Caffeine

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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C₈H₁₀N₄O₂  · H₂O                 212.21

C₈H₁₀N₄O₂                            194.19

1H-Purine-2,6-dione, 3,7-dihydro-1,3,7-trimethyl-;

1,3,7-Trimethylxanthine CAS RN: 58-08-2; UNII: 3G6A5W338E.

Monohydrate CAS RN: 5743-12-4.

1 DEFINITION

Caffeine is anhydrous or contains one molecule of water of hydration. It contains NLT 98.5% and NMT 101.0% of C₈H₁₀N₄O₂ calculated on the anhydrous basis.

2 IDENTIFICATION

Change to read:

A. SPECTROSCOPIC IDENTIFICATION TESTS (197), infrared Spectroscopy: 197M _{(CN 1-MAY-2020)}

B. The retention time of the caffeine peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

3 ASSAY

PROCEDURE

Buffer: 0.82 g/L of anhydrous sodium acetate

Mobile phase: Acetonitrile, tetrahydrofuran, and Buffer (25:20:955). Adjust with glacial acetic acid to a pH of 4.5.

System suitability solution: 0.02 mg/mL of Theophylline in Mobile phase. Shake, and sonicate if necessary, to dissolve.

Standard solution: Transfer 5.0 mg of USP Caffeine RS to a 25-mL volumetric flask. Add 5.0 mL of the System suitability solution and 10 mL of Mobile phase. Shake, and sonicate if necessary. Dilute with Mobile phase to volume, and filter.

Sample solution: 0.2 mg/mL of Caffeine in Mobile phase. [NOTE-Shake, and sonicate if necessary, to dissolve.]

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 275 nm

Column: 4.6-mm x 15-cm; packing L1

Flow rate: 1 mL/min

Injection size: 10 µL

System suitability

Sample: Standard solution

[NOTE-The relative retention times for theophylline and caffeine are 0.69 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 6.0 between theophylline and caffeine

Tailing factor: NMT 2.0 for theophylline and caffeine peaks

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of caffeine (C₈H₁₀N₄O₂) in the portion of Caffeine taken:

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

r_u = peak response of caffeine from the Sample solution

r_s = peak response of caffeine from the Standard solution

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

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

Acceptance criteria: 98.5%–101.0% on the anhydrous basis

4 IMPURITIES

RESIDUE ON IGNITION (281): NMT 0.1%

ORGANIC IMPURITIES

Mobile phase, Standard solution, Sample solution, Chromatographic system, and System suitability: Proceed as directed in the Assay.

Analysis

Sample: Sample solution

Calculate the percentage of each impurity in the portion of Caffeine taken:

Result = (r_u /r_T ) × 100

r_u = peak response for each impurity from the Sample solution

r_T = sum of the responses of all the peaks from the Sample solution

Acceptance criteria

Individual impurities: NMT 0.1%

Total impurities: NMT 0.1%

5 SPECIFIC TESTS

WATER DETERMINATION, Method III(921): Dry a sample at 80° for 4 h: the anhydrous form loses NMT 0.5% of its weight, and the hydrous form

loses NMT 8.5% of its weight.

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve hydrous Caffeine in tight containers. Preserve anhydrous Caffeine in well-closed containers.

LABELING: Label it to indicate whether it is anhydrous or hydrous.

USP REFERENCE STANDARDS (11)

USP Caffeine RS