International Journal of Biomedical and Advance Research

METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS

ESTIMATION OF OXYBENZONE, OCTINOXATE AND AVOBENZONE IN SUN-
SCREEN LOTION BY REVERSED PHASE HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Tapan Banker*, Prashant Kale, Ashok Peepliwal

ICPA Health Products Ltd, 286/287 G.I.D.C, Ankleshwar, Dist: Bharuch, Gujarat

E-mail of Corresponding author: bankertapan70@gmail.com

Abstract
A simple, precise, accurate, selective, and economic reversed-phase RP-HPLC method has
been developed for simultaneous analysis of Oxybenzone, Octinoxate and Avobenzone in
sun-screen lotion. Chromatographic separation was performed by using UV detector at
wavelength of 330nm. The separation was performed at ambient temperature by using C18
stationary phase followed by methanol: phosphate buffer (90:10) pH (3.0) as a mobile phase.
The mobile phase flow rate was 1.2ml/min and gradient elution was performed. Total run
time was 25min. The method was successfully applied on formulation and there has been no
interference of matrix components. The linearity of method was obtained in the range of 18 to
48µg/ml for Oxybenzone, 45 to 120µg/ml for Octinoxate and 12 to 32µg/ml for Avobenzone.
Recovery results obtained for all three components were within the range. All validation
parameters were done in accordance with International Conference on Harmonization (ICH).
The validation results and results from statistical analysis of the data, demonstrated the
method were reliable.

Keywords: Gradient elution, RP-HPLC, Oxybenzone, Octinoxate, Avobenzone

Introduction X-ray, ionizing, ultraviolet (UV), visible

A sunscreen cosmetic could be defined as
“any cosmetic product containing UV
filters in its formulation in order to protect
the skin from t the solar deleterious UV-
light, avoiding or minimizing the damage
that this radiation might cause on human
health” 1. Extraterrestrial sunlight includes
and infrared radiation, and radio waves.
Oxybenzone, Octinoxate and Avobenzone
are also called as UV filters as they
prevent damage to the skin from UV
radiation. UV spectrum is divided into
three bands. UVA (320-400nm), UVB
(290-320nm) and UVC (200-290nm).

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Research Article Banker et al 93

UVA radiation is more harmful then other protection and vitamin D deficiency from
two. UVB radiation is fully absorbed by overuse of sunscreen.
the stratum corneum and the top layers of Octinoxate is chemically ethyl
the epidermis, whereas up to 50% of hexyl methoxy cinnamate. It is frequently
incident UVA radiation penetrates used in combination with other UVB
2
Caucasian skin deep into the dermis. absorbers to achieve high SPF values in
Oxybenzone is chemically (2- the final product. Topical application of
hydroxy-4-methoxyphenyl)-phenyl - OMC is tolerated well: skin irritation is
methanone with a molecular weight almost negligible, and photocontact
228.24g/mol. It is an important compound dermatitis is rare. Upon exposure to
in organic photochemistry and organic sunlight, octinoxate degrades into a
synthesis. It is a white cryslline powder photoproduct with less UV-absorbing
which is insoluble in water and having a ability. Several studies suggest ways to
melting point 49 C. It is a derivative of improve the photostability of cinnamate
benzophenone. Benzophenones absorb Encapsulation of ethylhexylp-
UVB and some UVA (to approximately methoxycinnamate into nanoparticles
360 nm, with a peak at 290 nm). The most results in a reduction of the
7
popular benzophenone and one of the most photodegradation . Animal studies
common sunscreen ingredients is indicate that octyl methoxycinnamate may
benzophenone-3 or oxybenzophenone. It produce hormonal (estrogen-like) and
has been isolated in the blood and urine of possibly other adverse effects. Whether
3-5
humans after topical application. typical human use of octyl
Compared with other UV filters, methoxycinnamate may cause such effects
benzophenone-3 is the most bioavailable is unclear.
following topical application; however, Avobenzone is chemically 4-tert-
this bioavailability is not of toxicologic butyl-4’-methoxy-dibenzoyl methane with
3, 6
concern. Moreover, it has the highest a molecular weight 310.39. It is insoluble
reported incidence of photodermatitis. The in water but freely soluble in organic
photomutagenic properties of these solvent like methanol. Avobenzone having
compounds might be a contributing factor a phenyl ketone group and sterically
to the increased melanoma incidence that hindered group in a molecule. It absorbs
has been found in sunscreen users. Other wider range of UV wavelength. It absorbs
possibilities include consequent UVA (320-400nm, and it is associated
overexposure to sun without UVA with long term skin damage) and UVB
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Research Article Banker et al 94

(290-320nm, that causes sunburn rays).It is HPLC and it will take about 100min run-
one of most effective sun-screen time. But no method has been reported for
ingredient. The analytical control of the analysis and separation of Oxybenzone,
sunscreen cosmetics is necessary since the Octinoxate and Avobenzone in sun-screen
content of UV filters in the final product is lotion by RP-HPLC. 9
related to its sun protection efficacy that is Material & Methods
usually claimed by the labeled sun Chemicals: All reagents were HPLC
protection factor (SPF). grade. Methanol, Ortho Phosphoric Acid
The sun protection factor is a measure of and Potassium dihydrogen phosphate were
the ability of a sunscreen to protect against received from merck, Germany. HPLC
8
erythema, which is thus primarily a grade deionized filtered water was used to
measure of UVB protection. The SPF is a prepare buffer. All drug samples received
ratio of the dose of UV radiation required namely oxybenzone, octinoxate and
to produce a minimal erythema 24 hours avobenzone were received from ISP
after exposure in sunscreen-protected skin having % purity 98.59, 99.45 and
to the dose required to produce the same 99.00respectively.
degree of erythema in unprotected skin. In Instrumentation: The HPLC apparatus
other words: the SPF number defines how consisted of a Shimadzu
long you can stay in the sun before getting chromatogrimaaphic system (binary LC-
burnt. If it normally takes you 20 minutes pump series 2010, UV/Visible detector-
in the sun before you get burned, an SPF series 2010 software LC solution linked to
15 product will let you stay 15 times an autosampler with a 20-µ1 sample loop
longer in the sun: 20 min x 15 (SPF) = 300 and a chromatographic data processor. The
min (5 hours). Several methods have been software ran on HP computer operated
reported for analysis of sun-screen with Windows 2007 professional used for
ingredients by second order spectroscopy, this method.
flow injection isodiffertential derivative Chromatographic conditions: The
spectroscopy, UV spectroscopy and by detector was set to 330 nm and 20 µl
UPLC. Several methods also been reported volume of each sample solution and
for analysis of other sun-screen ingredient standard was injected into sample injector
like Homosalate, Octyl Dimethyl PABA, of HPLC and separated on waters C18
Octyl Salicylate, Camphor, column (250 x 4.6 mm i.d5µ) with
Phenylbenzimidazolesulphonic acid along methanol and phosphate buffer 20 mM and
with Oxybenzone and Octinoxate by RP- pH was adjusted 3.0 with Ortho
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Research Article
Phosphoric acid followed by gradient
elution method. The flow rate of a pump
was set 1.2ml/min.Change in gradient
elution with time is depicted in Table no 1.
Standard solution preparation: Pure
standards of Oxybenzone, Octinoxate &
Avobenzone were prepared and analyzed
under the optimized chromatographic
conditions. Accurately weigh 30mg of
Oxybenzone, 75mg of Octinoxate and
20mg of Avobenzone and transfer into
100ml volumetric flask and volume was
make up to mark with methanol.(Stock
Solution) Sonicate it for 15-30 min..Take
5ml from it and diluted up to 50ml with
methanol to obtain final concentration
30µgm/ml Oxybenzone, 75µgm/ml
Octinoxate and 20µgm/ml Avobenzone.
Filter it by using 0.2µ filter.
Sample solution preparation: For
preparing sample solution, 0.1gm of
sample were weighted (each containing
3.0% of Oxybenzone, 7.5% of Octinoxate
and 2.0% of Avobenzone) and transfer into
100ml volumetric flask. They were
dissolved in 100ml of methanol to obtain
final concentration 30µgm/ml
Oxybenzone, 75µgm/ml Octinoxate and
20µgm/ml Avobenzone. Sonicate it for
30min. Filter it by using 0.2µ filter.
Measurement of Isobestic Point:
Appropriate dilution of each standard
stock solution was made with methanol as
a solvent. Accurately weighted 20mg of
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Banker et al 95
Oxybenzone, 20mg of Octinoxate and
20mg of Avobenzone in three volumetric
flask and make volume up to 100ml using
methanol. Sonicate it for 15 min. And then
take 5ml from each and make the volume
up to 100ml with methanol. So
concentration of each of this will be
10µgm/ml. Then each solution was
scanned using double beam UV Visible
Spectrophotometer(Perkin-Elmer Lambada
25) between the range of 190 to 400 nm
and overlain spectra was obtained. The
wavelength selected was 330 nm which is
Isobestic Point. The overlain spectra of
Oxybenzone, Octinoxate and Avobenzone
are shown in figure below.
METHOD VALIDATION
Linearity: Six different concentrations
were prepared ranging from18µg/ml to
48µg/ml for Oxybenzone, 45µg/ml to
105µg/ml of Octinoxate and 12µg/ml to
32µg/ml for Avobenzone by diluting the
standard solution (stock solution) in
mobile phase. The calibration curve was
prepared by plotting peak areas against
concentration and r2 was found 0.999 for
all three components.
Precision: The precision study was carried
out by preparing at a concentration of
30µg/ml for Oxybenzone, 75µg/ml for
Octinoxate and 12µg/ml Of Avobenzone.
Precision study was carried out by
measuring repeatability and ruggedness.
Research Article
Accuracy: The accuracy of method was
established using recovery technique i.e.
by external standard method. The known
amount of standard was added in known
amount of sample as to get three different
levels (80%, 100% and 120%). Each
determination was done in triplicate.
System Suitability: System suitability
was performed by preparing standard
solution containing 30µg/ml
Oxybenzone, 75µg/ml of Octinoxate and
20µg/ml of Avobenzone and injecting six
replicate.
Robustness: Robustness of method was
studied by changing flow rate
±0.2ml/min and column temperature by
±5 c and % RSD was measured. It was
performed by preparing a concentration of
75µg/ml for Oxybenzone, 75µg/ml for
Octinoxate and 20µg/ml for Avobenzone
and injecting 3 replicate for
condition.
Results
Linearity: The calibration curve was
obtained by plotting peak areas against
concentration and showed linearity in
concentration range of 18µg/ml to48µg/ml
for Oxybenzone, 45µg/ml to 105µg/ml of
Octinoxate and 12µg/ml to 32µg/ml for
Avobenzone with 0.999 as the coefficient
of correlation. All the linearity data
depicted in Table 2.
Precision: The % assay for sun-screen
lotion was calculated for six replicate and
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Banker et al 96
% RSD was calculated. Precision was
performed by measuring ruggedness and
repeatability study. Results of repeatability
depicted in Table 3.
Accuracy: The % recovery was calculated
for triplicate samples and for all levels and
mean recovery was calculated. The mean
recovery was well within the acceptance
limit hence the method was accurate, as
of depicted in Table 4.
System Suitability: System Suitability
was performed by injecting six replicate of
Oxybenzone, Octinoxate and Avobenzone
respectively and all parameters are found
by to be within range.The result of system
suitability is depicted in Table 5.
Robustness: Robustness study was
performed by changing experimental
condition. In this study flow rate was
changed by ±0.2ml/min and column
each temperature by ±5 C and %RSD was
measured. The results obtained are
depicted in Table6.
Discussion
Many mobile phases were tried the initial
trial was performed on Methanol, Water
with 3.0 % THF Acetonitrile as mobile
phase in ratio (70:20:10) with isocratic
elution and the flow rate was kept
1.0ml/min. it produced a broad peak so a
second trial was necessary and was
performed by changing the of mobile
phase ratio to (65:25:10) of methanol,
water with THF 10% and acetonitrile
Research Article Banker et al 97

respectively. The peak shape produced by
the trial was not satisfactory hence method
was changed to gradient elution. The third
trial was performed by changing the
mobile phase as potassium dihydrogen
phosphate buffer with pH 3.5 and
methanol in the ratio (20:80), flow rate
was also changed from 1ml/min to
1.2ml/min but even then the results were
not satisfactory and finally mobile phase
containing 20mM Potassium dihydrogen
phosphate buffer and methanol, pH of
buffer adjusted to 3.0 with phosphoric acid
was optimized. Flow rates between 1.0 and
1.2ml/min were studied. A flow rate of 1.2
ml/min gave an optimal peak shape and
was selected. Using a reversed-phase C18
column, the retention times for
Oxybenzone, Octinoxate and Avobenzone
was found to be 3.7min, 9.3min and
16.9min respectively. Total time
analysis was kept 25min.The Isobestic
point was detected at 330 nm and this
wavelength was chosen for the analysis.
(Fig1). The developed method was linear
showing the coefficient of correlation of
0.999. Percentage RSD of accuracy study
for three levels (80, 100 and 120 %)
showed below 2.0% and precision was
found to be below 2.0%. All system
suitability parameters were found to be
within range. Percentage RSD
robustness study was found to be below
2.0 by changing parameters like column
temperature and flow rate.
Conclusion:
A new RP-HPLC method with UV
detector for simultaneous analysis and
separation of Oxybenzone, Octinoxate and
Avobenzone has developed for first time.
It is simple, precise, specific, sensitive and
economic with acceptable accuracy,
precision, correlation coefficient (r2). The
method has been successfully used to
determine concentration of oxybenzone,
octinoxate and avobenzone in sun-screen
lotion with shorter analysis time (<30min)
and also applied for qualitative and
quantitative analysis in formulated
preparation.
Acknowledgement
I am deeply thankful to ICPA Health
Product Ltd, Ankleshwar for providing
of research facilities to carry out this research
work. I am also thankful to Mr. Ashok
Peepliwal for providing guidance
regarding this work.
References
1 Salvador A, Chisvert A. A critical
survey on UV filters determination
Analytica Chimica Acta 2005; 537:1-
14.
2 Bruls WA, Slaper H, van der Leun JC.
Transmission of human epidermis and
of stratum corneum as a function of
thickness in the ultraviolet and visible

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3 wavelengths Photochem Photobiol sunscreens across skin. Am J Clin

1984; 40: 485–494. Dermatol 2000; 1: 217–224.
4 Janjua NR, Mogensen B, Anderssonl. 8 Perugini P, Simeoni S, Scalia S. Effect
Systemic absorption of the sunscreens of nanoparticle encapsulation on the
photostability of the sunscreen agent,
benzophenone-3, octylmetho-
2-ethylhexyl-p-methoxycinnamate. Int
xycinnamate, and 3-(4-methyl- J Pharm 2002; 246:37–45.
benzylidene) camphor after whole- 9 Lavker RM, Gerberick GF, Veres D,

body topical application and Irwin CJ, Kaidbey KH. Cumulative

reproductive hormone levels in effects from repeated exposures to

humans. J Invest Dermatol 2004; 123: suberythemal doses of UVB and UVA

57–61. in human skin. J Am Acad Dermatol

5 Gustavsson Gonzalez H, Farbrot A, 1995; 32: 53–62.

Larko O. Percutaneous absorption of 10 Sethi P.D (2001) High performance

benzophenone-3, a common Liquid Chromatography- Quantitative

component of topical sunscreens. Clin analysis of pharmaceutical

Exp Dermatol 2002; 27: 691–694. formulation, 1st ed; CBS Publication;

6 Hayden CG, Roberts MS, Benson pp 564-567.

HA.Systemic absorption of sunscreen

after topical application. Lancet
350:863–864.
7 Benson HA. Assessment and clinical
implications of absorption of

Table1 Change in concentration of mobile phase for gradient elution

Sr. Time(min) Concentration of Concentration of

No methanol (%) phosphate buffer
1 0.01 90 10
2 5.00 90 10
3 7.00 80 20
4 8.00 70 30
5 12.00 70 30
6 13.00 80 20
7 14.00 90 10
8 25.00 90 10

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Research Article Banker et al 99

Table2 Results of Linearity Study

Compound Equcation R2 Slope Intercept

Oxybenzone Y=43273x+27043 0.999 43273 27043
Octinoxate Y=39250x-81500 0.999 39250 -81500
Avobenzone Y=12310x+12113 0.999 12310 12113

Table 3 Results of repeatability study

Peak Area
Sr No.
Oxybenzone Octinoxate Avobenzone
1 1315944 3282218 1097999
2 1303567 3220927 1096159
3 1319462 3301695 1100626
4 1324364 3283106 1083128
5 1302983 3255871 1085468
6 1312633 3255852 1099967
7 1332691 3296816 1093423
8 1339654 3295485 1093458
9 1334587 3300250 1094754
10 1332548 3314512 1095878
Mean 1321843 3280673 1094086
SD 10769.36 28421.42 6967.47
%RSD 0.8147 0.8663 0.6368

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 Research Article Banker et al 100

Table4 Results of recovery study

Sr Label Claim Amount Taken Amount Recovered % Amount

% Assay
No. (µg/ml) (µg/ml) (µg/ml) Recovered
Oxy Octi Avo Oxy Octi Avo Oxy Octi Avo Oxy Octi Avo Oxy Octi Avo
1 24.2 50.2 15.8 100.8 98 99.37
2 24.4 50.5 15.8 101.6 98.4 99.37
80 24 60 16 24 51.3 15.9
3 24.3 50.2 15.7 101.2 98 98.74
1 30.2 64.4 19.6 100.6 99.0 100.5
2 100 30 75 20 30 65 19.5 30.2 64.9 19.7 100.6 99.8 101.0
3 30.2 64.5 19.7 100.6 99.2 101.0
1 36.2 77.3 23.6 101.6 99.3 98.33
2 36.3 77.9 23.6 101.9 100 98.33
120 36 90 24 35.6 77.8 24.0
3 36.1 77.4 23.6 101.4 99.4 98.33

%RSD 0.501 0.73 1.134

Table 5 Results of System Suitability Study

Compound % RSD Tailing Factor Theoretical Plate

Oxybenzone 0.150 1.21 4761
Octinoxate 0.650 1.47 7150
Avobenzone 1.027 1.03 12377

Table6 Results of Robustness Stuy

Compound %RSD %RSD %RSD %RSD

Oxybenzone Flow rate 0.295 ColumnTemp 0.030 Flow rate 0.599 Column Temp 0.990
Octinoxate (1.4ml/min) 0.225 (30⁰C) 0.642 (1ml/min) 0.619 (20⁰C) 0.959
Avobenzone 0.211 0.031 0.522 0.999

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Research Article Banker et al 101

Fig1 Overlain Spectra of Oxybenzone, Octinoxate and Avobenzone.

Fig2 Chromatogram of Standard Containing Oxybenzone, Octinoxate and Avobenzone

Fig3 Calibration Curve for Oxybenzone

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Research Article Banker et al 102

Fig4 Calibration Curve for Octinoxate

Fig5 Standard calibration curve of avobenzone

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