Académique Documents
Professionnel Documents
Culture Documents
Accepted
for publication
November
1, 2006.
Synopsis
A high-performance
liquid chromatographic methodwasdeveloped for quantitativeanalysis
of arbutin.The
arbutinwasseparated on an ODS Hypersil© %8 columnwith a mobilephaseof water:methanol:0.1 M
hydrochloric
acid(89:10:1,v/v/v).The levelof arbutinwasmeasuredby meansof UV detectionat 222 nm.
The optimum conditionsfor arbutin quantitativeanalysiswere investigated.The calibrationcurvewas
foundto be linearup to 1,000[tg/ml-• of arbutinconcentration,
andtheworkingcalibrationcurvefor
arbutindetermination overthe range0.5-30.0 lxg/ml-• of arbutin(r2 = 0.9999)wasestablished.
The
relativestandarddeviationsfor intradayand interdaywerefoundto be 0.98% and 1.15%, respectively.
A
detection
limit (3cr)andquantitation
limit(10cr)of0.02pg/ml-• and0.212g/ml
-•, respectively,
anda mean
percentagerecoveryof the spikedarbutinof 99.88 -+1.12% wereobtained.The proposed
methodhasbeen
appliedto the determination of arbutinin commercial skin-whitening
creams(Arbuwhite© cream,Super
Whitening
© cream,andShiseido
© cream)with average
contents
of 7.60, 5.30,and57.90mg/g-•, respec-
tively. It wasalsoappliedto the determinationof arbutin in medicinalplant extractsfrom Betu/aa/noides
Buch. Ham., Clerodendrum petasites
S. Moore, Curculigo latifo/ia Dryand. Var. latifolia, and HeJperethusa
crenulata
(Roxb.)Roem,levels
ofwhichwerefoundto be3.50,1.50,1.10,and0.12ing/g
-• , respectively
(no
articlereportedin the literatureaboutarbutinanalysis).
The proposed
HPLC methodis rapid,simple,and
selectivefor routineanalysis.
INTRODUCTION
Addressall correspondence
to BoonsomLiawruangrath.
35
36 JOURNAL OF COSMETIC SCIENCE
OH
• OH
CH20
H OH
Hydroquinone Arbutin
(a) (b)
Figure 1. Chemicalstructuresof hydroquinone
(a) and arbutin (b).
HPLC DETERMINATION OF ARBUTIN 37
EXPERIMENTAL
APPARATUS
REAGENTS
The followingstandardreagents
wereused:arbutinHPLC grade98% (Sigma,St Louis,
MO) and resorcinol98% (Fluka). The following reagentswere used:hydrochloricacid
(AR) (FarmitaliaCarlo Erba, Italy), glacial aceticacid (AR) (FarmitaliaCarlo Erba),
acetonitrile(HPLC grade) (Lab-ScanAnalytical Sciences,Ireland), ethyl acetate(AR)
(BDH laboratorysupplies,England),methanol(HPLC grade)(Lab-Scan AnalyticalSci-
ences),and ether (AR) (Lab-ScanAnalytical Sciences). De-ionizeddistilled water was
usedthroughout.
SAMPLES
Skin whitening
prodz/cts.
Arbuwhite© cream(Nature Best Health ProductCo., Ltd.,
Thailand),SuperWhitening© cream(Aunyamanee Herbs,Thailand),and Shiseido
©
cream(ShiseidoCo., Ltd., Tokyo,Japan)were used.
Plant materialand location.The bark of BetMaalnoideswas collectedat Bah Bae Village,
Mae Dtang District, ChiangMai Province,Thailand,in October2003 and wasiden-
tified by W. Thongchai1. Voucherspecimens havebeendepositedat CMU Herbarium,
ChiangMai University,ChiangMai, Thailand. The rootsof Clerodendrz/m petasites
S.
Moorewerecollectedfrom ChiangDow, ChaingMai, Thailand.The tubersof Curculigo
latij$liaDryand.Var. latifoliawerecollectedfrom Papae,Maetang,ChiangMai, Thai-
land. The trunk of HeJ;Oerethmacremdata(Roxb.) Roere. was collectedfrom Mae Sai,
Chiang Rai, Thailand.
38 JOURNAL OF COSMETIC SCIENCE
PROCEDURES
Samplepreparation
(skin-whitening
cream).About 0.5 g of each whitening cream was
accuratelyweighed and transferredinto three separate25-ml volumetricflasksand
dissolved
in methanol.
To eachflask50 t•g/ml-• of resorcinol
wasaddedasaninternal
standard.The solutionwassonicatedvigorouslyfor 30 min, centrifugedat 4000 rpm for
30 min, and filtered on a Millipore membrane(0.45 t•m) to obtain a transparent
solution.The supernatantliquid wasusedfor chromatographic analysis.
Extraction
of medicinal plants.The dried medicinalplantswerepowdered.Then 6 kg of
the powderwasextractedwith two successive portionsof 5.0 1 of de-ionizedwater and
methanol.They wereshakenin a wrist-actionshakerfor five hoursandfiltered.Then the
solventof the filtrate couldbe removedeither by usinga spray-driedtechnique(tem-
perature
100øCandflowrate1.0 ml/min-•) to givea brownpowder,
or by usinga
rotatoryevaporatorto give a dark browncruderesidue.
Preparation
ofstandard
soluions.
A 1,000tag/ml
-• stocksolution
ofarbutinstandard
was
preparedin methanol.A seriesof eachstandardsolutioncontaining0.5, 1.0, 3.0, 5.0,
10.0,and30.0lag/ml
-• wasprepared
fromthestockstandard
solution.
Preparationof samplesoluions.
Three setsof medicinalcrudeextracts(5 g) and cosmetic
samples(0.5 g) of eachset were extractedunderrefluxwith 100 ml of 75% methanol
for 30 min andfiltered.The flitrate wasevaporated
to about12 ml andtransferred into
a 250-ml separatorfollowedby addition of 50 ml of water. The mixture was then
extractedwith ether(2 x 30 ml). The combinedaqueouslayerwasextractedwith ethyl
acetate(3 x 50 ml). The combinedethyl acetateextractwasthen evaporated to dryness
and dissolved in 10 ml of methanol.
Preliminaryinvestigation.
A preliminary investigationwas carriedout to separatesome
chemicalconstituentsby TLC. The crude extract was extractedwith 75% methanol
underrefluxfor 30 min andthen filtered.The flitrate wasevaporated to about12 ml and
transferredto a 250-ml separatingfunnel togetherwith 50 ml of water.This solution
wasextractedthree times with 50 ml of ethyl acetate,and the combinedethyl acetate
extractswere evaporatedto drynessand the residuesdissolvedin 10% methanol.The
samplesolutionandthe standardsolutionswereseparated on a silicagel GF254(20 x 20
cm) glassplate, using ethyl acetate:methanol(9:1) as a developingsolvent.The crude
extractgavefive well-definedspots.The Rf valueof eachspotwasexactlythe sameas
that obtainedfrom eachspotof standard.
Optimizationof experimentalconditions
for RP-HPLC. RP-HPLC was performedunder
isocraticconditions.All experimentalconditionswereoptimizedby meansof a univari-
ate method as follows:
Analyticalwavelength.Optimum absorbance
of eachstandardsolutionwasdetermined
byinjection
ofthesame
amount
ofmixedstandard
solutions
(5.0lag/ml
-•) at different
wavelengths
from 200 nm to 400 nm. The mobilephasewasa mixtureconsisting
of
water:methanol
(80:20v/v)witha flowrateof 1.0ml/min-•.Astheoptimum
to obtain
the bestsensitivity,•maxwaschosen.
Mobilephase.Varioussolventsystems weretestedasthe mobilephasefor the separation
of arbutinin the samples,
e.g.,water:acetonitrile:0.1
M hydrochloricacid(94:5:1,v/v/v),
water:methanol:0.1M hydrochloricacid (89:10:1, v/v/v), and methanol:100mM phos-
phatebuffer,pH 2.1 (10:90 v/v).
HPLC DETERMINATION OF ARBUTIN 39
Recommended
RP-HPLC procedure.
A sampleand/orstandardsolutioncontainingarbutin
wasseparated
ona reverse-phase
ODSHypersil
© C•8column(125 mm x 4 mm, 5.0 pm)
and detectedat 222 nm. An aliquot of 100 pl of a seriesof arbutin standardsolutions
and 100 pl of sampleextractwasinjectedinto the LC systemand elutedwith the mobile
phase,water:methanol:0.1M hydrochloricacid (89:10:1, v/v/v) (flow rate = 1.0 ml/
min •). Theareaof thearbutinpeakwasmeasured.
Arbutinconcentration
in theplant
extractwas determinedby referenceto the calibrationcurvepreparedunder identical
experimentalconditions.
The optimal conditionsof HPLC for determiningarbutin were carriedout under iso-
cratic conditions.Variousmobile phasesystemswith differentcompositions were in-
vestigated.First, the optimal wavelengthfor the detectionof arbutin and other com-
pounds,as mentionedearlier,wasinvestigated,and the UV spectrumof eachstandard
compoundshowedthe absorptionmaximaat 222 nm. A wavelengthof 222 nm showed
the highestsensitivityfor arbutin. Second,amongthe mobile phasesstudied,a mixture
consistingof water:methanol:0.1 M hydrochloricacid (89:10:1, v/v/v) wasusedas the
mobilephase,and it wasfoundthat this mobile phasewasthe most suitablebecauseit
resultedin goodretentiontimes, resolution,and satisfactory peak profiles(Figure2).
Finally,theoptimumflowratewas1.0 ml/min-•, asit gavea goodresolution,
high
sensitivity,a shortanalysistime, etc. In the RP-HPLC analysis,arbutin and resorcinol
(internal standard)showedsinglesymmetricalpeaks(retentiontime 5.7 min and 10.7
min), respectively.
METHOD VALIDATION
mAU Resorcinol
2o.i
Arbutm
8O
4O
2 4 6 8 10 12 14
Figure2. HPLCchromatogram
of thearbutinstandard
(5 pg/ml •) andtheresorcinol
internalstandard
(5
pg/ml •).
Table I
LinearRegression
Analysis(n = 3) and Limits of Detection(S/N = 3) and Quantitation(S/N = 10)
Table II
Intraday and Interday PrecisionAnalysisof Arbutin StandardSolutions
Table III
Accuracyof the ProposedLC Method (n = 7)
mAU
_L
Figure 3. HPLC chromatogram
of a samplecontainingarbutinfrom skin-whiteningcream.
Table IV
Comparisonof Two Methodsfor MeasuringArbutin in Samplesof CommercialWhitening Cream
Cosmeticsample Spectrophotometric
(Arbuwhite© cream) RP-HPLC method• method• (20) Calculatedt-values
0.75 0.77
0.75 0.78
0.77 0.80
0.76 0.78 1.38
0.76 0.79
0.75 0.78
0.77 0.80
Average 0.76 0.79
and HeJ•perethusa
crenu/ata
(Roxb.) M. Roem.The sampleswereextractedwith water and
methanol. Then the amounts of arbutin in the sampleswere determined by the
RP-HPLC method. The amountsof arbutin found in the aqueousextract were 3.50,
1.50,1.10,and0.12pg/g-•,respectively.
Theamounts
ofarbutinfoundin themethanol
extractwere0.77,0.0002,and0.0012pg/g-•,respectively.
Themethod wassuccess-
fully applied to the determination of arbutin in three commercialskin-whitening
creams.The amountsof arbutin found in the samples(Arbuwhite© cream,Super
Whitening
© cream,andShiseido
© cream)were0.76, 0.58, and5.79 mg/g-•, respec-
tively.
CONCLUSION
The reversed-phase
high-performance liquid chromatographic
procedurehas beende-
velopedfor determiningarbutin in commercialskin-whiteningcreamsand someme-
HPLC DETERMINATION OF ARBUTIN 43
dicinalplant extracts,respectively.
The methodwasalsovalidatedfor limit of detection,
limit of quantitation,repeatability,reproducibility,and accuracy.The optimum con-
ditionsand analyticalcharacteristicsfor RP-HPLC determinationof arbutin exhibited
goodresolution,shortanalysistime, andratherhigh sensitivity.In the proposedmethod
for determiningarbutin in skin-whiteningcreams,the workingcalibrationcurvesover
theranges
of 0.5-30.0pg/ml-• wereestablished.
Themethod
wassuccessfully
applied
to the determinationof arbutinin threecommercialskin-whiteningcreams.The content
of arbutinfoundin the samples
(Arbuwhite
© cream,SuperWhitening© cream,and
Shiseido
© cream)
were0.76,0.58,and5.79mg/g-•, respectively.
Themethod
wasalso
appliedto the determinationof arbutin in somemedicinalplant extracts.The amounts
of arbutin in Bet•la alnoidesBuch. Ham., Clerodendr•m petasites
S. Moore., C•rc•/igo
latifoliaDryand.Var. latifolia,andHesperethma
cren•lata(Roxb.).Roemin the aqueous
extracts
were3.50, 1.50, 1.10,and 0.12 l•g/g-•, respectively.
Arbutinfromthese
medicinalplants can be used for the productionof skin-whiteningcosmetics.The
benefitsof the proposedmethodare simplicity, convenience,
rapidity, sensitivity,good
precision,and accuracy.The methodis suitablefor routineanalysisof arbutinin com-
mercial cosmeticsand raw plant materials.
ACKNOWLEDGMENTS
REFERENCES
All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immedi