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USP 35 Official Monographs / Caffeine 2427

Assay—[NOTE—Prepare solutions immediately before use, and ASSAY

protect from light.] • PROCEDURE
Buffer—Transfer 6.8 g of monobasic potassium phosphate Buffer: 0.82 g/L of anhydrous sodium acetate
to a 1-L volumetric flask. Dissolve the contents in 900 mL of Mobile phase: Acetonitrile, tetrahydrofuran, and Buffer
water. Adjust with phosphoric acid to a pH of 2.0. Dilute with (25:20:955). Adjust with glacial acetic acid to a pH of 4.5.
water to volume, add 0.2 mL of triethylamine, and mix well. System suitability solution: 0.02 mg/mL of theophylline in
Mobile phase—Prepare a mixture of Buffer and acetonitrile Mobile phase. Shake, and sonicate if necessar y, to dissolve.
(84:16), and degas. Make adjustments if necessar y (see System Standard solution: Transfer 5.0 mg of USP Caffeine RS to a
Suitability under Chromatography 〈621〉). 25-mL volumetric flask. Add 5.0 mL of the System suitability
solution and 10 mL of Mobile phase. Shake, and sonicate if
Standard preparation—Dissolve an accurately weighed quan- necessary. Dilute with Mobile phase to volume, and filter.
tity of USP Cabergoline RS in Mobile phase to obtain a solution Sample solution: 0.2 mg/mL of Caffeine in Mobile phase.
having a known concentration of about 0.25 mg per mL. [NOTE—Shake, and sonicate if necessar y, to dissolve.]
[NOTE—Sonication may be used to aid in the dissolution of Chromatographic system
cabergoline.] (See Chromatography 〈621〉, System Suitability.)
Assay preparation—Grind not fewer than 20 T ablets into a Mode: LC
fine powder. Transfer an accurately weighed portion of the Detector: UV 275 nm
powder, equivalent to about 2.5 mg of cabergoline based on Column: 4.6-mm × 15-cm; packing L1
the label claim, to a 10-mL volumetric flask. Dilute with Mobile Flow rate: 1 mL/min
phase to volume, and sonicate until completely dissolved. This Injection size: 10 µL
solution has a nominal concentration of about 0.25 mg per mL System suitability
of cabergoline, based on the label claim. [ NOTE—The Assay prep- Sample: Standard solution
aration may be passed through a PVDF type filter with a pore [NOTE—The relative retention times for theophylline and
size of 0.45 µm prior to analysis.] caffeine are 0.69 and 1.0, respectively.]
Chromatographic system (see Chromatography 〈621〉)—The Suitability requirements
liquid chromatograph is equipped with a 280-nm detector and Resolution: NLT 6.0 between theophylline and caffeine
a 4.0-mm × 25-cm column that contains 10- µm packing L1. Tailing factor: NMT 2.0 for theophylline and caffeine
The flow rate is about 1.3 mL per minute. Chromatograph the peaks
Standard preparation, and record the peak responses as directed Relative standard deviation: NMT 2.0%
for Procedure: the column efficiency is not less than 1000 theo- Analysis
retical plates; and the relative standard deviation for replicate Samples: Standard solution and Sample solution
injections is not more than 2.0%. Calculate the per centage of caffeine (C 8H10N4O2) in the por-
Procedure—Separately inject equal volumes (about 100 µL) tion of Caffeine taken:
of the Standard preparation and the Assay preparation into the
chromatograph, record the chromatograms, and measure the Result = ( rU/rS) × (CS/CU) × 100
peak responses. Calculate the per centage of the label claim of rU = peak response of caffeine from the Sample
C26H37N5O2 in the portion of T ablets taken by the formula: solution
100(CS / CU)(rU / rS) rS = peak response of caffeine from the Standard
solution
in which CS is the concentration of USP Cabergoline RS, in mg CS = concentration of USP Caffeine RS in the Standard
per mL, in the Standard preparation; CU is the nominal concen- solution (mg/mL)
tration of carbergoline, in mg per mL, in the Assay preparation, CU = nominal concentration of Caffeine in the Sample
based on the label claim; and rU and rS are the peak responses solution (mg/mL)
for cabergoline obtained from the Assay preparation and the Acceptance criteria: 98.5%–101.0% on the anhydrous basis
Standard preparation, respectively. IMPURITIES
• RESIDUE ON IGNITION 〈281〉: NMT 0.1%
• HEAVY METALS, Method II 〈231〉: NMT 10 ppm
• ORGANIC IMPURITIES
Mobile phase, Standard solution, Sample solution,
Caffeine
.

Chromatographic system, and System suitability:

Proceed as directed in the Assay.
Analysis
Sample: Sample solution
Calculate the per centage of each impurity in the portion of
Caffeine taken:
Result = ( rU/rT) × 100
C8H10N4O2 · H2O 212.21
C8H10N4O2 194.19 rU = peak response for each impurity from the Sample
1H-Purine-2,6-dione, 3,7-dihydro-1,3,7-trimethyl-; solution
1,3,7-Trimethylxanthine [58-08-2]. rT = sum of the responses of all the peaks from the
Monohydrate [5743-12-4]. Sample solution
Acceptance criteria
DEFINITION Individual impurities: NMT 0.1%
Caffeine is anhydrous or contains one molecule of water of hy- Total impurities: NMT 0.1%
dration. It contains NL T 98.5% and NMT 101.0% of
C8H10N4O2, calculated on the anhydrous basis. SPECIFIC TESTS
• WATER DETERMINATION, Method III 〈921〉: Dry a sample at
IDENTIFICATION 80° for 4 h: the anhydrous form loses NMT 0.5% of its
• A. INFRARED ABSORPTION 〈197M〉 weight, and the hydrous form loses NMT 8.5% of its weight.
• B. The retention time of the caffeine peak of the Sample
solution corresponds to that of the Standard solution, as ob-
tained in the Assay.

Official from May 1, 2012

Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.
 Accessed from [Link] by nEwp0rt1 on Tue Nov 22 03:37:46 EST 2011

2428 Caffeine / Official Monographs USP 35

ADDITIONAL REQUIREMENTS Procedure: the theophylline peak produces a discernible peak

• PACKAGING AND STORAGE: Preserve hydrous Caffeine in tight response at its retention time.
containers. Preserve anhydrous Caffeine in well-closed Procedure—Separately inject equal volumes (about 10 µL) of
containers. the Standard solution and the Test solution into the chromato-
• LABELING: Label it to indicate whether it is anhydrous or graph, record the chromatograms, and measure the peak re-
hydrous. sponses. Calculate the per centage of any related compound in
• USP REFERENCE STANDARDS 〈11〉 the portion of Injection taken by the formula:
USP Caffeine RS
100F(386.31/194.19)(CS / CW)(ri / rS)

in which F is the relative response factor and is equal to 0.878

.

for theobromine at a relative retention time of about 0.4, equal

Caffeine Citrate Injection to 1.10 for paraxanthine at a relative retention time of about
0.6, equal to 0.905 for theophylline at a relative retention time
» Caffeine Citrate Injection is a sterile solution of about 0.7, and equal to 1.0 for any other related compound;
containing Caffeine and citric acid in W ater for 386.31 and 194.19 are the molecular weights of caffeine citrate
and caffeine, respectively; CS is the concentration, in mg per
Injection. It contains not less than 90.0 per cent mL, of USP Caffeine RS in the Standard solution; CW is the caf-
and not more than 110.0 per cent of the labeled feine citrate concentration, in mg per mL, in the Test solution,
amount of caffeine citrate (C 14H18N4O9). It con- as obtained in the Assay; ri is the individual peak response for
tains no bacteriostat or other preser vative. each related compound obtained from the Test solution; and rS
is the caffeine peak response obtained from the Standard solu-
Packaging and storage—Preserve in single-dose, tight con- tion: not more than 0.10% of any individual related compound
tainers of Type I glass, and store at a temperature between 15 ° is found; and not more than 0.1% of total impurities is found.
and 30°. Other requirements—It meets the requirements under Injec-
tions 〈1〉.
USP Reference standards 〈11〉— Assay—
USP Caffeine RS
USP Endotoxin RS Mobile phase—Prepare a mixture of 0.01 M sodium acetate,
acetonitrile, and tetrahydrofuran (191:5:4). Adjust with glacial
Identification— acetic acid to a pH of 4.5, filter, and degas. Make adjustments
A: The retention time of the major peak in the chromato- if necessary (see System Suitability under Chromatography 〈621〉).
gram of the Assay preparation corresponds to that in the chro- Theophylline solution—Dissolve an accurately weighed quan-
matogram of the Standard preparation, as obtained in the Assay. tity of theophylline in water, and dilute quantitatively, and step-
B: It meets the requirements of the test for Citrate 〈191〉. wise if necessar y, with water, to obtain a solution having a con-
C: Transfer about 4 g of potassium iodide to a 100-mL volu- centration of about 0.02 mg per mL.
metric flask. Add 10 mL of water, and shake until the potassium Standard preparation—Transfer about 5 mg of USP Caffeine
iodide is dissolved. T ransfer 2 g of iodine to the volumetric flask, RS, accurately weighed, to a 25-mL volumetric flask. Add 5 mL
and shake until dissolved. Dilute with water to volume, and of Theophylline solution, dissolve in and dilute with water to vol-
mix. Transfer 5 drops of the solution so obtained to a 25-mL ume, and mix.
centrifuge tube containing 5.0 mL of the Injection, and mix. Assay preparation—Transfer an accurately measured volume
Add 0.5 mL of 2.0 M hydrochloric acid solution, and mix: a of Injection, equivalent to about 50 mg of caffeine, to a 250-
brown precipitate that dissolves on neutralization with 0.5 mL mL volumetric flask. Dilute with water to volume, mix, and pass
of sodium hydroxide TS is produced. through a polyvinylidene difluoride or equivalent membrane
Color and clarity—Transfer a suitable portion of the Injection having a 0.45- µm porosity.
to a clear glass test tube, and visually examine the solution in a Chromatographic system (see Chromatography 〈621〉)—The
well-lighted area: the solution is colorless and free of haze, obvi- liquid chromatograph is equipped with a 275-nm detector and
ous turbidity, and precipitate. a 4.6-mm × 150-cm column that contains 5- µm packing L1.
Bacterial endotoxins 〈85〉: not more than 0.25 USP Endo- The flow rate is about 1 mL per minute. Chromatograph the
toxin Unit per mg of caffeine. Standard preparation, and record the peak responses as directed
Sterility 〈71〉—It meets the requirements when tested as di- for Procedure: the relative retention times are about 0.7 for the-
rected for Membrane Filtration under Test for Sterility of the Prod- ophylline and 1.0 for caffeine; the resolution, R, between theo-
uct to be Examined. phylline and caffeine is not less than 6.0; the tailing factor,
pH 〈791〉: between 4.2 and 5.2. determined from the theophylline and caffeine peaks, is not
Particulate matter 〈788〉: not more than 150 particles are more than 2.0; and the relative standard deviation for replicate
equal to or greater than 10 µm, and not more than 25 particles injections, determined from the caffeine peaks, is not more than
are equal to or greater than 25 µm. 2.0%.
Related compounds— Procedure—Separately inject equal volumes (about 10 µL) of
the Standard preparation and the Assay preparation into the
Mobile phase and Theophylline solution—Proceed as directed chromatograph, record the chromatograms, and measure the
in the Assay. caffeine peak responses. Calculate the quantity, in mg, of caf-
Standard solution—Use the Standard preparation, prepared as feine citrate (C 14H18N4O9) in the volume of Injection taken by
directed in the Assay. the formula:
System sensitivity solution—Transfer 2.5 mL of the Standard
solution to a 100-mL volumetric flask, dilute with water to vol- 250(386.31/194.19)C(rU / rS)
ume, and mix.
Test solution—Use the Assay preparation, prepared as directed in which 386.31 and 194.19 are the molecular weights of caf-
in the Assay. feine citrate and caffeine, respectively; C is the concentration, in
mg per mL, of USP Caffeine RS in the Standard preparation;
Chromatographic system (see Chromatography 〈621〉)—Pro- and rU and rS are the peak responses obtained from the Assay
ceed as directed in the Assay. Chromatograph the System sensi- preparation and the Standard preparation, respectively.
tivity solution, and record the peak responses as directed for

Official from May 1, 2012

Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.