Journal of Pharmaceutical Research and Drug Information 2025; 00(00); 000–000
_________________________________________
*Correspondence: vuongttt@hup.edu.vn; 1
http://doi.org/10.59882/1859-364X/264
Journal homepage: jprdi.vn/JP
Journal of Pharmaceutical Research and Drug Information
An official journal of Hanoi University of Pharmacy
Research article
Simultaneous determination of two UV filters
(octylmethoxycinnamate, octylsalicylate) in sunscreen creams
by high performance thin layer chromatography (HPTLC)
Minh Thuy Ngoa, Thi Bich Dao Danga, Dinh Chi Leb, Thi Thanh Vuong Tonga,*
a Department of Analytical Chemistry and Drug Quality Control, Hanoi University of
Pharmacy, 13-15 Le Thanh Tong Street, Hoan Kiem District, Hanoi, Vietnam
b National Institute of Pharmaceutical Technology, Hanoi University of Pharmacy, 13-15 Le
Thanh Tong Street, Hoan Kiem District, Hanoi, Vietnam.
*Corresponding author: Thi Thanh Vuong Tong - vuongttt@hup.edu.vn
Article history:
Received 05 December 2024
Resived 10 January 2025
Accepted 17 January 2025
ABSTRACT
Octylmethoxycinnamate (OMC), octylsalicylate (OS) are UV filters commonly used in
sunscreen creams for skin protection against UV radiations. In this study, an HPTLC
method was developed and validated for simultaneous determination of OMC and OS in
sunscreen creams. The method was carried out using an silica gel 60 F254 thin layer plate
(20 cm × 10 cm) with a mixture of cyclohexane - aceton (10:1, v/v) as mobile phase.
Samples were applied on thin layer plate as 4-mm bands with applied volume at 7.0 µL per
band and the distance between two consecutive bands was 9 mm. The chromatograms
were developed over a distance of 80mm, then scanned and recorded at 307 nm for peak
detection and calculation of peak area. The method was fully validated according to
requirements on analytical method performance of AOAC International. The linearity
ranges of the method were from 17.9 to 32.2 µg/mL for OMC and from 16.3 to 29.4
µg/mL for OS, respectively, and its accuracy (recovery rate from 97.4% to 102.2% for
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000
OMC and from 98.0% to 102.4% for OS) and its precision (RSD ≤ 2.6% for OMC and ≤
2.4 % for OS) were sufficient for simultaneous determination of these two UV filters.
Therefore, validation results confirmed its reliability and suitability for intended
application. The method was employed to determine the content of OMC and OS in
commercial sunscreen creams.
Keywords: HPTLC, UV filter, sunscreen cream, octyl methoxycinnamate, octyl salicylate
INTRODUCTION
Exposure to ultraviolet (UV) radiation is harmful for human health. To minimize
direct exposure of human skin to UV radiation, many sunscreen products in different form
(creams, emulsions, etc.) are available on the market. The protective effects against UV
radiation of these products comes from the UV filters that they contain. UV filters are
chemical compounds capable of neutralizing harmful assaults of UV radiations on skin
surface by scattering them (physical mechanism) or by absorbing them (chemical
mechanism) [1]. Octylmethoxycinnamate (OMC), also known as octinoxate or
ethylhexylmethoxycinnamate, and octylsalicylate (OS), also known as octisalate or
ethylhexyl salicylate, are chemical UV filters with proven capacity in absorbing UV
radiation [2]. According to Annex VII of ASEAN Cosmetic Directive, both OMC and OS
are UV filters allowed to be use in cosmetic such as sunscreen creams with highest accepted
limits at 10% (w/w) and 5% (w/w), respectively [3,4]. These limits are also currently
imposed by Vietnamese authorities.
To control the quality of these UV filters in pure material, OMC and OS were
quantified by gas chromatography using dimethylpolysiloxane column and flame ionization
detector [5, 6]. OMC was determined in sunscreen products with UV-Vis absorption
spectroscopy [7] and with reversed-phase HPLC on C18 column [8, 9]. OS and OMC were
quantified simultaneously in sunscreen emulsion using HPLC [10]. LC-MS/MS was
employed to determine OS in human placeta tissue [11]. High performance thin layer
chromatography (HPTLC) was applied to analyze OMC [12] and to quantify OS in
sunscreen products [13].
Up to now, some authors have expressed concerns or reported cases on harmful
effects of OMC [14] and OS [15]. Therefore, to assure the protective effects of sunscreen
products, they must contain the right kind and amount of UV filters and the content of UV
filters in sunscreen products must not exceed authorized limit to avoid harmful effects that
may occur due to abusive use of UV filters. These practical requirements demand reliable
and available analytical tool for monitoring UV filters’ content in commercial cosmetic
products on market.
In this study, an HPTLC method was developed for simultaneous determination of
OMC and OS in sunscreen creams. The method was fully validated according to current
guidance of AOAC International [16] to assure its suitability and reliability for its intended
purpose.
MATERIALS AND METHODS
Reference standards, reagents and samples
Reference standards of OMC (purity: 97.9% w/w, batch No.: 20672909TO) and OS
(purity: 93.9% w/w, batch No: OS#1070220) were kindly provided by National Institute of
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000
Drug Quality Control (Hanoi, Vietnam).
Cyclohexane, aceton, diethyl ether, methanol PA grade were purchased from Merck
Vietnam (Ho Chi Minh City, Vietnam).
Representative placebo matrix was a commercial cream which did not contain OMC
and OS and was used as such after preliminary analysis confirmed that it did not contain
OMC and OS. The composition of the representative placebo consisted of water, zinc oxide,
caprylic/capric triglyceride, propylene glycol, cetyl alcohol, titanium dioxide, dimethicone,
ethylhexylglycerin, magnesium aluminum silicate, xanthan gum, disodium EDTA.
Samples were four commercial sunscreen creams containing at least one of the
analytes (according to label claim) purchased on market. Their label provided only the name
of components without any quantitative information about the content of each component in
product.
Standard solutions
Stock standard solutions of OMC and OS were prepared separately using methanol
as solvent at concentration of 1000 µg/mL. The mixed standard solutions for routine analysis
and method validation were prepared by diluting stock standard solutions with the same
solvent to targeted concentrations.
Sample preparation
About 0.25 g of sample was accurately weighed by using a weighing boat and
transferred into a 100-mL stoppered flask and dispersed with 30 mL of methanol by
sonicating in 30 minutes at 55-60oC to dissolve analytes. The stoppered flask was then
cooled down to room temperature and the content was transferred in to a 50-mL volumetric
flask and made to volume with the same solvent and mixed. The obtained liquid was left 15
minutes at 4oC, the liquid was then transferred into a centrifuging tube and centrifuged at
10000 rpm in 10 minutes at same temperature. The supernatant was applied on thin layer
plate.
Apparatus and Chromatographic conditions
Chromatographic separation was done on silica gel 60 F254 thin layer plates
purchased from Merck Vietnam (Ho Chi Minh City, Vietnam) and activated at 115oC in 15
minutes before use. HPTLC system Camag CAT No. 027.6200 of Camag (Muttenz,
Switzerland) consisted of semi-automatic sample application device Linomat 5, ADC2
developing chamber, TLC Scanner 4 device for thin layer plate scanning, and UV cabinet
with UV sources at 254 nm and 366 nm for observing thin layer plate. The system was
controlled by WinCAT software of Camag (Muttenz, Switzerland) for chromatographic
process and data collection.
The composition of mobile phase, the wavelength for scanning and recording
chromatograms were investigated during method development step to obtain suitable
conditions (the results were provided in Method development section).
For final method, the mobile phase was a mixture of cyclohexane and aceton (10:1,
v/v). The volume of mobile phase put into developing chamber was 10 mL. The relative
humidity inside developing chamber was maintained at 46-48% at room temperature during
developing procedure. The developing chamber was saturated with vapor of mobile phase
for 20 minutes before placing thin layer plate into developing chamber. Standard and sample
solutions were applied on plate in 4 mm-long bands. The distance between two adjacent
bands was 9 mm. The volume of sample applied on each band was 7.0 µL. Developing
distance of mobile phase was 80 mm. When the developing procedure finished, the
developed plate was taken out of developing chamber and left inside Hood cabinet for
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000
drying. The chromatogram of each band was scanned and recorded at 307 nm.
Method validation
For linearity validation, mixed standard solutions of OMC and OS were prepared at
5 different concentration levels for each analyte (from 17.9 to 32.2 µg/mL for OMC; from
16.3 to 29.4 µg/mL for OS).
For accuracy validation in spiked samples, exact quantities of OMC and OS were
added into representative placebo. The spiked samples were prepared in triplicate at each
spiking level and at 3 different spiking levels for each analyte (final concentrations for
applying on plate were about 20 µg/mL, 25 µg/mL, and 30 µg/mL for OMC; 18 µg/mL, 23
µg/mL and 28 µg/mL for OS) and processed as with sample preparations. The accuracy of
method was assessed by recovery rates between spiked quantities and detected ones of
analytes in spiked samples. The recovery rates and the RSD (%) of quantitative results at
each spiking level for each analyte must lie within ones recommended by AOAC
International [16].
For precision validation on real samples, 6 sample preparations of same commercial
product were analyzed in the same day and by same analyst for repeatability assessment, and
12 sample preparations of same commercial product were analyzed by two analysts and in
two different days for intermediate precision assessment. The repeatability and intermediate
precision were assessed by relative standard deviation (RSD) calculated from quantitative
results of each analytes in sample preparations. The values of RSD for each analyte must not
exceeded those recommended by AOAC International [16].
In order to estimate LOQ and LOD of the method, spiked samples were prepared by
adding OMC and OS at diferent concentrations into representative placebo and analyzed to
calculate peak height of analytes. The signal-to-noise ratios at LOQ and LOD levels were
calculated by comparing peak height obtained with each analyte on chromatograms of spiked
samples at corresponding concentrations and the peak height obtained on chromatogram of
representative placebo at the same Rf value of each analyte.
The limit of quantitation (LOQ) of each analyte was the lowest concentration at
which the linearity of the method was maintained and yielding the signal to noise ratio on
chromatogram not less than 10. The limit of detection (LOD) of each analyte was the lowest
concentration giving the signal to noise ratio not less than 3.
RESULTS AND DISCUSSIONS
Method development
To obtain good separation of OMC and OS on thin layer plate, several mobile phase
mixtures have been tried to test their suitability during preliminary studies, including:
Cyclohexane-aceton (5:1, v/v), cyclohexane-diethylether (5:1, v/v), cyclohexane-aceton
(7:1, v/v), cyclohexane-aceton (10:1, v/v), and cyclohexane-diethylether-aceton (15:1:2,
v/v/v). The Rf values of OMC and OS after chromatographic separation using these mobile
phases were summerized in Figure 1. Representative chromatograms obtained with several
mobile phases were provided in Figure 2 (A-C).
Minh Thuy Ngo et al. JPRDI 2025; 00(00); 000 - 000
Figure 1. Rf values of OMC and OS with different mobile phases.
(A)
(B)
(C)
Figure 2. Chromatograms obtained in preliminary studies with different mobile
phases: (A) Cyclohexane-diethyl ether-aceton (15:1:2); (B) Cyclohexane-aceton (10:1);
(C) Cyclohexane-diethyl ether (5:1).
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
Rf
Mobile phase
OMC OS