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Noninvasive detection by ATR and NIR-DR methods for skin-care ionic materials transported into the skin by iontophoresis
Journal of Molecular Structure 661-662 (2003) 391–396 www.elsevier.com/locate/molstruc
Noninvasive detection by ATR and NIR-DR methods for skin-care ionic materials transported into the skin by iontophoresisq Toyotoshi Uedaa,*, Yukio Watanabea, Ken-ichi Akaob, Harue Suzukic a
Department of Chemistry, Meisei University, Hodokubo 2-1-1, Hino, Tokyo 191-8506, Japan b JASCO, Ishikawa 2967-5, Hachioji, Tokyo 192-8537, Japan c Suzuki Plastic Surgery Clinic, Ohashi 89-1, Sanjodouri-Ohashi-Higashiiru, Higashiyama-ku, Kyoto 605-0009, Japan Received 30 April 2003; accepted 18 July 2003 Dedicated to Professor Bernhard Schrader
Abstract Two analytical methods without damage to the skin were proposed in order to detect and measure the quantity of the medication transported into the skin by the iontophoresis. The infrared attenuated total reflection (ATR) method was proven to be able to evaluate the content of such a substance as sodium all-trans-retinoate or magnesium ‘-ascorbyl-2-phosphate in the top (horny) layer of epidermis (about 1 mm under the skin surface), using characteristic bands to the above ion. Another method of near-infrared diffusive-reflection (NIR-DR) technique was shown probably to detect it in the dermis (1 mm under the surface), based on the shift of frequency and the change in intensity for the vibrational combination band of water molecules hydrating the ion. The quantity of the above material decreased monotonically in the horny layer for several hours after the treatment, while in the dermis it increased at first and then decreased via the maximum value. q 2003 Elsevier B.V. All rights reserved. Keywords: Iontophoresis; Noninvasive detection; ATR; NIR-DR; Retinoic acid
1. Introduction Iontophoresis is a painless method of delivering medication to a localized tissue area by applying a low-level electric direct current to a reservoir of the ionic medication. It is an effective and rapid method of delivering water-soluble ionized medication into the skin [1 – 3]. Like electrical charges repel. Therefore, a positively charged electrode transports the positive drug ions into the underlying tissue. q In honor of Professor Bernard Schrader. * Corresponding author. E-mail address: [email protected] (T. Ueda).
0022-2860/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2003.07.020
Similarly, a negatively charged electrode transports the negative drug ions into the skin. Skin-care materials would be suitable for the delivered medication in order to improve the skin appearance, removing pigmentations, freckles and wrinkles and tightening the skin [4 –7]. Recently, the iontophoresis of such cosmetic negative ions as sodium salt of all-trans-retinoic acid (I, vitamin A derivative) and magnesium ‘-ascorbyl-2-phosphate (II, vitamin C derivative), etc. has become very popular. The iontophoresis of these materials is more effective and faster to deliver the materials into the skin than their topical application to the skin.
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T. Ueda et al. / Journal of Molecular Structure 661-662 (2003) 391–396
However, it is very important to measure the quantity of transported ions into the skin and surrounding tissues so that the medication may become more effective and powerful for the improvement. Recently Miwa has reported the HPLC (coulometric ECD) detection of sodium ‘-ascorbyl-2-phosphate, and oxidized and reduced forms of ‘-ascorbic acid in the dermis and epidermis of extirpated skin for the biopsy after the iontophoresis [7]. However, this method gives serious damage and permanent scar to the skin of subjects. It is necessary to make use of the noninvasive detection for transported ions. The FTIRATR technique [8,9] has been used extensively for a variety of dermatological studies and blood glucose diagnosis focusing on the stratum corneum [10,11], and for drug penetration [12]. Near-infrared diffusivereflection (NIR-DR) spectroscopy [13] has also been applied for measuring tissue pH as an example of a noninvasive method [14]. As examples of such noninvasive techniques, we propose in this report the above two kinds of vibrational spectroscopy, i.e. attenuated total reflection (ATR) method for the top layer of epidermis and NIR-DR method for the dermis.
cutting off the terminal 500 cm21 region, because the first derivative is very similar to the normal absorption spectrum and the second derivative may give the right wavelength of a peak. We have often had the problem of the reproducibility in our measurements. Intensities depended sharply on the humidity, temperature, position of the skin and the subject. We tried to minimize the disturbance from these factors. The sampling position on the inner arm was marked with an oily marker pen. This point of the skin was pressed down on the flat surface of a ZnSe single crystal for ATR measurement, and it was tightly touched to the top of a reflection probe for NIR-DR measurement. In the ATR apparatus the infrared beam entered into ZnSe crystal, and it was totally reflected at the surface bound to on the sample since the angle of incidence was larger than the critical angle, but the wave-packet of this beam partially seeped into the sample through the surface and was absorbed by the sample. The depth (dp) of seeping was evaluated in Eq. (1). " 2 #21=2 l n l 2 dp ¼ ð1Þ sin u 2 2 < 2pn1 n1 6:45
2. Experimentals Iontophoresis and simple (topical) application were made on inner parts of right and left lower (or upper) arms. The skin of the arm was cleaned with a sheet of wet paper and then dry paper before the treatment and each measurement. ATR measurement was carried out in the range of 4000 –700 cm21 with a JASCO FT/IR-460 spectrometer and a MIRaclee single reflection HATR from PIKE Technologies. NIR-DR measurements were carried out in the range of 10,000 – 4000 cm21 with a JASCO VIR-9600 spectrometer and NIR reflectance probe of REMSPECK Corporation, which uses high quality low-water silica optical fiber and sophisticated optical design. Each spectrum was taken as an average of several scans. The ATR spectrum was transformed to the scale of absorbance proportional to the ion’s concentration. The NIR-DR spectrum was transformed to the K/M value based on Kubelka – Munk transformation. Another kind of NIR-DR spectrum was obtained from those differentiated by the wavenumber once, twice, three or four times
where the angle of incidence u was set to 458 and the refractive indices of ZnSe crystal ðn1 Þ and human skin ðn2 Þ were estimated to be approximately 2.4 and 1.35, respectively. The wavelength l of the infrared beam ranged from 2.5 to 14 mm (its corresponding wavenumber was 4000 – 700 cm21) and thus the seeping depth was about one sixth of the wavelength. It was estimated to be 0.4– 2 mm. In the NIR-DR experiment, the near-infrared beam is hardly absorbed because the absorption in this region is forbidden according to the selection rules of normal vibrations. The signal obtained was collected from the diffusive reflection light in dermis approximately 1 mm under the surface, which was estimated from the average path-length of diffuse reflected light by simulating the light propagation in skin tissue with the Monte Carlo method. The optical path-length was found to be 1.3– 2.5 mm at the wavelength range from 5500 to 7700 cm21 [15]. This signal is the relative index R of diffusive reflection, which is the intensity of reflected beam from the sample divided by that from the standard material (a kind of glass). This
T. Ueda et al. / Journal of Molecular Structure 661-662 (2003) 391–396
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relative index R was substituted for the absolute index R1 of diffusive reflection in Eq. (2). K ð1 2 R1 Þ2 ð1 2 RÞ2 ¼ < S 2R1 2R
ð2Þ
where K is the absorption coefficient and S is the scattering coefficient. If the scattering coefficient is constant in this region, KM function ðK=SÞ is proportional to the concentration of the material. Fig. 1 shows an example of ATR spectra for the skin before and after the iontophoresis and for material I. The spectrum of material I was obtained by subtracting the spectrum of pure water from that of 0.5% aqueous solution of material I. The two skin spectra were very similar except for a few regions affected by iontophoretic treatment. The strongest band of the skin was the so-called ‘amide I’ of skin protein (keratin) at 1644 cm21, which was assigned to the mixed mode of CyO stretching vibration and NH deformation vibration, and this was considered as a reference band of the relative intensity in these spectra. The difference between the spectra before and after the iontophoresis was shown as a spectrum (hereafter referred as a difference spectrum) in Fig. 2. The difference was very small but it was certainly observed. The difference spectrum was nearly
coincident with the reference spectrum of material I as seen in Fig. 2. Fig. 3 shows an example of KM spectra (the spectra of KM function), which was obtained from NIR-DR measurement of the skin taken before and 0– 6 h (every an hour) after the iontophoresis of material I (0.8%). These KM spectra of 0.8% aqueous solution of material I were very similar to each other
Fig. 1. ATR spectrum of Na salt of all-trans-retinoic acid (I) and ATR spectra of the skin before and after the iontophoretic operation. The ATR spectrum of Na retinoate was obtained by subtracting the spectrum of pure water from that of 0.5% aqueous solution of material I. The ATR spectrum before the iontophoresis was taken both before step 1 (the initial procedure) and after the massage operation of step 2 in a series of BEAULY procedures. The spectrum after the iontophoresis was taken just after the last lifting operation of step 4.
Fig. 3. NIR-DR spectra of the skin before and 0–6 h (every an hour) after the iontophoretic treatment of material I (0.8%). To calculate the difference spectra in Fig. 4, the skin should be in the similar condition. So, we used the spectrum after the massage operation of step 2 as ATR spectrum before the iontophoresis for the spectrum just after step 4, and used the spectrum before step 1 for the spectra 1, 2, 3, 4, 5 and 6 h after the iontophoretic operation, in a series of BEAULY procedures. All these spectra were transformed by Kubelka – Munk Eq. (2) and the terminal 500 cm21 regions were cut off.
Fig. 2. ATR spectrum of material I and the difference spectrum which showed the difference between the spectra before and after the iontophoretic operation. The difference spectrum was almost coincident with that of material I in water. The bands with an arrow were used to evaluate the transported quantity.
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and the difference among them was much smaller than that among the above ATR spectra, because these KM spectra were essentially due to water molecules under the effect of the transported ion into dermis and not due to the ion itself. Some peaks in the difference spectra were shifted to lower or higher wavenumber on the order of 50 cm21 and decreased or increased in intensity respectively, since water molecules were surrounding the transported ion and connected probably by hydrogen bonds. There were two main bands. The band at 5230 cm21 was assigned to the combination band of n2 þ n3 in a water molecule and the band at 7000 cm21 to the combination band of n1 þ n3 : Fig. 3 shows original KM spectra and Fig. 4 shows the difference spectra. As seen in Fig. 4, two major peaks in the difference spectrum were shifted downwards to 5180 and upward to 7100 cm21 respectively. The difference spectrum was calculated so that we could observe no shoulder at the original peak position in it. Beauly I and II instruments of BEULY CO., LTD. were used as a portable commercial apparatus for the treatment of iontophoresis. This treatment consists of four steps: deep electro-cleansing, massage treatment, nutrient (iontophoresis), and lifting processes. Each step required 4 min, and all the while the electrode face was pressed onto the skin of the treated area by the same person’s hand. In this circuit, major resisters (and condensers in parallel) were at two sites of the skin, which are the face (or arm in this experiment)
Fig. 4. Difference KM spectra between the KM spectra after and before the iontophoresis, part of which was given in Fig. 3. The proper difference was devised so that it could show no shoulder at the original peak position. The intensity of the combination band n1 þ n3 at 7100 cm21 was increased, while that of the combination band n2 þ n3 at 5180 cm21 was decreased, and this latter band was used for the quantity estimation.
and the hand, and the human body itself, which lies between the face and the hand, was rather conductive. For the reservoir dissolving ions, we used aqueous solutions of material I with concentrations of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 wt%, and of material II with concentrations of 2, 4, 6, 8, 10 and 12 wt%. The former solutions were made of all-trans-retinoic acid powder mixed with the equi-molar sodium hydroxide. Its sodium salt could be dissolved considerably in water. 3. Results and discussions In Figs. 5– 7, we showed the time dependence of the quantity of transported ions based on ATR and NIR-DR measurements for materials I and II in the similar experiments. As seen in Fig. 5, the transported quantity detected by ATR measurement decreased monotonically, which meant that the target ion was imported rapidly during the nutrient process of iontophoresis step and diffused slowly to the surrounding dermis after the treatment. On the other hand, the transported quantity of material II evaluated by NIR-DR measurement increased at first and then reached the maximum value and finally decreased, which meant that the ion did not yet reach the dermis just after the treatment and then it diffused deeply to the dermis from
Fig. 5. The considerable difference of the quantities of Na salt of alltrans-retinoic acid transported by the iontophoresis and by the simple application (diffusion). The quantities were evaluated by the peak absorbance of ATR difference spectra. The right and left arms of two subjects were examined for the iontophoresis using a Beauly II instrument and the simple application, respectively.
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Fig. 7. Transported ion’s quantity due to the concentration of material II in a reservoir. The quantity was evaluated by the absorbance of an ATR spectrum with the right and left arms of three subjects used for the iontophoresis and the simple application, respectively. Reservoir’s concentrations were 4, 8, 12% of magnesium ‘-ascorbyl-2-phosphate in aqueous solution.
Fig. 6. Time dependence of the transported quantities by iontophoresis evaluated by NIR-DR KM difference spectra. (a) Sodium all-trans-retinoate (1.2, 0.8, 0.2% aqueous reservoir), (b) magnesium ‘-ascorbyl-2-phosphate (II) (2, 8, 12% aqueous reservoir).
the epidermis. Finally it was diluted and decreased after the maximum value. In case of material I, the transported quantity increased for several hours and kept the maximum value. In the case of the high concentration of 1.2% the transported quantity increased even after 6 h, while in the low concentration of 0.2% the transported quantity began to decrease after only 3 h. This tendency can be seen clearly in Fig. 6(a). The negative charges of ions I and II were 2 1 and 2 2 respectively, and the electric repulsive force driving ion II into dermis was twice compared with the force to I. Therefore, ion II was transported to the dermis faster than ion I and the former quantity decreased after a few hours. In Fig. 7, the concentration dependences of transported ion’s quantity were shown for the iontophoresis and the simple application with ATR spectra of three subjects. The quantity of the ion
delivered into the skin by iontophoresis using Beauly II was seven times more than that by simple (topical) application. From the above ATR and NIR-DR results in Figs. 5– 7, the quantity transported into the skin was concluded to be larger by iontophoresis than by topical application, and the more the ions delivered, the more they should diffuse into the surrounding dermis subsequently. The concentrations of material II were changed to be 4, 8 and 12% both for iontophoresis and simple application. As shown in Fig. 7, the transported ion’s quantity was increased reasonably proportioning to the reservoir’s concentration of ion II. As was seen in Fig. 8, the quantity of the ions transported by a new type of Beauly II device was larger than that by an old type of Beauly
Fig. 8. The quantities transported by iontophoresis of sodium alltrans-retinoate with Beauly I and II instruments. The quantity was evaluated by the absorbance of ATR spectrum for right and left arms of two subjects.
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I. The performance of the new type was significantly improved more than the old type. The percentage of the improvement was approximately 30%. The following changes made in the new device may have contributed to this improvement. The direct current was changed to intermittent direct current and the alternate current of 1785 Hz in frequency was superimposed on the above direct current. Also the electrode surface to the skin was expanded three times for Beauly II compared to Beauly I. 4. Conclusion Noninvasive detection methods by ATR and NIRDR spectroscopy were reported to evaluate the quantity of the ionic medication transported into the skin of epidermis and dermis, respectively. Difference spectra, which meant the difference between the spectra before and after the iontophoresis, clearly showed the pattern characteristic to the transported ion itself in ATR result, whereas difference spectra in NIR-DR result reflected the water circumstance affected by the ion, and we estimated the quantity of the transported ion indirectly through it. The next step for us is to establish the quantitative analysis by comparing the above spectroscopic results with HPLC analysis of the skin extirpated by the biopsy in the same experiment. Acknowledgements The authors would like to thank Mr Tae-Yeol JEON, Ms Seong-Young HWANG in BEAULY CO.
LTD. (Yoksam-Dong 677-6, Kangnam-ku, Seoul, Korea) and Mr Chang-Kun LEE in AMAN TRADING CO. LTD. (Iriya 1-14-1, Taito-ku, Tokyo 110-0013, JAPAN) for kindly giving us their portable instruments of iontophoresis: Beauly I and II.
References [1] S. Leduc, Electric Ions and Their Use in Medicine, Rebman, London, 1908. [2] A. Strohl, J. Verne, J.C. Roucayrol, P.F. Ceccaldi, C. R. Soc. Biol. 144 (1950) 819. [3] P. Tyle, Pharm. Res. 3 (1986) 318. [4] Y. Imai, T. Usui, T. Matsuzaki, et al., Jap. J. Pharmcol. 17 (1967) 317. [5] M. Fujiwara, N. Nagao, K. Monnden, et al., Free Radical Res. 27 (1996) 97. [6] H. Suzuki, J. JSAPS 20 (1998) 46. [7] N. Nakashima, K. Asada, N. Akagi, N. Miwa, H. Suzuki, Fragrance J. 30 (2002) 27. [8] N.J. Harrick, J. Phys. Chem. 64 (1960) 1110. [9] J. Fahrenfort, Spectrochim. Acta 17 (1961) 698. [10] H.M. Heise, L. Kupper, W. Pittermann, M. Stucker, J. Mol. Struct. (2003) 651. [11] A. Christ, et al., J. Szurkowski. Anal. Sci. 17 (2003) 371. [12] J.-C. Tsai, C.-Y. Lin, H.-M. Sheu, Y.-L. Lo, Y.-H. Huang, Pharm. Res. 20 (2003) 632. [13] H.W. Siesler, Y. Ozaki, S. Kawada, M. Heise, Near Infrared Spectroscopy, Wiley-VCH, Weinheim, 2002. [14] B.R. Soller, R.H. Micheels, J. Coen, B. Parikh, L. Chu, C. His, J. Clin. Monit. 13 (1997) 279. [15] H. Arimoto, Y. Yamada, Spectroscopic blood component measurement by using reduced ranges of absorption spectra, European Conference on Biomedical Optics (ECBO2003), 2003, in press.
Noninvasive detection by ATR and NIR-DR methods for skin-care ionic materials transported into the skin by iontophoresisq Toyotoshi Uedaa,*, Yukio Watanabea, Ken-ichi Akaob, Harue Suzukic a
Department of Chemistry, Meisei University, Hodokubo 2-1-1, Hino, Tokyo 191-8506, Japan b JASCO, Ishikawa 2967-5, Hachioji, Tokyo 192-8537, Japan c Suzuki Plastic Surgery Clinic, Ohashi 89-1, Sanjodouri-Ohashi-Higashiiru, Higashiyama-ku, Kyoto 605-0009, Japan Received 30 April 2003; accepted 18 July 2003 Dedicated to Professor Bernhard Schrader
Abstract Two analytical methods without damage to the skin were proposed in order to detect and measure the quantity of the medication transported into the skin by the iontophoresis. The infrared attenuated total reflection (ATR) method was proven to be able to evaluate the content of such a substance as sodium all-trans-retinoate or magnesium ‘-ascorbyl-2-phosphate in the top (horny) layer of epidermis (about 1 mm under the skin surface), using characteristic bands to the above ion. Another method of near-infrared diffusive-reflection (NIR-DR) technique was shown probably to detect it in the dermis (1 mm under the surface), based on the shift of frequency and the change in intensity for the vibrational combination band of water molecules hydrating the ion. The quantity of the above material decreased monotonically in the horny layer for several hours after the treatment, while in the dermis it increased at first and then decreased via the maximum value. q 2003 Elsevier B.V. All rights reserved. Keywords: Iontophoresis; Noninvasive detection; ATR; NIR-DR; Retinoic acid
1. Introduction Iontophoresis is a painless method of delivering medication to a localized tissue area by applying a low-level electric direct current to a reservoir of the ionic medication. It is an effective and rapid method of delivering water-soluble ionized medication into the skin [1 – 3]. Like electrical charges repel. Therefore, a positively charged electrode transports the positive drug ions into the underlying tissue. q In honor of Professor Bernard Schrader. * Corresponding author. E-mail address: [email protected] (T. Ueda).
0022-2860/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2003.07.020
Similarly, a negatively charged electrode transports the negative drug ions into the skin. Skin-care materials would be suitable for the delivered medication in order to improve the skin appearance, removing pigmentations, freckles and wrinkles and tightening the skin [4 –7]. Recently, the iontophoresis of such cosmetic negative ions as sodium salt of all-trans-retinoic acid (I, vitamin A derivative) and magnesium ‘-ascorbyl-2-phosphate (II, vitamin C derivative), etc. has become very popular. The iontophoresis of these materials is more effective and faster to deliver the materials into the skin than their topical application to the skin.
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T. Ueda et al. / Journal of Molecular Structure 661-662 (2003) 391–396
However, it is very important to measure the quantity of transported ions into the skin and surrounding tissues so that the medication may become more effective and powerful for the improvement. Recently Miwa has reported the HPLC (coulometric ECD) detection of sodium ‘-ascorbyl-2-phosphate, and oxidized and reduced forms of ‘-ascorbic acid in the dermis and epidermis of extirpated skin for the biopsy after the iontophoresis [7]. However, this method gives serious damage and permanent scar to the skin of subjects. It is necessary to make use of the noninvasive detection for transported ions. The FTIRATR technique [8,9] has been used extensively for a variety of dermatological studies and blood glucose diagnosis focusing on the stratum corneum [10,11], and for drug penetration [12]. Near-infrared diffusivereflection (NIR-DR) spectroscopy [13] has also been applied for measuring tissue pH as an example of a noninvasive method [14]. As examples of such noninvasive techniques, we propose in this report the above two kinds of vibrational spectroscopy, i.e. attenuated total reflection (ATR) method for the top layer of epidermis and NIR-DR method for the dermis.
cutting off the terminal 500 cm21 region, because the first derivative is very similar to the normal absorption spectrum and the second derivative may give the right wavelength of a peak. We have often had the problem of the reproducibility in our measurements. Intensities depended sharply on the humidity, temperature, position of the skin and the subject. We tried to minimize the disturbance from these factors. The sampling position on the inner arm was marked with an oily marker pen. This point of the skin was pressed down on the flat surface of a ZnSe single crystal for ATR measurement, and it was tightly touched to the top of a reflection probe for NIR-DR measurement. In the ATR apparatus the infrared beam entered into ZnSe crystal, and it was totally reflected at the surface bound to on the sample since the angle of incidence was larger than the critical angle, but the wave-packet of this beam partially seeped into the sample through the surface and was absorbed by the sample. The depth (dp) of seeping was evaluated in Eq. (1). " 2 #21=2 l n l 2 dp ¼ ð1Þ sin u 2 2 < 2pn1 n1 6:45
2. Experimentals Iontophoresis and simple (topical) application were made on inner parts of right and left lower (or upper) arms. The skin of the arm was cleaned with a sheet of wet paper and then dry paper before the treatment and each measurement. ATR measurement was carried out in the range of 4000 –700 cm21 with a JASCO FT/IR-460 spectrometer and a MIRaclee single reflection HATR from PIKE Technologies. NIR-DR measurements were carried out in the range of 10,000 – 4000 cm21 with a JASCO VIR-9600 spectrometer and NIR reflectance probe of REMSPECK Corporation, which uses high quality low-water silica optical fiber and sophisticated optical design. Each spectrum was taken as an average of several scans. The ATR spectrum was transformed to the scale of absorbance proportional to the ion’s concentration. The NIR-DR spectrum was transformed to the K/M value based on Kubelka – Munk transformation. Another kind of NIR-DR spectrum was obtained from those differentiated by the wavenumber once, twice, three or four times
where the angle of incidence u was set to 458 and the refractive indices of ZnSe crystal ðn1 Þ and human skin ðn2 Þ were estimated to be approximately 2.4 and 1.35, respectively. The wavelength l of the infrared beam ranged from 2.5 to 14 mm (its corresponding wavenumber was 4000 – 700 cm21) and thus the seeping depth was about one sixth of the wavelength. It was estimated to be 0.4– 2 mm. In the NIR-DR experiment, the near-infrared beam is hardly absorbed because the absorption in this region is forbidden according to the selection rules of normal vibrations. The signal obtained was collected from the diffusive reflection light in dermis approximately 1 mm under the surface, which was estimated from the average path-length of diffuse reflected light by simulating the light propagation in skin tissue with the Monte Carlo method. The optical path-length was found to be 1.3– 2.5 mm at the wavelength range from 5500 to 7700 cm21 [15]. This signal is the relative index R of diffusive reflection, which is the intensity of reflected beam from the sample divided by that from the standard material (a kind of glass). This
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relative index R was substituted for the absolute index R1 of diffusive reflection in Eq. (2). K ð1 2 R1 Þ2 ð1 2 RÞ2 ¼ < S 2R1 2R
ð2Þ
where K is the absorption coefficient and S is the scattering coefficient. If the scattering coefficient is constant in this region, KM function ðK=SÞ is proportional to the concentration of the material. Fig. 1 shows an example of ATR spectra for the skin before and after the iontophoresis and for material I. The spectrum of material I was obtained by subtracting the spectrum of pure water from that of 0.5% aqueous solution of material I. The two skin spectra were very similar except for a few regions affected by iontophoretic treatment. The strongest band of the skin was the so-called ‘amide I’ of skin protein (keratin) at 1644 cm21, which was assigned to the mixed mode of CyO stretching vibration and NH deformation vibration, and this was considered as a reference band of the relative intensity in these spectra. The difference between the spectra before and after the iontophoresis was shown as a spectrum (hereafter referred as a difference spectrum) in Fig. 2. The difference was very small but it was certainly observed. The difference spectrum was nearly
coincident with the reference spectrum of material I as seen in Fig. 2. Fig. 3 shows an example of KM spectra (the spectra of KM function), which was obtained from NIR-DR measurement of the skin taken before and 0– 6 h (every an hour) after the iontophoresis of material I (0.8%). These KM spectra of 0.8% aqueous solution of material I were very similar to each other
Fig. 1. ATR spectrum of Na salt of all-trans-retinoic acid (I) and ATR spectra of the skin before and after the iontophoretic operation. The ATR spectrum of Na retinoate was obtained by subtracting the spectrum of pure water from that of 0.5% aqueous solution of material I. The ATR spectrum before the iontophoresis was taken both before step 1 (the initial procedure) and after the massage operation of step 2 in a series of BEAULY procedures. The spectrum after the iontophoresis was taken just after the last lifting operation of step 4.
Fig. 3. NIR-DR spectra of the skin before and 0–6 h (every an hour) after the iontophoretic treatment of material I (0.8%). To calculate the difference spectra in Fig. 4, the skin should be in the similar condition. So, we used the spectrum after the massage operation of step 2 as ATR spectrum before the iontophoresis for the spectrum just after step 4, and used the spectrum before step 1 for the spectra 1, 2, 3, 4, 5 and 6 h after the iontophoretic operation, in a series of BEAULY procedures. All these spectra were transformed by Kubelka – Munk Eq. (2) and the terminal 500 cm21 regions were cut off.
Fig. 2. ATR spectrum of material I and the difference spectrum which showed the difference between the spectra before and after the iontophoretic operation. The difference spectrum was almost coincident with that of material I in water. The bands with an arrow were used to evaluate the transported quantity.
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and the difference among them was much smaller than that among the above ATR spectra, because these KM spectra were essentially due to water molecules under the effect of the transported ion into dermis and not due to the ion itself. Some peaks in the difference spectra were shifted to lower or higher wavenumber on the order of 50 cm21 and decreased or increased in intensity respectively, since water molecules were surrounding the transported ion and connected probably by hydrogen bonds. There were two main bands. The band at 5230 cm21 was assigned to the combination band of n2 þ n3 in a water molecule and the band at 7000 cm21 to the combination band of n1 þ n3 : Fig. 3 shows original KM spectra and Fig. 4 shows the difference spectra. As seen in Fig. 4, two major peaks in the difference spectrum were shifted downwards to 5180 and upward to 7100 cm21 respectively. The difference spectrum was calculated so that we could observe no shoulder at the original peak position in it. Beauly I and II instruments of BEULY CO., LTD. were used as a portable commercial apparatus for the treatment of iontophoresis. This treatment consists of four steps: deep electro-cleansing, massage treatment, nutrient (iontophoresis), and lifting processes. Each step required 4 min, and all the while the electrode face was pressed onto the skin of the treated area by the same person’s hand. In this circuit, major resisters (and condensers in parallel) were at two sites of the skin, which are the face (or arm in this experiment)
Fig. 4. Difference KM spectra between the KM spectra after and before the iontophoresis, part of which was given in Fig. 3. The proper difference was devised so that it could show no shoulder at the original peak position. The intensity of the combination band n1 þ n3 at 7100 cm21 was increased, while that of the combination band n2 þ n3 at 5180 cm21 was decreased, and this latter band was used for the quantity estimation.
and the hand, and the human body itself, which lies between the face and the hand, was rather conductive. For the reservoir dissolving ions, we used aqueous solutions of material I with concentrations of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 wt%, and of material II with concentrations of 2, 4, 6, 8, 10 and 12 wt%. The former solutions were made of all-trans-retinoic acid powder mixed with the equi-molar sodium hydroxide. Its sodium salt could be dissolved considerably in water. 3. Results and discussions In Figs. 5– 7, we showed the time dependence of the quantity of transported ions based on ATR and NIR-DR measurements for materials I and II in the similar experiments. As seen in Fig. 5, the transported quantity detected by ATR measurement decreased monotonically, which meant that the target ion was imported rapidly during the nutrient process of iontophoresis step and diffused slowly to the surrounding dermis after the treatment. On the other hand, the transported quantity of material II evaluated by NIR-DR measurement increased at first and then reached the maximum value and finally decreased, which meant that the ion did not yet reach the dermis just after the treatment and then it diffused deeply to the dermis from
Fig. 5. The considerable difference of the quantities of Na salt of alltrans-retinoic acid transported by the iontophoresis and by the simple application (diffusion). The quantities were evaluated by the peak absorbance of ATR difference spectra. The right and left arms of two subjects were examined for the iontophoresis using a Beauly II instrument and the simple application, respectively.
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Fig. 7. Transported ion’s quantity due to the concentration of material II in a reservoir. The quantity was evaluated by the absorbance of an ATR spectrum with the right and left arms of three subjects used for the iontophoresis and the simple application, respectively. Reservoir’s concentrations were 4, 8, 12% of magnesium ‘-ascorbyl-2-phosphate in aqueous solution.
Fig. 6. Time dependence of the transported quantities by iontophoresis evaluated by NIR-DR KM difference spectra. (a) Sodium all-trans-retinoate (1.2, 0.8, 0.2% aqueous reservoir), (b) magnesium ‘-ascorbyl-2-phosphate (II) (2, 8, 12% aqueous reservoir).
the epidermis. Finally it was diluted and decreased after the maximum value. In case of material I, the transported quantity increased for several hours and kept the maximum value. In the case of the high concentration of 1.2% the transported quantity increased even after 6 h, while in the low concentration of 0.2% the transported quantity began to decrease after only 3 h. This tendency can be seen clearly in Fig. 6(a). The negative charges of ions I and II were 2 1 and 2 2 respectively, and the electric repulsive force driving ion II into dermis was twice compared with the force to I. Therefore, ion II was transported to the dermis faster than ion I and the former quantity decreased after a few hours. In Fig. 7, the concentration dependences of transported ion’s quantity were shown for the iontophoresis and the simple application with ATR spectra of three subjects. The quantity of the ion
delivered into the skin by iontophoresis using Beauly II was seven times more than that by simple (topical) application. From the above ATR and NIR-DR results in Figs. 5– 7, the quantity transported into the skin was concluded to be larger by iontophoresis than by topical application, and the more the ions delivered, the more they should diffuse into the surrounding dermis subsequently. The concentrations of material II were changed to be 4, 8 and 12% both for iontophoresis and simple application. As shown in Fig. 7, the transported ion’s quantity was increased reasonably proportioning to the reservoir’s concentration of ion II. As was seen in Fig. 8, the quantity of the ions transported by a new type of Beauly II device was larger than that by an old type of Beauly
Fig. 8. The quantities transported by iontophoresis of sodium alltrans-retinoate with Beauly I and II instruments. The quantity was evaluated by the absorbance of ATR spectrum for right and left arms of two subjects.
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I. The performance of the new type was significantly improved more than the old type. The percentage of the improvement was approximately 30%. The following changes made in the new device may have contributed to this improvement. The direct current was changed to intermittent direct current and the alternate current of 1785 Hz in frequency was superimposed on the above direct current. Also the electrode surface to the skin was expanded three times for Beauly II compared to Beauly I. 4. Conclusion Noninvasive detection methods by ATR and NIRDR spectroscopy were reported to evaluate the quantity of the ionic medication transported into the skin of epidermis and dermis, respectively. Difference spectra, which meant the difference between the spectra before and after the iontophoresis, clearly showed the pattern characteristic to the transported ion itself in ATR result, whereas difference spectra in NIR-DR result reflected the water circumstance affected by the ion, and we estimated the quantity of the transported ion indirectly through it. The next step for us is to establish the quantitative analysis by comparing the above spectroscopic results with HPLC analysis of the skin extirpated by the biopsy in the same experiment. Acknowledgements The authors would like to thank Mr Tae-Yeol JEON, Ms Seong-Young HWANG in BEAULY CO.
LTD. (Yoksam-Dong 677-6, Kangnam-ku, Seoul, Korea) and Mr Chang-Kun LEE in AMAN TRADING CO. LTD. (Iriya 1-14-1, Taito-ku, Tokyo 110-0013, JAPAN) for kindly giving us their portable instruments of iontophoresis: Beauly I and II.
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