Beta Carotene

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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C₄₀H₅₆               536.87

β.β-Carotene;

all-trans-ẞ-Carotene;

(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] CAS RN: 7235-40-7; UNII: 01YAE03M7J.

1 DEFINITION

Beta Carotene contains NLT 96.0% and NMT 101.0% of total carotenoids calculated as beta carotene (C₄₀H₅₆). It contains NLT 95% of all-trans-beta carotene in the total carotenoids content.

2 IDENTIFICATION

A.

Sample solution: Prepare as directed in the Sample solution in the test for Content of Total Carotenoids.

Analysis: Record the UV-Vis spectrum from 300-600 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 A₄₅₅/A₄₈₃ 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 Beta Carotene.

3 COMPOSITION

3.1 CONTENT OF TOTAL CAROTENOIDS

[NOTE-Use low-actinic glassware.]

Sample stock solution: 0.1 mg/mL of Beta Carotene in tetrahydrofuran

Sample solution: Transfer 3.0 mL of Sample stock solution to a 100-mL volumetric flask, and dilute with cyclohexane to volume.

Instrumental conditions

(See Ultraviolet-Visible Spectroscopy (857).)

Analytical wavelength: 456 nm

Cell path: 1 cm

Blank: Cyclohexane

Analysis

Sample: Sample solution

Calculate the percentage of total carotenoids (7) as beta carotene (C₄₀H₅₆):

T = A/(F × C)

A = absorbance of the Sample solution

F = 2505, coecient of extinction (E

1%) of pure all-trans-beta carotene in cyclohexane (100 mL · g⁻¹ · cm⁻¹)

C = concentration of the Sample solution (g/mL)

Acceptance criteria: 96.0%–101.0% of total carotenoids as beta carotene (C₄₀H₅₆)

3.2 CONTENT OF BETA CAROTENE

[NOTE-Use low-actinic glassware.]

Mobile phase: Transfer 50 mg of butylated hydroxytoluene to 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 µg/mL of butylated hydroxytoluene in alcohol

System suitability solution: Transfer 20 mg of USP Beta Carotene System 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 the suspension through a membrane filter of 0.45-um pore size, and use the clear filtrate.

Standard solution: 10 µg/mL of USP Beta Carotene RS in tetrahydrofuran and Diluent (1:9). Dissolve an appropriate amount of USP Beta Carotene RS in a volumetric flask first with tetrahydrofuran, using 10% of the volume of the flask, then dilute with Diluent to volume.

Sample solution: Dilute the freshly prepared Sample stock solution as prepared in the test for Content of Total Carotenoids (1 in 10) with

Diluent.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 448 nm

Column: 4.6-mm x 25-cm; 5-µm packing L68

Column temperature: 30°

Flow rate: 0.6 mL/min

Injection volume: 20 µl.

System suitability

Samples: System suitability solution and Standard solution

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 1.5 between all-trans-beta carotene and all-trans-alpha carotene; NLT 1.2 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

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

Analysis

Sample: Sample solution

Record the chromatograms, and 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 all-trans-beta carotene relative to total carotenoids in the sample taken:

Result = (r_U /r_T ) × 100

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

r_T = [(peak area of all-trans-alpha carotene × 1.0) + (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) + (sum of peak areas of other cis-isomers of beta carotene)] from the Sample solution

Acceptance criteria: NLT 95% of all-trans-beta carotene in the total carotenoids content

3.3 ALPHA CAROTENE AND OTHER RELATED COMPOUNDS

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

Analysis

Sample: Sample solution

Calculate the percentage of alpha carotene and other individual related compounds relative to total carotenoids in the portion of the

Sample taken:

Result = (r_U /r_T ) × 100

r_U = (peak area of all-trans-alpha carotene × 1.0) or (peak area response of other individual related compounds × appropriate relative response factor, Table 1) in the Sample solution

r_T = [(peak area of all-trans-alpha carotene × 1.0) + (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) + (sum of peak areas of other cis-isomers of beta carotene)] from the Sample solution

Acceptance criteria

Alpha carotene: NMT 1.0%

Total related compounds (including alpha carotene): NMT 5%

4 IMPURITIES

RESIDUE ON IGNITION (281): NMT 0.2%, 2 g of specimen being used

SPECIFIC TESTS

LOSS ON DRYING (731)

Analysis: Dry under vacuum over phosphorus pentoxide at 40° for 4 h.

Acceptance criteria: NMT 0.2%

5 ADDITIONAL REQUIREMENTS

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

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].

USP Beta Carotene System Suitability RS