Beta Carotene Capsules

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Change to read:

Beta Carotene Capsules contain NLT 90.0% _{(USP 1-May-2019)} and NMT 125.0% of the labeled amount of total beta carotene (C₄₀H₅₆). _{(USP 1-May-2019)}

2 IDENTIFICATION

A.

Sample solution: Dilute the Sample stock solution from the test for Content of Total Beta Carotene with cyclohexane to a final concentration of 1-5 µg/mL of beta carotene. Pass through a membrane filter of 0.45-µm pore size.

Analysis: Record the UV-Vis spectrum from 300 to 600 nm.

Acceptance criteria: The Sample solution shows a shoulder at about 427 nm, an absorption maximum at about 455 nm, and another maximum at about 483 nm. The absorbance ratio A/A is between 1.14 and 1.18.

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the test for Content of Total Beta Carotene.

3 ASSAY

Change to read:

CONTENT OF TOTAL BETA CAROTENE

[NOTE-Use low-actinic glassware.]

Mobile phase: Transfer 50 mg of butylated hydroxytoluene into a 1-L volumetric flask, and dissolve with 20 mL of 2-propanol. Add 0.2 mL of N-ethyldiisopropylamine, 25 mL of 0.2% ammonium acetate solution, 455 ml of acetonitrile, and about 450 mL of methanol. Allow the solution to reach room temperature, and dilute with methanol to volume.

Diluent: 50 mg/L of butylated hydroxytoluene in alcohol

System suitability solution: Transfer 20 mg of USP Beta Carotene Systern Suitability RS to a 50-ml volumetric flask. Add 1 ml of water and 4 mL of tetrahydrofuran, and sonicate for 5 min. Dilute with Diluent to volume, and sonicate for 5 min. Cool to room temperature, pass through a membrane filter of 0.45-um pore size, and use the clear filtrate.

Standard stock solution: 60 µg/mL of USP Beta Carotene RS in tetrahydrofuran. [NOTE-The USP Beta Carotene RS is subjected to the spectrophotometric purity test at the time of analysis; see the determination of the concentration of Standard solution A below.]

Standard solution A: Transfer 5.0 mL of the Standard stock solution into a 100-mL volumetric flask, add 5.0 mL of tetrahydrofuran, and dilute with Diluent to volume.

Determine the concentration of Standard solution A according to the Analysis of Standard solution B. [NOTE-The concentration of Standard solution B equals the concentration of Standard solution A.]

Standard solution B: Transfer 5.0 mL of the Standard stock solution into a 100-mL volumetric flask, and dilute with cyclohexane to volume.

Prepare in triplicate.

Instrumental conditions

(See Ultraviolet-Visible Spectroscopy (857).)

Analytical wavelength: 456 nm _{(USP 1-May-2019)}

Cell: 1 cm

Blank: Cyclohexane

Analysis

Sample: Standard solution B

Calculate the concentration of total beta carotene (µg/mL) as all-trans-beta carotene (C₄₀H₅₆) in Standard solution B:

Result = (A_u /a) × F

A_u = average absorbance of the three preparations of Standard solution B

a = absorptivity of pure all-trans-beta carotene in cyclohexane _{(USP 1-May-2019)} , 250

F = conversion factor, 1000 µg/mg

Sample stock solution: Randomly select a number of Capsules, equivalent to 10-50 mg of beta carotene, with a total weight not exceeding 5 g. For powder-containing Capsules, empty the shell, and transfer shell and contents into a 250-mL volumetric flask. For Capsules containing liquid formulations, place the Capsules directly into a 250-mL volumetric flask. Add 250 mg of butylated hydroxytoluene, 0.5 mL of alkaline protease R, and 15 mL of water. Swirl the flask gently to wet the entire contents. Sonicate the flask at 50 for 30 min, and swirl the flask every 10 min. Add 100 mL of alcohol to the warm suspension, and shake vigorously. Add 110 mL of methylene chloride, and shake vigorously again. Disperse any clumps with a homogenizer, and rinse the homogenizer probe with 15 mL of methylene chloride into the flask. Allow the solution to stand in the dark until it reaches room temperature (about 2 h), dilute with methylene chloride to volume, shake vigorously, and allow the solids to settle.

Sample solution: Dilute a volume of the Sample stock solution with a Diluent-methylene chloride mixture (1:1) so that the final concentration of beta carotene is 1-5 µg/mL. Pass through a membrane filter of 0.45-µm pore size.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 448 nm

Column: 4.6-mm x 25-cm; 5-um packing L.68

Column temperature: 30°

Flow rate: 0.6 mL/min

Injection volume: 20 µL

System suitability

Samples: System suitability solution and Standard solution A

[NOTE-The approximate relative retention times of the components in the System suitability solution are listed in Table 1.]

Table 1

Suitability requirements

Chromatogram similarity: The chromatogram from the System suitability solution is similar to the reference chromatogram provided with

the lot of USP Beta Carotene System Suitability RS being used.

Resolution: NLT 1.2 (USP 1-May-2019) between all-trans-beta carotene and all-trans-alpha carotene and between all-trans-beta carotene and 9-cis-beta carotene, System suitability solution

Tailing factor: NMT 2.0 for the all-trans-beta carotene peak, Standard solution A

Relative standard deviation: NMT 2.0% for the all-trans-beta carotene peak from replicate injections, Standard solution A

Analysis

Samples: Standard solution A and Sample solution

Identify the peaks of the relevant analytes of the Sample solution by comparing with those of the System suitability solution. Measure the peak area responses.

Calculate the percentage of the labeled amount of total beta carotene in the portion of Capsules taken:

Result = (Σr_u /r_s ) × (C_s /C_u ) × 100

r_u = (peak area of all-trans-beta carotene) + (peak area of 9-cis-beta carotene) + (peak area of 13-cis-beta carotene × 1.2) + (peak area of 15-cis-beta carotene × 1.4) from the Sample solution

r_s = peak area of all-trans-beta carotene from Standard solution A

C_s = concentration of all-trans-beta carotene in Standard solution A as determined above (µg/mL)

C_u = nominal concentration of total beta carotene in the Sample solution (µg/mL)

Calculate the percentage of all-trans-beta carotene in the portion of Capsules taken:

Result = (r_all-trans/Σr_u ) x 100

r_all-trans = peak area of all-trans-beta carotene from the Sample solution

r_u = (peak area of all-trans-beta carotene) + (peak area of 9-cis-beta carotene) + (peak area of 13-cis-beta carotene × 1.2) + (peak area of 15-cis-beta carotene × 1.4) from the Sample solution

Acceptance criteria: 90.0% (USP 1-May-2019) -125.0% of the labeled amount of total beta carotene (C₄₀H₅₆) _{(USP 1-May-2019)}

4 SPECIFIC TESTS

Change to read:

ALPHA CAROTENE AND OTHER RELATED COMPOUNDS

Mobile phase, System suitability solution, Sample solution, and Chromatographic system: Proceed as directed in the test for Content of Total Beta Carotene.

Analysis

Sample: Sample solution

Calculate the percentage of alpha carotene and other individual related compounds relative to total beta carotene in the portion of Capsules taken:

Result = (r_U/r_T) x 100

r_u = peak area of alpha carotene or other individual related compounds

r_T = sum of the areas of all the peaks

Acceptance criteria

Alpha carotene: NMT 1.0%

Total related compounds (including alpha

carotene): NMT 5% (USP 1-May-2019)

5 PERFORMANCE TESTS

UNIFORMITY OF DOSAGE UNITS (905): Meet the requirements

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight, light-resistant containers.

Change to read:

LABELING: The label states the name and content of any carriers and antioxidants added to the formulation and the content of total carotenoids as beta carotene. The label states the percentages of cis- and ali-trans-isomers in the total beta carotene at the time of product manufacture and release _{(USP 1-May-2019)}

USP REFERENCE STANDARDS (11)

USP Beta Carotene RS

(all-E)-1,1'-(3,7,12,16-Tetramethyl-1,3,5,7,9,11,13,15,17-octadecanonaene-1,18- diyl)bis[2,6,6-trimethylcyclohexene].

C₄₀H₅₆               536.87

USP Beta Carotene System Suitability RS