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ICPA Health Products Ltd, 286/287 G.I.D.C, Ankleshwar, Dist: Bharuch, Gujarat
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.
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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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Accuracy: The accuracy of method was % RSD was calculated. Precision was
established using recovery technique i.e. performed by measuring ruggedness and
by external standard method. The known repeatability study. Results of repeatability
amount of standard was added in known depicted in Table 3.
amount of sample as to get three different Accuracy: The % recovery was calculated
levels (80%, 100% and 120%). Each for triplicate samples and for all levels and
determination was done in triplicate. mean recovery was calculated. The mean
System Suitability: System suitability recovery was well within the acceptance
was performed by preparing standard limit hence the method was accurate, as
solution containing 30µg/ml of depicted in Table 4.
Oxybenzone, 75µg/ml of Octinoxate and System Suitability: System Suitability
20µg/ml of Avobenzone and injecting six was performed by injecting six replicate of
replicate. Oxybenzone, Octinoxate and Avobenzone
Robustness: Robustness of method was respectively and all parameters are found
studied by changing flow rate by to be within range.The result of system
±0.2ml/min and column temperature by suitability is depicted in Table 5.
±5 c and % RSD was measured. It was Robustness: Robustness study was
performed by preparing a concentration of performed by changing experimental
75µg/ml for Oxybenzone, 75µg/ml for condition. In this study flow rate was
Octinoxate and 20µg/ml for Avobenzone changed by ±0.2ml/min and column
and injecting 3 replicate for each temperature by ±5 C and %RSD was
condition. measured. The results obtained are
Results depicted in Table6.
Linearity: The calibration curve was Discussion
obtained by plotting peak areas against Many mobile phases were tried the initial
concentration and showed linearity in trial was performed on Methanol, Water
concentration range of 18µg/ml to48µg/ml with 3.0 % THF Acetonitrile as mobile
for Oxybenzone, 45µg/ml to 105µg/ml of phase in ratio (70:20:10) with isocratic
Octinoxate and 12µg/ml to 32µg/ml for elution and the flow rate was kept
Avobenzone with 0.999 as the coefficient 1.0ml/min. it produced a broad peak so a
of correlation. All the linearity data second trial was necessary and was
depicted in Table 2. performed by changing the of mobile
Precision: The % assay for sun-screen phase ratio to (65:25:10) of methanol,
lotion was calculated for six replicate and water with THF 10% and acetonitrile
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Research Article Banker et al 97
respectively. The peak shape produced by 2.0 by changing parameters like column
the trial was not satisfactory hence method temperature and flow rate.
was changed to gradient elution. The third Conclusion:
trial was performed by changing the A new RP-HPLC method with UV
mobile phase as potassium dihydrogen detector for simultaneous analysis and
phosphate buffer with pH 3.5 and separation of Oxybenzone, Octinoxate and
methanol in the ratio (20:80), flow rate Avobenzone has developed for first time.
was also changed from 1ml/min to It is simple, precise, specific, sensitive and
1.2ml/min but even then the results were economic with acceptable accuracy,
not satisfactory and finally mobile phase precision, correlation coefficient (r2). The
containing 20mM Potassium dihydrogen method has been successfully used to
phosphate buffer and methanol, pH of determine concentration of oxybenzone,
buffer adjusted to 3.0 with phosphoric acid octinoxate and avobenzone in sun-screen
was optimized. Flow rates between 1.0 and lotion with shorter analysis time (<30min)
1.2ml/min were studied. A flow rate of 1.2 and also applied for qualitative and
ml/min gave an optimal peak shape and quantitative analysis in formulated
was selected. Using a reversed-phase C18 preparation.
column, the retention times for Acknowledgement
Oxybenzone, Octinoxate and Avobenzone I am deeply thankful to ICPA Health
was found to be 3.7min, 9.3min and Product Ltd, Ankleshwar for providing
16.9min respectively. Total time of research facilities to carry out this research
analysis was kept 25min.The Isobestic work. I am also thankful to Mr. Ashok
point was detected at 330 nm and this Peepliwal for providing guidance
wavelength was chosen for the analysis. regarding this work.
(Fig1). The developed method was linear References
showing the coefficient of correlation of 1 Salvador A, Chisvert A. A critical
0.999. Percentage RSD of accuracy study survey on UV filters determination
for three levels (80, 100 and 120 %) Analytica Chimica Acta 2005; 537:1-
showed below 2.0% and precision was 14.
found to be below 2.0%. All system 2 Bruls WA, Slaper H, van der Leun JC.
suitability parameters were found to be Transmission of human epidermis and
within range. Percentage RSD of stratum corneum as a function of
robustness study was found to be below thickness in the ultraviolet and visible
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humans. J Invest Dermatol 2004; 123: suberythemal doses of UVB and UVA
Exp Dermatol 2002; 27: 691–694. formulation, 1st ed; CBS Publication;
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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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