Glycine

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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C₂H₅NO₂ 75.07

Glycine CAS RN^®: 56-40-6.

1 DEFINITION

Glycine contains NLT 98.5% and NMT 101.5% of glycine (C₂H₅NO₂), calculated on the dried basis.

2 IDENTIFICATION

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

3 ASSAY

PROCEDURE

Sample: 150 mg of Glycine

Blank: 100 mL of glacial acetic acid

Titrimetric system

(See Titrimetry (541).)

Mode: Direct titration

Titrant: 0.1 N perchloric acid VS

Endpoint detection: Visual

Analysis: Dissolve the Sample in 100 mL of glacial acetic acid, and add 1 drop of crystal violet TS. Titrate with the Titrant to a green endpoint.

Perform the Blank determination.

Calculate the percentage of glycine (C₂H₅NO₂) in the Sample taken:

Result = {[(V_S - V_B) x N x F]/W} x 100

V_S = Titrant volume consumed by the Sample (mL)

V_B = Titrant volume consumed by the Blank (mL)

N = actual normality of the Titrant (mEq/mL)

F = equivalency factor, 75.07 mg/mEq

W = Sample weight (mg)

Acceptance criteria: 98.5%-101.5% on the dried basis

4 IMPURITIES

4.1 RESIDUE ON IGNITION (281)

NMT 0.1%

4.2 CHLORIDE AND SULFATE (221), Chloride

Standard solution: 0.10 mL of 0.020 N hydrochloric acid

Sample: 1 g of Glycine

Acceptance criteria: NMT 0.007%

4.3 CHLORIDE AND SULFATE (221), Sulfate

Standard solution: 0.20 mL of 0.020 N sulfuric acid

Sample: 3 g of Glycine

Acceptance criteria: NMT 0.0065%

4.4 HYDROLYZABLE SUBSTANCES

Sample solution: 100 mg/mL of Glycine

Analysis: Boil 10 mL of the Sample solution for 1 min, and set aside for 2 h.

Acceptance criteria: The solution appears as clear and as mobile as 10 mL of the same solution that has not been boiled.

Change to read:

4.5 RELATED COMPOUNDS

Solution A: Transfer 2.16 g of octanesulfonic acid sodium salt to a 1000-mL volumetric flask, add 900 mL of HPLC grade water and 2.0 mL of perchloric acid, and mix to dissolve. Adjust with 5 N sodium hydroxide solution to a pH of 2.2. Dilute with HPLC grade water to volume. Pass the solution through a membrane filter of 0.2-µm pore size.

Solution B: Acetonitrile

Mobile phase: Gradient elution. See Table 1.

Table 1

Standard solution: A mixture of 0.005 mg/mL each of USP Glycine RS, USP Diglycine RS, USP Triglycine RS, and glycine anhydride,¹ and 0.0025 mg/mL of monochloroacetic acid² in HPLC grade water. [NOTE-Monochloroacetic acid may be omitted from the Standard solution if the article being tested does not contain this substance.]

Sample solution: Transfer 125 mg of Glycine into a 25-mL volumetric flask, dissolve in and dilute with HPLC grade water to volume.

Blank: HPLC grade water

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 200 nm

Column: 4.6-mm × 15-cm; 3-µm packing L1

Column temperature: 25°

Flow rate: 1 mL/min

Injection volume: 20 µL

System suitability

Samples: Standard solution and Blank

[NOTE-See Table 2 for the relative retention times.]

Suitability requirements

Interference peaks: Compare the chromatogram obtained from the Standard solution with that obtained from the Blank. Any peak area from the Blank that overlaps or co-elutes with the Amino acid peak from the Standard solution is NMT 2.0% of that amino acid peak area.

Resolution: NLT 2.0 between the diglycine and triglycine peaks, Standard solution

Relative standard deviation: NMT 5.0% each for the specified peaks, Standard solution

Analysis

Samples: Standard solution, Sample solution, and Blank

Separately inject the Blank, Standard solution, and Sample solution into the chromatograph. Compare the chromatogram from the Sample solution with that from the Blank. Disregard any peak observed in both the Sample solution and the Blank. Identify the amino acid impurities in the Sample solution by comparing with those specified in the Standard solution.

Separately calculate the percentage of each specified impurity in the portion of Glycine taken:

Result = (r_U/r_S) x (C_S/C_U) x 100

r_U = peak response of glycine anhydride, monochloroacetic acid, diglycine, or triglycine from the Sample solution

r_S = peak response of glycine anhydride, monochloroacetic acid, diglycine, or triglycine from the Standard solution

C_S = concentration of glycine anhydride, monochloroacetic acid, USP Diglycine RS, or USP Triglycine RS in the Standard solution (mg/mL)

C_U = concentration of Glycine in the Sample solution (mg/mL)

Separately calculate the percentage of iminodiacetic acid, hexamethylenetetramine, and any unspecified impurity in the portion of Glycine taken:

Result = (r_U/r_S) x (C_S/C_U) x 100

r_U = peak response of iminodiacetic acid, hexamethylenetetramine, or any unspecified impurity from the Sample solution

r_S = peak response of glycine from the Standard solution

C_S = concentration of USP Glycine RS in the Standard solution (mg/mL)

C_U = concentration of Glycine in the Sample solution (mg/mL)

Acceptance criteria: See Table 2. [NOTE-Disregard any impurity peak less than 0.05%.]

Table 2

5 SPECIFIC TESTS

LOSS ON DRYING (731)

Analysis: Dry at 105° for 2 h.

Acceptance criteria: NMT 0.2%

6 ADDITIONAL REQUIREMENTS

6.1 PACKAGING AND STORAGE

Preserve in well-closed containers at room temperature.

6.2 USP REFERENCE STANDARDS (11)

USP Diglycine RS

USP Glycine RS

USP Triglycine RS

¹ Analytical grade with purity NLT 99.0%.

² Analytical grade with purity NLT 99.0%.