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Transcript of Assay Method
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55 Yokohoonji, Kamiichi, Nakaniikawa, Toyama, 930-0397 Japan
Tel: +81-76-472-5549 Fax: +81-76-472-5417 Email: [email protected]
Spectrophotometric and HPLC Analysis Method
for Determining Astaxanthin Content in AstaREAL-P2AF
Contents
0.0 Introduction
1.0 Reagents and Equipment
2.0 Preparation of Cholesterol Esterase Solution for Hydrolysis of
Astaxanthin Esters.
3.0 Internal Standard Preparation
4.0 Astaxanthin Standard Preparation
5.0 Preparation of AstaREAL P2AF for Assay6.0 Spectrophotometric Analysis Method for Total Carotenoid
Content
7.0 Reversed-phase HPLC Analysis Method for Astaxanthin Content
Appendix A
1: Typical spectrophotometric profile of AstaREAL P2AF and
astaxanthin standard
2: Typical chromatograms of i) Astaxanthin standard with internal
standard and ii) AstaREAL P2AF with internal standard.
Appendix BTypical HPLC chromatograms at different degree of hydrolysis
stage.
Appendix CReference List
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0.0Introduction
BackgroundThere are two critical parts to the assay of astaxanthin extract powder prior to the HPLC reading.
The first is extraction of the astaxanthin from the powder matrix and the second is de-
esterification. Either one of these steps can easily be only partially complete which would result
in an incorrect and lower astaxanthin HPLC value. For spectrophotometric analysis, only the first
extraction step is critical.
Astaxanthin fromHaematococus pluvialis.
The astaxanthin molecule fromHaematococcus pluvialisalgae has optically pure chirality
(3S,3S) for all three forms in the following approximate ratio: monoester form ~ 82%, diester
form ~ 12%, free form ~ 6%. Free astaxanthin is the form in which the 3 and 3 hydroxyl groupsare not bonded (see chemical structure), an astaxanthin monoester has one fatty acid bonded to a
3 position, and an astaxanthin diester has a fatty acid bonded to each of the 3 positions.
3
3
Extraction of Astaxanthin from powdered matrix (Biomass, AstaREAL P2 and AstaREALP2AF).
The mono-esters and di-esters are relatively non-polar, whereas free form carotenoids are
relatively polar. The solvents used in this extraction and assay takes these factors into account,
thus variations to this procedure may affect the outcome.
In addition, the AstaREAL powder particles may naturally agglomerate during storage and
prevent full extraction. Inducting sufficient dispersion and repeated solvent extraction steps are
both necessary procedures to follow in order to minimize incomplete extraction.
Spectrophotometric Quantification
The spectrophotometric quantification of astaxanthin has been determined empirically and yields
a close estimation, but is not as accurate as HPLC. Spectrophotometric method is also influencedby other mixed carotenoids in the samples.
HPL C Analysis of Astaxanthin.
Accurate quantification of esterified astaxanthin by HPLC is not possible, however, various
systems have been developed for the analysis of free astaxanthin. Therefore, esterified
astaxanthin must first be hydrolyzed (de-esterified) completely by enzymatic procedure to yield
all free astaxanthin. Furthermore, chemical saponification is not recommended because these
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MoA_Spectrophotometric and HPLC Analysis for AstaREAL P2(AF) Ver. June 1918
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conditions oxidize astaxanthin and may introduce artifacts and results in a lower astaxanthin
HPLC value.
De-esterification is a particularly sensitive procedure and critical to accurate HPLC analysis. The
enzymatic hydrolysis of carotenoid esters is a slight modification of the procedure developed by
Jacobs P.B., R.D. LeBoeuf, S.A. McCommas, and J. D. Tauber,1982. Therefore, the HPLC assay
quantifies free astaxanthin by separation and absorbency using a standard curve.
Fuji usesPseudomonas fluorescenssource of enzyme, not porcine or bovine derived.
Pseudomonasfrom both Sigma and Wako Pure Chemical has been shown to work reliably. Each
enzyme has its own specificity so for astaxanthin, bacteria derived enzyme is more appropriate.
If de-esterification is incomplete, it will be evident because of small trans-astaxanthin peaks at
retention time of 11 mins and large collection of peaks between 20 and 30 minutes. (Also see
Appendix B)
A YMC C30 reversed-phased column, the Carotenoid ColumnTM, should be used for this
procedure. This column provides significantly better repeatability and ruggedness.
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MoA_Spectrophotometric and HPLC Analysis for AstaREAL P2(AF) Ver. June 1918
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1.0Reagents and Equipment
*0.05M Tris-HCl buffer (pH7.0)
*Cholesterol esterase: Wako Pure Chem., cat# 037-11221 or Sigma, cat#: C9281
*Trans-beta-apo-8-carotenal, Fluka cat#: 10829 [internal standard for HPLC analysis]
*Astaxanthin, Sigma, cat# A9335, analytical standard
*1% (v/v) phosphoric acid solution
*Acetone, Spectrophotometric grade
*Hexane, HPLC grade
*Petroleum ether
*Methanol, analytical grade
*MTBE: t-butyl-methyl-ether, spectrophotometric grade
*Sodium sulphate decahydrate
*Sodium sulphate anhydrous
*10mL centrifuge tubes
*30mL volumetric flasks
*100mL volumetric flasks
*200mL volumetric flasks
*0.45um syringe filter
*Water bath
*Analytical balance
*Centrifuge
*Sonicator
*Spectrophotometer
*HPLC equipped with a UV/VIS detector
*HPLC column: YMC-CarotenoidTMS5 micron, 250mm length x 4.6mm dia.
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2.0 Cholesterol Esterase Solution for hydrolysis of Astaxanthin Estersa) Dissolve an accurately weighed quantity of cholesterol esterase (Wako Pure Chem., Cat #:
037-11221 or Sigma, cat#: C9281) in 50 mM Tris-HCl (ph 7.0) having a known
concentration of 4 units per mL.
5 4
321
(!) In methods from older references, they may suggest freezing aliquots froma large stock of cholesterol esterase for later use. Do not do this.
(!)Prepare a fresh working cholesterol esterase solution each day.
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3.0Internal Standard Preparationa) Accurately weigh about 7.5 mg of trans-beta-apo-8-Carotenal (Fluka, Cat #: 10829, >20
%(UV-VIS) Apocarotenal), and transfer into a 200-mL volumetric flask.
I.S. solution
b) Dissolve in acetone, dilute with acetone to volume, and mix.
2 31 4
4.0 Standard Preparation
Standard stock solution
a)
volu
equ
b
Transfer about 5 mg each of Astaxanthin reagents (SIGMA, Cat #: A9335) to a 200-mL
metric flask, dissolve in about 100 mL of acetone, sonicate for a minute, and allow to
ilibrate to ambient temperature for 15 minutes.
321
) Dilute with acetone to volume and mix (Standard stock solution).1
(!) Measuring the exact I.S. and Standard weight mentioned is not important.Note the exact amount used.
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1 2
Standard
Solution A
c) Pipette 3.0 mL of Standard stock solutionto a 30-mLvolumetric flask, dilute with acetoneto volume, and mix (Standard solution A).
d) Pipette 3.0 mL of Standard stock solutionand 10.0 mL ofInternal standard solutionto a
30-mL volumetric flask, dilute with acetone to volume, and mix (Standard solution B).
21
Standar
Solution B
5.0Assay Preparationa) Shake well the AstaREAL P2AFsample for analysis to avoid powder aggregates.
Accurately transfer approximately 50 mg of powder to 10mL centrifuge tube, add 1mL of
purified water and vortex mix sufficiently to ensure an even dispersion and avoid
aggregates. Sonication may be used to disperse stubborn lumps.
5 4
2 31
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b) Add 10mL of acetone into the tube and sonicate for 5 minutes at 40-50 C.
1
c) Centrifuge at 3,000 rpm for 5 minutes and transfer supernatant to a 500-mL volumetricflask.
4321
d) Repeat steps b and c until supernatant is colourless (approximately 3-4 times).
Residue after extractionSupernatant at 3rd
extraction step.
Supernatant at 2nd
extraction step.
(!) Important steps for right quantification.APh
dequate extraction is necessary for right quantification.lease make sure that supernatant is colorless and residueas light ocher color.
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Assay
Solution A
e) Allow the supernatant to stand at room temperature for a minimum of 15 minutes, dilute
with acetone to volume and mix well.
f) Centrifuge at 3,000rpm for 5 minutes
g) Transfer 3.0 mLAssay solution A to a 10-mL glass centrifuge tube, add 1.0 mL ofI.S.
solution, and mix.
h) Set block heater at 37 oC, add 3.0 mL of Cholesterol esterase solution to the test tube,
and mix by gentle inversion.
1
(!)For total carotenoid analysis go to step 6.0.
(!)Follow steps g) to l) for HPLC samplepreparation.
21
21
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MoA_Spectrophotometric and HPLC Analysis for AstaREAL P2(AF) Ver. June 1918
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i) Allow to react at 37 oC for 45 minutes. Gently/slowly invert every 10 minutes, at least
twice, during the reaction.
j) Add 1 g of sodium sulfate decahydrate and 2 mL of petroleum ether, vortex for 30
seconds, and centrifuge at 3,000 rpm for 3 minutes.
k) Transfer the petroleum ether layer to a 10-mL glass centrifuge tube containing 1 g of
sodium sulfate anhydrate.
1
1 32
321
(!)Avoid to pipette the emulsive intermediate layer.
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l) Evaporate the petroleum ether layer in the stream of inert gas at room temperature, add 3
mL of acetone, sonicate to dissolve, and filter (Assay solution B).
1 2 3
4
Assay Solution B
(!)Now follow procedure 7.0 for HPLC analysis
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MoA_Spectrophotometric and HPLC Analysis for AstaREAL P2(AF) Ver. June 1918
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6.0 Spectrophotometric Analysis Method for Total Carotenoids Content
a) Determine the absorbance ofAssay solution Aat 474nm against an acetone blank on thespectrophotometer.
b) Calculate the Total Carotenoids Content (%) in AstaREAL-P2AF taken by the formula:
AAa* 500 / 210 / W* 100
in whichAAais the absorbance ofAssay solution A, 500 is dilution volume forAssay
preparation,210 is absorbance of a 1 (mg/mL) carotenoids solution in acetone, in a 1 cm
cuvette at 474 nm, Wis the weight, in mg, of the specimen taken forAssay preparation.
c) Refer to Appendix A. Data I: Figure 1., for typical absorbance profile for AstaReal-
P2AF and astaxanthin standard.
(!) Receive the absorbance value expected?Ex
= Car/100
If
pected absorbance ofAssay solution A* W/ 500 * 210
Car: Total carotenoids content, in % w/w (CoA value)W: sample weight, in mg
not, double check the extraction step; 5.0 (a) thru (f).
(!)Example data available on request.
21
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MoA_Spectrophotometric and HPLC Analysis for AstaREAL P2(AF) Ver. June 1918
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7.0Reversed-phase HPLC Analysis Method for Astaxanthin Content
a) Determine the absorbance of Standard solution Aat 474 nm, using acetone as the blank.
b) Analyze an aliquot of Standard solution B and Assay solution B by HPLC under the
following conditions:
Table-1.HPLC conditions:
Detector: UV/VIS detector, at 474 nm,
Column: YMC-CarotenoidTMS5, 4.6 x 250 mm,
Column temp: 25 oC,
Flow rate: 1.0 mL / minute,
Injection vol.: 20 L,
Mobile phase: Methanol, t-Butylmethylether, 1 % Phosphoric acid aqueous,
Mobile phase formula (%) is as follows:
Time (min.) Methanol t- Butylmethylether 1 % Phosphoric acid aqueous
0 81 15 415 66 30 4
23 16 80 4
27 16 80 4
27.1 81 15 4
35 81 15 4
Table-2.Retention time for Identification:
Components Retention time (min.)
13-cis-astaxanthin: 9
trans-astaxanthin: 10
9-cis-astaxanthin: 14
trans-beta-apo-8-carotenal: 16
(Internal standard)
c) Refer to Appendix A. Data II: Figure 2., and Figure 3., for typical chromatograms for
AstaREAL-P2AF with internal standard.
d) Calculate the concentration, in mg per mL, of astaxanthin in the Standard solutionA
taken by the formula:
ASa/ 210
in whichASais the absorbance of Standard solution A, and 210 is absorbance of a 1 (mg/mL)
astaxanthin solution in acetone, in a 1 cm cuvette at 474 nm.
(!) The expected absorbance of Standard solution Awould be ca. 0.525, with 5mg of astaxanthin standard reagent
in 2000 mL-dilution volume.
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e) Calculate the ratios of peak responses of total astaxanthin to I.S. obtained from theAssay
solution Band Standard solution Btaken by the formula:
(1.3P13-cis+ Ptrans+ 1.1P9-cis) / PI.S.
in which P13-cis, Ptrans, P9-cisand PI.S.are the peak responses of 13-cis-, trans-, 9-cis-astaxanthin
isomers and I.S., respectively, and 1.3 and 1.1 are the relative response coefficients of 13-cis-,
and 9-cis-astaxanthin to trans-astaxanthin, respectively.
f) Calculate the Astaxanthin Content (% w/w) in AstaREAL-P2AFtaken by the formula:
CSa(RAb/RSb) *500 / W* 100
in which CSais the concentration, in mg per mL, of astaxanthin in the Standard solution A,
500 is dilution volume for Assay preparation, W is the weight, in mg, of the AstaREALP2AFspecimen taken for theAssay solution preparation, andRAbandRSbare the ratios of the
peak responses of total astaxanthin to I.S. obtained from the Assay solution B and the
Standard solution B, respectively.
(!) Why not simple alkali saponification?Ad
staxanthin is not stable and readily undergo artifacts generation/egradation under alkaline condition.
(!) Reasonable good detection and separation in HPLC chromatograrm?See example graphs - Refer to Appendix A. Data II.
(!) Enzyme working properly?InF
stantly recognizable with your HPLC chromatograms.or more details refer to Appendix B.
(!)Example data available on request.
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Appendix A. Data I
AstaREAL-
P2AFAstaxanthin Standard
Fig-1. Spectrophotometric profiles of AstaREAL-P2AF and astaxanthin standard.
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Appendix A. Data II
Figure-2. Typical chromatogram of astaxanthin standard with internal standard.
Figure-3. Typical chromatogram of carotenoids from AstaREAL-P2AF with internal standard.
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Appendix B.
(!)Typical chromatogram of:
Mfa
Before enzymatic hydrolysis
ost of astaxanthin esterified withtty acid(s).
(!) Typical chromatogram of:
Ca
Insufficient hydrolysis
onsiderable amount of esterifiedstaxanthin remained.
(!) Typical chromatogram of:
M
frsa
Hydrolysis completed
ost of astaxanthin converted to
ee body astaxanthin and non-ignificant peaks of esterifiedstaxanthin.
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Appendix C.
Method References
1. Aquasearch Inc. Technical report, TR .1005.001. 1999. Comparison of HPLC andspectrophotometric analyses of astaxanthin content in Haematococcus pluvialis algalmeal.
2. BioAstin/NatuRoseTM Technical Bulletin #015, Cyanotech Corporation, 2000. HPLCand spectrophotometric analysis of carotenoids fromHaematococcusalgae powder.
3. Carotenoids, Volume 1B, Spectroscopy edited by G. Britton, S Lippen-Jense and HPtander, Birkhauseer & Verlag Basel 1995, p57-61.
4. Jacobs PB, LeBoeuf RD, McCommas SA, and Tauber JD. 1982. The cleavage ofcarotenoid esters by cholesterol esterase. Comp. Biochem.Physiol. 72B:157-160.
5. Kanemitsu A., 1958. Study of carotenoid pigment of salmon and trout 1, Identification ofmuscle pigment. Japanese Journal of Aquaculture (Nissuishi), 24: 209-215.
6. Schiedt K. and S. Liaaen-Jensen. 1995. Artifact. In: Carotenoids, Volume 1A, Isolationand Analysis, pp 84-91. Birkhauser Verlag Basel.
7. Schuep W. and J Schierle. 1995. Astaxanthin determination of stabilized, addedastaxanthin in fish feeds and pre-mixes. In: Carotenoids, Volume 1A, Isolation and
Analysis, pp 273-276. Birkhauser Verlag Basel.
8. Vecchi M., V. Muduna, and E. Glinz. 1987. HPLC separation and determination ofastacene, semi-astacene, and other keto-carotenoids. J. High Res. Chrom. & Chrom.
Commun. 10:348-351.
9. Wan, P.J., Zhang F., and R. J. Hron. 1995. Extraction, composition and stability ofpigments from crawfish shell waste. In, Nutrition and Utilization Technology in
Aquaculture (Eds: Lim, C. E., and Sessa D. J.), Chap 19, p255-268.
10.Weber S., 1990. Determination of added stabilized astaxanthin in fish feeds and premixeswith HPLC. Ed. H E Keller, Analytical methods for vitamins and carotenoids in feed,
Revised Supplement, Roche Publication, index No. 2264, pp 59-61.
11.Weber S., W. Hardi, and J. Shierle. Determination of Stabilized astaxanthin in CarophyllPink, Premixes. Hoffman-La Roche Ltd., publication, CH-Basle.
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MoA Spectrophotometric and HPLC Analysis for AstaREAL P2(AF) Ver June 1918