Introduction
Infrared spectroscopy records the molecu-lar vibrations of absorbing compounds, occur-ring as a result of their interaction with elec-tromagnetic energy in the mid-IR range, from 625 cm-1 (16 µm) to 4000 cm-1 (2.5 µm), which encompasses absorption bands corre-sponding to the fundamental vibrations of most functional groups. Each band in an IR spectrum can be characterized by the wavenumber of its absorption maximum, in addition to its intensity and bandwith. The wavenumber (usually expressed in cm-1) is the reciprocal of the wavelength and is related to the frequency of the absorbed radiation.
The wavenumber at which an absorbance occurs is characteristic of the underlying mo-lecular motion and consequently of the atoms participating in the chemical bond and on
their conformation and immediate environ-ment. The bandwidth, on the other hand, is related to the rates of motion of the molecule, and increases as motional rates increase.
FTIR spectrometer
The current IR spectrometers use Fourier transform (FT) algorythms for the analysis of the samples, thus offering decisive advantages over the earlier dispersive instruments [43]. In particular, they offer a better signal-to-noise ratio, an increased speed of wavelength scan-ning, and an improved sensivity and accuracy.
The crucial component of a FTIR spec-trometer is a Michaelson interferometer, which replaces the traditional monochromator in the dispersive earlier instruments. Its ad-vantage is that it enables simultaneous scan-ning of all the frequencies at significantly higher resolution. Two mirrors, one moving and the other fixed, reflect each a fraction of the radiation arriving from the IR source via a beamsplitter. The beams are recombined and interfere reciprocally, generating a resulting additive or substractive wave, the interfero-gram, prior to pass through the sample, where selective absorption takes place. As a result, the final signal arriving to the detector con-tains both the signature of the observed com-pound and that of the interferometer, thereby allowing identification of the specific bond vibrations.
Use in skin investigations
Use of a FTIR spectrometer for skin inves-tigations is not a very recent application of FTIR spectroscopy, since it has been used two decades ago for qualitative determinations of in vivo skin water content by Gloor et al.[44-47].
Direct in vivo measurements of the drug content at the surface of an opaque and non-rigid sample like the skin are difficult (if not impossible) to achieve with traditional IR instrumentation. Attenuated total reflectance (ATR) spectroscopy is an original infrared technique in which the infrared radiation is passed through an infrared transmitting crys-tal of high refractive index, allowing the ra-diation to reflect in the crystal one or more times (Figure 8).
In this way, an evanescent wave penetrates into the sample in contact with the crystal, producing a spectrum of it. Because of the
Figure 8: Schematic diagram of a ATR crystal (top) and its housing, mounted in the spectrometer com-partment (bottom). From [48].
dispersion of IR radiation, this technique al-lows however only semi-quantitative deter-minations.
In our studies, a trapezoidal ZnSe crystal measuring 7x1x0.2 cm was used. Cut at an angle of 45°, it allowed 12 IR beam reflec-tions in the frequency range 650-10000 cm-1.
Measurements of the TBF amount in the skin were made by placing the treated site of the arm directly on the crystal of the horizon-tal internal reflection element (Contact Sam-pler® , Spectra-Tech, Stamford, CT) of the ATR accessory. The latter was
placed in a Nicolet 730 FTIR spec-trometer (Nicolet, Madison, WI), equipped with a liquid nitrogen cooled mercury-cadmium-telluride detector. All measurements were performed at air conditioned labo-ratory conditions (21±1°C) and ambient relative humidity (50±10%). A rest period of 30 min was observed after the formulation
was removed, before the first FTIR measure-ment and TS, in order to allow the penetration of the non-absorbed formulaton. A spectrum of the skin was obtained by averaging 64 scans obtained in less than 1 min at a resolu-tion of 4 cm-1, over a range of 650-4000 cm-1 (Figure 9 and Table II).
Up to 15 spectra (corresponding to 15 data points for the concentration profile) were ob-tained for each formulation and subject: the first 10 before a single TS (up to 10 TS), and the last 5 before every dual TS (up to 20 TS),
Figure 9: ATR-FTIR spectrum of human skin in vivo, from the ventral forearm. Carbon dioxide and water vapour are environmental impurities.
Table II: Infrared band assignements for stratum corneum and its components. From [49].
Bond Vibrational motions Compound cm-1 region
O-H stretching lipida 3500-3400
N-H stretching proteina, lipidb 3400-3300
C-H asymmetric and symmetric stretching proteinb, lipida 3000-2700
C=O stretching lipida 1740
C=O stretching proteina (amide I), lipidb 1640
C-N stretching proteina (amide II) 1550
N-H bending
C-H bending, twisting proteinb, lipida 1470-1400
aPrimary component
bSecondary component
since the concentration of drug decreases rap-idly in the deeper layers of SC.
For the spectra analysis, the built-in spec-trometer software (Omnic® version 3.1a, Nicolet, Madison, WI) was used. For the semi-quantification of the drug, an intense distinct TBF absorbance at 774 cm-1 (Figure 10), due to the stretching vibration of the aromatic C-H (Figure 11), was selected,
where no major interfering SC absorbances were observed (Figure 12).
The relative amount of TBF was deter-mined using the area-under-the-curve (AUC) method. This method assumes that IR absorb-ance is linearly proportional to the concentra-tion of the absorbing species (Lambert-Beer's law), and a simple calibration allows to de-termine the absolute amount of it. In the in
vivo determinations, however, a direct calibration is obviously not possible, so we checked the linearity of the response in vi-tro, by spreading directly onto the ZnSe crystal 30 µl of a se-ries of TBF solutions in ETOH:IPM 50:50 at increasing concentrations, ranging from 100 to 300 mg/ml by incments of 50 mg. The linear re-gression yielded a correlation coefficient of 0.9933.
Since the contact of the arm on the ZnSe crystal is difficult to achieve in a reproducible way (provoking a variation of the spectra intensities independently of the amount of drug), the AUC of the TBF peak was nor-malized with respect to the AUC of the amides I+II band (originating, respectively, from the C-O and the N-H stretching
Figure 11: ATR-FTIR spectrum of the characteristic bands of the aromatic C-H absorbing at 774 cm-1.(only the right one was used for quantification).
Figure 10: ATR-FTIR spectrum of terbinafine, with the characteristic bands of the aromatic C-H absorbing at 774 cm-1.
of the amides contained in the SC proteins) located at 1645 cm
-1 and 1545 cm-1 (Figure 13), used as an internal standard. A similar standardization has al-ready been used for the in vivo quantification of water [45,46,50,51] and drugs [52-54]
in the skin.
On the othe hand, the values obtained after this normalization were not normalized with re-spect to the amount of SC sub-sequently removed, since the IR beam is supposed to penetrate, at the considered frequency, at a constant depht into the skin.
Finally, considering that the frequency of maximum absorb-ance of TBF peak (774 cm-1) was close to the inferior fre-quency range limit of the ZnSe crystal (650 cm-1) each spec-trum was automatically cor-rected for baseline and smoothed in the range of 760-790 cm-1 (TBF peak) and 1450-1750 cm-1 (amides I+II bands).
Figure 12: ATR-FTIR spectrum of human skin during tape stripping. The absorbances of stratum corneum do not interfere with terbinafine.
Figure 13: ATR-FTIR spectrum of human skin in vivo (ventral forearm), treated with terbinafine 15% in ethanol.