PRODUCT TEST METHOD

SUBJECT: Identification, Assay and

NUMBER: PTM
REVISION :
Uniformity of Dosage Unit (Content uniformity) SUPERSEDE: N/A PAGE 1 of
11
of Astaxanthin 4 mg capsules
Department / Section :
Prepare by
Signature

: .....................................

Date

Reviewed by : QC
Director

Next reviewed date

Signature

Date

Signature

Date

Approved by : QA
Director

Next reviewed date

PRODUCT TEST METHOD

SUBJECT: Identification, Assay and

NUMBER: PTM
REVISION :
Uniformity of Dosage Unit (Content uniformity) SUPERSEDE: N/A PAGE 2 of
11
of Astaxanthin 4 mg capsules
Department / Section :

Reference: In house method, Modified from Fuji chemical industry CO.,LTD

Reagent:

1. 0.05 M Tris-HCl buffer pH 7.0 : Preparation By dissolve

t33 303 mg of

into 50 ml volumetric flask dilute to volume with water and adjust pH to 7.0
with 1M HCl.

2.Trans-beta-apo-8-carotenal,Fluka cat #10829(internal standard for HPLC

analysis)

3. Cholesterol esterase ,Sigma cat # C9281

4. 1% phosphoric acid solution

5. 1 M HCl : mixture of 4.25 ml Hydrochloric acid in 50 ml of water

6. Acetone ,AR grade

PRODUCT TEST METHOD

SUBJECT: Identification, Assay and

NUMBER: PTM
REVISION :
Uniformity of Dosage Unit (Content uniformity) SUPERSEDE: N/A PAGE 3 of
11
of Astaxanthin 4 mg capsules
7. Hexane, HPLC grade
8. Petroleum ether, HPLC grade
9. Methanol , AR grade

10. MTBE : t-butyl- methyl-ether , HPLC grade

11. Water, HPLC grade

12. Sodium sulfate decahydrate, AR grade

13. Sodium sulfate anhydrous , AR grade

14. Standard: Astaxanthin Reference standard, Chromadex, Lot

no.00001695-010

Apparatus: High Performance Liquid Chromatographic system

1. HPLC Pump

2. UV / VIS Absorbance detector

3. Data integrator

4. Column : YMC Carotenoid TM S5 micron , 250 mmx 4.6 mm. cat no.
99S05-2546WT
Mobile phase :

Methanol ,

t-butyl- methyl-ether , 1% phosphoric acid solution

Cholesterol Esterase Solution for hydrolysis of Astaxanthin Ester

1.Dissolve an accurately weigh quantity of cholesterol esterase in 0.05 M Tris-

HCl (pH7.0) have a known concentration of 4 unit per ml (Preparation a fresh

PRODUCT TEST METHOD

SUBJECT: Identification, Assay and

NUMBER: PTM
REVISION :
Uniformity of Dosage Unit (Content uniformity) SUPERSEDE: N/A PAGE 4 of
11
of Astaxanthin 4 mg capsules
working cholesterol esterase solution each day)
Internal Standard Preparation
1. Accurately weigh about 7.5 mg of trans-beta-apo-8-carotenal and transfer
into 200 ml volumetric flask.

2. Dissolve in acetone , dilute with acetone to volume and mix.

Standard stock solution:

1. Weight quantity about 1.25 mg of Astaxanthin reference standard to a 50 ml

volumetric flask.

2. Add about 20 ml of Acetone and sonicate about 2 minutes and allow to

equilibrate to ambient temperature for 15 minutes.

3. Dilute to volume with Acetone.

4. Pipette 2.0 ml of Standard stock solution to a 20 ml volumetric flask ,dilute

with acetone to volume and mix (This is Standard solution A )

5. Pipette 2.0 ml of Standard stock solution and 10.0 ml of internal standard

solution to a 20 ml volumetric flask ,dilute with acetone to volume and mix

(This is Standard solution B )

( Volumetric pipet and volumetric flask must be used , Accuracy at this step
directly affects the result. )
Sample solution stock preparation (Assay solution A )

PRODUCT TEST METHOD

SUBJECT: Identification, Assay and

NUMBER: PTM
REVISION :
Uniformity of Dosage Unit (Content uniformity) SUPERSEDE: N/A PAGE 5 of
11
of Astaxanthin 4 mg capsules
1. Place 6 capsules (Equivalent to 24.0 mg of Astaxanthin) into a 100 ml
volumetric flask.

2. Add about 50 ml acetone and shake about 30 minutes and sonicate about
15 minutes until the capsules are fully dispersed.

3. Dilute to volume with acetone and mix.

4. Pipette 10 ml from this stock solution into a 100 ml volumetric flask and
dilute to volume with acetone.

5. Pipette 2 ml from this stock solution into a 20 ml volumetric flask and dilute
to volume with acetone.

(Final concentration 0.0024 mg/ml of Astaxanthin)

Sample preparation (Assay solution B)

1. Pipet 2.0 ml of the sample stock Assay solution A into a 10 ml glass

centrifuge tube .

2. Add 1.0 ml of Internal standard solution and mix well.

(Must be use volumetric pipette, Pipetting accuracy at this step directly affects
the result)

3. Set water bath at temp. 37 C heat the solution test tube in the water bath
and add 3.0 ml of Cholesterol esterase solution to the test tube and mix by
gentle inversion.

4. Allow to react at 37 C for 45 minutes, gentle slowly invert every 10 minutes,

at least twice, during the reaction.

5. Until 45 minutes, add 1 g of sodium sulfate decahydrate and 2.0 ml of

PRODUCT TEST METHOD

SUBJECT: Identification, Assay and

NUMBER: PTM
REVISION :
Uniformity of Dosage Unit (Content uniformity) SUPERSEDE: N/A PAGE 6 of
11
of Astaxanthin 4 mg capsules
petroleum ether, vortex for 30 seconds, and centrifuge at 3,000 rpm for 3
minutes.

6. Transfer the petroleum ether layer to a 10 ml glass centrifuge tube

containing 1 g of sodium sulfate anhydrate.

7. Evaporate the petroleum ether layer in the stream of inert gas at room
temperature, add 3 ml of Acetone, sonicate and filter into vial HPLC.

Chromatographic conditions:
Mobile phase:

As described

Column:

YMC Carotenoid TM S5 micron , 250 mmx 4.6

mm. cat no. 99S05-2546WT

Flow rate:

1.0 ml / minute

Wavelength:

474 nm

Injection

20 L

volume:
Column
temperature
Profile Program

30 C

PRODUCT TEST METHOD

SUBJECT: Identification, Assay and

NUMBER: PTM
REVISION :
Uniformity of Dosage Unit (Content uniformity) SUPERSEDE: N/A PAGE 7 of
11
of Astaxanthin 4 mg capsules

Time (min)

Methanol

t-butyl- methyl-

1% phosphoric

81

15

15

66

30

23

16

80

27

16

80

27.1

81

15

35

81

15

ether

Retention time for Identification

Component

13-cis-astaxanthin :

acid solution

Retention time (min)

trans-astaxanthin :

10

9-cis-astaxanthin :

14

trans-beta-apo-8-carotenal (Internal standard)_ 17

Procedure:

After equilibrating the HPLC system with mobile phase

1. Inject the standard solution for assay and uniformity dosage unit for 6
injections

PRODUCT TEST METHOD

SUBJECT: Identification, Assay and

NUMBER: PTM
REVISION :
Uniformity of Dosage Unit (Content uniformity) SUPERSEDE: N/A PAGE 8 of
11
of Astaxanthin 4 mg capsules
2. Inject the sample solution for assay test for 2 injections
Identification:
The retention time of the Astaxanthin peak in the assay preparation

corresponds to that of the standard preparation

System suitability for Assay and Uniformity of dosage unit test:

1. The relative standard deviation (RSD) of the Astaxanthin peak areas for 6
injections of the Astaxanthin must be not more than 2.0 %

2. The tailing factors in Astaxanthin peak not more than 2.0

Standard solution A ( UV method)

1. Determine the absorbance of standard solution A at 474 nm, used acetone

as the blank

2. Calculate the concentration , in mg per ml , of astaxanthin in the standard

solution A taken by the formula:
Asa / 210
Where :
Asa

210 =

Absorbance of standard solution A

Absorbance of 1 (mg/ml) astaxanthin solution in acetone

PRODUCT TEST METHOD

SUBJECT: Identification, Assay and

NUMBER: PTM
REVISION :
Uniformity of Dosage Unit (Content uniformity) SUPERSEDE: N/A PAGE 9 of
11
of Astaxanthin 4 mg capsules
in a 1 cm cuvette at 474 nm.
Calculation:
1. For calculate the ratio of peak responses of total astaxanthin to I.S .
obtained from the Assay solution B and
Standard solution B taken by
(1.3 P

13-cis

+P

trans

+ 1.1 P

9-cis

)/P

I.S.

Where
P

13-cis

Peak responses of 13-cis-astaxanthin

Peak responses of trans-astaxanthin

Peak responses of 9-cis-astaxanthin

Peak responses of Internal standard

1.3

Relative responses coefficients of 13-cis-astaxanthin

1.1

Relative responses coefficients of 9-cis-astaxanthin

tran
s

9cis

I.S.

PRODUCT TEST METHOD

SUBJECT: Identification, Assay and

NUMBER: PTM
REVISION :
Uniformity of Dosage Unit (Content uniformity) SUPERSEDE: N/A PAGE 10 of
11
of Astaxanthin 4 mg capsules

2. Calculate the Astaxanthin content (%)

mg of Astaxanthin / capsule =

RAb x Csa x D
RSb

% of Astraxanthin

RAb

x 100
N

mg of Astraxanthin / capsule
LA

x 100

Ratio of the peak responsesof total astaxanthin to I.S.

obtained from Assay solution B

RSb

Ratio of the peak responsesof total astaxanthin to I.S.

obtained from Standard solution B

CSa

Concentration of Astraxanthin standard in the Standard

solution A

Diluting of Assay solution B

Number of capsule use in Assay solution

PRODUCT TEST METHOD

SUBJECT: Identification, Assay and

NUMBER: PTM
REVISION :
Uniformity of Dosage Unit (Content uniformity) SUPERSEDE: N/A PAGE 11 of
11
of Astaxanthin 4 mg capsules
LA

Label amount of Astraxanthin in mg /capsule