of natu ral astax anthin derived fro m haematococcus algae ... · cus biom erald r. c cyanotech....
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Analysisin Asta
For questioCyanotech
1.0 IntroducThe carotenodiesters of acanthaxanth
Figure 1. C
Accurate quanumber of dianalysis of fror enzymatict is simple, cverify the acc(trans-ß-apo
s of Natuaxanthin
ns or commCorporatio
ction oid fraction ostaxanthin, 5in, and lutein
arotenoid C
antification oifferent fatty ree astaxantc procedure complete ancurate extra-8’-carotena
astadies
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ral AstaxOleoresiand Hae
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of Haematoc5% free astan as shown
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monoesters oting of β-caro
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astaxanthmonoeste
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anthin
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of astaxanthotene,
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SSaa HAqpp SPgwi GAtkg 2
3
SpectrophoSpectrophotoas accurate any degrada
HPLC AnalyAstaxanthin quantificationpresent in thprior to HPLC
Safety Proper safetyglasses and working condngestion, an
General ConAll manipulato light, oxyg
keeping the tglassware sh
2.0 Reagent• Tris H• 1.0 N• Meth• DI or • pH bu• Aceto• Trans• Astax• Chole• Petro• Anhy• Hexa• Aceto• Dichl• Dime
3.0 Equipme• Gl• Ma• Gr• gla• pH• Fu
otometric Quometric quaas HPLC. S
ation product
ysis analysis by n of astaxane sample. HC analysis.
y precautionsolvent-resi
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nsiderationtions should
gen and heattemperaturehould be use
ts HCl ( EMD P
N NaOH soluanol (VWR PDistilled wa
uffer calibratone (minimus-ß-apo-8’-cxanthin stanesterol Esteroleum Ether ydrous sodiuanes, HPLC one, HPLC Goromethane
ethylsulfoxide
ent ass bottles wagnetic stir praduated cylass beakersH meter unnel
uantificationtification of
Spectrophotots of astaxan
High Performnthin as well However, it i
ns should bestant glovessures shouldact.
s d be performt. It is recom
es at 20° C ifed.
P/N 9310) ution for pH aP/N JT9093
ater tion solution
um technical carotenal (Sigdard (Chromrase (1 vial o(such as VWm sulfate (sGrade (such
Grade (suche (DCM) as de (DMSO, su
with stir bar,plate inders, 500 , 250 mL an
n f astaxanthinometric quannthin presen
mance Liquias mixed cas essential t
e followed. Ps. Extractiond be taken to
ed in low ligmmended thf possible. C
adjustment (-3) with 0.05
grade) cont
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dissolution such as VWR
125 mL, 25
mL and 100d 500 mL
2
n yields a clontification is t in the sam
id Chromatoarotenoids athat all ester
Protective gens should beo minimize s
ht and temphat the assayCarotenoids
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ASB-000016s enzyme pe0-08)
R P/N SX076/N HX 0290
/N 9002-03)solvent (suchR P/N BDH1
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ose approximinfluenced bple.
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ear should ine performedsolvent expo
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S318-1) gma P/N B1
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h as VWR P/115-4LP)
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/N BDH1113
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4
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tion of 0.05er is added tfor astaxan
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fer, pH 7.0 matic digest o
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7.00 ±0.01 on to 7.00 u
at 4º C for upd, check intethe old one a
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8
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y to facilitate ly cut open tose the vial
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ide to dry foht.
he volumetricroom temper
n, dry test tuter which ma
b
aematococc
p sample red 50 mL gl
Then fill the aps’ content the remova
the gelcap intightly and p
metric flask uis colorless.
ally solid or sdissolution inric flask in thely disperse
r at least 30
c flask and trature (20° C
ube and cenay have carr
i
b,c
w
cus Algae P
lass beakers
beaker withis partially sl of all the con a 20 mL scplace in the s
using a Paste. Add acetosemi-solid ann the acetonhe sonicator d.
0 minutes. A
the gelcap cC), then brin
trifuge for 3 ried over in t
d
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Products.
s and record
h 20 mL acetsolid or semontent. If socrew cap viasonicator ba
eur pipette. ne to a finalnd a sonicate solution.
r bath and so
Accurately we
ontent is comng the flask t
minutes at 3the transfers
h.com
d the
tone to i-solid
onication al. Add ath for 15
Rinse volume tion step If onicate
eigh the
mpletely to
3800-s. This is
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88.03 Extract
Noa) Weig
cleanappeburne
b) Add 0c) Add 2
heate
i. PivoTh
ii. Opaglin
iii. Pitu
iv. Tostasy
v. PrPr
tion of Caroote: Performh approxima
n and dry 10 arance of thed or discolo0.5 grams of2-3 mL of DMed water bat
e
pette 1 mL oolume with ahis is the secptional: Reagainst an acenear betweenpette accurabe. o the test tubandard (tran
yringe is rinsroceed to 9.0roducts.
otenoids fro duplicate exately 25 mg mL centrifu
he sample anored materiaf glass beadMSO to the th at 43-46°C
f
g
of extract intcetone to prcond dilutiond and recordetone blankn 0.50 and 1ately 3 mL o
be containingns-ß-apo-8’-ced after use0 Hydrolysi
om Astaxanxtractions foof astaxanthge tube. Rend note any al. ds to the tubecentrifuge tuC for 15 min
g.ii
10
to a clean, drepare a 1/1n. d the absorbon the Spec
1.50). f the second
g 3 mL of thcarotenal) us
e with acetons of Carote
thin Beadleor each samphin beadletsecord the weunusual phy
e. ube, cap tighutes. Mix th
g
ry 10 mL vo0 dilution. S
bency at 474ctrophotome
d dilution into
e second dising the 50 µne. enoids from
ets or Driedple or dried Ha
eight to the nysical charac
htly and mix;he sample a
g.iii,iv
w
olumetric flasStopper the fl
4 nm of the seter. (Absorb
o a separate
lution add 5µL glass syr
m Haematoc
Haematoco
ematococcunearest 0.1 mcteristics suc
; then place few times d
g.i
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sk and bring lask and mix
second dilutbency readin
e clean, dry t
0 µL of interringe. Make
occus Alga
occus Biom
us biomass img. Observch as clump
the tube in turing this ste
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to x well.
ion ngs are
test
rnal e sure the
ae
mass
nto a ve the ps,
the pre-ep.
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d) Remothe c
e) Transf) Add 2
3800flask.
g) Repeflask.
h) After
tempi) Trans
4200the fi
j) Prepavolumsecon
a
k) Readthe Scalcuprocethrouone o
a
d
ove the tubeell material. sfer the supe2-3 mL of ac-4200 rpm fo. eat steps d-f . Four to five
all the supeerature (20°sfer an aliqu rpm to remorst dilution. are a 1/5 dilu
metric flask. nd dilution. . Typically
total volumsample in
d and recordSpectrophotoulations beloweeding. If thgh 8.0Ck. If
of the two ini
e from the wa ernatant to acetone to theor 3 minutes
until the supe extractions
ernatant is co° C), then briuot (8-10 mLove any part
ution by pipe Bring the fla
a 1/5 dilutiome) are requ
n 10 mL total the absorbe
ometer. (Absw, verify thae duplicate s
f still not withitial samples
b
ater bath an
a 25 mL or 5e centrifuge s to pellet the
pernatant is s with aceto
ollected in thing the flask) into a cleaticulate matt
etting accuraask to volum
n is adequatuired for lessl volume) areency at 474 sorbency reaat the duplicasamples are
hin 3%, eithes, or redo du
e
11
d centrifuge
50 mL volumtube, mix vige solid mate
colorless, trne are usua
he volumetrick to volume wn, dry test tuter which ma
ately 2 mL ome with aceto
te. Occasios concentrate required fonm of the se
adings are linate samples e not within 3er prepare a uplicates.
c
f
e at 3800-42
metric flask ugorously for
erial. Transf
ransferring thally enough.
c flask, let thwith acetoneube and cenay have carr
of extract intoone, stopper
nally dilutionted samplesor more conecond dilutionear betweeare within 3
3% of each othird sample
c
w
00 rpm for 3
sing a Paste30 seconds
fer supernat
he supernat
he solution ee and mix wetrifuge for 3
ried over in t
o a clean anr and mix fla
ns of 1/2 (5 ms or dilutions centrated saon against aen 0.50 and 3% of each oother, repeae that should
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3 minutes to
eur pipette.s. Centrifugetant to the vo
ant to the vo
equilibrate toell. minutes at 3
the transfers
d dry 10 mLask well. This
mL sample iof 1/10 (1 m
amples. n acetone b1.50). Using
other before at steps 8.0Cd fall within 3
c
f
h.com
pellet
e tube at olumetric
olumetric
o room
3800-s. This is
L s is the
n 10 mL mL
lank on g the
Cj 3% of
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8
9Hfac
l) Once
followNote:
m) To thapo-8aceto
n) Proce
8.1 Calculat
• Total
• Carot
• Appro
w
• Relat
RP
wh
9.0 HydrolysHaematococfatty acids neastaxanthin ocholesterol e
h
e it is confirmwing: Pipette: Make suree test tube c
8’-carotenal)one. eed to 9.0 H
tions
Carotenoid
tenoids (mg wh
oximate Asta
Percent A
where 85% is
tive percent
PD = ⏐R1 –
here ⏐R1 – R
sis of Carotccus algae peed to be reonly quantifi
esterase and
i
med that the e accuratelye to note whicontaining 3) using the 5
Hydrolysis o
Quantificati
g) extracted
here 210 is t
axanthin Pe
Astaxanthin =
s the percen
difference (R
R2 ⏐ x 100 R
R2 ⏐ = abso
tenoids fromroducts commoved comes ‘free’ (no
d is employe
duplicates ay 3 mL of theich duplicatemL of the s
50 µL glass s
of Caroteno
on
= Abs max
the extinctio
rcentage
= Carot
tage of asta
RPD) of dup
,
olute differen
m Haematomprise astaxapletely, sinc
on-esterified)ed here.
j
12
are within 3%e second dilue is used at tecond dilutiosyringe. Ma
id from Hae
X volume 210
n coefficient
tenoids (mg sample
xanthin of th
plicates
nce between
coccus Alganthin that he the HPLC ) astaxanthin
k
%, select ONution to a sethis time. on add 50 µke sure the
ematococcu
of acetone0 t of astaxant
) extracted wt (mg)
he total caro
the duplicat
gae Producthas been est
method emn. This react
w
NE of the dupparate clean
L of internalsyringe is ri
us Algae Pr
X dilution
thin in aceto
X 85% ,
otenoid conte
tes
ts terified with
mployed for thtion is cataly
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plicates for tn, dry test tu
standard (trnsed after u
roducts.
,
one
ent
fatty acids. he analysis oyzed by the e
l,m
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the ube.
rans-ß-se with
These of enzyme
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a) To thß-apo
b) To thestergelcacholeis com
c) Placefrequ
d) Remoether
e) Centr
f) Remolayerthis s
g) Againh) Centri) Remo
layerno wa
j) Repesepa
a
d
e test tube co-8’-carotenis test tube,
rase stock soap products sesterol esterampletely thae the test tubently.
ove the test r. Cap the terifuge the tu
ove the test , transferring
step. n add 2 mL prifuge at 350ove the test , transferringater is transf
eat steps g) trate addition
containing thal), add 2 madd 600 µL
olution contashould receiase stock sowed before be in the wat
tube from thest tube andbe at 3500 t
tube from thg it to a sepa
petroleum et00 to 4200 rptube from th
g it to the tesferred in thisthrough i) unns are usual
he 3 mL of thL of 0.05M T
L of cholesteains 2 units oive 900 µL oolution contadispensing.ter bath set
he water bat mix vigorou
to 4200 rpm
he centrifugearate clean,
ther to the tepm for <30 she centrifugest tube contas step. ntil the uppely necessary
b
d
13
he second dTris buffer, prol esteraseof enzyme). of cholesteroains 3 units o
at 35-37° C
th and add 0usly. for <30 seco
e and removdry test tube
est tube. Caseconds. e and removaining the fir
r (petroleumy.
b
dilution with apH7.0. Cap t stock soluti Cap the tes
ol esterase sof enzyme).
for 45 minu
0.5 g of sodiu
onds.
ve the uppere. Make sur
p the test tu
ve the upperrst petroleum
m ether) laye
b
e
w
added internthe test tubeion (600 µL st tube and m
stock solution Note: Mak
tes, mixing t
um sulfate a
r, colored (pere that no wa
be and mix
r, colored (pem ether solut
er is colorless
c
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nal standard e and mix weof cholesteromix well. (Bn (900 µL of
ke sure the s
the solution
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vigorously.
etroleum ethtion. Make s
s. Three to
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BioAstin f solution
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her) ferred in
her) sure that
four
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k) Turn
nitrogsure flow bfrom
l) Oncetube
m) Inspecarrie
a
n) If no Runnsolvesamp
f
on the nitroggen manifoldthe pipette dby opening othe test tube
e the petroleand turn off
ect the test tued over in th. Note: It is
water is ppetroleumwith the wether watsodium supetroleum- m)
water is prening Solvent ent’ and mix ple is now re
gen gas flowd into the tesdoes not reaor closing the. um ether is the nitrogen
ube containie petroleum
s important thpresent in them ether to thewater, forminter free. Traulfate with p
m ether solut
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f
k
w to the nitrost tube contaach into the se control va
completely en gas flow.
ing the driedm ether extrac
hat NO watee test tube, ae test tube a
ng ‘clumps ‘ onsfer the peetroleum ethtions. Evapo
er the extracne:18%Acetofer the solut
LC analysis.
14
ogen manifolaining the cosolution or slve to facilita
evaporated,
d petroleum ction.
er is present add <0.5 g o
and mix gentof hydrated
etroleum etheher until coloorate the pet
ct quantitativone). Bring ion to a clea
h
l
ld. Place onombined petplashing ma
ate the evap
remove the
ether residu
t in the sampof anhydroustly. The anhsodium sulfaer to a cleanorless, transtroleum ethe
vely into a 3 the 3 mL fla
an and dry te
j
w
ne of the Pasroleum ethe
ay occur. Adjporation of th
e nitrogen ma
ue for water t
ple prior to ths sodium suhydrous sodifate and leavn, dry test tusferring and cer solution a
mL volumetrask to volumest tube for H
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steur pipetteer solutions. just the nitro
he petroleum
anifold from
that may hav
he HPLC anulfate and 1.5ium sulfate wving the petrbe and rinsecombining th
as outlined in
ric flask usine with ‘runnHPLC analys
h.com
es of the Make
ogen gas m ether
the test
ve been
nalysis. If 5 mL will react roleum e the he n steps k
ng HPLC ing sis. The
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1DCgCFIM RCBTCASDDT911L ( W
10.0 HPLC ADetector: UColumn: Luguard columColumn temFlow rate: 1njection vo
Mobile phas
Retention tiCompound Beta-CarotenTrans-ß-ApoCanthaxanthAstacene Semi-AstaceDi-Cis AstaxDi-Cis AstaxTrans Astaxa9-Cis Astaxa13-Cis Astax15-Cis AstaxLutein
(See Appen
With HPLC s
1. First with dcarotother
2. Run tnm. R5.6 marea.
Analysis V/Vis detect
una 3µ Silican (Phenome
mperature: A.2 mL/minut
olume: 1.2 µse running s
mes for Ide
ne o-8’-Carotenahin
ene xanthin #1 xanthin #2 anthin anthin xanthin xnthin
dix A for re
system runn
run triplicatedetection at tenal at a retr. Calculate a
triplicate tranReview chrominutes. Pea
This is PST
n
tor at 474 nma(2), 100Å 1enex P/N AJAmbient (20 te µL solvent: 82%
entification
al (internal S
epresentativ
ing and stab
e diluted tran458 nm. Re
tention time average pea
ns-astaxanthmatograms
ak area of reTA . (Peak are
m and 458 n50 x 4.60 mO 4348 & K- 25°C)
% Hexane:1
Standard)
ve HPLC ch
ble under co
ns-β-apo-8’-ceview chromof 1.9 minut
ak area. Thi
hin standardand record plicates shoea trans-asta
15
nm m, Phenome
KJO 4282)
8% Acetone
Retenti 1 1 2 4 4 5 5 5 6 6 7 8
romatogram
nditions liste
carotenal stamatograms ates. Peak ares is PIS . (Pe
ds prepared peak area fould be withinaxanthin sta
n
enex (P/N 0
e, Isocratic
on time (mi1.4 1.9 2.9 4.0 4.4 5.2 5.4 5.6 6.3 6.6 7.1 8.6
m)
ed above:
andards prend record peea of replica
eak area inte
under sectioor trans-astan 3% of eacandard)
w
0F-4162-E0
inutes)
epared undeeak area forates should bernal standa
on 7.0-f withaxanthin at ah other. Calc
www.cyanotec
0) with suita
r section 6.0r trans-β-apobe within 3%rd)
detection aa retention ticulate avera
h.com
able
0 e iii o-8’-% of each
t 474 me of
age peak
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a
3. Run h474 n
a
b
4. Run hRecoastaxP15CA
a
PA
W1.11.6PS
b.
WCSD W
11.0 Refere• Jacob
carot• Quac• ORA
Versi
. Calculateadjustmen(Concentr
hydrolyzed anm. . Record pe
(Peak are
. PRIS mustCaroteno
hydrolyzed aord the peak xanthin, PTA,A.
. Calculate
ARAX = (PDCA
here: 133 is the re60 is the resSTA is the pea
Calculate th
AXP = (P
here: STA is the tranis the total sis the weigh
ences bs P.B., R.Dtenoid estersckenbush, FLaboratory on 1.1, 10-0
Spectrophont to the Conration standa
astaxanthin
eak area of ea internal st
1. Perceninsure from Hsample
t be 97±5. Ifoids from H
astaxanthin areas of di- 9-cis astaxa
the peak ar
A1 + PDCA2 +
esponse factsponse factoak area of th
he per cent a
ARAX x CSTA
ns astaxanthsample dilutiht of sample
D. LeBoeuf, Ss by choleste.W., JournalProcedure,
01-03.
otometric Puncentration oard trans-as
sample prep
trans-β-apo-tandard samnt recovery ocarotenoids
Haematococe is calculate
PRI
f PRIS is outHaematococ
sample prepcis astaxantanthin, P9CA
rea ratios of
PTA + 1.133x
tor for 9-cis aor for 13-cis a
e trans asta
astaxanthin
A x D x 100)
hin concentron in sectionused in sec
S.A. McComerol esterasel of Liquid ChFood and D
16
rity as showof Standard
staxanthin)
pared under
-8’-carotenample) of the internas are recoveccus Algae ed as:
IS = (PISS X 1
side this limccus Algae P
pared under thin #1, PDCA, 13-cis asta
astaxanthin
xP9CA + 1.60
astaxanthinastaxanthin
axanthin stan
in the sampl
÷ W
ation of the n 8. f abovection 8. a abo
mmas, and Je. Comp. Bihromatograp
Drug Adminis
wn in sectionas shown in
section 9.0
al peak deter
al standard, red during 9Products.
100) ÷ PIS
it, repeat proProducts.
section 9.0 A1, di-cis astaaxanthin, P13
, PARAX as
0x P13CA + P1
ndard from 2
le, AXP, as:
standard so. ove.
. D. Tauber.iochem. Phyphy, 10:643-stration. Doc
w
7.1 b and mn section 7.1
n with detec
rmined at 45
PRIS, is use9.0 Hydrolys
PRIS, in the
ocedure 9.0
n with deteaxanthin #2,3CA, and 15-
:
15CA) ÷ PSTA
2. above.
olution from 2
1982. Theysiol. 72B: 1-653, (1987)cument No.:
www.cyanotec
make any req1 c. This is C
ction at 458n
58 nm. This
ed as a measis of Carote astaxanthi
Eq (1)
Hydrolysis
ection at 474, PDCA2, trans-cis astaxant
Eq (3)
2.a above.
e cleavage o57-160. ) ORA-LAB.5
h.com
quired CSTA.
nm and
is PISS.
sure to tenoids n
)
s of
4 nm. s thin,
Eq (2)
)
of
5.9,
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1Ac
TH
• RensCaro
• VeccSemi10:34
• WebePrem
12.0 ExampAn analysis oconducted fo
• The aproce
• The w• The f
This
The standardHPLC Analy
From From Perce PRIS Whic From
On
clostatra
strom B. andtenols. Comhi M., V. Muiastacene, a48-351. er S., W. Ha
mixes. Hoffm
le of BioAstin Sollowing the
average absedure 7.0 f rweight of olefirst dilution iresults in a t
d solutions aysis and pro
m chromatogr
PIS =113,
m chromatogr
PISS = 112
ent recovery
= (112,751x
h meet the c
m chromatogr
PSTA = 772
ne other pea
772,789 ÷
ose to 100%andard as prans astaxant
CSTA = 0.7
d S. Liaaen-Jmp. Biochemuduna, and End other Ke
ardi, and J. Smann-La Roc
SCE5, 5% nprocedures
sorbance of tead at 474 n
eoresin usedin proceduretotal dilution
and sample soduced the c
ram 1, the p
954
ram 2, the p
2,751
y of the inter
x 100) ÷ 113
criteria that P
ram 3, the p
2,789
ak was detec
÷ (772,789 +
% purity. Therepared in pthin standard
757 ÷ (217 x
Jensen. 198m. Physiol. BE. Glinz. 198to-carotenoi
Schierle. Deche Ltd. pub
atural astaxoutlined abo
three samplenm was 0.75d in procedure 8.0 e was
of D = 1000
solutions wechromatogra
peak area int
peak area int
nal standard
3,954= 98.9
PRIS = 97± 5
peak area for
cted with an
+ 272) = 1.00
e average abrocedure 7.0d is calculate
x 1.0) = 0.003
17
81. Fatty Ac., Comp. Bio87. HPLC Sids. J. High
etermination lication, CH-
anthin oleorove. The de
es of trans a57 re 8.0 was W100 and the 0.
ere run on anms presente
ternal standa
ternal standa
d, PRIS, is ca
5
r the trans-a
area of 272
00 (essentia
bsorbance of0 f read at 4ed from 7.1
349 mg/mL
cid Compositochem. 69: Separation a
Res. Chrom
of Stabilized-Basle.
resin extracteetails of the a
astaxanthin s
W = 33.8 mg second dilu
n HPLC as ded in Appen
ard is:
ard sample i
alculated from
astaxanthin s
2. The purity
ally 100% pu
f three samp474 nm was c:
= 3.49 µg/l
w
tion of some625-627.
and Determinm. And Chrom
d Astaxanthi
ed from Haeanalysis are
standard as
g ution in proce
describe in pndix A.
is:
m Eq (1) in s
standard is:
is then:
re)
ples of trans0.757. The
www.cyanotec
e Esterified
nation of Astm. Commun
in in Caroph
ematococcus:
prepared in
edure 8.0 f w
procedure 10
section 10 3
s astaxanthinconcentratio
h.com
tacene, n.
hyll Pink
s, was
n
was 10.
0.0
3.a.
n on of the
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F PPPPPP T P P T A
A
W1.0
From chroma
PDCA1 = 4,50PDCA2 = 4,69PTA = 300,66P9CA = 47,00P13CA = 24,08P15CA = 3,904
The peak are
PARAX = (4,5
PARAX = 0.52
The per cent
AXP = 0.528
AXP = 5.46
here: 0 is purity of
atogram 4 th
07 1
67 02 80 4
ea ratios of a
507 + 4,691
289
t astaxanthin
89 x 0.00349
% (w/w)
f 100%.
he peak area
astaxanthin,
+ 300,667 +
n in the sam
9 x 1,000 x 1
a of the vario
PARAX, is c
+ 1.133 x 47
ple is calcul
100 ÷ 33.8
18
ous isomers
calculated fr
,002 + 1.6 x
ated from Eq
s of astaxant
rom Eq (2) in
x 24,080 + 3,
q (3) in sect
w
thin in the sa
n section 10
,904)÷ 772,7
tion 10 4.b.
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A Appendix 1 HPLC Chro
Cpr
omatograms
hromatograrepared unde
s
am 1.Trans-er section 6.0
19
β-apo-8’-car0 e iii. Detec
rotenal interction at 458 n
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rnal standardnm
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Chromatostandard insection 9.0
ogram 2, Trn astaxanthin f. Detection
20
rans-β-apo-8n sample as pn at 458 nm
8’-carotenal iprepared und
w
internal der
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Cp
Chromatogrprepared und
ram 3, Trander section 7
21
ns-astaxanth7.0 f. Detecti
hin standard aion at 474 nm
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as m
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Chromunder s
matogram 4.ection 9.0 f.
22
Astaxanthin Detection a
n sample as at 474 nm
w
prepared
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