Received: 1 August 2019 Revised: 29 August 2019 Accepted: 2 September 2019

DOI: 10.1002/tbio.201900004

FULL ARTICLE

Method to quantify the in vivo skin penetration of topically

applied materials based on confocal Raman spectroscopy

Peter J. Caspers1,2 | Claudio Nico1 | Tom C. Bakker Schut1,2 | Johanna de Sterke1 |

Paul D. A. Pudney3 | Patricia R. Curto1 | Abigail Illand1 | Gerwin J. Puppels1,2

1
RiverD International B.V., Rotterdam, The
Netherlands
2
Center for Optical Diagnostics and
Therapy, Department of Dermatology,
Erasmus MC, University Medical Center,
Rotterdam, The Netherlands
3
Beauty and Personnel Care Science and
Technology, Unilever R&D Port Sunlight,
Wirral, UK
Correspondence
Gerwin J. Puppels, Erasmus MC, University
Medical Center Rotterdam - Center for
Optical Diagnostics and Therapy,
Department of Dermatology, Rotterdam,
The Netherlands.
Email: gpuppels@riverd.com
Present address
Patricia R. Curto, Sartorius Stedim Biotech,
Epsom, UK.
Abigail Illand, ISMO, University Paris Sud,
Orsay, France
Abstract
This article describes a unique nonin-
vasive capability to determine the con-
centration (in mg/cm3) and total
amount of topically applied materials
in the skin (in μg/cm2 of skin surface).
It is based on in vivo confocal Raman
spectroscopy. A theoretical derivation
is given of a general method to calcu-
late a concentration ratio from a Raman spectrum of a material in a medium, which
can be a solvent or other matrix, such as the skin. A practical implementation of
the method is then presented along with a clarification of the assumptions used and
applied to a quantitative analysis of the in vivo skin penetration of trans-retinol and
propylene glycol (PG). A comparison was made between the concentrations pro-
files of retinol and PG found in the skin and the concentrations of retinol and PG
that had been applied to the skin. Determination of the amount of these materials in
the skin at different timepoints after topical application also enabled a straightfor-
ward calculation of the flux of materials into the skin (in μg cm−2 h).

KEYWORDS
flux, human, in vivo, noninvasive, penetration, quantitative, stratum corneum

1 | INTRODUCTION properties is important for development, optimization and

The skin is the largest organ of the body with a total surface
area of about 2 m2 in adults. It fulfills regulatory and sensory
functions. The skin also protects the body against the outside
world, preventing water loss as well as the entry of harmful
substances and microorganisms. In particular, the stratum
corneum, the outermost skin layer, is a formidable barrier
which must also be overcome for the delivery of active mol-
ecules through the skin. A good understanding of the barrier
testing of topical products in cosmetics and trans-dermal
drug delivery, as well as for monitoring the potentially harm-
ful penetration of materials from the (work) environment.
In vivo measurement of trans-epidermal water loss
(TEWL), when performed under well-controlled environmental
conditions, provides quantitative information about the barrier
function of the skin against water loss from the body. However,
to date, no noninvasive in vivo method exists to quantitatively
determine the penetration of topically applied materials into the

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https://doi.org/10.1002/tbio.201900004
2 of 10
skin. An invasive method to study the stratum corneum is tape
stripping: removal of cell layers with adhesive tapes. The
removed skin material can be analyzed by various analytical
methods. A common method for quantitative analysis is tape
stripping and analysis with high performance liquid chromatog-
raphy (TS-HPLC) [1]. Depth-related information is obtained
ordering the analysis results according to tape strip number.
The method requires several analytical steps, including solvent
extraction of skin components from the tape and measurement
of the optical density of the tape to determine the protein mass.
These steps, together with the variations in the exact depth and
amount of stratum corneum removed with each tape, have
prevented the development of TS-HPLC into a reliable and
reproducible routine method for the investigation of skin pene-
tration of topically applied products.
A widely used ex vivo technique for investigating skin
permeation of materials is the Franz cell [2–4], although the
conditions under which such experiments are performed are
far from the actual in vivo situation.
Sarri et al have recently demonstrated in vivo quantitative
penetration of deuterated glycerol in the skin using coherent
anti-Stokes Raman scattering (CARS) [5]. Results were
presented in concentration by mass as a function of depth
into the skin. Because the method required deuterated active
molecules to create a distinctive signal detectable by CARS,
this might limit its implementation.
Since its introduction, about 20 years ago, in vivo confocal
Raman spectroscopy has become a well-established technology
for skin analysis, providing a wealth of information about the
molecular composition of the skin with high spatial resolution.
Iconic examples are the profiles showing water concentration
(in mass% of wet tissue weight) as a function of distance to the
skin surface [6, 7], which illustrate the water barrier function of
the skin, and the demonstration of strongly reduced concentra-
tions of natural moisturizing factor (NMF) in the stratum cor-
neum individuals with loss-of-function mutations in the
filaggrin gene [8–10]. Nevertheless, with a few exceptions,
most of the information about molecular skin composition
obtained by Raman spectroscopy has been semiquantitative or
qualitative by nature. Pot et al have reported semiquantitative
concentration profiles of p-phenylenediamine in the stratum
corneum in milligrams of p-phenylenediamine per grams of
keratin [11]. Pudney et al have studied the delivery of trans-
retinol to the skin using confocal Raman spectroscopy. Results
in the retinol papers are shown in concentrations of trans-retinol
in mass percentage of wet tissue [12, 13]. In these papers,
results were not yet presented fully quantitatively in actual con-
centrations (mg/cm3) or total content per skin area (μg/cm2)
and no generalized approach was presented, which can be
applied to different components and solvents.
Here, we introduce a simple comprehensive method,
based on confocal Raman spectroscopy, which enables
CASPERS ET AL.
determination of in vivo skin penetration of materials in
μg/cm2 of skin area for water-soluble, lipid-soluble, and
alcohol-soluble materials.
2 | MATERIALS AND METHODS
2.1 | Theoretical background
The quantitative analysis is an extension of the previously
described semiquantitative analysis, which was based on
spectral fitting of the in vivo skin Raman spectrum with the
Raman spectra of intrinsic skin constituents and the spec-
trum of the material to be analyzed. The ratio of the fit coef-
ficient of the material and the fit coefficient of keratin, the
major stratum corneum protein, was used as a semiquantitate
measure of the material-to-protein ratio in the skin [7].
The fully quantitative analysis presented here is realized
by the following steps:
1. an in vitro calibration step in which Raman spectra are
measured of solutions of the material in a suitable sol-
vent, at different concentrations. This calibration enables
calculation of the mass ratio mmaterial:msolvent from the
measured Raman signal intensity ratio Rmaterial:Rsolvent;
2. an in vitro calibration step for the solvent and stratum cor-
neum protein. This enables calculation of the mass ratio
mmaterial:mprotein in the stratum corneum as a function of
Rmaterial:Rprotein; (expressed in mg material/g protein);
3. multiplication of the mass ratio mmaterial:mprotein by the
protein concentration in the stratum corneum. This results
in the material concentration in the skin (in mg/cm3);
4. integration of the material concentration over the stratum
corneum thickness. This results in the total amount of mate-
rial in the stratum corneum per skin surface area (μg/cm2).
The intensity of the Raman signal of a material is propor-
tional to the concentration of that material in the measurement
volume. It furthermore depends on the Raman scattering
properties of the material and on experimental factors such as
the size of the measurement volume, laser intensity, and mea-
surement geometry. In an inhomogeneous medium like the
human skin, the surface roughness, skin layers and structures,
and cellular structures unpredictably affect the experimental
factors and thereby affect the intensity of the detected Raman
signal. As a result, the absolute intensity of the Raman signal
of the material in the skin cannot be directly related to its
concentration. However, the ratio of the signal intensities
of two materials is proportional to the ratio of their concen-
trations in the measurement volume. This holds true for
materials in a homogenous solution as well as for materials
in a heterogenous medium. For a material dissolved in a
solvent we can write:
CASPERS ET AL. 3 of 10

Rmaterial γ The Raman signal intensity ratio RA/Rprotein can be deter-

= cmaterial:solvent ∙ material ð1Þ
Rsolvent γsolvent mined from a Raman spectrum of the skin containing mate-
rial A. This method can be generalized to materials which
in which Rmaterial/Rsolvent is the ratio of the Raman signal dissolve in other solvents using Equation (3).
intensities, γ material/γ solvent is the concentration ratio, and Multiplication of the mass ratio (mA/mprotein) with the pro-
cmaterial:solvent is the proportionality constant for the material tein concentration in the skin (γ protein) yields the concentration
in the solvent. of the material in the skin (γ material). Mass ratio and concentra-
Because the material and the solvent share the same mea- tion are functions of the distance z to the skin surface.
surement volume, the ratio between the concentrations is


equal to the mass ratio. mA  
γ A ðzÞ = ∙γ protein ðzÞ μg=cm3 ð6Þ
mprotein z
Rmaterial mmaterial
= cmaterial:solvent ∙ ð2Þ
Rsolvent msolvent Integrating γ A(z) over the full penetration depth results in
the total amount MA of material A per area of skin surface:
cmaterial:solvent can be determined by plotting the Raman
intensities Rmaterial/Rsolvent against mmaterial/msolvent for solu- ð skin thickness
tions in a range of mmaterial/msolvent ratios. The slope of the MA = γ A ðzÞ∙dz ð7Þ
graph is equal to cmaterial:solvent. 0

By carrying out this procedure for solutions of a material

A in a solvent and for solutions of a material B in the same By definition, protein concentration is:
solvent, proportionality constants cA:solvent and cB:solvent are
mprotein
determined. From this we obtain the proportionality constant γ protein = ð8Þ
V
cA : B between materials A and B.
in which mprotein is the mass of protein in a given volume V.
−1
cA:B = cA:solvent ∙cB:solvent ð3Þ One can consider the volume V to be comprised of the
partial volumes Vi occupied by each of the materials present
This step implies that a proportionality constant between (protein, other dry mass constituents, water, topically applied
Raman signal intensity ratio and mass ratio can be determined product):
for two materials separately, even if the two materials were
never measured together in the same solution. Another impli- X
n X
n
mi
cation of Equation (3) is that the proportionality constant can V= Vi = ð9Þ
i i
ρi
be determined between materials dissolved in different sol-
vents. For example, for a material A dissolved in ethanol and
where ρi is the density and mi is the mass of material
material B dissolved in water the proportionality constant for
i present in the mixture (see also the Section 4 under
A relative to B, cA:B, can be calculated by combining three
Section 4.1 where the current approximations that are used
constants:
in the quantification method, are discussed).
Therefore, the concentration of protein as a function of
−1
cA:B = cA:ethanol ∙cethanol:water ∙cB:water ð4Þ the distance z to the skin surface can be expressed as:

where cethanol:water is the proportionality constant of ethanol
X  − 1
X −1

n mi n1 mi
in water. γ protein ðzÞ = mprotein ∙ i ρ
= i ρ m
∙
i i protein
Here, this approach is used to determine the mass ratio
between a material A in the skin and the protein fraction in ð10Þ
the skin from the ratio of their Raman intensities RA/Rprotein.
In case of a water-soluble material, this requires determina-
tion of cA:water and of cprotein:water. Substitution in Equa- 2.2 | Implementation
tion (2) and reversing the order gives:
The proportionality constant of protein in water was deter-
mA RA mined from the Raman spectra of aqueous solutions of pro-
−1
= cprotein:water ∙cA:water ∙ ð5Þ tein in known protein/water mass ratios. Stratum corneum
mprotein Rprotein
protein, which consists predominantly of keratin, is insoluble
4 of 10
and cannot be used to determine the proportionality constant
cprotein:water directly. Bovine serum albumin (BSA) is highly sol-
uble in water and was used instead. Using Equation (3), the
proportionality constant cprotein:water follows from multiplication
of the proportionality constant between protein and BSA and
the proportionality constant between BSA and water.
cprotein:water = cprotein:BSA ∙cBSA:water
The Raman signal intensity of BSA is comparable to that
of stratum corneum protein [7], which implies that equal
concentrations of BSA and stratum corneum protein give
approximately the same Raman signal intensities, so cprotein:
BSA is approximately 1.
For the quantification of retinol, which dissolves well in
ethanol, the proportionality constants were determined for
retinol in ethanol and for ethanol in water. From Equa-
tions (4) and (5), the combination of cprotein:BSA, cBSA:water,
cethanol:water, and cretinol:water were then used to determine the
retinol/protein mass ratio from Raman intensity ratios
Rretinol/Rprotein calculated from in vivo Raman spectra of
the skin.
mretinol −1 −1 −1
= cBSA:protein ∙cBSA:water ∙cethanol:water ∙cretinol:ethanol ∙
mprotein
The Raman signal intensities in Equation (5) were calcu-
lated by an ordinary least squares fit of the intensity-
normalized reference spectrum of the material and the
intensity-normalized reference spectrum of the solvent to the
Raman spectrum of the solution. The Raman signal intensi-
ties were calculated as the fit coefficients b from the least
squares estimate, b = (XTX)−1XTy, in which y is the depen-
dent Raman spectrum, X is a matrix with in the columns the
independent reference spectra, and b is a vector of fit coeffi-
cients for each column in matrix X. The superscript T is the
matrix transpose of X and (XTX)−1 is the matrix inverse of
(XTX). The dependent Raman spectrum was the spectrum of
the solution, the independent Raman spectra were a refer-
ence Raman spectrum of the material and a reference Raman
spectrum of the solvent.
In this experiment, the amounts of propylene glycol
(PG) in the stratum corneum were not negligible. Therefore,
applying Equation (10) the protein concentration as a func-
tion of distance z to the skin surface is:
1 mdry SC 1 mwater ðzÞ
γ protein ðzÞ = ∙ + ∙ BSA in water were prepared with mass ratios of 0.0, 10.1,
ρdry SC mprotein ρwater mprotein ðzÞ
CASPERS ET AL.
1 mPG ðzÞ − 1
+ ∙ Þ ð13Þ
ρPG mprotein ðzÞ
Equation (13) is comprised of known factors:
• The density of the dry stratum corneum [11] is
1.15 g/cm3; the densities of water and PG are, respec-
ð11Þ tively, 1.0 g/cm3 and 1.036 g/cm3.
• The dry mass of stratum corneum consists of 15% lipids
and between 20% and 30% natural moisturizing factor
[12–16]. Therefore, the mass ratio between protein and
the dry stratum corneum (mprotein/mdry SC) is about 60%.
• The ratio mwater/mprotein, was determined by multiplying
mdry SC/mprotein by the water to dry stratum corneum mass
ratio mwater/mdry SC, which follows directly from the well-
known water concentration profiles [7, 17–21]. The water
concentration profile was approximated by a linear gradi-
ent, ranging from 25% at the skin surface to 65% at the
boundary between stratum corneum and living epidermis.
• The mass ratio mPG/mprotein is determined experimentally
by applying Equation (5).
Substitution of Equation (13) in Equation (5) yields MA;
Rretinol the amount of material A in the skin per cm2 of skin surface.
Rprotein This is calculated in μg/cm2. The volar forearm stratum cor-
ð12Þ neum thickness was approximated by a constant value of
15 μm [17, 18, 22, 23] in all calculations.
An estimate of the flux JA of a topically applied material
A into the skin in (μg cm−2 h−1) is obtained by determining
the decrease of applied material A in the skin over time;
JA = −ΔMA/Δt.
2.3 | Sample preparation
Ethanol 99.8% was purchased from Acros Organics (Geel,
Belgium). Bovine serum albumin (BSA) and retinol were
purchased from Sigma-Aldrich (Zwijndrecht, The Nether-
lands). All chemicals were used as purchased without further
purification. The solutions were prepared by weight using an
analytical balance and homogenized by placing the sample
tubes on a roller mixer RM5 (Karl Hecht, Sondheim, Ger-
many). Solutions of trans-retinol in ethanol were prepared
with mass ratios of 0.0, 9.6, 19.8, and 48.9 mg/g retinol:eth-
anol. Solutions of PG in water were prepared with mass
ratios of 0.0, 10.0, 30.0, 49.9, and 99.9 mg/g PG:water.
Solutions of ethanol in water were prepared with mass ratios
of 0.0, 5.0, 10.0, 20.2, 52.9 mg/g ethanol:water. Solutions of
30.9, 51.5, 75.4 mg/g BSA:water.
CASPERS ET AL.
2.4 | Raman spectroscopy
In vitro Raman measurements were performed with a
gen2-SCA confocal Raman spectrometer with RiverICon
4 instrument control and data acquisition software (RiverD
International B.V., Rotterdam, The Netherlands). The laser
wavelength was 785 nm, laser power on the sample was
25 mW. For solution measurements, 30 μL was pipetted in a
stainless-steel sample cup which was then placed directly on
the measurement window of the instrument (Fig. 1). The sample
cup shielded the measurement from environmental light
and prevented evaporation from the sample. Of each sample,
100–200 Raman spectra were measured with an exposure
time of 5 seconds per spectrum.
In vivo Raman spectroscopy data from the paper by
Pudney et al [12] were reanalyzed. Briefly, in this study a
trans-retinol solution in PG and ethanol was prepared in the
following mass ratio: 70% ethanol:30 PG:0.3% trans-retinol.
A 4 cm × 4 cm test area on the central part of the volar fore-
arm was marked. A dose of 70 μL of the solution was
applied on the skin and spread gently, without rubbing, over
the test area using the tip of a micropipette. Raman measure-
ments started 10 minutes after the application of solution to
the skin. Baseline measurements were taken from the marked
area prior to application of the solution. in vivo Raman mea-
surements were performed using a model 3510 Skin Compo-
sition Analyzer (RiverD International, Rotterdam, The
Netherlands). Spatial resolution in the axial direction (perpen-
dicular to the skin surface) [7] was 5 μm. At each timepoint,
measurements were performed at eight different locations
within the skin area to which the retinol solution was applied.
At each location Raman spectra were recorded from the skin
surface to a depth of at least 30 μm with a step size of 2 μm.
The spectral resolution, spatial resolution, and laser wave-
lengths (671 and 785 nm) of the Model 3510 SCA, used in
the in vivo study of Pudney et al [12] and of the gen2-SCA,
used for the in vitro calibration measurements, are the same.
Both systems use a spectrometer design which renders signal
throughput from sample to CCD-detector virtually
FIGURE 1 Sample cup for
measuring calibration solutions. The
sample was pipetted in a small cavity in
the sample cup. The sample cup was,
sample facing down, placed on the
measurement window
5 of 10
wavelength independent and polarization independent. This
enables data preprocessing (wavenumber calibration, correc-
tion for wavenumber dependent system signal detection effi-
ciency, system background signal subtraction), which renders
the spectra of both the Model 3510 SCA and the gen2-SCA
free of any instrument signatures. This in turn enables the use
of spectra obtained with gen2-SCA for the analysis of spectra
obtained with a Model 3510 SCA system (and vice versa).
2.5 | Data preprocessing
All Raman spectra were corrected for instrument back-
ground and instrument wavelength dependent response and
interpolated on a calibrated relative wavenumber axis by the
RiverICon 4 software.
2.6 | Data processing
High quality Raman spectra of solutions were obtained. A
reference spectrum of the material dissolved in a solvent was
created by subtraction of the solvent spectrum from the solu-
tion spectrum. A Raman signal intensity ratio Rmaterial/Rsolvent
was calculated on the basis of an ordinary least squares fit to
the solution spectra by the reference spectrum of the material
and the solvent spectrum. A first order polynomial was
included in the fit to account for slight variations in spectral
backgrounds. The proportionality constant cmaterial:solvent was
calculated as the slope of the linear regression of Raman sig-
nal intensity vs mass ratio [Equation (2)].
In vivo spectra of the skin were fitted with a set of spec-
tra of intrinsic stratum corneum constituents. The set con-
sists of reference spectra of keratin, natural moisturizing
factor (NMF), urocanic acid, urea, lactate, ceramide, and
cholesterol and has been described in detail elsewhere [7].
For analysis of the skin penetration of a topically applied
material, the reference spectrum of the material was added to
the set of spectra of intrinsic stratum corneum constituents.
A third order polynomial was included in the least squares
fits to account for signal background. The mass ratio
6 of 10
mmaterial:mprotein as a function of distance to the skin surface
was calculated from the fit coefficients of the material and
keratin from the fit model, using Equation (5) for PG and or
Equation (12) for trans-retinol.
The total amount of material present in the skin, expressed
in mass of material per unit skin area, was calculated by
numerical integration of the material concentration over the
average stratum corneum thickness of 15 μm [17, 18, 22, 23].
The results obtained at the eight measurement locations
for each timepoint were averaged, to yield a single retinol
concentration profile and a single PG concentration profile
for each timepoint.
Adding reference spectra of PG and retinol to the set of
fit spectra, will also result in small nonzero fit contributions
to the spectra of untreated skin. The baseline fit
(A)
(C)
FIGURE 2 Raman spectra of calibration solutions. A, Bovine serum albumin in water; B, PG in water; C, ethanol in water; D, trans-retinol in
ethanol. The spectra have been offset vertically for clarity. Concentrations of the solutions and Raman signal collection times are mentioned in the Section 2
CASPERS ET AL.
contributions of retinol and PG were subtracted from the
results at time points after topical application of the retinol-
solution.
SkinTools 3 analysis software (RiverD International
B.V., Rotterdam, The Netherlands) was used to quantify
penetration of topically applied materials in the skin
according to the method described above. This includes the
method to determine the proportionality constants and refer-
ence spectra of materials in different solvents, as well as
quantitative analysis of the in vivo Raman measurements.
3 | RESULTS
Figure 2 shows the Raman spectra of the calibration solu-
tions used to determine the proportionality constants for
(B)
(D)
CASPERS ET AL. 7 of 10

BSA, ethanol, PG, and retinol. The Raman intensity ratios
between material and solvent were calculated from the spec-
tra and plotted against their mass ratio in Figure 3, together
with the linear regression lines for each material. The slopes
of the regression lines, which are the proportionality con-
stants in Equation (2), are given in the figure caption.
The distribution and total amount of retinol and PG pene-
trated into the skin were determined from the original
in vivo Raman spectra measured by Pudney et al [12]. Mass
ratios of retinol and PG with respect to protein were calcu-
lated using the proportionality constants for retinol and for
PG (Figure 3), and multiplied by the protein concentration to
obtain the concentration as a function of distance to the skin
surface. Figure 4 shows the concentration profiles and total
amount of trans-retinol in the skin measured over a period of
6 hours after application. Figure 5 shows the concentration
profiles and total amount of PG determined from the same
Raman measurements.

4 | DISCUSSION

In this article, we describe a novel and unique method to fully

FIGURE 3 Raman intensity ratio vs mass ratio. The slope
proportionality constants are the slopes of the fitted line [see
Equation (2)]. The slopes ± least squares errors are: trans-retinol in
ethanol 35.8 ± 0.3; ethanol in water 22.2 ± 0.1; PG in water
15.3 ± 0.2; bovine serum albumin in water 10.65 ± 0.04. Coefficient
of determination for all lines: R2 > .999
quantify components in the human stratum corneum using
in vivo confocal Raman spectroscopy. This is demonstrated by
the quantification of PG and retinol using the original Raman
spectra which were obtained by Pudney et al in 2006 [12].
At time 0-1 hour after the 10 minutes application period,
a total amount of 4.4 μg/cm2 trans-retinol was detected in
the skin, which amounts to 40% of the applied dose
(11 μg/cm2). For PG this was 1.8 × 102 μg/cm2, which
amounts to 16% of the applied dose (1.12 × 103 μg/cm2).
These percentages of recovered retinol and recovered PG
both seem reasonable. Also, the concentrations measured
near the skin surface at time 0-1 hour are comparable to the
concentrations of trans-retinol and PG in the applied solu-
tion. Although ethanol readily penetrates into the skin when
applied under circumstances that prevent evaporation, for
example, when the treated area is covered, no traces of etha-
nol were found in the skin in this study. Therefore, it is clear
that the ethanol evaporated after application of the retinol:

(A) (B)

FIGURE 4 Quantitative analysis of the concentration and total amount of retinol in the skin of the volar forearm after topical application of a
0.3% solution of retinol in 30% PG:70% ethanol. A, Concentration profiles as a function of distance to the skin surface in mg/cm3 tissue measured
over a period of 0-6 hours after application. B, Total amount in μg/cm2 skin area. The error bars show the SD of the eight repeated measurements at
different locations within the treated skin area. The dashed line is a linear fit to determine the flux of PG. in vivo data were reused from Pudney
et al [12]
8 of 10 CASPERS ET AL.

(A) (B)

FIGURE 5 Quantitative analysis of the concentration and total amount of PG in the skin of the volar forearm after topical application of a
0.3% solution of retinol in 30% PG:70% ethanol. A, Concentration profiles as a function of distance to the skin surface in mg/cm3 tissue measured
over a period of 0-6 hours after application. B, Total amount in μg/cm2 skin area. The error bars show the SD of the eight repeated measurements at
different locations within the treated skin area. The dashed line is a linear fit to determine the flux of PG. in vivo data were reused from Pudney
et al [12]

PG:ethanol mixture on the skin. The volume ratio of the PG:
ethanol mixture was 1:3. Therefore, evaporation of the etha-
nol effectively reduced the volume by a factor of 4, and
increased the concentration of retinol in the remaining PG
by the same factor. Hence, taking into account the evapora-
tion of ethanol, the calculated concentrations in the applied
solution are 10 mg/cm3 for trans-retinol and 1.0 g/cm3 for
PG. The detected concentration in the skin, near the skin sur-
face, at time 0-1 hour, was 11 mg/cm3 for retinol (Figure 4),
which is slightly higher than the concentration in the applied
solution after evaporation of the ethanol. Retinol dissolves
well in ethanol and lipids. In the applied solution retinol was
dissolved in ethanol, which evaporated after application to
the skin. Because trans-retinol has a larger affinity with the
lipids in the stratum corneum than with PG, we speculate
that this difference in affinity has increased the concentration
in the skin slightly above the value of the concentration in
the solution after evaporation of ethanol. The detected con-
centration of retinol after product application, is 3-to-4
orders of magnitude higher than endogenous retinol levels
reported for human stratum corneum [24, 25]. The detected
concentration in the skin for PG was 0.36 g/cm3 (Figure 5),
which was 36% of the concentration in the applied solution
after evaporation of ethanol. The SDs in Figures 4 and 5
show differences between the measurement locations within
the treated skin area, that is, variations in the distribution of
retinol and PG due to biological variation. Repeated mea-
surements on one location of the skin surface typically have
a very high reproducibility (results not shown). Therefore, a
reduction of the SD can be achieved by averaging over a
larger number of measurements at different locations.
The retinol and the PG were applied to the skin in a mass
ratio of about 1:100. The retinol flux was 0.6 μg cm−2 h−1
and the flux of PG was 8 μg cm−2 h−1, yielding a flux ratio of
about 1:13. The development of the concentration profiles of
retinol (Figure 4A) and PG (Figure 5A) suggests that the
boundary between the stratum corneum and the living epider-
mis present a bigger barrier to PG permeation than diffusion
through the stratum corneum itself, unlike the case for retinol.
In this section, we describe prerequisites, assumptions,
and approximations used in a practical implementation of
the method, and we highlight its most noticeable advantages.
A prerequisite of the method is a robust and reproducible
definition of the Raman signal intensity. We have defined
Raman intensity of a material as the fit coefficient of the ref-
erence spectrum of that material using an ordinary least
squares regression. This requires that the Raman spectrum of
the material in skin is the same as the reference Raman spec-
trum of the material in solution. If this is not the case, for
example, due to molecular interactions in the solution or in
the skin, the fitted spectrum will not accurately represent the
spectrum of the material in the skin. This may lead to a less
accurate determination of the amount of material present in
the skin. Here, we have neglected this potentially con-
founding effect. Another requirement of this implementation
is a fit model to accurately represent the in vivo skin spec-
trum, that is, a set of reference spectra for which the residual
of the least squares fit to the in vivo skin spectrum is small.
The fit model should therefore be representative of the
intrinsic skin components and of the topically applied mate-
rials. We have used an established fit model that accurately
represents in vivo Raman spectra of the stratum corneum
CASPERS ET AL.
[7]. The method to determine the proportionality constant of
a material requires that the material is soluble in a suitable
solvent. Solubility must be sufficient to enable the Raman
intensity ratio between material and solvent at a range of
concentrations. Because, the method is not limited to partic-
ular solvents, this requirement can be easily met.
4.1 | Approximations
The approximation is used that the Raman signal intensity of
the BSA molecule is equal to the signal intensity of the pro-
tein in the skin, meaning that their proportionality constant
is equal to 1. The uncertainty of this approximation was esti-
mated to be less than 15%, based on measurements of differ-
ent proteins [7]. This may give rise to a systematic error in
the calculation of the amount of a material in the skin [see
Equations (11) and (12)].
Referring to Equation (9), we have assumed that the total
volume of the materials present in the skin is equal to the
sum of the volumes of the individual materials. This approx-
imation holds true, except in cases in which solids dissolve
in liquids. Of the dry stratum corneum components (pro-
teins, lipids, and NMF) the proteins and lipids do not dis-
solve in water. The amino acids, amino acid derivatives, and
salts making up the NMF, will dissolve to varying degrees.
This might introduce a small error, which we have not fur-
ther addressed.
Additionally, converting the quantification of a material
from g/gprotein to μg/cm3, as expressed in Equation (6),
requires a protein concentration profile in the skin. For this,
we have approximated the water mass percentage in the stra-
tum corneum by a linear gradient of a water mass percentage
of 25% at the skin surface to 65% at a depth of 15 μm, which
is based on numerous in vivo confocal Raman profiles mea-
sured on the volar forearm in a variety of studies [7, 17–21].
We have approximated the protein fraction to be 60% of
the stratum corneum dry mass. This approximation is based
on literature that reports 15% of the dry stratum corneum
mass to consist of lipids, and 20%-30% to consist of natural
moisturizing factor (NMF), leaving a remaining 60% for pro-
tein [14–16]. A value of 1.15 g/cm3 density of the stratum
corneum dry mass was used [26].
Finally, for the calculation of the total amount of a mate-
rial in stratum corneum [Equation (7)], we need to integrate
the depth profile over its thickness. Stratum corneum thick-
ness varies between measurement locations. In this article,
we approximated the volar forearm stratum corneum thick-
ness by a constant value of 15 μm [17, 18, 22, 23].
Refinement of these approximations is of course possible.
Instead of using a fixed stratum corneum thickness and
approximating the water mass percentage by a fixed linear
gradient, the actual local water concentration profiles (and
9 of 10
the actual stratum corneum thickness, derived from these
profiles) could be measured and used in the calculations.
Choe et al have made suggestions for further improvement
of protein concentration determination in the stratum cor-
neum [27].
5 | CONCLUSIONS
We describe a novel and unique method to fully quantify
materials in the human stratum corneum as well as the flux of
materials through the stratum corneum using in vivo confocal
Raman spectroscopy. We have clarified the assumptions that
have been made at this stage of development of the method.
Apart from the intrinsic characteristics of the confocal
Raman spectroscopy technique, which can be used in vivo
and nondestructively, the method presented here enables
(simultaneous) quantification of many different types of mate-
rials in the skin. The required calibration process of a material
in a solvent is straightforward. We have determined the cali-
bration constants of water, ethanol, and oleic acid (not shown)
against protein. Many materials are soluble in these solvents
but, if necessary, different solvents might be used as well.
In this article, we have focused on the quantitative analysis
of topically applied material penetration into the stratum cor-
neum. We are currently developing additional fit models that
will allow extension to the living epidermis and to the dermis.
To our knowledge, to date no other technique can readily
provide a direct quantitative in vivo analysis of penetration
of materials into the skin or the flux of materials into the
skin. We expect this method to become of value in a wide
array of applications including development, optimization
and testing of topical products in cosmetics and trans-dermal
drug delivery and in risk assessments of occupational skin
exposure to materials.
A C KN O W L E D G E M E N T S
The authors thank Mr. R. Puppels and Mr. F. Pelizza for
their contributions to the in vitro calibration measurements.
OR CI D
Peter J. Caspers https://orcid.org/0000-0002-6122-5049
Gerwin J. Puppels https://orcid.org/0000-0001-8017-1923
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How to cite this article: Caspers PJ, Nico C, Bakker
Schut TC, et al. Method to quantify the in vivo skin
penetration of topically applied materials based on
confocal Raman spectroscopy. Translational
Biophotonics. 2019;1:e201900004. https://doi.org/10.
1002/tbio.201900004