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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

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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

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

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

and 2 mL pet

etroleum ethater is transf

vigorously.

etroleum ethtion. Make s

s. Three to

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(trans-ell. ol

BioAstin f solution

troleum

her) ferred in

her) sure that

four

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

sent, transfe(82%Hexanwell. Transf

eady for HPL

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

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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

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

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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

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,

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

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.

www.cyanotec

ample solutio

0 4.a.

789

h.com

on are:

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

w

rnal standardnm

www.cyanotec

ds

h.com

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

www.cyanotech.com

Cp

Chromatogrprepared und

ram 3, Trander section 7

21

ns-astaxanth7.0 f. Detecti

hin standard aion at 474 nm

w

as m

www.cyanotech.com

Chromunder s

matogram 4.ection 9.0 f.

22

Astaxanthin Detection a

n sample as at 474 nm

w

prepared

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