D-785

Beta Carotene Determination by HPLC using UV-Vis Spectroscopy

Section D — Laboratory Operations and Specifications 8 pages

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1.0 Purpose

The purpose of this procedure is to define the method for the quantitation and/or identification

of Beta Carotene in raw materials and finished products using HPLC and UV/VIS

spectrophotometry.

2.0 Scope

This procedure applies to the quantification and identification of Beta Carotene in raw materials

and finished products. Beta Carotene is an excellent chromophore and was measured at 454nm.
Other wavelengths can be used with justification if interferences are present.

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 AD-— Analytical Development

43 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums

4.4 CofA — Certificate of Analysis

[SOP D-785 | Page 2 of 8]

Standard Operating Procedure SOP No | Rev
Beta Carotene Determination by HPLC using UV/VIS | 2-785 4 : oa

Spectroscopy

5.0 References

5.1 MV-LAB-19-121, Protocol, Beta Carotene Determination by HPLC using UV/Vis
Spectroscopy.

6.0 Supplies

6.1 Chemicals: All reagents are HPLC grade or better.

6.1.1 Ethanol (denatured with 5% isopropanol and 5% methanol)

6.1.2 Acetonitrile

6.1.3 Methanol

6.1.4 Chloroform

6.1.5 Beta-Carotene reference standard

6.2 Glassware

6.2.1 HPLC vials, 12mm x 32mm with screw cap enclosures with septa

6.2.2 Scintillation Vials

6.2.3 1L Mobile Phase Container

6.2.4 50mL Volumetric Flask

6.2.5 100mL Volumetric Flask

6.3 Disposables

6.3.1 10mL Pipette Tips

6.3.2 ImL Pipette Tips

6.3.3 200uL Pipette Tips

6.3.4 1.5mL microfuge tubes

6.3.5 16mL Test Tubes

6.3.6 Disposable Plastic Luer Lock Syringe — 3mL, 6mL, or 10mL

6.3.7 Nylon Syringe Filters, 0.45um

[SOP D-785 | Page 3 of 8]

Standard Operating Procedure SOP No | Rev
Beta Carotene Determination by HPLC using UV/VIS | -785 , : aoe

Spectroscopy

6.3.8 Weigh paper

6.4 Equipment

6.4.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven

and UV detector with a chromatographic data handling system

6.4.2 Analytical Balance

6.4.3. Ultrasonic bath

6.4.4 Vortex

6.4.5 Stir Plate

6.4.6 Eppendorf Centrifuge

6.4.7. 10mL Pipette

6.4.8 ImL Pipette

6.4.9 200uL Pipette

7.0 Preparation of Mobile Phase, Dissolution Buffer, Samples, and Standards

7.1 Mobile Phase (Acetonitrile: Methanol:Chloroform 60:25:15)

7.1.1. Transfer 600 mL acetonitrile to a 1000-mL mobile phase bottle.

7.1.2 Add 250 mL methanol.

7.1. Add 150 mL chloroform, and mix well.

7.2 Diluent (Ethanol:Acetonitrile: Methanol:Chloroform 70:18:7.5:4.5)

7.2.1. Transfer 700 mL ethanol to a 1000-mL mobile phase bottle.

7.2.2 Add 180 mL acetonitrile.

7.2.3. Add 75 mL methanol.

7.2.4 Add 45 mL chloroform, and mix well.

7.3 Standard Preparation

7.3.1 Beta-carotene is light sensitive. Prepare solutions in low-actinic glassware.

[SOP D-785 | Page 4 of 8]

Standard Operating Procedure SOP No | Rev
Beta Carotene Determination by HPLC using UV/VIS | 2-785 z rp

Spectroscopy

7.3.2 The linear range of the method is 0.2 — 20 mcg/mL. All standard and sample

preparations must be within the linear range.

7.3.3 Use the actual purity from the CofA or the standard certification for Beta

Carotene reference material for calculations. The stock standard preparation
reflects 100% content for the analyte assayed.

7.3.4 The standard is prepared by weighing no less than the minimum weight of the

analytical balance, then bringing up to the final volume using Diluent in an
appropriately sized volumetric flask, then sonicating for 10 minutes.

7.3.5 Dilutions are prepared using Diluent. Dilutions can be made using volumetric

flasks or using 10mL, 1mL, and 200uL variable pipettes. Specific standard

concentrations will approximate the concentration expected to be found in the
product being tested based on the sample dilution and calculated from the label
claim or raw material potency. Dilutions can be prepared in HPLC vials.

7.3.6 Alternative standard preparations are acceptable as long as the preparations are
within the linear range of this method, 0.2 — 20 mcg/mL.

7.4. Sample Preparation

7.4.1. Specific sample testing details are provided in each products profile. If a
specific testing details section is not available, then follow preparation
procedure as described below, maintaining concentration within the linear range

of this method.
7.4.2 For finished products, at least 10 dosage units are pooled and ground by mortar

and pestle as necessary. Work quickly to avoid exposure to light.

7.4.3. Samples can be dissolved in Diluent at any volume starting from 50 mL and any
weight greater than the minimum weight of the analytical balance..

7.4.4 Based on the label claim and fill or tablet weight for finished products or

expected potency for raw materials, accurately weigh and transfer an amount of
sample into an appropriately sized low-actinic volumetric flask to generate a

concentration that is within the linear range of the method.

[SOP D-785 | Page 5 of 8]

Standard Operating Procedure SOP No | Rev
Beta Carotene Determination by HPLC using UV/VIS | 0-785 2 . a

Spectroscopy

7.4.5 Add H20 to 10% of the flask volume, and swirl or vortex to ensure that the entire

sample is wetted.

7.4.6 Dilute to the final volume using Diluent, and sonicate for 10 minutes.

7.4.7 Before injection, insoluble matter should be removed via filtration using a nylon

syringe filter. Discard at least 0.5mL of the sample before collecting filtrate.
Dilute filtrate as needed then add 1mL of the final dilution to an HPLC vial for

analysis.

7.4.7.1. Alternatively, samples and standards can also be centrifuged at 6000
RPM for 5 minutes in an Eppendorf centrifuge to pellet insoluble

matter.

8.0 Test Conditions

8.1 Isocratic

Time YoA %B Gradient Type

0.00 100 0.0

20.00 100 0.0 Isocratic

8.1.1 Column — Kinetex XB-C18, Sum, 100A, LC column, 250mm x 4.6mm, or
equivalent

8.1.2 Flow Rate — 1.0mL/min

8.1.3 UV Detection — 454nm

Injection Volume - 10uL

Column Temperature — 25°C

8.1.6 Recommended 3-D Spectral Range — 300 to 600 nm

8.2 Recommended Sequence

8.2.1 Make at least 2 injections of a Blank (Diluent).

8.2.2 Make five injections of the Working Standard.

[SOP D-785 | Page 6 of 8]

Standard Operating Procedure SOP No | Rev
Beta Carotene Determination by HPLC using UV/VIS | 2-755 2 ; a

Spectroscopy

8.2.3 Make a single injection of each Sample Preparation.

8.2.4 Make a single injection of the Working Standard after every six samples and at

the end of the run.

8.3 System Suitability

8.3.1 The %RSD of five consecutive injections of Working Standard is NMT 5.0%.

8.3.2. The %RSD of all Working Standard injections is NMT 5%.

8.4 Column Wash and Storage

8.4.1 Column wash is not required.

8.4.2 Store the column with mobile phase.

9.0 Example Calculations

x= x CF x 100
9.1 Ru Sample peak area

Rg Mean standard peak area

Wtsiq Weight of reference standard in mg

Vetq Volume of the standard preparation accounting for dilutions in mL

P Purity of the reference standard in decimal format

SA Sample amount in mg (solids) or mL (liquids)

Vsn. | Wolume of the sample preparation accounting for dilutions in mL

SS Serving size: Average Weight of a single dosage unit in mg for tablets, capsules,

and gummies. Volume of a single serving from the theoretical formula in mL for
liquids or mg for powders, or 1 for raw materials.

LA Label amount in mg per dose or 1 for raw materials

CF Conversion factor (1 if label claim is for Beta Carotene, 0.5 is label claim is for

Retinol A Equivalents or Vitamin A)

[SOP D-785 | Page 7 of 8]

Standard Operating Procedure SOP No | Rev
Beta Carotene Determination by HPLC using UV/VIS | 2-785 , : are
Spectroscopy

10.0 Example Chromatography

10.1. Blank

blank

10.2 Standard

DAD1A,Sig=454.0,4.0 Ref=360.0,100.0
20-
13.158
17.67
-carotene
15-
12.5-
Z 10-
E 75
2.5"
a eo no la 0 4 2 3 4 5 6 7 8&8 9 0 4 12 #13 «14 «15 1 17 «18 «19 = 20
Time [min]

10.3. Sample

190420 0.01mg/mli

Zz 410- 13.196
-carotene

0 4 2 3 4 5 6 7 8&8 8 0 1 12 13 #14 #«15 «1 «#617 «18 «6«19~=«20
Time [min]

[SOP D-785 | Page 8 of 8]

Standard Operating Procedure SOP No | Rev ‘
Beta Carotene Determien atie on by HPLC usie ng UV/VIS | 2-785 2 8 aae

Spectroscopy

11.0 Revision History

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 02/20/20 | New N/A J. Maignan Update for consistency with current methods, add linear range, add note about light sensitivity, make treatment of sample with I 06/20/22 water mandatory, add recommended sequence, replace CC-22-0281 S. Sassman requirements with system suitability, add conversion from beta- carotene to retinol equivalents, add example chromatography. | - | - |
| 2 | 12/20/22 | Added test details. Minor edits. CC- | 22-0475 | J. Sassman |