Determination of Astaxanthin and Fucoxanthin in Raw Materials by HPLC-UV

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1.0 Purpose 
 
 This document describes the analytical procedure for the determination of free Astaxanthin and
 
 Fucoxanthin in raw materials. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of free Astaxanthin and
 
 Fucoxanthin in raw materials. Astaxanthin is quantified as the sum of the all-trans, 9-cis and
 13-cis geometric isomers of Astaxanthin. This method does not apply to raw materials
 
 containing fatty acid esters of Astaxanthin and/or Fucoxanthin — but only to quantification of
 the free forms. This method was validated under protocol PRTCL-21-0043.
 
 3.0 Responsibility 
 
 3.1 It is the responsibility of QC and Analytical chemists who have verified their ability to
 execute this procedure to follow this procedure. 
 
 3.2 ‘It is the responsibility of QC Laboratory Management to implement this procedure and
 to ensure that the procedure is being followed. 
 
 3.3. ‘It is the responsibility of QC Laboratory Management and/or Analytical Development
 Personnel to keep this procedure current with the associated monographs and laboratory
 
 practices. 
 
 4.0 Definitions 
 
 4.1 QC — Quality Control 
 
 4.2 AXN — Astaxanthin 
 
 
 

[SOP D-100 | Page 2 of 10]

 Standard Operating Procedure SOP No | Rev 
 Determination of Astaxanthin and Fucoxanthin in Raw | D-1007 0 Page 2 of 10 
 Materials by HPLC-UV 
 
 4.3. FXN- Fucoxanthin 
 
 4.4 MeOH — Methanol 
 
 4.5. ACS— American Chemical Society 
 
 4.6 HPLC — High Performance Liquid Chromatography 
 
 4.7 UV-Vis — Ultraviolet-Visible (Detection) 
 
 5.0 References 
 
 5.1 PRTCL-21-0043, Protocol, Validation of an Analytical Method for the Determination
 
 of Astaxanthin and Fucoxanthin by HPLC / UV-Vis 
 
 5.2 USP Monograph, Astaxanthin Esters 
 
 6.0 Supplies 
 
 6.1 Chemicals — All reagents are ACS grade or better 
 
 6.1.1 Milli-Q Water 
 
 6.1.2 MeOH 
 
 6.1.3 Acetone 
 
 6.1.4 AXN Reference Standard (all-trans, free form) 
 
 6.1.5 FXN Reference Standard (all-trans, free form) 
 
 6.2 Supplies and Glassware 
 
 6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa 
 
 6.2.2 Volumetric glassware 
 
 6.2.3. Weigh paper 
 
 

[SOP D-100 | Page 3 of 10]

 Standard Operating Procedure SOP No Rev 
 Determination of Astaxanthin and Fucoxanthin in Raw D-1007 Page 3 of 10 
 Materials by HPLC-UV 
 
 6.2.4 Syringes with 0.45u glass fiber syringe filters 
 
 6.3 Equipment 
 
 6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven
 and UV-Vis detector with a chromatographic data handling system
 
 6.3.2 Analytical Balance 
 
 6.3.3. Analytical Micro Balance 
 
 6.3.4 Sonicator Bath 
 
 7.0 Procedure 
 
 7.1 Mobile Phase & Diluent Preparation 
 
 7.1.1. Mobile Phase 
 
 7.1.1.1. Mobile Phase A: Water 
 
 7.1.1.2 Mobile Phase B: MeOH 
 
 7.1.2 Extraction Solvent / Diluent 
 
 7.1.2.1 Acetone 
 
 Note: Actives spray-dried onto water-soluble carriers require pre-treatment with water,
 
 see Sample Preparation below. 
 
 7.2 Standard Prep 
 
 7.2.1 AXN: Accurately weigh and transfer ~3 mg of reference standard into a 100
 mL volumetric flask using ~60 mL of acetone. Swirl to dissolve then QS to
 
 volume. Sonicate for 10 minutes then mix well. Sonicate for an additional 10
 minutes and mix well. 
 
 

[SOP D-100 | Page 4 of 10]

 Standard Operating Procedure SOP No | Rev 
 Determination of Astaxanthin and Fucoxanthin in Raw | D-1007 0 Page 4 of 10 
 Materials by HPLC-UV 
 
 7.2.2 FXN: Accurately weigh and transfer ~2 mg of reference standard into a 100 mL
 
 volumetric flask using ~60 mL of acetone. QS to volume then sonicate for 5
 
 minutes and mix well. 
 
 7.3. Sample Preparation 
 
 7.3.1 The validated linear ranges for the analytical method are 19.2 — 44.8 ug/mL
 AXN, 1.37 — 3.13 pg/mL 9-cis-AXN, 4.9 — 10.3 wg/mL 13-cis-AXN and 11.7 —
 
 27.3 ug/mL FXN. 
 
 7.3.2 Extract sufficient sample (based on the raw material manufacturer assay value)
 
 with Extraction Solvent in order to generate a concentration that is within the
 
 validated linear range. Mechanically shake at ~1/2 volume for 15 minutes, then
 QS to volume and sonicate for 10 minutes. Shake vigorously then filter a 5ml
 
 aliquot for analysis, discarding the first 3-4ml of filtrate.
 
 7.3.3. When analyzing samples that are spray-dried onto water-soluble carriers (e.g.
 
 maltodextrin), pre-treatment with water is required. Transfer the powder to the
 bottom of a 100mL volumetric flask and add SmL of water. Thoroughly wet the
 
 material with gentle, intermittent swirling over the course of 5 minutes. QS to
 
 volume with Extraction Solvent and sonicate for 10 minutes. Shake vigorously
 then sonicate for an additional 10 minutes. Shake vigorously then filter a Sml
 
 aliquot for analysis, discarding the first 3-4ml of filtrate.
 
 7.3.4 For materials being analyzed for the first time using this method, an in process
 
 validation is required to demonstrate spectral purity and extraction efficiency as
 a part of system suitability before data can be reported using this method
 
 74 HPLC Parameters 
 
 7.4.1. Column: HALO C30, 4.6 x 150mm, 160A, 2.7m SPP (Or Equivalent)
 
 7.4.2 Column Temperature: 35°C 
 
 7.4.3. Flow rate: 0.8 mL/min 
 
 

[SOP D-100 | Page 5 of 10]

 Standard Operating Procedure SOP No | Rev 
 Determination of Astaxanthin and Fucoxanthin in Raw | D-1907 0 Page 5 of 10 
 Materials by HPLC-UV 
 
 7.4.4 Mobile Phase: Gradient 
 
 7.4.4.1 Time,min %B 
 
 0.0 90 
 
 5.00 90 
 
 15.00 100 
 
 20.00 100 
 
 20.10 90 
 
 25.00 90 
 
 7.4.5 Wavelength: 477 nm (AXN), 450 nm (FXN) 
 
 7.4.6 Injection Volume: 5 wL 
 
 7.4.7. Run Time: 25 minutes 
 
 7.4.8 3-D Spectral Range (for Identification): 340nm - 650nm 
 
 7.5 | Recommended Sequence 
 
 7.5.1 Make at least 2 injections of the Diluent. 
 
 7.5.2 Make at least five (5) injections of Working Standard. 
 
 7.5.3. Make a single injection of each Sample Preparation. 
 
 7.5.4 Make a single injection of the Working Standard after every ten (10) sample
 injections or at the end of the run. 
 
 7.6 System Suitability Requirements 
 
 7.6.1 The %RSD of five (5) consecutive standard injections is NMT 3%.
 
 

[SOP D-100 | Page 6 of 10]

 Standard Operating Procedure SOP No | Rev 
 Determination of Astaxanthin and Fucoxanthin in Raw | D-1907 0 Page 6 of 10 
 Materials by HPLC-UV 
 
 7.6.2 The %RSD of all standard injections is NMT 5.0%. 
 
 7.6.3 If present, any interference in the diluent should be subtracted out of the sample
 
 and standard peak areas. 
 
 7.7 Example calculations for determining % assay: 
 
 7.71 % = Be y MtsaX? y “¥ elxy 400 
 
 R Sample peak area 
 ‘a 
 [Note: Add 1.3(13-cis peak area) + 1.1(9-cis peak area) to Ry if present in AXN sample.]
 
 R Mean (n=5) standard peak area 
 a 
 Wt. Weight of the reference standard 
 
 Va Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount 
 
 Vin5i Volume of the sample preparation accounting for dilutions in mL
 
 7.8 System Wash, Column Wash and Column Storage 
 
 7.8.1 Wash and store the column in 75:25 ACN / Water 
 
 

[SOP D-100 | Page 7 of 10]

 Standard Operating Procedure SOP No | Rev 
 Determination of Astaxanthin and Fucoxanthin in Raw | D-1007 0 Page 7 of 10 
 Materials by HPLC-UV 
 
 8.0 Chromatograms 
 
 8.1 Typical Diluent Chromatograms 
 
 3.00 *§ Astex RMS0G0212 Run 06-06-22 #2 Acetone UV_VIS_1 WVL-477 nm
 mAU 
 
 0.00 — 1.2a5 na aie aaeel 2.50 aan Sa 3> .7S8A LA AAA3 .0P0 ELL RE RAL I8PE.D2S5U RLANEELO7A. R5P0NA REELD 8.n75 d 101. 0Sa0 a n Rn 41.2PO5S Dae IN Dae 1N2G,. 5ap0e hee ee ieee 13.7Si5pe See eee mie ¥S5a .0E0S ARNEL ER 1P6.2P5 =17,. 5P0S A 1P8.75c 2P9.O00P Anne P21.P28 2P2.5P0 R an DO23. 75 +25.00
 gan. 2 Fucox Devel 12-22-21 #2 Acetone UV_VIS_1 WVL:450 nm
 ; mAU 
 
 

[SOP D-100 | Page 8 of 10]

 Standard Operating Procedure SOP No Rev 
 Determination of Astaxanthin and Fucoxanthin in Raw D-1007 Page 8 of 10 
 Materials by HPLC-UV 
 
 8.2 Typical Standard Chromatograms 
 
 400. AIX RMS000212 Run 08-06-22 #3 WSTD UV_ViS_4 VWVL:477 nm
 4] MAU 
 
 eee ; ES pees ie cae Joe ree, ae ee Soa neeee aed———— "4750 1875 20.00 21.25 : Dene, AGES] QRS ZO. ae
 
 70.0 - fb Fucox Devel 12-22-21 #3 wWSTD UV_VIS_1 WVL:450 nm
 ~ 4/mau 
 650- 
60.04 1 -Fucox - 6.827

 550-4 
 500-4 
40.0 nt

30.04 2504

 2004 
 1504 
 10.0~ 
 
 $.0- 
 a CM SAU (a) Ah, pT Aes MS, SARL SO IDI 475000 18.75 24.25
 
 

[SOP D-100 | Page 9 of 10]

 Standard Operating Procedure SOP No Rev 
 Determination of Astaxanthin and Fucoxanthin in Raw D-1007 Page 9 of 10 
 Materials by HPLC-UV 
 
 8.3 Typical Raw Material Chromatograms 
 
 'B Astax RMS0R0un0 062-061-22 2#8 Precision-1 UV_VIS_1 WV:477 nm
 4} MAU 
 ,%a 
 
 aeer a “750 | «1875 2~——(té«i SC(‘ié‘i TS 2.50 2375 = 25.00
 
 ng a Fucox Devel 12-22-21 #10 UV_VIS_1 WV.:450 nm
 4} MAU 
 6504 
 3 - Fucox - 6.827 
 c00] 
 01 
 $00 
 4504 
 4004 
 100: 
 
25.0 200

 1504 
 
 10.0- 
 504 
 
 504 
 te Te tT ams 55@ Jos Sook Se eee ad Fee ee pee foes Sey ers eee eee eae Je aes Deke ee cee oaes pate coer Gee gees oes ee moe see eee | Eee ceo eee Sere Dem SR pS eae cores nee Wis Bowe eae ones ees Ee pees eae poe
 
 

[SOP D-100 | Page 10 of 10]

 Standard Operating Procedure SOP No | Rev 
 ae : ee D-1007 0 Page 10 of 
 Determination of Astaxanthin and Fucoxanthin in Raw 10 
 Materials by HPLC-UV 
 
 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 08/02/22 | New procedure. N/A C. Perry | - | - |