Determination of Beta Carotene by Visible Spectroscopy

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1.0 Purpose 
 
 This document describes the analytical procedure for the determination of Beta Carotene (when
 
 present as beadlets encapsulated in alginate or gelatin) in raw materials and finished products.
 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of Beta Carotene (when present
 
 as beadlets encapsulated in alginate or gelatin) in raw materials and finished products. This
 method was validated under protocol PRTCL-24-0024. 
 
 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 H20 — Deionized Water (>18MQ:-cm) 
 
 
 

[SOP D-102 | Page 2 of 5]

 Standard Operating Procedure SOP No Rev 
 
 Determination of Beta Carotene by Visible D-1023 Page 2 of 5 
 Spectroscopy 
 
 4.3 NHsOH — Ammonium Hydroxide 
 
 4.4 THF — Tetrahydrofuran 
 
 4.5 BHT — Butylated Hydroxytoluene 
 
 4.6 H3POs — Phosphoric Acid 
 
 4,7 ACS — American Chemical Society 
 
 5.0 References 
 
 Sal PRTCL-24-0024, Validation of an Analytical Method for the Determination of Beta
 Carotene by Visible Spectroscopy 
 
 6.0 Supplies 
 
 6.1 Chemicals — (All reagents are ACS grade or better.) 
 
 6.1.1 H20 
 
 6.1.2 NH4sOH 
 
 6.1.3. H3PO4 
 
 6.1.4 THF 
 
 6.1.5 BHT 
 
 6.1.6 Alkaline Protease (Use BioCat Alkaline Protease R, or equivalent.)
 
 6.1.7 Cyclohexane 
 
 6.2 Supplies and Glassware 
 
 6.2.1 lcm Cuvettes 
 
 
 

[SOP D-102 | Page 3 of 5]

 Standard Operating Procedure SOP No | Rev 
 Determination of Beta Carotene by Visible D-1023 0 | Page 3 of 5 
 
 Spectroscopy 
 
 6.2.2 Low Actinic Volumetric Glassware 
 
 6.2.3 Weigh Paper 
 
 6.2.4 Syringes with 0.45 Teflon or Glass Fiber Syringe Filters 
 
 6.3 Equipment 
 
 6.3.1 Spectrophotometer 
 
 6.3.2 Analytical Balance 
 
 6.3.3. pH Meter 
 
 6.3.4 Heated Ultrasonic Bath 
 
 6.3.5 Digital Timer 
 
 6.3.6 Digital Thermometer 
 
 7.0 Procedure 
 
 7.1 Extraction Solvent & Diluent Preparation (Preparations may be scaled.)
 
 7.1.1 Extraction Solvent —- 4% Ammonium Hydroxide Buffer 
 
 7.1.1.1 Add 36ml Ammonium Hydroxide to 214ml H20 and mix well. Adjust
 pH to 9.5 with H3PO4. The accuracy of the pH adjustment is of critical
 
 importance! 
 
 7.1.2 Diluent — Stabilized THF Solution (0.1% BHT) 
 
 7.1.2.1 Add 500mg BHT to 500ml THF and mix well. 
 
 7.2 Sample Preparation 
 
 Use low actinic glassware! Minimize sample exposure to light and o
 
 
 

[SOP D-102 | Page 4 of 5]

 Standard Operating Procedure SOP No | Rev 
 Determination of Beta Carotene by Visible D-1023 0 | Page 4 of 5 
 
 Spectroscopy 
 
 7.2.1 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.
 
 db ebede The validated linear range for the analytical method is 0.26 — 4.23 g/mL.
 
 (Use low actinic glassware! Minimize sample exposure to light and oxygen!)
 
 12 Pool at least 20 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.) To prepare the stock sample, weigh
 
 sufficient sample (based on the raw material manufacturer assay value / finished
 product profile) to deliver ~10mg active into a 100ml volumetric flask.
 
 7.2.4 Add 20m] Extraction Solvent and two drops of alkaline protease and swirl
 vigorously to thoroughly wet the sample. Place in a heated ultrasonic bath
 
 preheated to 55°C. Submerge and immobilize the flask carefully, making sure
 that the level of the water in the water bath is below the level of the sample in
 
 the flask. Sonicate for 20 minutes, swirling vigorously every 5 minutes.
 
 tds 
 Remove the sample from the sonicator and allow to equilibrate to room
 temperature. QS to volume with diluent then add a stir bar and mix on a stir
 plate for 20 minutes. If undissolved beadlets remain, add two drops of NHsOH
 and continue to stir for 10 minutes more. 
 
7.2.6 Allow the mixture to settle then dilute 3:100 with cyclohexane. Shake

 vigorously then filter a Sml aliquot for analysis, discarding the first 1-2ml before
 directing the filtrate into a cuvette. 
 
 7.3 Spectrophotometer Parameters 
 
 TL Assay Wavelength: 456nm 
 
 
 

[SOP D-102 | Page 5 of 5]

 Standard Operating Procedure SOP No Rev 
 
 Determination of Beta Carotene by Visible D-1023 Page 5 of 5 
 Spectroscopy 
 
 7.3.2 Identification Wavelength (Absorbance Ratio): Aassnm / A4g3nm
 
 7.3.3 Cell Path: 1cm 
 
 7.3.4 Blank: Cyclohexane 
 
 7.4 Calculation for determining percentage: 
 
 74.1 T=AMF*C) 
 
 Where: 
 
 T Percentage of Total Carotenoids as Beta Carotene (C4oHs6)
 
 A Absorbance of the sample solution 
 
 F 2505, Coefficient of Extinction (E'”) of pure all-trans-beta carotene
 
 in Cyclohexane (100ml * gTM! * cm’!) 
 
 C Concentration of the sample solution (g/ml) 
 
 8.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 03/12/24 | New procedure. N/A C. Perry | - | - |