Urea C 13

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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(¹³CH₄N₂O) 61.05

Urea [13C] CAS RN: 58069-82-2.

1 DEFINITION

Urea C 13 contains NLT 98.0% and NMT 102.0% of urea C 13 (¹³CH₄N₂O).

2 IDENTIFICATION

A. SPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscopy: 197A or 197K

B. The intensity of the major peak with mass-to-charge (m/z) ratio of 190 in the Sample solution corresponds to that of the System suitability solution, as obtained in the test for Isotopic Purity.

3 ASSAY

PROCEDURE

Mobile phase: Acetonitrile, methanol, and water (89:10:1)

System suitability solution: 2.5 mg/mL of USP Urea C 13. RS and 0.003 mg/mL of biuret in Mobile phase

Standard solution: 2 mg/mL of USP Urea C 13 RS in Mobile phase

Sample solution: 2 mg/mL of Urea C 13 in Mobile phase

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 200 nm

Column: 4.6-mm x 25-cm; 5-µm packing L8

Flow rate: 0.8 mL/min

Injection volume: 20 µL

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 1.5 between urea and biuret, System suitability solution

Relative standard deviation: NMT 1%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of urea C 13 (¹³CH₄N₂O) in the portion of Urea C 13 taken:

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

r_u = peak response of urea C 13 from the Sample solution

r_s = peak response of urea C 13 from the Standard solution

C_s = concentration of USP Urea C 13 RS in the Standard solution (mg/mL)

C_u = concentration of Urea C 13 in the Sample solution (mg/mL)

Acceptance criteria: 98.0%–102.0%

4 IMPURITIES

Residue on Ignition 〈281〉: NMT 0.1%

4.1 Limit of Biuret

Standard solution: 0.033 mg/mL of biuret in water

Sample solution: 33.3 mg/mL of Urea C 13 in water

Analysis: Treat 3 ml. of the Sample solution and 3 mL of the Standard solution separately as follows. To each solution add 3 mL of sodium hydroxide solution (10 in 100) and 3 drops of copper sulfate solution (0.5 in 100), and allow to stand for 5 min.

Acceptance criteria: NMT 0.1%; any reddish-violet color in the Sample solution is not more intense than that in the Standard solution.

Change to read:

4.2 ISOTOPIC PURITY

Control stock solution: 2.0 mg/mL of USP Urea RS in dimethylformamide

Control solution: Transfer 0.5 mL of the Control stock solution to a vial. Add 0.5 mL of bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (TMS) derivatizing agent. Cap the vial and shake about 10 times. Heat at 90-110° on a heating block for about 60 min.

System suitability stock solution: 2.0 mg/mL of USP Urea C 13 RS in dimethylformamide

System suitability solution: Transfer 0.5 mL of the System suitability stock solution to a vial. Add 0.5 mL of TMS derivatizing agent. Cap the vial and shake about 10 times. Heat at 90°-110° on a heating block for about 60 min.

Sample stock solution: 2.0 mg/mL of Urea C 13 in dimethylformamide

Sample solution: Transfer 0.5 mL of the Sample stock solution to a vial. Add 0.5 mL of TMS derivatizing agent. Cap the vial and shake about 10 times. Heat at 90-110° on a heating block for about 60 min.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: GC

Detector: Mass spectrometer, positive ionization

Monitoring mode: Selected ion monitoring set up to monitor all ions individually in the m/z ratio range of 188-193

Solvent delay: Suitable time. [NOTE-Time may vary depending on the instrument and manufacturer's instructions.]

Column: 0.25-mm x 30-m capillary; coated with a 0.25-µm film of phase G27

Temperatures

Detector: 280°

Injection port: 280°

Column: See Table 1.

Table 1

Carrier gas: Helium

Flow rate: 1 mL/min _{(ERR 1-Sep-2023)}

Injection volume: 1 μL

Injection type: Split, split ratio 16:1

System suitability

Samples: Control solution and System suitability solution

Suitability requirements

Most abundant ion: Fragment at m/z 189, Control solution

Relative standard deviation: NMT 5%, Control solution

Most abundant ion: Fragment at m/z 190, System suitability solution

Relative standard deviation: NMT 5%, System suitability solution

Carbon-13: NLT 99% in USP Urea C 13 RS, System suitability solution

Analysis

Samples: Control solution, System suitability solution, and Sample solution

Inject each solution in triplicate. [Note—Calculate the average responses of all the fragments between m/z 188 and m/z 193 from triplicate injections of each solution.]

Carbon-13 enrichment calculation

Calculate the contribution of carbon-13 (C) due to natural abundance to the response of derivatized Urea C 13:

C = (A × D)/E

A = average response of the m/z 190 fragment in urea C 13, System suitability solution or Sample solution

D = average response of the m/z 188 fragment in urea, Control solution

E = average response of the m/z 189 fragment in urea, Control solution

Calculate the percentage of carbon-13 enrichment in USP Urea C 13 RS and Urea C 13:

Result = [A/(A + B − C)] × 100

A = average response of the m/z 190 fragment in urea C 13, System suitability solution or Sample solution

B = average response of the m/z 189 fragment in urea C 13, System suitability solution or Sample solution

C = contribution due to natural abundance of carbon-13, System suitability solution or Sample solution

Oxygen-18 enrichment calculation

Step 1: Determine the measured mass of urea TMS in each injection of the Control solution:

Result = (Ʃ M_i × r_i)/Ʃr_i

M_i = mass of the fragment for m/z 189, 190, 191, 192, and 193

r_i = response of the corresponding m/z fragment

Calculate the average measured mass (M ) of urea TMS derivative.

Determine the mass correction factor (Δ):

Result = M_c,ave − M_c,theor

M_c,ave = average mass of urea TMS derivative

M_c,theor = theoretical mass of urea TMS derivative, 189.385

Step 2: Determine the measured mass of urea C 13 TMS in each injection of the Sample solution:

Result = (Ʃ M_i × r_i)/Ʃr_i

M_i = mass of the fragment for m/z 189, 190, 191, 192, and 193

r_i = response of the corresponding m/z fragment

Calculate the average measured mass (M ) of the urea C 13 TMS derivative characteristic ion from the triplicate injections of the

Sample solution.

Step 3: Calculate the measured molecular weight (M ) of urea C 13 in the Sample solution:

Result = M_u,ave − Δ − M_TMS

M_u,ave = average mass of urea C 13 TMS derivative in the Sample solution from Step 2

Δ = correction factor from Step 1

M_TMS = theoretical mass of the Si C H fragment, 129.329

Step 4: Calculate the average mass of oxygen (O ) in the Sample solution:

Result = M_U − M_T

M_U = average mass of urea C 13 in the Sample solution from Step 3

M_T = theoretical mass of the ¹³CN₂H₄ fragment, 45.044

Step 5: Calculate the percentage of enrichment of oxygen-18 in the Urea C 13 taken:

Result = [(O_u − A₁ )/(A₂ − A₁ )] × 100

O_u = average mass of oxygen in the Sample solution from Step 4

A₁ = theoretical mass of the natural isotope of oxygen, 15.995

A₂ = theoretical mass of the oxygen-18 isotope, 17.999

Acceptance criteria

Carbon-13: NLT 99%

Oxygen-18: NMT 15%

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers at room temperature.

USP Reference Standards 〈11〉

USP Urea RS

USP Urea C 13 RS