Cholesterol

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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C₂₇H₄₆O          386.65

Cholest-5-en-3-ol, (3β)-;

Cholest-5-en-3β-ol CAS RN: 57-88-5

1 DEFINITION

Cholesterol is a steroid alcohol containing NLT 95.0% and NMT 102.0% of cholest-5-en-3ẞ-ol (C₂₇H₄₆O), calculated on the dried basis. It may contain suitable antioxidants.

2 IDENTIFICATION

A. SPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscoру: 1974 ог 197K

B. It meets the requirements of the test for Optical Rotation (781S), Procedures, Specific Rotation.

C. It meets the requirements of the test for Melting Range or Temperature (741).

3 ASSAY

PROCEDURE

Standard solution: 1.0 mg/mL of USP Cholesterol RS and 1.0 mg/mL of pregnenolone isobutyrate (internal standard) in heptane

Sample solution: 1.0 mg/mL of Cholesterol and 1.0 mg/mL of pregnenolone isobutyrate (internal standard) in heptane

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: GC

Detector: Flame ionization

Column: 0.25-mm x 30-m capillary bonded with a 0.25-µm layer of phase G2

Temperatures

Injection port: 285°

Column: 275°

Detector: 300°

Carrier gas: Helium

Flow rate: 2.0 mL/min

Injection volume: 1.0 µL

Injection type: Split ratio, 25:1

Liner: Cup splitter liner (4 mm x 6.3 x 78.5) with deactivated glass wool

System suitability

Sample: Standard solution

[NOTE-The relative retention times for pregnenolone isobutyrate and cholesterol are 1.0 and 1.2, respectively.]

Suitability requirements

Resolution: NLT 10 between pregnenolone isobutyrate and cholesterol

Relative standard deviation: NMT 2.0% for the peak response ratio of cholesterol to the internal standard

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of cholesterol in the portion of sample taken:

Result = (R_u /R_s ) × (C_s /C_u ) × 100

R_u = peak response ratio of cholesterol to the internal standard (peak response of cholesterol/peak response of the internal standard) from the Sample solution

R_s = peak response ratio of cholesterol to the internal standard (peak response of cholesterol/peak response of the internal standard) from the Standard solution

C_s = concentration of USP Cholesterol RS in the Standard solution (mg/mL)

C_u = concentration of Cholesterol in the Sample solution (mg/mL)

Acceptance criteria: 95.0%–102.0% on the dried basis

4 IMPURITIES

RESIDUE ON IGNITION (281): NMT 0.1%

LIMIT OF RELATED STEROLS AND OTHER ORGANIC IMPURITIES

Internal standard solution: 0.02 mg/mL of pregnenolone isobutyrate (internal standard) in heptane

System suitability solution: 0.02 mg/mL of USP Cholesterol RS, 0.04 mg/mL of desmosterol, and 0.02 mg/mL of lathosterol in Internal

standard solution

Sample solution: 2.0 mg/mL of Cholesterol in Internal standard solution

Chromatographic system: Proceed as directed in the Assay.

System suitability

Sample: System suitability solution

[NOTE-See Table 1 for the relative retention times.]

Table 1

Suitability requirements

Resolution: NLT 2.0 between desmosterol and lathosterol

Relative standard deviation: NMT 5.0% for peak response ratio of desmosterol to the internal standard

Analysis

Samples: System suitability solution and Sample solution

Three more related sterols may be observed (see Table 2).

Table 2

Calculate the percentage of desmosterol or lathosterol in the portion of Cholesterol taken:

Result = (R_u1 /R_s1 ) × (C_s1 /C_u ) × 100

R_u1 = peak response ratio of desmosterol or lathosterol to the internal standard (peak response of desmosterol or lathosterol/peak response of the internal standard) from the Sample solution

R_s1 = peak response ratio of desmosterol or lathosterol to the internal standard (peak response of desmosterol or lathosterol/peak response of the internal standard) from the System suitability solution

C_s1 = concentration of desmosterol or lathosterol in the System suitability solution (mg/mL)

C_u = concentration of Cholesterol in the Sample solution (mg/mL)

Calculate the percentage of β-cholestanol, 24-dehydrolathosterol, 4-methylcholest-5-en-3β-ol, or any other unspecied organic impurity in the portion of Cholesterol taken:

Result = (R_u2 /R_s2 ) × (C_s2 /C_u ) × 100

R_u2 = peak response ratio of β-cholestanol, 24-dehydrolathosterol, 4-methylcholest-5-en-3β-ol, or any other unspecied impurity to the internal standard (peak response of β-cholestanol, 24-dehydrolathosterol, 4-methylcholest-5-en-3β-ol, or any other unspecied impurity/peak response of the internal standard) from the Sample solution

R_s2 = peak response ratio of cholesterol to the internal standard (peak response of cholesterol/peak response of the internal standard) from the System suitability solution

C_s2 = concentration of USP Cholesterol RS in the System suitability solution (mg/mL)

C_u = concentration of Cholesterol in the Sample solution (mg/mL)

Acceptance criteria: See Table 3. Disregard peaks less than 0.05% for any unspecied impurities and any peaks due to solvent.

Table 3

5 SPECIFIC TESTS

Melting Range or Temperature 〈741〉: 147°–150°

Optical Rotation 〈781S〉, Procedures, Specific Rotation

Sample solution: 20 mg/mL, undried, in dioxane

Acceptance criteria: -34° to 38°

Change to read:

5.1 ACIDITY

Sample: 1.0 g

Analysis: Dissolve the Sample in 10 mL of ether in a small flask, add 10.0 mL of 0.10 N sodium hydroxide, and shake for about 1 min. Heat gently to expel the ether, then boil for 5 min, Cool, dilute with 10 mL of water, add phenolphthalein TS, and titrate with 0.10 N sulfuric acid until the pink color just disappears, stirring the solution vigorously throughout the titration. Perform a blank determination (see Titrimetry (541) _{(CN 1-Aug-2024)}).

Acceptance criteria: The difference between the number of mL of 0.10 N sulfuric acid consumed in the blank and the number of mL consumed in the Sample is NMT 0.3 mL.

LOSS ON DRYING (731)

Analysis: Dry under vacuum at 60° for 4 h.

Acceptance criteria: NMT 0.3%

5.2 SOLUBILITY IN ALCOHOL

Sample: 500 mg

Analysis: Dissolve the Sample in 50 mL of warm alcohol in a stoppered flask or cylinder, and allow to stand at room temperature for 2 h.

Acceptance criteria: No deposit or turbidity is formed.

6 ADDITIONAL REQUIREMENTS

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

LABELING: Label it to indicate whether cholesterol is derived from animal, synthetic, or vegetable sources. For animal-derived sources, indicate the species and tissue used (for example, bovine brain and spinal cord, wool fat, or chicken eggs). Indicate the names and amounts of any added antioxidants.

USP REFERENCE STANDARDS (11)

USP Cholesterol RS