AAPS PharmSciTech (2022) 23: 73

DOI: 10.1208/s12249-022-02224-w

Research Article

In Vitro Assessment of Sunscreen Efficacy Using Fourier Transform Infrared

(FTIR) Spectroscopy on Synthetic Skin

Farah Mahdi Abd Ali Al-Saeedi1 and Eman Zmaily Dahmash1,2,3

Received 13 August 2021; accepted 20 January 2022 ; published online 11 February 2022
Abstract Although there are several methods for assessing the sun protection factor (SPF) of
sunscreen products, there is no standard and reliable in vitro method. Each test entails
limitations and drawbacks. Therefore, this study aimed to assess the employability of FTIR as
an alternative and quick method to evaluate the efficacy of various sunscreen formulations,
their concentrations, and the timing of their application. Infrared radiation has longer
wavelengths than ultraviolet, penetrates deeply into the skin, and hence enables the
understanding of sunscreens’ ability to block the transmission of radiation. The FTIR
transmission using synthetic skin to study the effect of sunscreen agents (oxybenzone, octyl
methoxycinnamate, titanium dioxide (TiO2), and zinc oxide (ZnO)) was conducted in the
range 450–4000cm−1. Comparison studies were made at the peak of 805cm−1. After 2 h of
sunscreen application, using the maximum concentrations, the FTIR peak at wavenumber
805cm−1 demonstrated a significant reduction of transmission from 96.55 to 60.09%, 57.59%,
32.02%, and 37.1% for oxybenzone, octyl methoxycinnamate, TiO2, and ZnO respectively
(P<0.05). A significant reduction in transmission was observed (P<0.05) with increasing
sunscreen concentrations after 2 h of application. Nevertheless, the upper limit of
concentration showed no appreciable change from the middle level of concentration, and
hence it is cost-effective to employ the middle concentration. Inorganic sunscreens showed a
higher protection level than organic. Fixed-dose combinations of sunscreens showed an
enhanced effect yet were not synergistic. In conclusion, the use of FTIR spectroscopy with
synthetic skin is a quick and user-friendly technique that enables the assessment of the
efficacy of sunscreen formulations.
KEY WORDS: sunscreen formulations; FTIR spectroscopy; oxybenzone; octyl methoxycinnamate;
topical delivery.

INTRODUCTION
The prevalence of skin cancers and photodamaging
effects caused by ultraviolet radiation has increased the use
of sunscreen agents, which have shown valuable effects in
reducing the symptoms and reoccurrence of these problems
(1). However, manufacturers’ labeling of sunscreens varies
greatly, confusing the consumers regarding the efficacy and
the appropriateness of photoprotection provided by their
products.
The solar spectrum consists of ultraviolet (UV), infrared
(IR), and visible (VIS) radiation. The UV radiation is divided
into three regions: (UVA), (UVB), and (UVC) (2). The UVC
1
Faculty of Pharmacy, Isra University, Amman, Jordan.
2 tion into the skin layers (1).
Department of Applied Pharmaceutical Sciences and Clinical
Pharmacy, School of Pharmacy, Isra University, P. O. Box 33, Isra
University Office, Amman, 11622, Jordan.
3
To whom correspondence should be addressed. (e–mail: eman.
zmaily@iu.edu.jo)
radiation is blocked by the ozone layer and does not reach
the earth’s surface. While the UVA radiation has a longer
wavelength, therefore, its rays penetrate deeper into the skin
and cause photoaging, immunosuppression, and
photocarcinogenesis (3) . The UVB radiation is not
completely filtered out by the ozone layer and has a positive
and negative effect. The negative effect of UVB is that it
causes erythema (sunburn), but the positive effect is its
contribution to the production of vitamin D (4). On a
summer’s day, UV radiation consists of 96.5% UVA and only
3.5% of UVB (5–7). The visible part of the solar spectrum
ranges from 400 to 780 nm, followed by the infrared part from
780 to 3000 nm. UV, VIS, and IR differentiate themselves
with their energy and ability of their penetration depth into
the skin; the longer the wavelength, the deeper the penetra-
The core ingredients of sunscreen products are UV
filters; they can reduce the intensity of UV light reaching
the skin. Chemical/organic filters play an important role in
absorbing UVB radiation, but physical inorganic filters are

1530-9932/22/0200-0001/0 # 2022 The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists
73 Page 2 of 11
able to block UVA/UVB sunlight through reflecting and
scattering. Organic filters contain chromophores that absorb
wavelengths greater than 200 nm. These compounds absorb
high-energy UV rays and release the energy as lower-energy
rays, thus preventing the skin-damaging UV rays from
reaching the skin (8, 9). Inorganic agents (physical sunblock)
block sunlight by reflecting or scattering UV radiation over a
broad spectrum of wavelengths (10). The most common
physical UV filters are TiO2 and ZnO (11). These metal
oxides are generally less toxic, more stable, and safer for
humans than those of organic ingredients (11).
The effectiveness of a sunscreen is usually expressed by
the sun protection factor (SPF), which is characterized as the
UV energy required to create a minimal erythema dose
(MED) on protected skin, divided by the UV energy required
to produce a MED on unprotected skin (1, 2). This means
when a sunblock with SPF 30 is applied, it will protect the
skin from UVB radiation 30 times longer than the time
needed to burn unprotected skin (1).
There are several tests for SFP efficacy, in vivo, in vitro,
and in silico. The in vivo test for the SPF is done by applying
an artificial UV-light dose and gradually increasing it on areas
of human volunteers’ backs, on sunscreen protected and
unprotected zones. This method is considered the gold
standard for the evaluation of efficacy (12). However, the
evaluation of the in vivo test performance is reliant on using a
light source with spectral irradiance that differs from original
sunlight. Testing the SPF under solar simulated light source
exposure showed that the UVB is overestimated compared to
natural sunlight exposure (4). The key disadvantages of the
in vivo method are that it is expensive, does not provide any
information on the absorbance profile of the sunscreen study,
and may violate the ethical consideration of human testing
(13). Therefore, several in vitro models were explored.
In vitro assessment of SPF is based chiefly on two
approaches. The first one involves the measurement of
absorption or the transmission of UV radiation through the
sunscreen product spread as a film on a suitable UV
transparent substrate such as quartz plates or biomembranes
(14). The second method involves determining the absorption
characteristics of the sunscreens’ agents based on spectro-
photometric analysis of dilute solutions (12), which is based
on the work of Mansur et al. (15). They developed a very
simple mathematical equation to estimate the sun protection
factor by an in vitro method using UV spectrophotometry
(13). The in vitro methods can be used during the production
process, and in the analysis of the final product, to provide
important information before proceeding to the in vivo tests.
The assessment of the SPF value using the Mansur equations
requires the sunscreen agent to dissolve and is not applicable
for inorganic sunblock agents (13).
Models for the calculation of the SPF in silico employ the
same algorithm used with in vitro SPF measurements.
Nonetheless, this algorithm replaces the transmission mea-
surement by the calculation of the combined extinction of the
UV filters in an uneven sunscreen film. Therefore, the
simulations require a database with UV spectra of the
relevant UV filters, in addition to a mathematical description
of the film irregularity among other tedious requirements,
making it a user-unfriendly method (16).
AAPS PharmSciTech (2022) 23: 73
Unlike biological skin, synthetic skin is easy to obtain and
store. The use of synthetic skin also minimizes the variability in
permeations that are associated with the utilization of biological
skin (17). These advantages, besides the ethical concerns, have
directed scientists to look for synthetic membranes. Strat-M®
(Merck Millipore, USA) is a skin-mimic synthetic membrane,
recently launched and is now commercially available. This
membrane is made of multiple layers of polyester sulfone (18).
Infrared radiations can penetrate the skin at various
levels depending on the wavelength (19). Fourier transmis-
sion infrared (FTIR) analysis is a simple, non-invasive
technique that has been proven to successfully assess collagen
in the skin (20), and determine water content in the stratum
corneum in the skin (21). Similarly, Binder and coworkers
(22) used FTIR on a pig’s ear to study the penetration of
drugs and additives. Permeation through a synthetic mem-
brane and human skin was investigated for model formula-
tions and a commercial ibuprofen formulation FTIR (23).
Furthermore, using FTIR on human skin in situ, attracted
many medical (especially melanoma) and dermatological
researchers, since it is safer than other UV-Vis techniques
used in the cosmetics industry (20, 24–27).
Despite the presence of several methods to assess the
SPF, there is no standard in vitro method that is reliable and
user-friendly. Each test entails limitations and drawbacks. The
effect of different sunscreen agents’ concentration and
duration of administration was not addressed comprehen-
sively. Therefore, this project will employ FTIR as an
alternative method to evaluate the effect of various sunscreen
agents, their concentrations, and the timing of their applica-
tion on IR transmission through validated synthetic skin
(START M®). Although IR has a higher wavelength than
that of UVA, and UVB, it penetrates the skin and hence
blocking IR penetration (transmission) could be used to
relate to sunscreens’ effect on the skin. This study further
aims to investigate the employability of FTIR spectroscopy
for evaluating the changes in concentration, type of sunscreen
agent, and the application time on light transmittance.
MATERIALS AND METHODS
Materials
Oxybenzone (Benzophenone-3) (Symrise, Holzminden,
Germany), octyl methoxycinnamate (Aktin Chemicals, China),
titanium dioxide (TiO2), and zinc oxide (ZnO) (The Ten
Development Limited, China) were donated by Advance Pharma
Care (Amman, Jordan). The HPLC-grade water was purchased
from Labchem (USA). Glycerin was purchased from BBC
Chemicals (Oldenburg, Germany). The Strat-M® (size -x-
diameter 25 mm) membrane was purchased from Merck
Millipore LTD (Germany). All chemicals and reagents were used
as supplied. The commercial cream used for the cream prepara-
tion was Natural Glow® basic cream and was purchased locally
from Natural Glow Health and Beauty (Amman, Jordan).
AAPS PharmSciTech (2022) 23: 73 Page 3 of 11 73

Methods RESULTS AND DISCUSSION

Preparation of Sunscreens Containing Cream Formulations The Effect of Sunscreen Agents’ Concentration, Type, and
Using Organic and Inorganic Materials Application Timing on FTIR Transmission

The required amount of the sunscreen was accurately This manuscript was focused on investigating the change
weighed using an analytical 4-digit balance (Sartorius, of FTIR transmission through synthetic skin (Strat-M®) upon
Goettingen, Germany). Then 5.0 g of the basic cream was the application of sunscreen agents. Initial attempts were
also accurately weighed using an analytical 4-digit balance made to assess the change in the FTIR transmission when the
(Sartorius, Goettingen, Germany). The sunscreen was dis- blank cream was placed on the synthetic skin, then key
persed and mixed with the basic cream using the trituration troughs were selected and monitored for a change in
method. The concentrations used of the sunscreens are listed transmission intensity upon the application of the sunscreen
in Table I. formulas. Furthermore, the effect of the application time on
the peak transmission intensity was evaluated.
This FTIR transmission is a spectroscopic method that
Preparation of Samples for FTIR Measurements can be used to monitor the transmission of IR radiation at
different wavenumbers. The FTIR spectra of the Strat-M®
The first step in sample preparation was the application synthetic membrane range are from 450 to 4000 cm−1.
of the sunscreen formula on the synthetic skin, where 2.0 mg/ Figure 1 shows several bands with transmission exceeding
cm2 from each formula was applied on a 25-mm (diameter) 90%, which makes it a good substrate for performing the
disc of Strat-M® and spread manually. The skin was placed FTIR experiments. Any change in the transmission of the
on the FTIR spectrometer. The FTIR spectra were recorded selected troughs could indicate the presence of a blockage of
on the synthetic membrane and components using a Perkin the transmission, and hence, an indirect indication of the
Elmer FTIR spectrometer (OH, USA), coupled with Spec- sunscreen effect.
trum 10 software that is used to operate and treat FTIR Strat-M® membrane has multiple layers with different
spectra. A sample was loaded on the sample holder above the diffusivity. The outer layer consists of two layers of polye-
laser lens and held in place by screwing down the relevant thersulfone, which is more resistant to diffusion, whereas the
adaptor. For each sample’s FTIR spectrum, scans were bottom layer is a more diffusive polyolefin layer. The rate-
obtained over the range of 450–4000 cm−1 with a resolution limiting layer for permeation via the skin is the outermost
of 2 cm−1. The measurements of the transmittance triplicate epidermal layer. However, in synthetic membranes, the rate-
samples using the FTIR were assessed at three different time limiting layer is not clear. As can be seen from Figure 1(a),
points of the application of the sunscreen: zero time, after 20 the transmission of the IR showed FTIR spectra with troughs
min, and after 2 h. and peaks that tally with the properties of the Strat-M®. The
two sharp troughs located at 2847 and 2912 cm−1 are assigned
to C-H stretching. The other bands, 1462, 1375, 719, and 805
Statistical Analysis cm−1, can be attributed to C-H and C-C, C-N, and/or C-O
stretching or bending of the olefinic polymer of the Strat-M®
All data was generated, replicated, and analyzed statis- (28, 29).
tically by one-way or two-way ANOVA from Minitab v. 18 Initially, the FTIR spectra were taken for the Strat-M®
statistical pack. The level of significance was quoted as membrane alone (Figure 1(b) A) and with the basic cream
P<0.05, with a confidence interval of 95%. that does not contain any sunscreen agent (see Figure 1(b)
B). As can be seen from the spectra, the transmittance was
Table I Summary of the Percentage w/w of Each Sunscreen Agent almost the same. The peaks that represented the Strat-M®
Used in Each Formulation membrane did not change in both location and intensity.
Therefore, there was no interference due to the components
of the basic cream in the Strat-M®.
Formula Sunscreen agent used in the formulation As can be seen from Figure 1(a), transmittance exceeded
94%, and hence it is expected that the light can penetrate the
ZnO TiO2 Oxybenzone Octyl methoxycinnamate
skin and have a harmful effect. The FTIR spectroscopy was
F1C 3% 6% employed as a quick tool to assess the ability of sunscreen
F2C 16% 3% agents to block the transmission of light (IR). Theoretically,
F3C 16% 6% the light should be able to transfer through the skin layers to
F4C 16% 3% some extent. Longer wavelengths produce less excitation but
F5C 16% 6% could penetrate deeper into the skin. Hence, FTIR was
F6C 16% 16% 3% investigated to understand the effect of the type of sunscreen
F7C 16% 16% 6% agent and its concentration on transmittance. Also, the effect
F8C 16% 3% 6% of the application time on the transmittance through the
F9C 16% 3% 6% Strat-M® was examined.
F10C 16% 16%
F11C 16% 16% 3% 6%
73 Page 4 of 11
Fig. 1 a Strat-M® FTIR spectrum over a wavenumber from 450 to 4000 cm−1; b a comparison of the FTIR spectra of (A) Strat-M® membrane
alone and (B) Strat-M® membrane with basic cream; and c the mechanism of action for the physical sunscreen versus chemical sunscreen
Chemical Sunscreen
The difference in the mechanism of the action of the
organic (chemical) sunscreen in comparison with the inor-
ganic (physical) sunscreen is shown in Figure 1(c). Organic
sunscreens are absorbed into the skin and then it will absorb
UV rays, convert the rays into heat, and release them from
the body. Physical sunblock sits on top of the skin and reflects
the sun’s rays. Oxybenzone Cream. Oxybenzone
(benzophenone-3) is an organic sunscreen of type UVA.
The FTIR spectrum of pure benzophenone-3 (Figure 2(a))
was conducted using a few crystals of the compound. The
band at 1633 cm−1 is assigned to C=O stretching; the two
bands at 1593, 1506, and 1434 cm−1 are due to aromatic C=C;
the rest of the bands are due to the bending vibrations of the
bonds of the compound.
Cream containing oxybenzone (benzophenone-3) was
prepared and applied onto the Strat-M® membrane and then
the assessment of the FTIR transmission was carried out. The
presence of oxybenzone reduced the percentage of transmit-
tance when assessed on the trough at 805 cm−1, starting from
2% up to 5%. The maximum concentration was based on the
maximum allowed concentration in sunscreen products
(CosIng Annex VI) (30). The relationship between the
transmittance (%T) and absorbance (A) is an inverse
relationship according to the following equation: A = log10
(1/T). Therefore, as the concentration of the sunscreen
increases, the absorbance of light increases, and a reduction
of the harmful effect of light on the lower parts of the skin’s
layers is anticipated (31).
Figure 2(b) shows the FTIR spectra of various concen-
trations of oxybenzone containing formulas obtained after 2 h
of application on Strat M® membrane. The peak at 805 cm−1
was used for comparison, as a change in the transmittance
percentage at all concentrations prepared in this study was
AAPS PharmSciTech (2022) 23: 73
noticed. Other peaks were showing a similar pattern (e.g.,
1261, 1013 cm−1); nevertheless, the calculations were based
on the peak at wavenumber 805 cm−1.
As the concentration of the oxybenzone increased,
moving from 2 to 5%, the lines became steeper, as seen in
Figure 2(c), which revealed that the rate of the absorbance
increased as the concentration of the agent increases. For the
three concentrations prepared for the sunscreen agent, after
20 min, the absorbance was less than after 2 h. In another
mean between 0 min and 20 min, no significant effect was
observed. Blockage of transmission was noted after 20 min.
However, after 2 h, a significant effect was noticed (transmis-
sion dropped from 95 to 60.09%). Two-way ANOVA analysis
demonstrated that there is a significant difference (P = 0.027)
after 2 h, whereas the effect in blocking transmittance could
not be observed at 20 min, and the difference between them
was statistically insignificant. Hence, it could be proposed that
for oxybenzone going from the lowest to the highest
approved concentration, the product requires 120 min to
demonstrate optimal effect (i.e., absorb the light and hence
reduce the damage on the underlying tissues). The two-way
ANOVA analysis further revealed that there is no statistically
significant effect of concentration (P=0.116).
Figure 2(d) illustrates the percentage transmission versus
concentration at wavenumber 805 cm−1 after 2 h for the
oxybenzone containing cream at various concentrations (0–
5%). The relationship between transmission and
concentration was found to be non-linear, where the curve
concaved upwards, suggesting that the difference in %T
between the last two concentrations, namely the 3% and 5%,
was less than the smaller concentration. This could be the
reason why commercially available formulations usually use
the average concentration and not the maximum. Using
average concentration is cost effective, as employing the
maximum concentration of benzophenone will not increase
AAPS PharmSciTech (2022) 23: 73 Page 5 of 11 73

Fig. 2 a FTIR spectrum of oxybenzone highlighting the troughs and peaks of transmission of IR light over the range of wavenumber 400–4000
cm−1; b FTIR spectra of benzophenone-3 containing cream with increased concentrations from (A) 0%, (B) 2%, (C) 3%, and (D) 5% after 2 h;
c comparison of the effect of the time and oxybenzone concentration on the transmittance (%T) of sunscreen containing oxybenzone at
wavenumber 805 cm−1 (mean ± SD, n=3); and d plot of %T versus concentration at wavenumber 805 cm−1 after 2 h for oxybenzone cream for
0%, 2%, 3%, and 5%

the sun protection level. However, one-way ANOVA analysis
revealed a significant influence of oxybenzone concentration
on transmission after 2 h of application (P=0.000), and the
Tukey pairwise comparison test showed that there is a
significant difference in transmission between all individual
concentrations (P<0.05). Several commercially available
products contained oxybenzone within the medium range of
3%, such as Rosy Tone Broad Spectrum Sunscreen by
L’Oreal Paris (32). Furthermore, sun protection products
include a variety of sunscreen agents aiming at broadening
the sun protection range, improving the SPF, and reducing
the concentration of individual sunscreen agent to reduce
their toxic side effects (33). Other studies showed that high
levels of oxybenzone exposure could be associated with
various endocrine problems (34, 35).
Octyl Methoxycinnamate Cream. Octyl
methoxycinnamate or ethylhexyl methoxycinnamate is an
organic sunscreen of type UVB. It is the most popular
sunscreen agent in the USA owing to its low irritation
p ot e n t i a l ( 3 6 ) . T h e I R s p e c t r u m o f p u r e o c t y l
methoxycinnamate (Figure 3(a)) was conducted using the
liquid compartment. The two bands located at 2959 and 2932
cm−1 are assigned to C-H stretching; the band at 1706 cm−1 is
assigned to C=O stretching; the two bands at 1603 and 1512
cm−1 are due to aromatic C=C; the rest of the bands are due
to the bending vibrations of the bonds of the compound.
The second chemical sunscreen used was octyl
methoxycinnamate. It reduced the percentage of
transmittance starting from concentrations of 3% up to
10%. By increasing the concentration of octyl
methoxycinnamate in each formula, the slope of each line
decreased concurrently (Figure 3(b)). Moving from 3% up to
10%, the slope of the line became steeper, similar to the
behavior of the oxybenzone cream. Two-way ANOVA
analysis demonstrated that the time factor produces a
statistically significant effect (P= 0.024), whereas the concen-
tration impact was not significant (P= 0.41). However, using
the Tukey pairwise comparison test, the effect of time was not
significant between 0 and 20 min, but was significant
otherwise.
By plotting %T versus concentration at wavenumber 805
cm−1 after 2 h of applying various concentrations of octyl
methoxycinnamate cream (0–10%), the relationship was
found to be non-linear (Figure 3(c)). The curve was found
to be concaved upwards, suggesting that the difference in %T
between the last two concentrations, namely the 6% and
10%, was less than the smaller concentration. The use of the
sunscreen at lower concentrations up to medium concentra-
tion resulted in a linear relationship and a proportional
reduction in the transmission of the IR radiation. However, at
higher concentrations, the change was not proportional.
Similar to oxybenzone, both one-way ANOVA analysis (P =
0.000) and Tukey pairwise analysis showed a significant
difference among different concentrations after 2 h of
application.
73 Page 6 of 11
Fig. 3 a FTIR spectrum of octyl methoxycinnamate; b comparison of the effect of the time and octyl methoxycinnamate concentration on the
transmittance (%T) of sunscreen containing octyl methoxycinnamate at wavenumber 805 cm−1 (mean ± SD, n=3); and c plot %T versus
concentration at wavenumber 805 cm−1 after 2 h for octyl methoxycinnamate cream for 0%, 3%, 6%, and 10%
Physical Sunscreen
Titanium Dioxide Cream. The UV spectroscopic method
using the Mansur equation will not assess the sun protection
capability of sunscreens containing physical agents due to
solubility issues (13). Therefore, using the FTIR method as a
tool to assess sunscreen agents’ ability to block light
transmission was investigated. Physical sunscreens are applied
on the surface of the skin and scatter UV rays (Figure 1(c)).
This is accomplished via mineral filters and the important
physical filters are TiO2 and ZnO.
Due to the inorganic nature of TiO2, the FTIR spectra of
TiO2 powder showed no sharp characteristics peaks within the
range of 450–4000 cm−1 (37). The minimum concentration of
TiO2 used was 8%, and the slope of this concentration was
found to be 0.2479, as depicted in Figure 4(a), which was close to
the second concentration of the two chemical sunscreens
mentioned previously. The slope of the 3% oxybenzone and
6% octyl methoxycinnamate was 0.258. The second concentra-
tion of TiO2 revealed a double change in the slope, which
suggests better protection than relevant chemical counterparts.
The change of the slope of the third concentration of TiO2
(25%) gave nearly the same effect as the 16%. Two-way
ANOVA analysis demonstrated the significant effect of time
on transmission (P= 0.032) but not concentration.
By plotting %T versus concentration at wavenumber 805
cm−1 after 2 h for the three TiO2 cream concentrations applied
to Strat-M after 2 h of application, the relationship was found to
be non-linear (Figure 4(b)). Similar to the results obtained from
chemical sunscreens, the curve concaved upwards, suggesting
AAPS PharmSciTech (2022) 23: 73
that the difference in %T between the last two concentrations,
namely the 16% and the 25%, was less than the smaller
concentration. One-way ANOVA analysis showed that there is
a significant difference between concentrations (P = 0.000), and
the Tukey stepwise comparison showed a significant difference
among various concentrations.
Zinc Oxide Cream. Similarly, the FTIR spectrum for
ZnO showed no peak in the range of 450–4000 cm−1, due to
its inorganic compounds. The second most important physical
sunscreen is ZnO, and similar concentrations to the TiO2
formulas were prepared; they showed the same behavior as
the TiO2 (Figure 4(c)). A sharp and significant reduction in
transmission was obtained after 2 h (two-way ANOVA, P =
0.042). The concentration-factor results demonstrated an
insignificance effect on transmission with time (P = 0.435).
When plotting %T versus concentration at wavenumber
805 cm−1 after 2 h for the three ZnO cream concentrations
plus the 0%, the relationship was found to be non-linear
(Figure 4(d)). It looks like a curve in a graph and has a
variable slope value. The curve was concaved upwards,
suggesting that the difference in %T between the last two
concentrations, namely the 16% and 25%, was less than the
smaller concentration. The one-way ANOVA test results
showed that the effect of concentration after 2 h is significant
(P= 0.000), and the Tukey stepwise comparison showed a
significant difference between all concentrations. Despite the
slight reduction in transmission between the middle and high
concentrations, it is still significant.
AAPS PharmSciTech (2022) 23: 73 Page 7 of 11 73

Fig. 4 a Comparison of the effect of the time and TiO2 concentration on the transmittance (%T) of sunscreen containing TiO2 at wavenumber
805 cm−1 (mean ± SD, n=3); b plot %T versus concentration of TiO2 at wavenumber 805 cm−1 after 2 h for TiO2 cream for 0%, 8%, 16%, and
25%; c comparison of the effect of the time and ZnO concentration on the transmittance (%T) of sunscreen containing ZnO at wavenumber
805 cm−1 (mean ± SD, n=3); and d plot %T versus concentration of ZnO at wavenumber 805 cm−1 after 2 h for ZnO cream for 0%, 8%, 16%,
and 25%

Overall, for both physical and chemical sunscreens, the
effect of time is critical on transmission. Unfortunately, the
practice of using sunscreen formulations is focused on
applying it and then going outside, not giving the product
ample time to produce its anticipated effect by either blocking
light transmission (in physical sunscreens) or absorbing the
light and preventing its transmission deeper into the skin.
Regulators do not require manufacturers to specify the time
required for sunscreen products to produce an optimal effect
(38). Therefore, clients may be exposed to UV radiations
while they are not fully protected. However, after the effect is
established, the concentration of the sunscreen product is
critical.
Although the maximum reduction of transmission was
observed at 120 min, there was a substantial reduction in
transmission at 60 min that ranged from 50 to 70% of the
reduction at 120 min, which varies according to the sunscreen
type and concentration. Such results suggest that the sun-
screen formulations applied to the skin could produce
protection after 60 min, while maximum protection could be
observed after 120 min of application. It is worth mentioning
that the upper limit of each sunscreen agent was based on the
maximum allowed concentration based on Annex VI of the
EU regulations of cosmetics-sunscreen agents (30). The lower
limit was based on the search within commercially available
products and the middle concentration was between the
upper limit and the lower limit.
Figure 5 shows the % transmission of chemical and
physical sunscreens. From the graph, it is noted that physical
sunscreens demonstrate superior sun protection over the
chemical sunscreens. Most sunscreen products use more than
one sunscreen material in their formulations; and hence this
part of the investigations aimed at assessing the effect of a
combination of chemical and/or physical sunscreens on IR
transmission. The two chemical sunscreens used at their
maximum allowed concentrations, oxybenzone (5%) and
octyl methoxycinnamate (10%), showed similar screening
behavior (Figure 5A). The same behavior was noticed at the
medium concentrations of both the octyl methoxycinnamate
(6%) and the oxybenzone (3%) (Figure 5B). Overall, there
was no statistically significant difference between oxybenzone
and octyl methoxycinnamate transmission at both concentra-
tions (one-way ANOVA, P>0.05).
The two physical sunscreens used at their maximum and
medium allowed concentrations, TiO2 and ZnO, showed
similar screening behavior (Figure 5C and D). Despite a
slightly lower transmission obtained by TiO2 at its maximum
concentration (25%) than the ZnO (25%), there was no
statistically significant difference between the two physical
sunscreens (one-way ANOVA, P>0.05).
The two physical sunscreens beside the two chemical
counterparts are drawn in Figure 6(a), which revealed the
superiority of the physical sunscreens versus the chemical
sunscreens. These metal oxides are generally less toxic, more
stable, and safer for humans than those of organic ingredients;
73 Page 8 of 11
Fig. 5 Comparison of the FTIR transmission spectra (%T) over time of: A oxybenzone 5% and octyl methoxycinnamate 10%; B oxybenzone
3% and octyl methoxycinnamate 6%; C TiO2 25% and ZnO 25%; and D TiO2 16% and ZnO 16% with the basic cream at wavenumber 805
cm−1 (mean ± SD, n=3)
yet, they are not preferred by the consumer due to their
aesthetic appearance and poor cosmetic appearance (7, 35,
39). Two-way ANOVA analysis revealed an overall statistically
significant difference among sunscreen agents (P<0.05). The
Tukey stepwise analysis showed the effect to be significant at 120
min, whereas, between 0 and 20 min, there is no significant
difference. In line with the findings, physical sunscreens (ZnO
and TiO2) are considered to be more safe and effective than
other products in the USA, as per the Environmental Working
Group’s 2010 Annual Sunscreen Guide (1). .
Fixed-Dose Combinations
The two chemical sunscreens (oxybenzone and octyl
methoxycinnamate) were mixed as F1C, and this formula was
introduced to the FTIR instrument to study its behavior at
wavenumber 805 cm−1. Another formula, F10C, was prepared
from the two physical sunscreens (ZnO and TiO2) and was
analyzed by the FTIR.
Figure 6(b) shows that the formula with combined
chemical sunscreen agents (F1C) demonstrated a higher level
of absorbance when compared to the formula with combined
physical sunscreen agents (F10C). This can be observed from
the difference in the slope of the two lines (0.2518 versus
0.4283 for F1C and F10C respectively). There is a statistically
significant difference between F1C and F10C after 2 h of
application (Tukey post-test P<0.05). However, it was noted
that combining the physical sunscreen agents did not result in
a synergistic effect and/or additive effect. The percentage
transmission of the combined products did not vary
AAPS PharmSciTech (2022) 23: 73
significantly from the individual products set at medium
concentrations. When the slope is compared, the combined
physical agents resulted in a slope of 0.4283, while the 16%
TiO2 slope was 0.4885. The extra value of adding two physical
sunscreen agents, which is a trend with cosmetics’ manufac-
turers, needs to be addressed.
Fixed-dose combinations of physical and chemical
sunscreen agents were developed (F2–F5). The % trans-
mittance was conducted for the formulations and the
results are illustrated in Figure 6(c). The formulations
containing the physical sunscreens, namely the ZnO and
the TiO2 showed a better performance in absorbing more
incident rays when mixed with octyl methoxycinnamate
(F3C and F5C) rather than oxybenzone (F2C and F4C)
(Figure 6(c)). Studies have reported the synergistic effect
of combining organic with inorganic sunscreen (40). Using
combined products at a lower concentration to obtain
better protection (lower transmission) is favorable, as it
will produce cost-effective formulations and enables
broad-spectrum protection (42, 43).
The % transmittance of 3-component formulas was
conducted, and the results are illustrated in Figure 6(d).
When octyl methoxycinnamate was mixed with any other two
components, namely F7C, F8C, and F9C, the performance of
the sunscreen formula was better. The two physical sun-
screens when combined with octyl methoxycinnamate pro-
duced the best reduction in transmission. However, two-way
ANOVA analysis indicated that there is no significant
difference among the combined 3-component formulations.
It is a regular practice in the cosmetics industry to use a
combination of organic and inorganic sun protection mate-
rials. However, some studies have reported the occurrence of
AAPS PharmSciTech (2022) 23: 73 Page 9 of 11 73

Fig. 6 a Comparison of the FTIR transmission spectra (%T) over time of the two chemical (oxybenzone 5%, octyl methoxycinnamate 10%)
and two physical (TiO2 25% and ZnO 25%) sunscreens at wavenumber 805 cm−1 (mean ± SD, n=3); b comparison of the two mixed formulas’
transmission (%T) at wavenumber 805 cm−1, F1C and F10C, where F1C is the mixture of two chemical sunscreens, and F10C is the mixture of
two physical sunscreens, transmittance (%T) (mean ± SD, n=3); c combined two-component formulas with one physical and one chemical
sunscreen agent in each formula: F2C, F3C, F4C, and F5C, with the transmittance (%T) at wavenumber 805 cm−1 (mean ± SD, n=3);
d combined three-component formulas of three sunscreen agent in each formula: F6C, F7C, F8C, and F9C, with the transmittance (%T) at
wavenumber 805 cm−1 (mean ± SD, n=3); and e combined four-component formulas of sunscreen agents F11C, the transmittance (%T) at
wavenumber 805 cm−1 (mean ± SD, n=3)

photolysis of organic sunscreen agents upon use with
inorganic sunscreens such as TiO2 (41). Therefore, careful
consideration needs to be made upon the use of fixed-dose
combination materials.
Several studies have reported a good correlation be-
tween permeation behavior of human skin and Strat-M®, and
that such synthetic membranes were developed to be used as
alternatives to human skin. Furthermore, Strat-M® synthetic
membranes were designed to share similar structural and
chemical characteristics of the human epidermis (44–46).
Hence, this developed method can be assessed on human/
animal cadaver skin. However, there are a few issues of
concern when using human or animal skin, such as limited
availability, the complex sample preparation process, strict
storage conditions, biohazard issues, and expensive study
costs (47). Additional concerns are related to humans, such as
biological variabilities in terms of skin thickness, hair follicles’
density, lipid content, and composition (41, 48). Furthermore,
excised skin may be subject to artifacts upon storage (below
−20°C), which may result in increased permeation of mate-
rials (49). Therefore, the developed method in this work
produced a simple and reproducible model for assessing the
transmission blockage of various sunscreen agents.
CONCLUSIONS
This research project aimed to employ FTIR as a quick
tool to assess the performance of physical and chemical
sunscreen materials using a validated synthetic membrane
(Strat-M®). Also, the effect of concentration, duration of the
73 Page 10 of 11
application, and fixed-dose combinations were investigated.
The IR spectra of both chemical sun blockers at three
concentrations revealed a direct relationship between the
concentration and the absorbance of the incident light. The
physical sun blockers showed better absorbance than the
chemical counterparts. Furthermore, the mixed two-
component physical sunscreen showed a better performance
than the mixed organic counterparts. The key finding is
pertinent to the effect of time on transmission through the
synthetic skin. All the assessed sunscreens required time to
demonstrate the required effect to a maximum after 2 h.
Overall, this study enabled the identification of a quick,
simple, and reproducible method to assess the sunscreen
behavior of various sunscreen agents.
AUTHOR CONTRIBUTION
E.Z. Dahmash: conceptualization, methodology, valida-
tion, writing of original draft, supervision, research project
management, final revision of original draft. F. Al-Saeidi:
investigation, preparation and characterization, writing, orig-
inal draft preparation.
FUNDING
Isra University (Jordan) provided funding for Farah
Mahdi Abd Ali AL-Saedi towards her MSc.
DECLARATIONS
Conflict of Interest The authors declare no competing interests.
REFERENCES
1. Latha MS, Martis J, Shobha V. 2013. Sunscreening agents: a
review. J Clin Aesthet Dermatol. 2013;6(1):16–26.
2. Dutra EA, Almança D, Kedor ERM, Inês M, Miritello R.
Determination of sun protection factor ( SPF ) of sunscreens by
ultraviolet spectrophotometry. Farm J Pharm Sci. 2004;40
(3):381–5.
3. Rezende SG, Dourado JG, De FMA, Vinhal DC, Silva EC, Gil
EDS. Methods used in evaluation of the sun protection factor of
sunscreens factor. Rev Eletrônica Farmácia. 2014;11(2):37–54.
4. Sohn M. In vitro biorelevant and in silico sunscreen perfor-
mance evaluation on the basis of film thickness frequency
distribution of formulations and UV filter repartition. 2016.
5. Diffey BL. What is light ? Photodermatol Photoimmunol
Photomed. 2002;18(2):68–74.
6. Battie C, Verschoore M. Cutaneous solar ultraviolet exposure
and clinical aspects of photodamage. Indian J Dermatology,
Venereol Leprol. 2012;78(7):9–14.
7. Latha MS, Martis J, Shobha V, Sham Shinde R, Bangera S,
Krishnankutty B, Bellary S, Varughese S, Rao P, Naveen Kumar
BR. Sunscreening agents: a review. J Clin Aesthet Dermatol.
2013 Jan;6(1):16–26.
8. Young AR. Chromophores in human skin. Phys Med Biol.
1997;42:789–802.
9. Gabard B. Sun protection and sunscreens. In: Barel AO, Paye
M, Maibach HI, editors. Handbook of cosmetic science and
AAPS PharmSciTech (2022) 23: 73
technology, Third Edition. 3rd ed. New York: Informa
Healthcare USA, Inc.; 2009. p. 323–330.
10. Serpone N, Dondi D, Albini A. Inorganic and organic UV
filters: their role and efficacy in sunscreens and suncare
products. Inorg Chim Acta. 2007;360(3):794–802.
11. Kockler J. Sunscreens: photostability, formulation and skin
penetration. 2014.
12. Yang SI, Liu S, Brooks GJ, Lanctot Y, Gruber JV. Reliable and
simple spectrophotometric determination of sun protection
factor: a case study using organic UV filter-based sunscreen
products. J Cosmet Dermatol. 2018;17(3):518–22.
13. Sudhahar V, Balasubramanian V. Sun production factor (SPF)
determination of marketed sunscreen formulation by in-vitro
method using UV-VIS spectrophotometer. Sch Res Libr Arch
Appl Sci Res. 2013;5(5):119–22.
14. Springsteen A, Yurek R, Frazier M, Carr KF. In vitro measure-
ment of sun protection factor of sunscreens by diffuse transmit-
tance. Anal Chim Acta. 1999 Feb;380(2–3):155–64.
15. Mansur JS, Breder MNR, Mansur MCA, Azulay RD.
Determinação do fator de proteção solar por
espectrofotometria. An Bras Dermatol, Rio Janeiro.
1986;61:121–4.
16. Herzog B, Osterwalder U. In silico determination of topical sun
protection. Cosmet Sci Technol. 2011 Jan;62:62.
17. Neupane R, Boddu SHS, Renukuntla J, Babu RJ, Tiwari AK.
Alternatives to biological skin in permeation studies: current
trends and possibilities. Pharmaceutics. 2020;12(2).
18. Uchida T, Kadhum WR, Kanai S, Todo H, Oshizaka T,
Sugibayashi K. Prediction of skin permeation by chemical
compounds using the artificial membrane. Strat-MTM Eur J
Pharm Sci. 2015;67:113–8.
19. Barolet D, Christiaens F, Hamblin MR. Infrared and skin:
friend or foe. J Photochem Photobiol, B. 2015/12/21. 2016
Feb;155:78–85.
20. Riaz T, Zeeshan R, Zarif F, Ilyas K, Muhammad N, Safi SZ,
Rahim A, Rizvi SAA, Rehman IU. FTIR analysis of natural
and synthetic collagen. Appl Spectrosc Rev. 2018 Oct;53(9):703–
46.
21. Lucassen GW, Caspersb PJ, Gerwin J. Puppels. Water content
and water profiles in skin measured by FTIR and Raman
spectroscopy. In: Proceedings of SPIE. 2000. p. 39–45.
22. Binder L, Kulovits EM, Petz R, Ruthofer J, Baurecht D, Klang
V, Valenta C. Penetration monitoring of drugs and additives by
ATR-FTIR spectroscopy/tape stripping and confocal Raman
spectroscopy – a comparative study. Eur J Pharm Biopharm.
2018;130(May):214–23.
23. Russeau W, Mitchell J, Tetteh J, Lane ME, Hadgraft J.
Investigation of the permeation of model formulations and a
commercial ibuprofen formulation in Carbosil® and human
skin using ATR-FTIR and multivariate spectral analysis. Int J
Pharm. 2009;374(1–2):17–25.
24. Shakya BR, Shrestha P, Teppo HR, Rieppo L. The use of
Fourier transform infrared (FTIR) spectroscopy in skin cancer
research: a systematic review. Appl Spectrosc Rev 2020;0(0):1–
33.
25. Movasaghi Z, Rehman S, Rehman IU. Fourier transform
infrared (FTIR) spectroscopy of biological tissues. Appl
Spectrosc Rev. 2008;43(2):134–79.
26. Kyriakidou M, Anastassopoulou J, Tsakiris A, Koui M,
Theophanides T. FT-IR spectroscopy study in early diagnosis
of skin cancer. In Vivo (Brooklyn). 2017;31(6):1131–7.
27. Sakuyama S, Hirabayashi C, Hasegawa JI, Yoshida S. Analysis
of human face skin surface molecules in situ by Fourier-
transform infrared spectroscopy. Ski Res Technol. 2010;16
(2):151–60.
28. Nandiyanto ABD, Oktiani R, Ragadhita R. How to read and
interpret FTIR spectroscope of organic material. Indones J Sci
Technol. 2019;4(1):97–118.
29. Coates J. Interpretation of infrared spectra, a practical ap-
proach. Encycl Anal Chem. 2000;12:10815–37.
30. EU portal. CosIng Annex VI, List of UV filters allowed in
cosmetic products. 2020. p. 1–8.
31. Geoffrey K, Mwangi AN, Maru SM. Sunscreen products:
rationale for use, formulation development and regulatory
considerations. Saudi Pharm J. 2019;27(7):1009–18.
AAPS PharmSciTech (2022) 23: 73
32. Bhattacharjee D, Patil A, Jain V, Preethi.s P. A comparison of
natural and synthetic sunscreen agents: a review. Int J Pharm
Res. 2021;13.
33. Chatelain E, Gabard B, Surber C. Skin penetration and sun
protection factor of five UV filters: effect of the vehicle. Skin
Pharmacol Physiol. 2003;16(1):28–35.
34. Ghazipura M, McGowan R, Arslan A, Hossain T. Exposure to
benzophenone-3 and reproductive toxicity: a systematic review
of human and animal studies. Reprod Toxicol [Internet].
2017;73:175–83 Available from: https://www.sciencedirect.com/
science/article/pii/S0890623817302277.
35. DiNardo JC, Downs CA. Dermatological and environmental
toxicological impact of the sunscreen ingredient oxybenzone/
benzophenone-3. J Cosmet Dermatol. 2018;17(1):15–9.
36. Sambandan DR, Ratner D. Sunscreens: an overview and
update. J Am Acad Dermatol [Internet]. 2011;64(4):748–58
Available from: https://www.sciencedirect.com/science/article/
pii/S0190962210000228.
37. Kumar PM, Badrinarayanan S, Sastry M. Nanocrystalline TiO2
studied by optical, FTIR and X-ray photoelectron spectroscopy:
correlation to presence of surface states. Thin Solid Films.
2000;358:122–30.
38. FDA. Labeling and effectiveness testing: sunscreen drug
products for over-the-counter human use — small entity
compliance guide. 2012.
39. Palm MD, O’Donoghue MN. Update on photoprotection.
Dermatol Ther. 2007;20(5):360–76.
40. Lademann J, Schanzer S, Jacobi U, Schaefer H, Pflücker F,
Driller H, et al. Synergy effects between orga and inorganic UV
filters in sunscreens. J Biomed Opt. 2005 Jan;10:14008.
41. Kim EJ, Kim MJ, Im NR, Park SN. Photolysis of the organic
UV filter, avobenzone, combined with octyl methoxycinnamate
by nano-TiO2 composites. J Photochem Photobiol B Biol.
2015;149:196–203.
42. Maier T, Korting HC. Sunscreens – which and what for? Skin
Pharmacol Physiol [Internet]. 2005;18(6):253–62. Available
from:. https://doi.org/10.1159/000087606.
Page 11 of 11 73
43. Gaspar LR, Maia Campos PMBG. Evaluation of the
photostability of different UV filter combinations in a sun-
screen. Int J Pharm [Internet]. 2006;307(2):123–8 Available
from: https://www.sciencedirect.com/science/article/pii/
S0378517305006496.
44. Milanowski B, Wosicka-Frąckowiak H, Główka E, Sosnowska
M, Woźny S, Stachowiak F, et al. Optimization and evaluation of
the in vitro permeation parameters of topical products with non-
steroidal anti-inflammatory drugs through Strat-M(®) mem-
brane. Pharmaceutics [Internet]. 2021 Aug 20;13(8):1305. Avail-
able from: https://pubmed.ncbi.nlm.nih.gov/34452264
45. Schmook FP, Meingassner JG, Billich A. Comparison of human
skin or epidermis models with human and animal skin in in-vitro
percutaneous absorption. Int J Pharm. 2001;215(1–2):51–6.
46. Simon A, Amaro MI, Healy AM, Cabral LM, de Sousa VP.
Comparative evaluation of rivastigmine permeation from a
transdermal system in the Franz cell using synthetic membranes
and pig ear skin with in vivo-in vitro correlation. Int J Pharm.
2016;512(1):234–41.
47. Haq A, Goodyear B, Ameen D, Joshi V, Michniak-Kohn B.
Strat-M® synthetic membrane: permeability comparison to
human cadaver skin. Int J Pharm. 2018;547(1–2):432–7.
48. Moloney FJ, Collins S, Murphy GM. Sunscreens. Am J Clin
Dermatol [Internet]. 2002;3(3):185–91. Available from: https://
doi.org/10.2165/00128071-200203030-00005
49. Dancik Y, Kichou H, Eklouh-Molinier C, Soucé M, Munnier E,
Chourpa I, Bonnier F. Freezing weakens the barrier function of
reconstructed human epidermis as evidenced by Raman spec-
troscopy and percutaneous permeation. Pharmaceutics. 2020;12
(11):1041.
Publisher’s Note Springer Nature remains neutral with regard
to jurisdictional claims in published maps and institutional
affiliations.