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HPLC Analysis of Natural Astaxanthin 031513 Cyanotech
HPLC Analysis of Natural Astaxanthin 031513 Cyanotech
HPLC Analysis of Natural Astaxanthin 031513 Cyanotech
www.cyanotech.com
Analysis
s of Natural Astax
xanthin Derived fro
om Haem matococc cus Micro
oalgae
axanthin Oleoresin, Astaxa
in Asta anthin Geelcaps, A
Astaxanthhin Beadlets,
and Haematococ ccus Biommass (3/155/2013)
F
For questions or comm
ments pleasse contact Gerald
G R. C
Cysewski, P
Ph.D., Chieff Science O
Officer,
C
Cyanotech Corporation, email: gc
cysewski@cyanotech..com ; Phon ne: (808) 33
34-9420
11.0 Introducction
TThe caroteno oid fraction of
o Haematoccoccus algae e contains a
about 70% mmonoesters o of astaxanthin, 10%
ddiesters of astaxanthin, 5%
5 free asta nder consistting of β-caro
axanthin, witth the remain otene,
ccanthaxanthin, and lutein n as shown in Figure 1 below.
b
asta
axanthin
astaxantthin lutein
dies
ster
free 5% 4% canthaxaanthin
10%
1
5%
b
beta-carottene
6
6%
a
astaxanth
hin
m
monoesteer
70%
F
Figure 1. Carotenoid Complex
C in Haematoco
occus
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S
Spectropho otometric Qu uantification
S
Spectrophotoometric quantification off astaxanthin
n yields a cloose approximmation of astaxanthin, but is not
a
as accurate as HPLC. Spectrophoto
S ometric quan ntification is influenced b
by other mixxed caroteno
oids and
a
any degrada nthin present in the sam ple.
ation productts of astaxan
H
HPLC Analy ysis
Astaxanthin analysis by High Perform
A mance Liquiid Chromato ography (HPLC) provides the most a accurate
q
quantification
n of astaxan
nthin as well as mixed ca arotenoids a nd degradattion productss of astaxannthin
p
present in the sample. However,
H it is essential that
t all esterrified astaxanthin be com
mpletely hyd
drolyzed
p
prior to HPLCC analysis.
S
Safety
P
Proper safetyy precaution
ns should be
e followed. Protective
P ge nclude but iss not limited to safety
ear should in
g
glasses and solvent-resistant gloves
s. Extraction
ns should bee performed in a fume ho ood that is in
n proper
w
working conddition. Meassures should
d be taken to
o minimize ssolvent expo
osure through h inhalation,,
ingestion, an
nd skin conta
act.
2
2.0 Reagentts
• Tris HCl
H ( EMD P/N P 9310)
• 1.0 N NaOH solu ution for pH adjustment
a (Fisher
( P/N SS318-1)
• Methanol (VWR P/N P JT9093-3) with 0.05 5% BHT (Sig gma P/N B1253)
• DI or Distilled waater
• pH bu uffer calibrattion solution
• Acetoone (minimu um technical grade) conttaining 500 m mg/L BHT (SSigma B 12553)
• Transs-ß-apo-8’-c carotenal (Sig gma P/N:10810) as inte ernal standarrd
• Astaxxanthin standard (Chrom madex P/N ASB-000016
A 695-005)
• Choleesterol Esterrase (1 vial ofo 1000 units
s enzyme pe er vial, VWR
R P/N IC 105
543991)
• Petrooleum Ether (such as VW WR P/N 4980-08)
• Anhyydrous sodium sulfate (such as VWR R P/N SX076 61-1)
• Hexaanes, HPLC Grade (such h as VWR P/N HX 0290 -1)
• Acetoone, HPLC Grade
G (such as VWR P//N 9002-03)
• Dichloromethane e (DCM) as dissolution
d solvent
s (such
h as VWR P//N BDH1113 3)
• Dimeethylsulfoxide e (DMSO, su uch as VWR
R P/N BDH1 115-4LP)
3
3.0 Equipme
ent
• Glass bottles with
w stir bar, 125 mL, 25 000 mL
50 mL and 10
• Maagnetic stir plate
p
• Grraduated cylinders, 500 mL and 10000 mL
• gla
ass beakers, 250 mL and 500 mL
• pHH meter
• Fuunnel
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• An
nalytical bala
ance (0.1 mg g accuracy)
• Ceentrifuge cappable of 4200 rpm
• Vo
olumetric flassks with stop
pper (3 mL, 10 mL, 50 m mL, or 100 m
mL)
• 10
0 mL centrifuuge tubes with caps
• 12
2 mL test tubbes with lids
• 0 mL glass sample vials
20
• Sm
mall spatula
• Vo
ortex mixer
• Vo
olumetric pippettes (1 mL, 2 mL)
• Pip
pette, 5 mL adjustable
• Pa
asteur pipettes with bulbbs
• MeeOH squirt bottle
b
• 5 mL
m amber viials with tops s
• Gaas-tight glass syringes, 50
5 µL, 100 µLµ
• Sp
pectrophotom meter and coontrolling computer
• Water bath set at 35-37° C
• Nittrogen manifold
• Fu
ume hood
• Alu
uminum foil
• Pa
arafilm
• HPPLC column: Luna 3µ Siilica(2), 100Å Å 150 x 4.60
0 mm, Pheno omenex (P/N
N 00F-4162-E0) with
suitable guardd column (Ph henomenex P/N AJO 43 348 & KJO 4282)
• HPPLC system with UV/VIS S detector
4
4.0 Preparattion of 0.05M Tris Bufffer, pH 7.0
Tris buffe
er is added to
t the enzymmatic digest of
o astaxanthhin in the qua antitative an
nalysis of Bio
oAstin
products for astaxanthin and other carotenoiids. Tris bufffer helps prrovide a neuttral pH for th
he
enzyme cholesterol
c esterase
e to function
f and
d is prepared
d at a concen ntration of 0.05 molar (MM) with
the pH addjusted to 7..0.
a) Rinse the folloowing glassw ware first with MeOH the en secondly with DI wate er: glass botttle with
stir bar, graduated cylinde er, and funneel.
b) Weigh out 7.88 g of Tris HClH and trans ntitatively to the 1000 mL glass
sfer the whitte solid quan
boottle containing a stir barr.
c) Addd 1000 mL DI water to the t glass boottle and stir on a stir pla
ate until all so
olid materiall has
beeen dissolved d.
d) Caalibrate pH meter
m to pH 7.00
7 ±0.01
e) Addjust the pH of the solution to 7.00 using 1.0 N N NaOH solutio on.
f) Store under re efrigeration at
a 4º C for up p to 3 monthhs. When un nsatisfactory enzymatic d digestion
of astaxanthin is observed d, check inte
egrity and pHH of solution, when nece essary prepa are a
fre
esh solution, discarding thet old one appropriatel
a y.
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a b c e
5
5.0 Choleste erol Esteras
se Preparattion
Cholesteerol esterase
e is commerc cially availabble as a solid
d. In order tto be able too use the enzzyme for
the HPLCC analysis of astaxanthinn, it has to be
b solubilizedd in a bufferr that stabilizzes the enzyyme
during storage but dooes not affec
ct its activity
y during the h
hydrolysis (dde-esterification) step off the
analysis. For ease of
o use, pre-mmeasured aliquots of the e enzyme solution are prrepared and kept at
–20º C. A cholesterrol esterase solution is prepared
p so as to contain 3.33 enzym me units perr mL.
a) Rinse 500 mL beaker, stirr bar, and 50 00 mL gradu ated cylinde er three times with MeOH H using
squirt bottle.
b) Rinse items in n step 1 six time with DI. water.
c) Rinse 500 mL beaker with h a small ammount of Tris buffer.
d) Meeasure 300 mL m of Tris buffer into thee 500 mL graaduated cyliinder.
e) Sin
nce the amo ount of enzym v is quite ssmall, open carefully and place the cap
me in each vial
up
pside down on o the counter (small am mounts of ennzyme may b be adhering to the inside e of the
ca
ap), and pipe ette 3.0 mL Tris
T buffer fro om graduate ed cylinder in
nto the vial a and 0.5 mL T Tris
bu
uffer into the caps. Pourr the Tris bufffer from thee rinses of th
he vial into thhe 500 mL beaker
an
nd remove th he Tris buffer from caps using the pi pette, transfferring it to the beaker as well.
epeat 3 times.
Re
f) Ge
ently stir till dissolved
d (~15 min).
sing the 5 mL pipette sett at 3.6 mL, fill as many 5 mL amberr vials as po
g) Us ossible with tthe
en
nzyme solutio on. Seal thee vials with caps,
c and sto
ore in freezeer at < -20° C for up to th hree
moonths until used. Label with
w the date e prepared, a
assigned me edia lot number.
d e f g g
6
6.0 Preparattion of Tran ns-β -Apo-8’’-Carotenal Internal Staandard Solu ution
T
The preparattion of standdard solution
ns and subse e HPLC are iintegral parts of the
equent calib ration of the
q
quantitative analysis
a of microalgae
m products
p by HPLC.
H
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a b b b
c d e f
7
7.0 Preparattion of Tran ns-Astaxantthin Daily Calibration
C C
Check Stand dard
T
The preparattion of standdard solution
ns and subseequent calib ration of the
e HPLC are iintegral parts of the
q
quantitative analysis
a of microalgae
m products
p by HPLC.
H
a) Ad
dd approximately 120 mL of HPLC Running
R Solvvent (82% HHexane: 18% % Acetone) to o the
50 mL glass bottle containing a smalll stir bar and
25 d place on sstir plate with
h moderate sstirring.
b) Obbtain the tran
ns-astaxanth
hin standard
d compound,, and with a glass pipette (no bulb), transfer
1 mg
m or less ofo the compoound to a tes
st tube (dip ppipette into ccompound bottle, this sh hould
supply ample amount for the
t preparattion). Gentlyy blow out th he air from thhe bottle with
h N2,
qu
uickly cap.
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c) Too dissolve, add 1.5 mL (aabout 1 Pastteur pipette volume) of d dichlorometh hane (DCM)) to the
tes
st tube contaaining the standard com mpound. Makke sure that a all of the com
mpound is pproperly
ssolved. This is the Com
dis mpound Solu ution AX. N ote: Organicc solvents (ssuch as DCM M and
die
ethyl ether) have
h been shown
s to cattalyze the isoomerization of carotenoids. Thus it is
im
mportant to minimize
m the time the standard comp pounds are ‘iin contact’ w
with these so
olvents.
a b c d
d) Foor the following steps the e use of the spectrophottometer is re equired. Use e HPLC Run nning
Soolvent (82% Hexane:18% % Acetone) as a a blank w when determ mining the abbsorbance off the
sta
andard soluttions.
e) Too the 250 mL L bottle contaaining Runn ning Solvent (82% Hexan ne:18% Ace etone) add enough of
the
e Compound d Solution AX to yield an n absorbancce of approxiimately 1.0 a at the wavele ength of
47
74 – 476 nm. Once an absorbance
a of
o approxima ately 1.0 is a
achieved, taake at least three
meeasurements s and record d the correspponding abssorbances an nd calculate and record their
av
verage value e including th
he lot # of the compound d found on th he bottle lab
bel.
f) Noow that the standard
s solution is preppared, into sseparate clea an and dry ttest tubes, ppreviously
wrrapped with aluminum foil, pipette as many 1 0 mL aliquots of the sstandard so olution as
po
ossible. Cap bes tightly. Inject and an
p the test tub nalyze the prepared stan ndard solutioon on the
HPPLC System m following procedure
p 10
0.0 HPLC A Analysis be elow for the determinatio on of the
sta
andard comp pound’s spe ectrophotome etric purity.
i. Spectropho otometric Pu urity: After th e standard ssolution has been run on n the
HPLC, analyze it to sho ow the peakk areas of all detectable peaks and
determine the
t spectrop photometric p purity of the standard coompound acccording
to 7.1 Calcculations be elow, enterin g the pertine ent informatiion in the resspective
HPLC Note ebooks. Mak ke sure that only detecta able peaks a are included by
reviewing thhe chromato ogram used to quantitate e the standaard compoun nd and
making app propriate chaanges to the e integration of the chrommatogram.
ii. With the spectrophoto
s ometric purrity determin ned, calcula ate and record the
concentration of the Trans-Astaxa
T anthin Daily Calibration Check Standard as
shown in 7.1 Calculations below.
iii. Inject and analyze att least five replicates of the Tran ns-Astaxanth hin Daily
Calibration Check Stan ndard and re eport the ave erage HPLC C concentrattion of all
replicates.
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e f g
7
7.1 Calculattions
a) Avera
age Absorba
ance = (ab
bsorbance1 + absorban ce2 + absorrbance3)
3
b) Spec
ctrophotomettric Purity = HPLC Peak Area oof Standard Compound_ _
HPLC Peak
P Areas o
of All Detecttable Peaks
c) Conc
centration of Standard (in
n mg/mL) = average absorbance
e of standard
d solution x p
purity
217
wheree purity is
(a) th
he claimed purity of the compound as per th he Certificatte of Analyssis, expresssed as a
decimal (for example: pu urity as per CoA
C = 98.6% % Æ 0.986) o or
(b) th
he spectroph hotometric purity as calc culated in ste
ep above.
(c) 21
17 is the exttinction coeffficient for Trans-Astaxan nthin at a wa
ave length off 474 nm in Running
Solvent
S (82%% Hexane:18 8% Acetone)) .
N
Note: Use th
he spectroph urity when a standard co
hotometric pu ompound’s C Certificate off Analysis do
oes not
shhow an expiration date nor a stated purity,
p or the
e expiration d
date of a staandard comp pound is
exxpired and th
he standard compound
c shows
s a puriity >90%.
8
8.01 Extracttion of Caro otenoids fro om Astaxanthin Oleore esin
Note:: Perform du uplicate extra actions for each
e sample
a) Using g the small spatula,
s stir the
t oleoresin n sufficientlyy, then weigh
h approximaately 25 mg oof
astax
xanthin oleorresin into a clean
c and drry 20 mL gla ass sample vvial. Recordd the weight to the
neareest 0.1 mg
b) Pipettte acetone into the vial to t wash the resin off the e sides and ddissolve the oleoresin.
c) Trans sfer the acettone solution n into a 100 mL volumettric flask usin ng a Pasteur pipette. Waash the
tube with acetone e until all ole moved and tthe acetone is colorlesss, transferring
eoresin is rem g the
rinsattes to the 10
00 mL volum metric flask.
d) After all the rinsee acetone is collected in the volumettric flask, let the solutionn equilibrate to room
temperature (20°° C), then briing the flask k to volume w with acetonee and mix weell.
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a b c,d
e e,g g
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i i
j) Proce
eed to 9.0 Hydrolysis
H of
o Carotenoids from Ha cus Algae P
aematococc Products.
8
8.02 Extracttion of Caro otenoids fro om Astaxanthin Gelcap p
No
ote: Perform quadruplica ate extractions for each sample
a) Weigh out accura ately four geelcaps into inndividual, tarred 50 mL gllass beakerss and record d the
weighht to the neaarest 0.1 mg.
b) Using g small scisssors carefullyy cut open th he gelcap. T Then fill the beaker with h 20 mL acettone to
avoidd splashing of
o gelcap content. Note: Some gelca aps’ content is partially ssolid or semi-solid
and a sonication step may be e necessary y to facilitate the removal of all the co ontent. If so
onication
is ind
dicated, using small sciss sors carefullly cut open tthe gelcap in n a 20 mL sccrew cap via al. Add
enough acetone to cover the e gelcap. Clo ose the vial tightly and p place in the ssonicator baath for 15
min.
c) Rinse e the scissorrs with acetoone into the beaker.
d) Trans sfer the rinsaate quantitattively to a 10
00 mL volum metric flask u using a Paste eur pipette. Rinse
the beaker and gelcap
g with acetone
a until the rinsate is colorless.. Add acetone to a final volume
of ca. 80 mL. No ote: Some gelcaps’
g conttent is partiaally solid or ssemi-solid annd a sonicattion step
may be necessarry to facilitate complete dispersion/d dissolution inn the acetone solution. If
soniccation is indic
cated, placee the stopperred volumetrric flask in th he sonicatorr bath and soonicate
for 20
0 minutes orr until the geelcap contentt is complete ely dispersed.
a b b
b,c d
he beaker with
e) Set th w the emptty gelcap aside to dry fo r at least 30 0 minutes. A Accurately we eigh the
dried, empty gelc cap and recoord the weight.
f) After all the supe ernatant is co
ollected in th
he volumetricc flask and tthe gelcap content is com mpletely
dispeersed, let the e solution eq
quilibrate to room
r temperrature (20° C C), then brin
ng the flask tto
volumme with acettone and mix x well.
g) Transsfer an aliqu uot (8-10 mL) into a clean, dry test tu
ube and centrifuge for 3 minutes at 3 3800-
4200 rpm to remo ove any partticulate mattter which maay have carrried over in tthe transferss. This is
the first dilution.
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e f g g.i
g.ii
g g.iii,iv
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a b c c c
d) Remo ove the tubee from the waater bath and centrifuge e at 3800-4200 rpm for 3 minutes to pellet
the cell material.
e) Transsfer the supe ernatant to a 25 mL or 505 mL volum metric flask using a Paste
eur pipette.
f) Add 2-3
2 mL of ac cetone to the
e centrifuge tube, mix viggorously for 30 secondss. Centrifuge e tube at
3800-4200 rpm fo or 3 minutess to pellet the
e solid mate
erial. Transffer supernattant to the vo
olumetric
flask..
g) Repe eat steps d-f until the sup
pernatant is colorless, trransferring th
he supernatant to the voolumetric
flask.. Four to five
e extractions
s with acetone are usua ally enough.
d e f f
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h i j k l,m
l) Once e it is confirm
med that the duplicates are
a within 3% %, select ON NE of the dupplicates for tthe
follow
wing: Pipette e accurately
y 3 mL of thee second diluution to a separate clean
n, dry test tu
ube.
Note:: Make sure e to note whiich duplicate
e is used at tthis time.
m) To the test tube containing
c 3 mL of the second dilutio on add 50 µL of internal standard (trrans-ß-
apo-88’-carotenal)) using the 50
5 µL glass syringe.
s Ma ke sure the syringe is rinsed after use with
acetoone.
n) Proceeed to 9.0 Hydrolysis
H o Carotenoid from Hae
of ematococcu us Algae Prroducts.
8
8.1 Calculattions
• Appro
oximate Asta
axanthin Percentage
Percent Astaxanthin
A = Carottenoids (mg ) extracted X 85% ,
sample wt (mg)
where
w s the percentage of astaxanthin of th
85% is he total caro
otenoid conte
ent
PD = ⏐R1 – R2 ⏐ x 100 ,
RP
R
here ⏐R1 – R2
wh R ⏐ = abso
olute differen
nce between the duplicattes
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a b b c
d d e
f) Remo ove the test tube from th he centrifuge e and removve the upperr, colored (pe etroleum ethher)
layer, transferringg it to a sepaarate clean, dry test tube e. Make surre that no wa ater is transfferred in
this step.
s
g) Againn add 2 mL petroleum
p etther to the te
est tube. Ca p the test tube and mix vigorously.
h) Centrrifuge at 350 00 to 4200 rp pm for <30 seconds.
s
i) Remo ove the test tube from th he centrifuge e and removve the upperr, colored (pe etroleum ethher)
layer, transferringg it to the tes
st tube conta aining the firrst petroleum
m ether soluttion. Make ssure that
no wa ater is transfferred in this
s step.
j) Repe eat steps g) through
t i) un
ntil the upper (petroleum m ether) laye er is colorlesss. Three to four
separate addition ns are usually necessary y.
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f f h j
k l
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n n
1
10.0 HPLC Analysis
A
Detector: UV/Vis detecttor at 474 nm
D m and 458 nm
n
C
Column: Lu una 3µ Silica
a(2), 100Å 150 x 4.60 mm, Phenome
enex (P/N 00F-4162-E0
0) with suita
able
g
guard column (Phenome enex P/N AJO 4348 & KJO
K 4282)
C
Column tem mperature: Ambient
A (20 - 25°C)
F
Flow rate: 1.2 mL/minutte
Injection vo
olume: 1.2 µL
µ
M
Mobile phasse running solvent:
s 82%
% Hexane:18% Acetonee, Isocratic
R
Retention times for Ide
entification
C
Compound Retenti on time (miinutes)
B
Beta-Carotenne 1
1.4
T
Trans-ß-Apoo-8’-Carotena
al (internal Standard)
S 1.9
1
C
Canthaxanthhin 2
2.9
A
Astacene 4
4.0
S
Semi-Astaceene 4
4.4
D
Di-Cis Astax
xanthin #1 5
5.2
D
Di-Cis Astax
xanthin #2 5
5.4
T
Trans Astaxaanthin 5
5.6
9
9-Cis Astaxa
anthin 6
6.3
1
13-Cis Astax
xanthin 6
6.6
1
15-Cis Astax
xnthin 7
7.1
L
Lutein 8
8.6
W
With HPLC system
s running and stab
ble under conditions liste
ed above:
2. Run triplicate
t tran
ns-astaxanthhin standard
ds prepared under sectio on 7.0-f with detection at 474
nm. Review
R chromatograms and record peak area fo or trans-asta
axanthin at a retention time of
5.6 minutes.
m Peaak area of replicates should be within
n 3% of each other. Calcculate avera age peak
area. This is PSTTA . (Peak are
ea trans-asta
axanthin sta
andard)
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3. Run hydrolyzed
h astaxanthin
a sample preppared under section 9.0 n with detecction at 458nnm and
474 nm.
n
a. Record pe eak area of trans-β-apo--8’-carotenaal peak deterrmined at 4558 nm. This is PISS.
(Peak areea internal sttandard sammple)
1. Percen nt recovery of
o the interna ed as a measure to
al standard, PRIS, is use
insure carotenoidss are recove red during 9
9.0 Hydrolys sis of Carottenoids
from Haematococ
H ccus Algae Products. PRIS, in the e astaxanthin
samplee is calculate
ed as:
PRIS
I = (PISS X 1
100) ÷ PIS Eq (1))
b. PRIS mustt be 97±5. Iff PRIS is outside this lim it, repeat pro
ocedure 9.0 Hydrolysis
s of
Caroteno
oids from Haematococ
H ccus Algae P Products.
4. Run hydrolyzed
h astaxanthin
a sample prep pared under section 9.0 n with dete ection at 474 4 nm.
Recoord the peak areas of di-cis astaxantthin #1, PDCAA1, di-cis asta
axanthin #2,, PDCA2, transs
astaxxanthin, PTA, 9-cis astaxa
anthin, P9CA, 13-cis asta
axanthin, P133CA, and 15--cis astaxantthin,
P15CAA.
PA
ARAX = (PDCAA1 + PDCA2 + PTA + 1.133x
xP9CA + 1.60
0x P13CA + P115CA) ÷ PSTA Eq (2)
Where:
1.1
133 is the re
esponse facttor for 9-cis astaxanthin
a
1.6
60 is the res
sponse factoor for 13-cis astaxanthin
a
PSTA
S is the pea
ak area of the trans asta
axanthin stan
ndard from 2
2. above.
b. Calculate th
he per cent astaxanthin
a in the samplle, AXP, as:
Where:
CSTA
S is the tran
ns astaxanth hin concentration of the standard so
olution from 2
2.a above.
D is the total sample
s dilution in section
n 8. f above .
W is the weigh ht of sample used in sec ction 8. a abo
ove.
11.0 Refere
ences
• Jacob bs P.B., R.D
D. LeBoeuf, S.A.
S McCom mmas, and J . D. Tauber. 1982. The e cleavage o
of
carottenoid esters
s by choleste e. Comp. Biiochem. Phyysiol. 72B: 157-160.
erol esterase
• Quac ckenbush, F.W., Journall of Liquid Chhromatograpphy, 10:643--653, (1987))
• ORA Laboratory Procedure, Food and Drug D Adminisstration. Doccument No.: ORA-LAB.55.9,
Version 1.1, 10-001-03.
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• Rensstrom B. and
d S. Liaaen-JJensen. 198 81. Fatty Accid Composittion of some
e Esterified
Carotenols. Commp. Biochem m. Physiol. B., Comp. Bioochem. 69: 625-627.
• Vecchi M., V. Mu
uduna, and E.E Glinz. 19887. HPLC S Separation aand Determin
nation of Asttacene,
Semiiastacene, and other Keto-carotenoiids. J. High Res. Chrom m. And Chrom m. Commun n.
10:34
48-351.
• Webeer S., W. Ha
ardi, and J. Schierle.
S De
etermination of Stabilized
d Astaxanthiin in Carophhyll Pink
mixes. Hoffm
Prem mann-La Roc che Ltd. publication, CH--Basle.
112.0 Example
AAn analysis of
o BioAstin SCE5,
S 5% natural astaxanthin oleorresin extracte
ed from Haeematococcuss, was
ollowing the procedures outlined abo
cconducted fo ove. The deetails of the a
analysis are:
• The average
a abssorbance of three
t sample
es of trans a
astaxanthin sstandard as prepared in
n
proce
edure 7.0 f read at 474 nm n was 0.75 57
• The weight
w of ole
eoresin usedd in procedurre 8.0 was W = 33.8 mgg
• The first
f dilution in
i proceduree 8.0 e was 100 and the second diluution in proce
edure 8.0 f w
was 10.
This results in a total
t dilution of D = 1000
0.
T
The standard
d solutions and
a sample solutions
s we
ere run on an
n HPLC as ddescribe in p
procedure 10
0.0
H
HPLC Analyysis and prooduced the chromatogra
c ms presenteed in Appen
ndix A.
From
m chromatogrram 1, the peak
p area intternal standa
ard is:
PIS =113,954
From
m chromatogrram 2, the peak
p area intternal standa
ard sample iis:
PISS = 112
2,751
Perce
ent recovery
y of the internal standard
d, PRIS, is ca
alculated from
m Eq (1) in ssection 10 3
3.a.
PRIS = (112,751x
x 100) ÷ 113
3,954= 98.9
From
m chromatogrram 3, the peak
p area forr the trans-a
astaxanthin sstandard is:
PSTA = 772
2,789
On
ne other pea cted with an area of 272
ak was detec 2. The purity is then:
clo
ose to 100%
% purity. Thee average ab bsorbance off three samp
ples of transs astaxanthin
n
sta
andard as prrepared in procedure 7.0 0 f read at 4
474 nm was 0.757. The concentratio on of the
tra
ans astaxantthin standard ed from 7.1 c:
d is calculate
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Where:
0 is purity off 100%.
1.0
F
From chroma
atogram 4 th
he peak area
a of the vario
ous isomerss of astaxantthin in the sa
ample solutio
on are:
PDCA1 = 4,5007
PDCA2 = 4,691
PTA = 300,6667
P9CA = 47,0002
P13CA = 24,08
80
P15CA = 3,904
4
T
The peak are
ea ratios of astaxanthin,
a PARAX, is calculated
c frrom Eq (2) in
n section 10
0 4.a.
P
PARAX = 0.52
289
T
The per centt astaxanthin
n in the sample is calculated from Eq
q (3) in secttion 10 4.b.
A
AXP = 0.528
89 x 0.00349
9 x 1,000 x 100
1 ÷ 33.8
A
AXP = 5.46 % (w/w)
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A
Appendix 1 HPLC Chro
omatograms
s
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Chromatogr
C ram 3, Tran ns-astaxanthhin standard aas
prepared
p und
der section 7.0
7 f. Detectiion at 474 nm
m
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