USP 36 Official Monographs / Pentobarbital 4735

Mobile phase—Prepare a mixture of 1600 mL of water,

Pentobarbital
.

40 mL of glacial acetic acid, 30.4 mL of 0.5 M dodecyl-

triethylammonium phosphate, and 20 mL of Cupric acetate
solution. Adjust with 1 N sodium hydroxide to a pH of 4.0,
dilute with water to obtain 2000 mL of solution, filter
through a filter having a 0.5-µm or finer porosity, and de-
gas. Make adjustments if necessary (see System Suitability
under Chromatography 〈621〉).
Stock standard solution—Transfer about 50 mg of nitrilo-
triacetic acid, accurately weighed, to a 100-mL volumetric C11H18N2O3 226.27
flask, dilute with Cupric acetate solution to volume, and mix. 2,4,6(1H,3H,5H)-Pyrimidinetrione, 5-ethyl-5-(1-methylbu-
Standard solution—Transfer 1.0 mL of the Stock standard tyl)-, (±)-;
solution to a 25-mL volumetric flask, dilute with Cupric ace- (±)-5-Ethyl-5-(1-methylbutyl)barbituric acid [76-74-4].
tate solution to volume, and mix. This solution contains
about 0.02 mg of nitrilotriacetic acid per mL. DEFINITION
Test solution—Transfer about 2 g of Pentetic Acid, accu- Pentobarbital contains NLT 98.0% and NMT 102.0% of
rately weighed, to a 100-mL volumetric flask. Add about C11H18N2O3, calculated on the dried basis. Where the ma-
70 mL of Cupric acetate solution, and swirl to dissolve. Soni- terial is labeled as intended solely for veterinary use, Pen-
cate, if necessary, to dissolve. Dilute with Cupric acetate solu- tobarbital contains NLT 97.0% and NMT 102.0% of
tion to volume, and mix. C11H18N2O3, calculated on the dried basis.
Resolution solution—Transfer 1.0 mL of the Stock standard IDENTIFICATION
solution to a 25-mL volumetric flask, dilute with Test solution • A. INFRARED ABSORPTION 〈197S〉
to volume, and mix. Sample solution: 7 in 100
Chromatographic system (see Chromatography 〈621〉)—The Medium: Chloroform
liquid chromatograph is equipped with a 290-nm detector • B. The retention time of the major peak of the Sample
and a 4.6-mm × 25-cm column that contains 5-µm packing solution corresponds to that of the Standard solution, as
L1 that has been highly deactivated (carbon loading of obtained in the Assay.
about 30%). The flow rate is about 1 mL per minute. Equili-
brate the column by passing, in sequence, water, methanol, ASSAY
and water for about 15 minutes each, and then Mobile • PROCEDURE
phase for about 45 minutes. Chromatograph the Resolution Mobile phase: 0.01 M monobasic potassium phosphate
solution, and record the peak responses as directed for Pro- and acetonitrile (65:35). Adjust the pH to 3.5.
cedure: the resolution, R, between pentetic acid and nitrilo- Standard solution: 0.1 mg/mL of USP Pentobarbital RS
triacetic acid is not less than 2.0, and the relative retention in Mobile phase
times are about 0.6 for pentetic acid and 1.0 for nitrilo- Sample stock solution: 1 mg/mL of Pentobarbital in
triacetic acid. Chromatograph the Standard solution, and re- Mobile phase (sonicate until dissolved)
cord the peak responses as directed for Procedure: the rela- Sample solution: Transfer 10.0 mL of the Sample stock
tive standard deviation for replicate injections is not more solution to a 100-mL volumetric flask, and dilute with
than 5.0%. Mobile phase to volume.
Procedure—Separately inject equal volumes (about 20 µL) Chromatographic system
of the Standard solution and the Test solution into the chro- (See Chromatography 〈621〉, System Suitability.)
matograph, and measure the responses for the major peaks. Mode: LC
Calculate the percentage of nitrilotriacetic acid in the por- Detector: UV 214 nm
tion of Pentetic Acid taken by the formula: Column: 4.6-mm × 25-cm; 5-µm packing L1
Flow rate: 1 mL/min
10,000(C / W)(rU / rS) Injection size: 10 µL
System suitability
of which C is the concentration, in mg per mL, of nitrilo- Sample: Standard solution
triacetic acid in the Standard solution, W is the weight, in Suitability requirements
mg, of Pentetic Acid taken to prepare the Test solution, and Column efficiency: NLT 15,000 theoretical plates
rU and rS are the nitrilotriacetic acid peak responses obtained Tailing factor: NMT 1.5
from the Test solution and the Standard solution, respectively. Relative standard deviation: NMT 2.0% for
The limit is 0.1%. pentobarbital
Iron—Using 1.5 g of specimen, proceed as directed in the Analysis
test for Iron under Edetic Acid. The color of the test solution Samples: Standard solution and Sample solution
is not deeper than that of the solution containing the stan- Calculate the percentage of C11H18N2O3 in the portion
dard iron solution (0.01%). of Pentobarbital taken:
Assay—Transfer about 200 mg of Pentetic Acid, accurately Result = (rU/rS) × (CS/CU) × 100
weighed, to a 125-mL conical flask, add 50 mL of water and
1.5 mL of 1 N sodium hydroxide, and swirl to dissolve the rU = peak area from the Sample solution
specimen. Add 10 mL of 0.1 N ammonium thiocyanate, and rS = peak area from the Standard solution
mix. Add about 40 mL of methyl ethyl ketone, mix, and CS = concentration of USP Pentobarbital RS in the
allow the layers to separate. Titrate with 0.05 N ferric am- Standard solution (mg/mL)
monium sulfate VS, stirring continuously. As the titration CU = concentration of Pentobarbital in the Sample
proceeds, the aqueous phase turns from colorless to yellow, solution (mg/mL)
and the organic phase remains colorless. As the endpoint is Acceptance criteria: 98.0%–102.0% of C11H18N2O3 on
approached, stop the titration, mix, and allow the layers to the dried basis; and 97.0%–102.0% of C11H18N2O3 on
separate. Add 0.1-mL increments of 0.05 N ferric ammo- the dried basis, where the material is labeled as in-
nium sulfate VS, mixing and allowing the layers to separate tended solely for veterinary use
after each addition, until the organic layer turns from color-
less to pink. Each mL of 0.05 N ferric ammonium sulfate
consumed is equivalent to 19.668 mg of C14H23N3O10.
4736 Pentobarbital / Official Monographs USP 36

IMPURITIES ADDITIONAL REQUIREMENTS

Inorganic Impurities • PACKAGING AND STORAGE: Preserve in tight containers.
• RESIDUE ON IGNITION 〈281〉: NMT 0.1% • USP REFERENCE STANDARDS 〈11〉
• HEAVY METALS, Method II 〈231〉: NMT 20 ppm USP Pentobarbital RS
Organic Impurities
• PROCEDURE
Mobile phase: Prepare as directed in the Assay.
Standard solution: 0.001 mg/mL of USP Pentobarbital
RS in Mobile phase
Pentobarbital Sodium
.

Sample solution: 1 mg/mL of Pentobarbital in Mobile

phase C11H17N2NaO3 248.25
Chromatographic system 2,4,6(1H,3H,5H)-Pyrimidinetrione, 5-ethyl-5-(1-methyl-
(See Chromatography 〈621〉, System Suitability.) butyl)-, monosodium salt.
Mode: LC Sodium 5-ethyl-5-(1-methylbutyl)barbiturate [57-33-0].
Detector: UV 214 nm
Column: 4.6-mm × 25-cm; 5-µm packing L1 » Pentobarbital Sodium contains not less than
Flow rate: 1 mL/min 98.0 percent and not more than 102.0 percent of
Injection size: 10 µL C11H17N2NaO3, calculated on the dried basis.
System suitability
Sample: Standard solution Where the material is labeled as intended solely
Suitability requirements for veterinary use, Pentobarbital Sodium contains
Column efficiency: NLT 15,000 theoretical plates not less than 97.0 percent and not more than
Tailing factor: NMT 1.5 102.0 percent of C11H17N2NaO3, calculated on
Relative standard deviation: NMT 15.0% for the dried basis.
pentobarbital
Analysis Packaging and storage—Preserve in tight containers.
Samples: Standard solution and Sample solution
Calculate the percentage of any impurity in the por- USP Reference standards 〈11〉—
tion of Pentobarbital taken: USP Pentobarbital RS
Completeness of solution—Mix 1.0 g with 10 mL of car-
Result = (rU/rS) × (CS/CU) × (1/F) × 100 bon dioxide-free water: after 1 minute, the solution is clear
and free from undissolved solid.
rU = peak area for any impurity from the Sample Identification—
solution A: Ultraviolet Absorption 〈197U〉—
rS = peak area for pentobarbital from the
Standard solution Solution: 10 µg per mL.
CS = concentration of USP Pentobarbital RS in the Medium: dilute ammonium hydroxide (1 in 200).
Standard solution (mg/mL) B: The retention time of the major peak in the chromato-
CU = concentration of Pentobarbital in the Sample gram of the Assay preparation corresponds to that in the
solution (mg/mL) chromatogram of the Standard preparation, as obtained in
F = relative response factor of the impurity (see the Assay.
Impurity Table 1) C: Ignite about 200 mg: the residue effervesces with
Acceptance criteria: See Impurity Table 1. acids, and meets the requirements of the tests for Sodium
〈191〉.
Impurity Table 1 pH 〈791〉: between 9.8 and 11.0, in the solution prepared
Relative Relative Acceptance in the test for Completeness of solution.
Retention Response Criteria, Loss on drying 〈731〉—Dry it at 105° for 6 hours: it loses
Name Time Factor NMT (%) not more than 3.5% of its weight.
6-Imino-5-ethyl-5- Heavy metals, Method II 〈231〉: 0.003%.
(1-methyl butyl) Related compounds—
barbituric acid 0.39 1.5 0.2
Mobile phase—Prepare as directed in the Assay.
5-Ethyl-5-(1-ethyl-
propyl) barbituric
Standard solution—Dissolve an accurately weighed quan-
acida 0.93 1.0 0.1
tity of USP Pentobarbital RS in Mobile phase, and dilute
quantitatively, and stepwise if necessary, with Mobile phase
Pentobarbital 1.0 — — to obtain a solution having a known concentration of about
5-Ethyl-5-(1,3- 0.001 mg per mL.
dimethylbutyl)
Test solution—Transfer about 110 mg of Pentobarbital So-
barbituric acid 1.5 0.9 0.3
dium, accurately weighed, to a 100-mL volumetric flask,
Unknown impurities — 1.0 0.1 add about 80 mL of Mobile phase, and sonicate until dis-
Total — — 0.5 solved. Dilute with Mobile phase to volume, and mix.
a Where the material is labeled as intended solely for veterinary use, the Chromatographic system (see Chromatography 〈621〉)—The
limit of 5-ethyl-5-(1-ethylpropyl) barbituric acid is 3.0%. liquid chromatograph is equipped with a 214-nm detector
SPECIFIC TESTS and a 4.6-mm × 25-cm column that contains 5-µm packing
• LOSS ON DRYING 〈731〉: Dry a sample at 105° for 2 h: it L1. The flow rate is about 1.0 mL per minute. Chromato-
loses NMT 1.0% of its weight. graph the Standard solution, and record the peak responses
as directed for Procedure: the capacity factor, k′, is not less
than 2.5; the column efficiency is not less than 15,000 theo-
retical plates; the tailing factor is not more than 1.5; and
the relative standard deviation for replicate injections is not
more than 15.0%.
Procedure—Separately inject equal volumes (about 10 µL)
of the Standard solution and Test solution into the chromato-
graph, record the chromatograms, and measure the areas