stripping

The tape stripping procedure, using an adhesive film (tesa film number 5529, Beiersdorf, Hamburg, Germany, width 1.9 cm), started 1 h after the application of the emulsion. In agreement with the standard protocol described previously,^{7 11} the tape strips with a length of 6 cm were applied centrally inside the treated skin area. The longer side of the tesa films were arranged parallel to the 10-cm borderline of the treated skin area. The tape strips were pressed ten times using a roller and removed with one quick move afterward.

2.4.

UV/VIS Spectroscopic Measurements

After removal from the skin, the tape strips were fixed to a frame taken from a commercially available slide holder. With a razor blade, the edges of the strips were removed to a resultant thickness of 1.5 cm. This was done to avoid edge effects caused by a lack of perfect overlap of each tape strip take from the same area.⁷

2.5.

HPLC Measurements

The concentrations of the UV filter substance in the extracts used for the UV/VIS spectroscopic measurements were determined by HPLC measurements.¹³ The concentration was calculated based on the peak values. Standard solutions containing 4-methylbenzylidene camphor were measured for calibration. The HPLC system used consisted of a pump L-6200A, a detector L-4500 (Diode Array), an autosampler AS-2000A, and an interface D-6000 (all Merck-Hitachi, Darmstadt, Germany). The column LiChrospher 60 RP-select B (5 μm, 250×4 mm) was used (Merck, Darmstadt, Germany). A mixture of 82% acetonitrile (gradient grade), 17.9% water (for chromatography), and 0.1% phosphoric acid (pro analysi) (all Merck, Darmstadt, Germany) was applied as an isocratic eluent with a flow of 1 ml/min. A volume of 25 μl was injected. For the detection, a wavelength of 300 nm was used.

2.6.

Investigation of the Corneocyte Aggregates on the Tape Strips by Laser Scanning Microscopy

Laser scanning microscopy was utilized to measure the distribution of the topically applied substances on the tape strips removed from the treated skin.¹⁴ Therefore, 0.2% curcumin (MERCK-Schuchardt, Hohenbrunn, Germany) was added to the o/w emulsion containing the UV filter substance. The emulsion was applied onto the skin. After a penetration time of 1 h, tape strips were removed and investigated at different times by transmission and fluorescence measurements using a laser scanning microscope (LSM 2000, Carl Zeiss, Jena, Germany). The curcumin distribution was measured using the fluorescence signal at wavelengths above 600 nm after excitation with 488 nm radiation.

2.7.

Investigation of the Corneocyte Aggregates on the Tape Strips Using Light Microscopy

Corneocyte aggregates on tape strips removed from treated skin were investigated by light microscopy. A light microscope (System microscope BX60, Olympus Optical Company, Hamburg, Germany) was used to take images of the tape strips at different storage times in the transmission mode. The microscope was equipped with a digital camera SIS ColorView 12 and the appropriate software analySIS AUTO 3.1 (both Soft Imaging System GmbH, Mu¨nster, Germany).

2.8.

Statistics

The method of linear regression was used to determine the relationship between the spectroscopic data and the HPLC values. The coefficients of correlation R ² were calculated using the software program Microsoft® Excel 97.

3.1.

Time-Dependent Spectroscopic Changes of the Absorption of the UV Filter Substance

Figure 1 shows the typical spectra of a tape strip measured at different time intervals after removal from the skin treated with an o/w emulsion, containing the UVB filter 4-methylbenzylidene camphor. The absorbance in the visible range above 400 nm was characterized by a small change in the spectra, measured immediately and after 30 min. Later on, this value remained constant. In contrast, the typical absorption of the filter substance at the UV region increased in time. After a time period of 24 h, the absorption remained constant. A comparable result was obtained for curcumin. The absorbance was enhanced with increasing time after removal by a factor f _curcumin =A_6h /A _immediately =1.5. The comparable factor for the UV filter substance was found at f _UVB filter =A_6h /A _immediately =1.7.

Figure 1

UV/VIS spectra of the second tape strip, measured at different times after removal from the skin, which was treated 1 h before with an o/w emulsion containing 4% 4-methylbenzilidene camphor.

Laser scanning microscopy was applied to check whether the spectral changes observed for the UV filter substance were caused by changes in the distribution of the filter substance on the removed tape strips. The experiments were carried out using the o/w emulsion containing the fluorescence dye curcumin.

Typical distributions of the dye on the same tape strip at different times are given in Fig. 2 as a superposition of fluorescent and transmission images. The fluorescent dye is characterized by the light structures on the background of the dark images of the corneocyte aggregates. The distribution was not homogeneous if the tape strips were measured immediately after removal [Fig. 2(a)]. After periods of 1 and 6 h, the areas covered with curcumin increased [Figs. 2(b) and 2(c)], however, 24 h after removal (figure not presented) the dye was distributed homogeneously on the tape.

Figure 2

Time dependence of the distribution of curcumin (light structures) on a tape strip determined by laser scanning microscopy: (a) Measured immediately, (b) measured 1 h after removal from the skin, and (c) measured 6 h after removal from the skin.

3.2.

Absorbance of the UV Filter Substance Measured for the Removed Tape Strips and in Solution

In the following experiments, we investigated whether the absorption of the UV filter substance measured on the removed tape strips could be used directly to determine the concentration. Therefore, the absorption spectra of the tape strips were measured 24 h after removal, when the absorbance remained constant. After the direct spectroscopic measurements, the tape strips were extracted and the absorbance of the UV filter substance in solution was determined. In Table 1, the results found for the absorbance of 4-methylbenzylidene camphor are summarized.

Table 1

3.3.

Comparison of Spectroscopic Data and HPLC Measurements

To estimate the accuracy of the spectroscopic method for the quantification of the concentration of 4-methylbenzylidene camphor, dependent on the concentration range, the results of the spectroscopic measurements were compared with HPLC measurements carried out on the same samples (Table 2). The data obtained are separated into two concentration ranges. In the upper part of the table, the tapes containing more than 1 μg/ml (corresponding to 1 μg/cm² tape) were summarized. The data of tapes with a lower or equal content are shown next.

Table 2

The deviation of the concentrations of the UV filter substance determined by UV spectroscopy and HPLC was ⩽5% for more than 95% of the total amount recovered, corresponding to 87.9 μg/ml. In less than 5% of the total amount recovered (corresponding to 3.7 μg/ml), the deviation was ⩽25%. This result was confirmed correlating graphically the HPLC values with the data determined by UV/VIS spectroscopy. A linear regression line with a high coefficient of correlation was found (R ²⩾0.99).

3.4.

Spectral Changes in the Pseudoabsorption of the Corneocytes

To obtain additional information on the time behavior of the pseudoabsorption at 430 nm, caused by the corneocyte aggregates, light microscopic measurements were performed. The results of the investigations of the tapes, stripped 1 h after application of the sunscreen, are given in Fig. 3. A pronounced edge structure of the corneocyte aggregates was observed, if the investigations were performed immediately after removal [Fig. 3(a)]. Later on, this structure disappeared [Fig. 3(b)]. Regarding the extension of the horny layer cells [Fig. 3(c)], no further changes were observed during an additional 23 h. The pseudoabsorption measured 1 h after removal of the tape strips was used to calculate the horny layer profile.

Figure 3

Time-dependent changes in the pattern of the corneocyte aggregates after tape stripping determined by light microscopy: (a) Measured immediately, (b) measured 1 h after removal from the skin, and (c) measured 24 h after removal from the skin.

3.5.

Penetration Profile of the UV Filter Substance

The results of the spectroscopic measurements characterizing the amount of the corneocytes removed by each tape and the corresponding concentration of the UV filter substance were used to calculate the penetration profile of a typical UVB filter. Figure 4 shows the local distribution of 4-methylbenzylidene camphor applied in the o/w emulsion inside the SC. The length of the black bars represents the amount of UVB filter substance found on the corresponding tape strips. The absorbing species are positioned in the uppermost part of the horny layer.

Figure 4

Local distribution of the UVB filter substance 4-methylbenzylidene camphor, 1 h after application of an o/w emulsion.

4.1.

Time-Dependent Spectroscopic Changes

It is a well-known spectroscopic fact that the absorbance of a system containing identical amounts of absorbers is reduced if the substance does not cover the measuring area homogeneously. Lambert-Beer’s law, correlating absorbance and concentration linearly, is valid only if the absorbing species are distributed homogeneously. The lower absorbance found for the tape strips measured directly after stripping (see Fig. 1) reflects such a nonhomogeneous distribution of the UV filter substance on the tapes. This is in agreement with a theoretical consideration given, especially taking into account the behavior of UV filter substances in sunscreens.¹⁵

Later on, the absorbance measured increased because the distribution became more homogeneous, reaching an endpoint after 24 h. These changes are reflected by the increasing absorbance of the characteristic curcumin band described by the factor f _curcumin =A_6h /A _immediately (see Sec. 3.1). Both results obtained with laser scanning microscopy and optical spectroscopy confirm a diffusion of the filter substance into and inside the adhesive layer of the tape strips. This confirms the given interpretation for the time-dependent spectral changes observed for the absorption of the UV filter substance (see Fig. 1).

4.2.

Filter Absorbance Measured for the Removed Tape Strips and in Solution

The identical absorbances of the UV filter substance measured on the tape strips 24 h after removal and in the corresponding extracts (see Table 1) demonstrate that the absorbing species on the tapes are distributed homogeneously to the same extent as in solution, in agreement with the results of the Laser Scanning Microscopic experiments. This means that the tape strips removed can be used after a sufficient time period, 24 h in the investigated system, to determine the amount of UV filter substance. This may be important for screening investigations, as the direct use of the tape strips is less time consuming than an extraction followed by HPLC. However, a general application of this protocol requires a UV/VIS absorption band and checking of each individual system (active substance, formulation, and tape) to ensure that sufficient diffusion into and inside the adhesive layer occurs.

4.3.

Spectral Changes of the Corneocyte Pseudoabsorption

Referring to Fig. 3, it is obvious that the horny layer particles fixed to the tape strips have a pronounced edge structure immediately after removal, which disappears by 1 h. This surface phenomenon influences the scattering, reflection, and diffraction behavior responsible for the measured pseudoabsorption of the corneocytes.⁷

A simple explanation would be that the emulsion previously applied is concentrated on the surface of the corneocyte aggregates under in vivo conditions. This effect is transferred to the tape strips and can be detected immediately after removal. Diffusion processes then came into action, resulting in an increased homogeneous distribution of the emulsion and in a disappearance of the edge structures. This explains the changes in corneocyte-determined absorbance in the visible range, observed immediately after removal (see Fig. 1). As a result of matching the refractive index, the edge structure disappears and the corneocyte-determined absorbance remains constant.

To avoid disturbances arising from this effect, the spectroscopic determination of the amount of corneocytes on the removed tape strips does not start earlier than 30 min after the removal of the tape strips.¹¹

4.4.

Comparison of Spectroscopic Data and HPLC Measurements

The comparison of the data obtained with spectroscopic measurements and HPLC exhibits that the differences are distributed statistically. Thus, a systematic error can be excluded.

Considering the mean values of the deviation at the higher concentration range (see Table 2) and the correlation coefficients (R ²⩾0.99), it is obvious that the spectroscopic data are well suited to measure the concentration of the UV filter substance quantitatively at a concentration range of >1 μg/ml. The relative error increases with the decreasing absolute concentration of the UV filter substance. Therefore, the exact measurements in this concentration range require HPLC measurements.

4.5.

Penetration Behavior of UV Filter Substance

The penetration profile typically obtained for 4-methylbenzylidene camphor is given to illustrate the information available from the described method (see Fig. 4). In agreement with the demand on an effective sunscreen, the filter substance is positioned in the uppermost part of the horny layer. This behavior is in agreement with the results of other studies also detecting the UV filter substances in the uppermost part of the stratum corneum.^{7 9 16 17}