Supercritical Fluid Extraction and High

Performance Liquid Chromatography

Determination of Homosalate in Lipsticks

2001,54,795-797

A. Salvador* /I. Gadea /A. Chisvert/M. C. PascuaI-Marff

Departamento de Quimica Analitica, Facultad de Quimica, U niversitat de Val@ncia, Doctor Mohner 50, 46100-Bu rjassot,Val@ncia, Spain;
E-Maih Am paro.Salvador@ uv.es.

[10], whereas the US Federal Drug Ad-

KeyWords ministration [11] authorizes 15%.
Homosalate has been determined in
Column liquid chromatography sunscreen creams and lotions by HPLC
Supercritical fluid extraction [12 13] and GC-MS [14]. The samples
Homosalate in cosmetic products were treated with organic solvents in an
Green chemistry ultrasonic bath and filtered. Analytical
methods [13 14] were validated by appli-
cation to cosmetic creams without homo-
Summary salate which were spiked with known
amounts of pure homosalate added to the
Supercritical fluid extraction of homosalate is reported for the first time. Extraction parameters finished product.
such as pressure, time of extraction, use of modifier, temperature and sample mass were stu- Supercritical fluids are used in analysis
died. The proposed method was assayed for the extraction of homosalate in sunscreen lip- because of their interesting solvent prop-
sticks. Efficient recoveries were obtained after 5 min of dynamic extraction with supercritical erties in extraction [15] or as mobile
C02 and 15% ethanol as modifier at 300 bar and 60 ~ flow rate 4 mL min 1. Extracts were phases in chromatography [16]. Critical
dissolved in ethanol, and homosalate determined by HPLC using water - acetic acid - ethanol reviews on the use of supercritical fluid ex-
mobile phase and flow rate of 1 mL min 1. UV detection was at 309 nm using homosalate in traction (SFE) and supercritical fluid
ethanol solutions as standards, limit of detection 4 I~g mL 1. The RSD was 0.4- 1.0%. The re- chromatography that help explain the sta-
sults were comparable to real concentrations of samples and values by HPLC after clissolution tus and the role of these techniques today
of samples using tetrahyclrofuran. The method, based on SFE,involves low solvent consumption have recently been published by Luque de
and, can therefore be considered a green procedure. Castro [17] and Poole [18]. One of the
most active areas of green chemistry re-
search is that of analytical methodology
development [19 21]. New methods and
techniques that reduce the use of organic
Introduction Homosalate (3,3,5 trimethylcyclohexyl solvents throughout the chemical analysis
salicylate) can give substantial skin pro- cycle, such as the use of supercritical
Exposure to UV radiation from the sun tection against UVB (280 315 nm) and fluids, have been proposed as green che-
plays a causal role in skin disorders [1]. UVA II (315 340 nm) radiation and it is mical procedures [22].
UV radiation-induced effects are mani- one of the eight UV-filters, the use of In a recent article Scalia et al. [23] pro-
fested as acute responses such as inflam- which in sunscreen formulations is per- posed for the first time that supercritical
mation, i.e. sunburn [2], hyperplasia [3] mitted worldwide. CO2 be used to analyze sunscreen formu-
and immunosuppression [4 5] and as The concentration of the UV filters in lations; UV-filters (2-ethylhexyl-p-dime-
chronic responses, primarily photocarci- sunscreen formulations must be moni- thylaminobenzoate, 2-hydroxy-4-metho-
nogenesis [6] and photoaging [7 9]. The tored to assure the labelled Sun Protection xybenzophenone, 2-ethylhexyl-p-metho-
use of sunscreen products has been advo- Factor in commercial preparations with- xycinnamate, 4-methylbenzylidene cam-
cated by many health care practitioners as out exceeding authorized levels. The max- phor and 4-t-butyl-4'-methoxydibenzoyl-
a means of reducing skin damage pro- imum authorized level for homosalate is methane) were determined in the supercri-
duced by UV radiation from sunlight [1]. 10% according to European Community tical extracts by HPLC using a methanol-

Short Communication Chromatographia 2001, 54, December (No. 11/12) 795

0009-5893/00/02 795- 03 $ 03.00/0 9 2001 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH
acetonitrile-tetrahydrofuran-water(45:10:
10:35) with 0.5% acetic acid as eluent, and
very good results were obtained.
The aim of this study is to propose a
SFE method which allows rapid leaching
of homosalate from samples with a slow
or complicated dissolution such as lip-
sticks. The proposed analytical method
only requires the use of CO2 and small vo-
lumes of ethanol and could be considered
as a green chemical method.
To our knowledge there are no pub-
lished reports focusing on the determina-
tion of homosalate in solid formulations
such as lipsticks. This is also the first study
in which SFE of homosalate is examined.
Experimental
Apparatus
An SFX 3560 Isco (Lincoln, NE, USA)
supercritical fluid extractor was used. This
system is provided with two pumps allow-
ing mixture of CO2 and a modifier.
The homosalate extraction yield was
monitored by UV absorption, using a
8453 Hewlett-Packard UV-VIS diode ar-
ray spectrophotometer.
A Hitachi liquid chromatograph,
equipped with Hitachi L-7100 high pres-
sure pump and Hitachi L-7420 UV-VIS
detector was used to determine homosa-
late in sunscreen lipsticks. A LiChro-
spher | RP-18 (Merck) column (125 •
4 mm, 5 #m packing) was used.
Reagents and Samples
Ethanol (EtOH) (Scharlau, Barcelona,
Spain) was used to dissolve the sample ex-
tracts. A mixture of deionized water,
EtOH (Scharlau) and acetic acid (HAc)
(Panreac, Barcelona, Spain) was used as
mobile phase for homosalate determina-
tion.
Tetrahydrofuran (THF) (Scharlau)
was used for sample pre-treatment for the
HPLC method used as reference. A mix-
ture of deionized water, THF and HAc
(Panreac) was used as mobile phase for
homosalate determination.
Pure homosalate provided by Chemir
(Barcelona, Spain) was used as standard.
Siliceous earth (Scharlau) was used as
inert support for supercritical extraction.
The first pump of the equipment was
filled with CO2 (Air Liquid, Valencia,
Spain) and the second pump with EtOH
796
(Scharlau). The extraction system allowed
the flows from the C O 2 pump and the
EtOH pump to be mixed to modify the
polarity of the solvent.
Lipstick samples containing known
concentrations of homosalate were pre-
pared according to a standard procedure
followed in cosmetic industries (provided
by Laboratories Guinama, Valencia,
Spain). These formulations also contained
other ingredients such as: beeswax, cocoa
butter, coconut oil, isopropyl myristate,
butyl hydroxy toluene, castor oil and pre-
servatives.
Procedures
StudyofSFEParameters
The effect of the supercritical extraction
variables on the extraction yield of homo-
salate was studied to select adequate para-
meters for homosalate extraction. 1 g silic-
eous earth and 0.01 g pure homosalate
were introduced into the extraction car-
tridge in all experiments. The extract ob-
tained was diluted with EtOH, and homo-
salate was determined by UV at 239 nm,
using standard solutions of pure homosa-
late in ethanol (8 18 ixgmL 1).
ProposedMethod
Three sample replicates were prepared
and analyzed as follows: 1 g siliceous earth
was weighed into the extraction cartridge,
and 0.05 g lipstick added. SFE was carried
out for 5 min at 300 bar and 60 ~ using a
4 . 5 m L m i n 1 CO2:EtOH (85:15) flow
rate. The residue collected was dissolved
in ethanol, filtered (if necessary), and di-
luted appropiately with ethanol. 20 ixL of
the sample solution was injected into the
HPLC, and separated using a flow rate of
1 m L m i n 1 and H20:HAc:EtOH (24.9:
0.1:75.0) as mobile phase. UV detection
was at 309 nm using homosalate in etha-
nol solutions (25 751xgmL 1) as stan-
dards.
ReferenceMethod
No published methods for the determina-
tion of homosalate in solid samples have
been reported. We have modified a proce-
dure proposed by DiNunzio and Gadde
[24] for determining other UV-filters and
it has been used here as the reference
method. Three sample replicates were pre-
pared and analyzed as follows: 0.05 g of
the lipstick sample was weighed into a
Chromatographia 2001, 54, December (No. 11/12)
beaker, 10 mL THF was added, the bea-
ker was introduced into an ultrasonic bath
for 15 min, filtered (if necessary), and the
contents transferred to a 25 mL volu-
metric flask and diluted with THF. Sam-
ple solutions 20 ixL were injected into the
HPLC, and eluted using a 1.5mLmin 1
flow rate and a solution of THF:HA-
c:H20 (55.00:0.09:44.91) as mobile phase.
UV detection was at 309 nm using homo-
salate in T H F solutions (25 75 ixgmL 1)
as standards.
Results and Discussion
Selectionof Supercritical
Parameters
Figure 1 shows the effect of pressure, ex-
traction time, and percentage of modifier
(ethanol) on the extraction yield, at 40 ~
Extraction yields under these conditions
were ;r 90% using CO2 nor using ethanol-
CO2 mixtures. However, at 60 ~ extrac-
tion yields >90% were obtained in all
cases, being >95% if mixtures of CO2
with 5 15% ethanol were used.
The effect of the weighed mass on the
homosalate yield was studied by extract-
ing a lipstick sample containing a known
concentration of homosalate. It was trea-
ted under the selected conditions and ana-
lyzed by HPLC. The extraction yield in-
creased as the sample mass decreased. A
99 • 1% yield was obtained by weighing
0.05 g of sunscreen sample.
Validation of ProposedMethod
Homosalate was determined in lipstick
samples by the above method and by the
reference procedure. Results and standard
deviation (3 replicates per sample) are
shown in Table I where real values are
also indicated. The relative standard de-
viations of the results were 0.4 1%.
The calibration curve was linear over
the working range (r = 0.99996). The sen-
sitivity estimated from the slope of the ca-
libration curve was 15030 • 80 mLixg 1.
The limit of detection estimated by 3. s y/x
/ b was ca. 4 ixg mL 1, where s y/x is the
standard deviation of the calibration line
and b the slope.
No contamination of the LC column
from other constituents of the sample was
observed probably because most of them
were retained in the extraction cartridges
under the working conditions.
Short Communication
 loo[
60
40
o~ 100
"~
.~,
.~
80
60
40
[5] Whitmore, S.E.; Morison, W.L. Arch.
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">'
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Homosalate concentration / mg.g 1 London, UK, 1988.
Sample
[17] Luque de Castro, M.D.; Jimenez Carmo-
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A paired Student's t-test [25] which is Acknowledgments [21] de la Guardia, M.; Khalaf, K.D.; Carbo-
nell, V.; Morales-Rubio, A.E. Anal. Chim.
appropiate when two methods of analysis
Acta 1995, 308, 462 468.
have to be compared by studying test sam- The authors acknowledge the financial [22] Hjeresen, D.L. Crit. Rev. Anal. Chem.
ples containing subtantially different support of the Spanish Ministry of Educa- 1998,28,9 12.
amounts of analyte was used to determine tion and Culture for our research project [23] Scalia S., J. Chromatogr. 2000, 870, 199
the accuracy of the results. The results ob- 205.
on the development of analytical methods [24] DiNunzio, J.E.; Gadde, R.R.J. Chroma-
tained were consistent with the real con- for quality control of sunscreen formula- togr. 1990, 519, 117 124.
centrations and with those obtained by tions (PM-98 0210). A. Chisvert ac- [25] Miller, J.C.;Miller, J.N. StatisticsfbrAna-
the reference procedure (95% confidence knowledges the predoctoral grant for re- lytical Chemistry, Prentice-Hall, Scarbor-
ough, Ont., Canada, 1993.
level). search on sunscreens analysis given by the
Conselleria d'Educaci6 i Cultura (Valen- Received: Nov 8, 2000
cia) and by the Spanish Education Minis- Revised manuscripts
Conclusions try.
received: Feb 19 and Aug 13, 2001
Accepted: Sep 18, 2001
The proposed method allows extraction
of homosalate in lipstick samples in 5 min
with supercritical CO2, using a 4 m L References
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Short Communication Chromatographia 2001, 54, December (No. 11/12) 797