Assay Method

7/21/2019 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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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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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



*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.

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(!) 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.

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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

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(!)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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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.

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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

Assay Method

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