Determination of Lutein & Zeaxanthin by HPLC-UV

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1.0 Purpose 
 
 This document describes the analytical procedure for the determination of Lutein & Zeaxanthin
 
 in raw materials and finished products. 
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of Lutein & Zeaxanthin in raw
 
 materials and finished products. This method was validated under protocol PRTCL-23-0009.
 
 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 ACN — Acetonitrile 
 
 4.3 THF — Tetrahydrofuran 
 
 
 

[SOP D-101 | Page 2 of 7]

 Standard Operating Procedure SOP No Rev 
 Page 2 of 7 
 Determination of Lutein & Zeaxanthin by HPLC-UV D-1017 
 
 4.4 MeOH — Methanol 
 
 4.5 ACS — American Chemical Society 
 
 4.6 HPLC — High Performance Liquid Chromatography 
 
 47 UV-Vis — Ultraviolet-Visible (Detection) 
 
 5.0 References 
 
 5.1 PRTCL-23-0009, Protocol, Validation of an Analytical Method for the Determination
 of Lutein & Zeaxanthin by HPLC-UV 
 
 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 ACN 
 
 6.1.4 THF 
 
 6.1.5 Lutein Reference Standard - USE SIGMA-ALDRICH P/N PHR-1699 
 
 6.2 Supplies and Glassware (Note: USE RED GLASSWARE) 
 
 6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ UNSLIT septa
 
 6.2.2 Volumetric glassware (red low actinic stained glass) 
 
 6.2.3 Weigh paper 
 
 6.2.4 Syringes with 0.45p glass fiber syringe filters 
 
 
 

[SOP D-101 | Page 3 of 7]

 Standard Operating Procedure SOP No | Rev 
 Determination of Lutein & Zeaxanthin by HPLC-UV | D-1017 Q | Page 3 of7 
 
 6.3 Equipment 
 
 6.3.1 Suitable gradient HPLC system consisting of a pump, AUTOSAMPLER
 
 WITH NEEDLE WASH, COLUMN COMPARTMENT CAPABLE OF 
 
 MAINTAINING 10°C, and UV-Vis detector with a chromatographic data
 handling system 
 
 6.3.2 Analytical Balance 
 
 6.3.3. Wrist Action Shaker 
 
 7.0 Procedure 
 
 7.1 Mobile Phase & Diluent Preparation 
 
 7.1.1 Mobile Phase 
 
 7.1.1.1 Combine 990mL MeOH + 10mL water and mix well. 
 
 7.1.2 Extraction Solvent 
 
 7.1.2.1 Use THF. 
 
 7.1.3 Diluent 
 
 7.1.3.1 Use MeOH. 
 
 7.2 Standard Prep (Prepare in red glassware.) 
 
 7.2.1 Prepare Lutein standard stock at ~0.5 mg/mL in THF. Shake stock for 15
 
 minutes at ~ 4 volume then QS to volume. Dilute stock 2:50 with Diluent using
 
 volumetric glassware. Shake vigorously then filter a SmL aliquot for analysis,
 discarding the first 3-4mL of filtrate. 
 
 
 

[SOP D-101 | Page 4 of 7]

 Standard Operating Procedure SOP No | Rev 
 Determination of Lutein & Zeaxanthin by HPLC-UV | )-1017 0 | Page 4of7 
 
 7.2.2 A Zeaxanthin standard is not prepared. Zeaxanthin is quantified against Lutein
 at 446 nm, taking advantage of a coincident detector response at this
 
 wavelength. 
 
 7.2.3 Be sure to use Sigma-Aldrich P/N PHR-1699. This material contains
 
 Zeaxanthin impurity in such quantity as to facilitate its identification in raw
 
 materials and finished products. 
 
 7.3 Sample Preparation (Prepare in red glassware.) 
 
 7.3.1 Specific sample testing details are provided in each product profile. If a specific
 testing details section is not available, follow preparation procedure as described
 
 below, maintaining concentration within the linear range of this method.
 
 Fedaz The validated linear ranges for the analytical method are 13.7 — 36.5 yg/mL
 
 Lutein and 2.8 — 7.4 ug/mL Zeaxanthin. 
 
 7.3.3 Pool at least 10 dosage units and homogenize as appropriate (e.g. grind tablets /
 
 capsule fill / powders / stick pack contents by mortar and pestle, cryogenically
 
 powder and dissolve gummies, etc.) Extract sufficient sample (based on the raw
 material manufacturer assay value / product profile) with Extraction Solvent in
 
 order to generate a sample stock that is ~0.2 mg/mL Lutein. Shake for 15
 minutes at ~ 4 volume then QS to volume. Dilute stock 1:10 with Diluent using
 
 volumetric glassware. Shake vigorously then filter a 5mL aliquot for analysis,
 discarding the first 3-4mL of filtrate. 
 
 7.4 HPLC Parameters 
 
 74.1 Column: Halo C30, 2.7um (SPP), 160A, 4.6 x 150mm 
 
 7.4.2 Column Temperature: 10°C 
 
 7.4.3 Flow rate: 0.8 mL/min 
 
 
 

[SOP D-101 | Page 5 of 7]

 Standard Operating Procedure SOP No | Rev 
 Determination of Lutein & Zeaxanthin by HPLC-UV | D-1017 0 | Page5 of 7 
 
 7.4.4 Mobile Phase: Isocratic 
 
 7.4.5 Wavelength: 446 nm 
 
 7.4.6 Injection Volume: 5 wL (Use 1500u] ACN Needle Wash) 
 
 7.4.7 Run Time: 15 minutes 
 
 7.4.8 3-D Spectral Range (for Identification): 350nm - 550nm 
 
 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 2%.
 
 7.6.2 The %RSD of all standard injections is NMT 3%. 
 
 7.7. Example calculations for determining % Assay / LC: 
 
 7.71 % = Re x Weak? oe x 10x SS0/LA 
 
 Re Veta 
 
 R,, Sample peak area 
 
 R, Mean (All) standard peak area (Lutein) 
 
 Wt. Weight of the reference standard, mg 
 
 Fas Volume of the standard preparation accounting for dilutions, mL
 
 
 

[SOP D-101 | Page 6 of 7]

 Standard Operating Procedure SOP No | Rev 
 Determination of Lutein & Zeaxanthin by HPLC-UV | D-10917 0 | Page 6 of 7 
 
 = Purity of the reference standard in decimal format 
 
 SA Sample amount, g 
 
 Vet Volume of the sample preparation accounting for dilutions, mL
 
 SS Serving Size from Product Profile, mg (Use “1g” for Raw Materials.)
 
 LA Label Amount, mg (Use “1” for Raw Materials.) 
 
 7.8 System Wash, Column Wash and Column Storage 
 
 7.8.1 Wash and store the column in Mobile Phase 
 
 8.0 Chromatograms 
 
 8.1 Typical Diluent Chromatogram 
 
 * Lzeladcn Re NaH WWISIW4em 
 j 
 rAd 
 
 
 

[SOP D-101 | Page 7 of 7]

 Standard Operating Procedure SOP No | Rev 
 Determination of Lutein & Zeaxanthin by HPLC-UV | D-1017 0 | Page7ot7 
 
 8.2 Typical Standard Chromatogram 
 
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 8.3. Typical Raw Material Chromatogram 
 
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 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 03/09/23 | New procedure. N/A C. Perry | - | - |